CPA/600/R-01/027
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NATIONAL CENTER FOR ENVIRONMENTAL RESEARCH
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2001 STAR Drinking Water Progress Review Workshop
Table of Contents
Introduction vii
Section 1. Chemical Contaminants: Exposure, Effects, & Assessment
Development of Biomarkers for Haloacetonitriles-Induced Cell Injury in Peripheral Blood 3
Ahmed Elsayed Ahmed
Metabolic Fate of Halogenated Disinfection Byproducts In Vivo and Relation to Biological Activity 5
L.M. Ball
Genotoxicity and Occurrence Assessment of Disinfection Byproducts in Drinking Water 6
Roger A. Minear, MichaelJ. Plewa
Assessment of Human Dietary Ingestion Exposures to Water Disinfection Byproducts via Food 8
James H. Raynier, E.D. Pe/lizzari, Y. Hu, B. Chi Ids, K. Briggs, H. Weinberg, V. Unnam
Bioavailability of Haloacetates in Human Subjects 10
Irvin R. Schultz, Robert Shangraw, Torka Poet
Physiologically Based Pharmacokinetic Modeling of Haloacid Mixtures in Rodents and Humans 12
Irvin R. Schultz, Robert D. Stenner, Richard J. Bull
Inhalation and Dermal Exposure to Disinfection Byproducts of Chlorinated Drinking Water 14
Clifford P. Weisel, Jeffrey Laskin
NHEERL Research on Carcinogenic Contaminants in Drinking Water 16
Douglas C. Wolf
Aluminum Toxicokinetics: Oral Absorption From Drinking Water and Brain Retention 17
Robert A. Yokel, Patrick J. McNamara, David Elmore
Section 2. Chemical Contaminants: Arsenic
Overview of EPA's Arsenic in Drinking Water Regulation 21
Irene S. Dooley
Arsenic-Glutathione Interactions and Skin Cancer 23
Catherine B. Klein, Elizabeth T. Snow
A Dose-Response and Susceptibility Investigation of Skin Keratoses and Hyperpigmentation
Caused by Arsenic in Drinking Water 25
Allan H. Smith, Reina Haque, Joyce Chung, Lee Moore, D.N. Guha Mazumder,
Binay K. De, Nilima Ghosh, Soma Mitra, David Kalman
Trivalent Methylated Arsenicals: Novel Biomarkers of Arsenic Toxicity in Humans 26
Miroslav Styblo, Luz M. Del Razo, Shan Lin, William R. Cullen, David J. Thomas
The Office of Research and Development's National Center for Environmental Research '"
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2001 STAR Drinking Water Progress Review Workshop
Section 3. Chemical Contaminants: Formation of DBFs
Use of Differential Spectroscopy To Study DBF Formation Reactions 31
Mark M. Benjamin, Oregon' V. Korshin
Brominated DBF Formation and Speciation Based on the Specific UV Absorbance
Distribution of Natural Waters 32
James E. Kilduff, Tanju Karanfil
Molecular Weight Separation and HPLC/MS/MS Characterization of Previously
Unidentified Drinking Water Disinfection Byproducts 35
Roger A. Minear, Sylvia Barrett
Membrane Introduction Mass Spectrometry Studies of Halogenated Cyano
Byproduct Formation in Drinking Water 37
Terese M. Olson
Mechanisms and Kinetics of Chloramine Loss and Byproduct Formation in the Presence
of Reactive Drinking Water Distribution System Constituents 39
Richard L. Valentine, Junghoon Choi, Steve Duirk
Development of a New, Simple, Innovative Procedure for the Analysis of Bromate
and Other Oxy-Halides at Sub-ppb Levels in Drinking Water 41
Howard S. Weinberg, Carrie Delcomyn
Formation and Stability of Ozonation Byproducts in Drinking Water 42
Howard S. Weinberg, Alice Harris, Shikha Bhatnagar
Kinetic-Based Models for Bromate Formation in Natural Waters 44
Paul Westerhoff
Mechanistic-Based Disinfectant and Disinfectant Byproduct Models 46
Paul Westerhoff, David Reckhow, Gary Amy, Zaid Chowdhwy
Section 4. Drinking Water Treatment Studies
Evaluation of the Efficacy of a New Secondary Disinfectant Formulation Using Hydrogen Peroxide
and Silver and the Formulation of Disinfection Byproducts Resulting
From Interactions With Conventional Disinfectants 51
Stuart A. Batterman, Khalil H. Mancy, Shuqin Wang, Lianzhong Zhang, James Warila,
Ovadia Lev, Mil lei Shuval, Badri Fattal, An-Tsun Huang
Evaluation of Ozone Byproduct Formation Under Ozone Dose, Temperature, and pH Variation 55
Michael S. Elovitz, Jehng-Jyun Yao, Dick J. Miltner
Integrated Approach for the Control of Cryptosporidium parvum Oocysts and Disinfection
Byproducts in Drinking Water Treated With Ozone and Chloramines 57
Benito J. Marinas, Roger A. Minear, Jaehong Kim, Hongxia Lei, Jason L. Rennecker,
Amy M. Driedger, Benito Corona-Vasquez
iv The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Pilot Studies of an Ozonation/FBT Process for the Control of DBPs in Drinking Water 59
Susan J. Masten, Kyung-Hyuk Lee. Kuan-Chung Chen, Alexander A. Yavich
Section 5. Microbial Contaminants
Prevalence and Distribution of Genotypes of Cryptosporidium parvitm in United States Feedlot Cattle 63
Edward R. Atwill, C. Elmi, W.P. Epperson, DM. Grotelueschen, J. Kirkpatrick.
B. Hoar, W.M. Sischo, L.V. Carpenter, D. Brewster, W. Riggs
Development of Detection and Viability Methods for Waterborne Microsporidia
Species Known To Infect Humans 64
David A. Battigelli, MM. Marshall, M. Borchardt
Meaningful Detection of Mycobacterium avium Complex in Drinking Water:
Are Disinfectant-Resistant Morphotypes Virulent? 65
Gerard Cangelosi, Christine Palermo, Shawn Faske
Detection of Emerging Microbial Contaminants in Source and Finished
Drinking Water With DNA Microarrays 67
Darrell P. Chandler, Ricardo DeLeon, Timothy M. Straub
Infectivity and Virulence of Cryptosporidium Genotype 1/H Oocysts in Healthy Adult Volunteers 68
Cynthia L. Chappell, Pablo C. Okhuysen, Saul Tzipori, Giovanni Widmer
Mycobacterium avium Complex in Drinking Water: Detection, Distribution, and Routes of Exposure 70
Timothy E. Ford, Anand Patel, Yu-Rong Chu
NERL Microbial Program With Emphasis on Protozoan Methods 73
H.D. Alan Lindquist
Studies of the Infectivity of Norwalk and Norwalk-Like Viruses 74
Christine L. Moe, Lisa Lindesmith, Ralph S. Baric, Paul Stewart,
William Heizer, Jeffrey A. Frelinger
Development and Evaluation of Procedures for Detection of Infectious Microsporidia in Source Waters 76
Paul A. Rochelle
Attachment and Inactivation During Virus Transport in Ground Water 78
Joseph N. Ryan, Menachem Elimelech, Ronald W. Harvey
Molecular Detection of Vegetative and Coccoid Forms of H. pylori 80
Manoucher Shahamat, C. Povlick, J. Hind, F. Robb, K. Sowers, M. Levin, B. Bradley
Detection and Occurrence of Human Caliciviruses in Drinking Water 81
Mark D. Sobsey
Development and Evaluation of Methods for the Concentration, Separation, and Detection
of Three Protozoa From Large Volumes of Water 83
Saul Tzipori, Udi Zuckerman, Michael Buckholt, Giovanni Widmer, Abhineet Sheoran
Risk Factors for Cryptosporidium parvum Infection and Disease 84
Lucy A. Ward, Y. Wang, S. Pereira, S. Clarke
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Development of a Rapid, Quantitative Method for the Detection of Infective
Coxsackie and Echo Viruses in Drinking Water 85
Marylynn V. Yates, W. Chen, A. Mulchandani
Section 6. Contaminant Sources
Molecular Tracers of Contaminant Sources to Surface Water Drinking Supplies 89
Laurel J. Standley, Louis A. Kaplan, J. Denis Newbold
Appendix: STAR Drinking Water Progress Review Workshop Agenda 93
Index of Authors 99
vi The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Introduction
The mission of the U.S. Environmental Protection Agency (EPA) is to protect public health and to safeguard
and improve the natural environmentCair, water, and land. Achieving this mission requires applying sound science
to assessing environmental problems and evaluating possible solutions. The National Center for Environmental
Research (NCER) at EPA is committed to providing the best products in high-priority areas of scientific research
through significant support for long-term research.
One high-priority research program identified by EPA's Office of Research and Development (ORD) is
Drinking Water. The Safe Drinking Water Act (SDWA) was originally passed in 1974 to protect public health by
regulating the Nation's public drinking water supply. The law was amended in 1986 and 1996. The 1996 amend-
ments greatly enhanced the existing law by recognizing source water protection, operator training, funding for water
system improvements, and public information as important components of safe drinking water. This approach
ensures the quality of drinking water by protecting it from source to tap. The SDWA applies to every public water
system in the United States, and the responsibility for making sure these public water systems provide safe drinking
water is divided among EPA, states, tribes, water systems, and the public.
Research described in this progress review was funded through EPA's Science to Achieve Results (STAR)
Program, which is managed by NCER. Grants were awarded through an open, peer-reviewed competition of pro-
posals submitted in response to Requests for Application (RFA) for each of the years from 1997 to 1999. The
research being supported by these grants, along with work conducted in the EPA laboratories and other outside
research institutions, is addressing key research questions identified in the Research Plan for Arsenic in Drinking
Water, the Research Plan for Microbial Pathogens and Disinfection By-Products in Drinking Water (both available
at http://www.epa.gov/ORD/WebPubs/final/), and research on drinking water contaminants identified as priorities
for additional research (the Contaminant Candidate List, CCL).
This progress review provided an opportunity for investigators to interact with one another and to discuss the
progress and findings of their research with EPA and other interested parties. If you have any questions regarding
the program, please contact the program manager, Cynthia Nolt-Helms, at 202-564-6763 or nolt-helms.cynthia@
epamail.epa.gov.
The Office of Research and Development's National Center for Environmental Research vu
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Section 1.
Chemical Contaminants
Exposure,
Effects, & Assessment
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2001 STAR Drinking Water Progress Review Workshop
Development of Biomarkers
for Haloacetonitriles-Induced Cell Injury in Peripheral Blood
Ahmed Elsayed Ahmed
The University of Texas Medical Branch, Galveston, TX
Drinking waters are contaminated with a mixture
of halogenated hydrocarbons that are disinfection by-
products. Among these are a number of toxic and car-
cinogenic halogenated acetonitriles (HANs)^that are
known to induce a variety of acute and chronic adverse
effects in humans and laboratory animals. The goal of
this project is to develop unique biomarkers, in a
readily accessible compartment such as blood, for HAN
exposure and HAN-induced cell injury. This injury may
result from HAN-induced alkylative or oxidative dam-
age to cellular macromolecules. To evaluate the mech-
anism of HAN-induced DNA damage, the effect of
HANs on the conversion of native supercoiled plasmid
DNA to circular or linear forms was quantified electro-
phoretically. The results indicated HAN-induced oxida-
tive DNA damage in vitro. H2O2 mediation of dibromo-
acetonitrile (DBAN) induced DNA damage is a possi-
ble mechanism of HAN genotoxicity.
In a dose-response and time course study, fibro-
blasts were exposed to various concentrations of the
water disinfection byproduct, DBAN. The results indi-
cate that there was a concentration and time-dependent
depletion of cellular antioxidants, while markers for
oxidative stress and cellular damage were significantly
increased (see Figure 1). The effect progressed and was
followed by an increase in membrane damage and cell-
ular death. Treated cells exhibited features of apoptosis
at low exposure levels and severe membrane damages
and necrosis at higher concentrations of the chemical.
The ability of fibroblasts to withstand DNA dam-
age induced by DBAN was evaluated by determining
the magnitude of DNA repair processes in the cell using
a base excision repair (BER) assay. BER was upregu-
lated in cells exposed to low concentrations of DBAN,
and was downregulated at higher concentrations and
longer exposure periods (see Figure 2). These studies
indicate that DBAN activates oxidative stress. Depend-
ing on its magnitude, oxidative stress signals induce up-
or downregulation of gene expression of BER enzymes.
The existence of human diseases associated with im-
paired DNA repair graphically illustrates the impor-
tance of these processes of quality control. Chemically
induced disruption of BER might be found in humans,
and may lead to a decrease in defense mechanisms
against various types of carcinogenic DNA damage.
Currently, the goal is to closely investigate the
sequence of the signals leading to the regulation of
BER induced by the drinking water disinfectant by-
products and how to intervene in their progression.
These signals will constitute an array of mechanism-
based biomarkers for HAN exposure and effects. Future
studies will focus on the characterization and quanti-
tative determination of oxidative and alkylative dam-
ages to cellular macromolecules following inhalation
exposure of animals to HANs. These studies should
provide a basis for the development of regulatory
guidelines and policies governing the tolerance levels
for chronic human exposure to HANs.
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
04
•«
OJ
•*H
O 03.
£
M
if*
•—
f-f
. 2hr
• 1 hr
0 25 50 75 100 125 150 175 200 225
Control EtOH 10 20
120 240
DBAN (fiM)
Time (Min.)/100 MM
Figure 1. Effect of dose-response and time on DBAN-mduced lipid peroxidation
Time Course
lit 20 mi».
iiM& JR. es|i
10
Figure 2. Effect of DBAN on DNA base excision repair.
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Metabolic Fate of Halogenated Disinfection
Byproducts In Vivo and Relation to Biological Activity
L.M. Ball
University of North Carolina at Chapel Hill, Chapel Hill, NC
Halogenated byproducts of drinking water disinfec-
tion are of concern because of their widespread human
consumption and the current uncertainty over their
health effects. Haloacids as a class are the most abun-
dant of the halogenated disinfection byproducts. Many
of these compounds are suspect carcinogens, and in
addition, may form macromolecular adducts that could
be useful as biomarkers of exposure to disinfectants.
This project attempts to elucidate mechanisms of bio-
logical activity and disposition of haloacids.
The initial hypothesis was that dihaloacetic acids
could be metabolically activated to genotoxic interme-
diates by a glutathione-dependent enzyme pathway, and
that tricyclic adducts would be formed on the DNA
bases. The goals were to: (1) use the Ames plate incor-
poration assay to measure the production of genotoxic
species in the form of mutations, and manipulate the
metabolic activation conditions to favor glutathione
conjugation, cytochrome P450-linked oxidation, or
NAD+-dependent oxidation, by addition of the appro-
priate cofactors, alone or in combination, to define the
metabolic pathway(s) that produce genotoxic products,
with the intent of using that information to prioritize the
synthesis of potential DNA adducts towards those
molecules that were plausibly produced by pathways
linked to mutagenicity; (2) synthesize such potential
DNA adducts; and (3) investigate the formation of
DNA adducts following treatment with haloacids in
vivo.
Evidence was obtained from mutagenicity assays
that glutathione-mediated pathways did not lead to
mutagenic products from dichloroacetic acid—in fact,
they were more likely to enhance the toxicity of this
compound. However, NAD+ enhanced the mutagenicity
of dichloroacetic acid, hypothetically by acting as co-
factor for oxidation of an intermediate alcohol to a
carboxyl group. This conclusion was investigated to see
whether it held true for the brominated analogues. The
purpose at this stage was to prioritize the different
haloacetic acids for in vivo metabolic studies; prefer-
ence was given to the most mutagenic compounds for
investigation of potential in vivo DNA adduct forma-
tion.
The brominated haloacids each exhibited some
interesting features. Monobromoacetic acid clearly was
toxic to the bacteria, because few or no colonies (either
revertant or background) were able to grow at the two
highest doses, and this toxicity was mitigated when
glutathione was included in the assay mix. NAD+ did
not appear to have any effect on plate counts with or
without glutathione. With dibromoacetic acid, plate
counts increased at low doses, just barely reached a
doubling of the background rate, and fell off at the
highest dose. A similar pattern was seen when gluta-
thione was added alone. In the presence of NAD+,
either alone or in combination with glutathione, no in-
crease in plate counts was seen, or was toxicity
especially marked. The only compound of those tested
to exhibit a clear mutagenic response was the mixed
haloacid, bromochloroacetic acid (BCA). BCA more
than doubled the background rate, consistently, either
alone or in the presence of NAD*. This increase in plate
counts was not observed when glutathione was added,
either alone or in combination with NAD+. Synthesis of
putative DNA adducts has been unproductive to date
(results not shown).
The original hypothesis developed was that a
glutathione-dependent pathway could contribute to
formation of genotoxic metabolites from dihaloacetic
acids. The results presented here confirm earlier find-
ings that the presence of glutathione does not enhance
the mutagenicity of haloacetic acids in the Ames plate
incorporation assay. However, while glutathione ap-
peared to increase the toxicity of dichloroacetic acid,
for the brominated species bromoacetic acid and bro-
mochloroacetic acid, the effects of glutathione amount-
ed to detoxication, in the form of a decrease in toxicity
for MBA and of mutagenicity for BCA. Thus, inter-
action with glutathione is biologically important for this
class of compounds; the outcome of that interaction
would appear to differ depending on the nature of the
halogen substituent.
Earlier conclusions that thioether conjugates de-
rived from haloacetic acids are unlikely to contribute
greatly to the genotoxicity of these compounds, and that
synthesis of DNA adducts inferred to be derived from
this route can be assigned a lower priority, remain
unchanged. Nevertheless, sufficient evidence has been
developed to show that glutathione-dependent pathways
play important, and perhaps different, roles in the over-
all biological activity of the haloacetic acids. BCA,
although not the most abundant dihaloacetic acid pro-
duced from drinking water disinfection, would appear
to be the most mutagenic haloacetic acid; hence, it is
expected to offer the highest chance of observing DNA
adducts in any quantity, in addition to the high likeli-
hood that it will form adducts similar to those arising
from dichloroacetic acid.
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Genotoxicity and Occurrence Assessment
of Disinfection Byproducts in Drinking Water
Roger A. Minear and Michael J. Plewa
Department of Civil Engineering, University of Illinois at Urbana-Champaign, Urbana, IL
The objectives of this project were to: (1) calibrate
short-term genotoxicity assays based on mutation in
Salmonella typhimurium and direct DNA damage in
cultured mammalian cells using regulated disinfection
byproducts (DBFs); (2) compare the relative genotoxic-
ities of chlorinated versus brominated DBFs; (3) com-
pare the relative genotoxicities of chlorination byprod-
ucts versus brominated byproducts; (4) compare the rel-
ative genotoxicities of DBFs derived from singular
versus combination (sequential) use of ozonation and
chlorination; and (5) provide a DBF occurrence data-
base for extrapolating genotoxicity results to practice.
This project merged quantitative chemistry and
biology components. A new method was developed to
evaluate brominated and chlorinated DBFs based on an
ion chromatograph system as the detector, instead of a
coulometric titration cell. Gas phase HBr and HC1 that
corresponded to brominated and chlorinated com-
pounds were dissolved in water, and the aqueous sam-
ple was applied to ion chromatography for separation
and quantification.
Comparison among chlorination, chloramination,
and chlorine dioxide treatment of Suwannee River ful-
vic acid with bromide ions showed that the ratio of the
brominated fraction of total organic halogen (TOX) to
the chlorinated fraction of TOX during chlorine dioxide
treatment was much higher than those during chlorina-
tion and chloramination. The cytotoxic and mutagenic
properties of known DBFs were quantitatively compar-
ed using bacterial and mammalian cell systems. A rapid
microplate cytotoxicity assay using S. typhimurium was
developed and calibrated. The cytotoxicity data were
incorporated into the analysis of the genotoxic potency
of each DBF. Selected DBFs were assayed for muta-
genicity in strains TA98, TA100, and RSJ100 under
preincubation test conditions. The rank order of de-
creasing cytotoxicity in TA100 was 3-chloro-4-
(dichloromethyl)-5-hydroxy-2-(5H)-furanone (MX) »
bromoacetic acid (BA) » bromoform (BF) > dibro-
moacetic acid (DBA) » tribromoacetic acid (TEA) >
chloroform (CF) » dimethylsulfoxide. The rank order
of the mutagenic potency of the DBFs from the highest
to lowest was MX >» BA » BF > DBA » dichloro-
acetic acid (DCA) > chloroacetic acid (CA) » TEA ~
trichloroacetic acid (TCA). Rapid, quantitative mam-
malian cell cytotoxicity and genotoxicity assays using
cultured Chinese hamster ovary (CHO) cells also were
developed and calibrated. The CHO cell cytotoxicity
rank order from highest to lowest was bromonitro-
methane (BNM) > dibromonitromethane (DBNM) >
BA > MX > DBA > CA > potassium bromate > TBA >
DCA > TCA. Genotoxicity analyses of the DBFs were
conducted using single-cell gel electrophoresis (SCGE),
which detects genomic DNA damage at the level of the
individual nucleus. Using SCGE genotoxic potency, the
rank order was BA > DBNM > tribromonitromethane
> trichloronitromethane > BNM > MX > CA > di-
chloronitromethane > DBA > ethylmethane-sulfonate
([EMS] positive control) > TBA » TCA ~ DCA (see
Figure 1).
Comparative toxicological analyses of DBFs and
DBF mixtures isolated from disinfected water using a
variety of biological endpoints are important for risk
assessment and assist the formulation of public regula-
tory policies that protect the environment and the public
health. Data to date show that there was no significant
correlation between the relative cytotoxicity and geno-
toxicity data of the DBFs when compared between S.
typhimurium and CHO cell assays. Order ranking and
magnitude of responses of some DBFs are quite differ-
ent between the bacterial and mammalian assays.
Complex DBF mixtures have been isolated from
real waters after chlorination, chloramination, or ozona-
tion. Cytoxicity and genotoxicity analysis of these
samples currently are underway and will be the focus of
the work in the final project year.
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Bromoacetic Acid
Dibromoacetic Acid
Tribromoacetic Acid
Chloroacetic Acid
Dichloroacetic Acid
Trichloroacetic Acid
Bromonitromethane
Dibromomtromethane
Tnbromonitromethane
Dichloronrtromethane
Trichloronitromethane
MX(EMX)
Ethylmethanesulfonate
0.01
0.1
10
Water Disinfection By-Product (mM)
Figure 1. Single-cell gel electrophoresis analysis of genomic DNA damage in CHO cells induced by selected DBFs. For comparison of the
relative genotoxic activities of the DBFs, the standard chemical mutagen ethylmethyl-sulfonate was included. The halonitro-
methanes were provided by Dr. Susan Richardson, U.S. Environmental Protection Agency
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Assessment of Human Dietary Ingestion
Exposures to Water Disinfection Byproducts via Food
James H. Raynter', E.D. Pellizzari1, Y. Hu', B. Childs', K. Briggs', H. Weinberg2, and V. Unnam 2
'Research Triangle Institute, Research Triangle Park, NC; 2 School of Public Health, University of North Carolina,
Chapel Hill, NC
The overall objective of this research project is to
estimate the magnitude of exposure to disinfection
byproducts (DBFs) in drinking water via their ingestion
after uptake into food during cooking or home pro-
cessing. Although the potential for contamination has
been shown, the magnitude of this contamination has
not been studied. This research will specifically ad-
dress the uptake of compounds known to arise from the
process of water disinfection (ozonation in conjunction
with a secondary process such as chloramination),
including nonhalogenated aldehydes, ketones and acids,
trihalomethanes, haloacetic acids, bromate, chloro-
picrin, and haloacetonitriles. The main hypotheses to be
tested are: (1) foods prepared using contaminated water
become contaminated; (2) food is a significant source
of DBF exposure: (3) DBF concentrations in food can
be predicted with knowledge of DBF concentrations in
tap water and foods consumed; and (4) dietary expo-
sures of children are higher than for adults living in the
same household.
Work to date has focused on the investigation of a
method for nonhalogenated aldehydes and ketones in
composite foods and beverages, the stability of halo-
acetonitriles and haloacetic acids (HAAs) to boiling, the
partition of HAAs into foods during cooking and
rinsing, and on a method for the determination of
bromate in food. A method for nonhalogenated alde-
hydes and ketones based on derivatization of carbonyl
compounds with o-(2,3,4,5,6pentafluorobenzyl)-hydro-
xylamine to form the corresponding oxime was devel-
oped and applied to foods and beverages. The sample
extracts were very complex and contained many of the
target analytes without the addition of any known
DBFs. During boiling of spiked water, haloaceto-
nitriles were lost very rapidly, while some HAAs were
more persistent. Chloroacetic, dichloroacetic, bromo-
chloroacetic, and dibromoacetic acids were unaffected
following boiling for 60 minutes while bromodichlo-
roacetic, chlorodibromoacetic, and tribromoacetic acids
were lost quickly (within 10 minutes); trichloroacetic
acids were of intermediate stability.
The partition of HAAs into carrots, green beans,
pinto beans, chicken, and lettuce following cooking or
contact with water spiked to contain HAAs was studied.
The foods selected for study were spaghetti, dried
beans, carrots, green beans, chicken, and lettuce and
were chosen based on different cooking conditions,
different chemical compositions (i.e., starch, vegetable,
protein), and the fact that they are commonly consumed
by children. Foods were cooked according to package
directions using either reagent water or water spiked to
contain HAAs. Following cooking, foods were homo-
genized and extracted as previously described (Raymer
et al., J Exposure Anal Environ Epidemiol 2000; 10:
808-15).
In some cases, more than 60 percent of HAAs in
the cooking water were taken up by the food during
cooking (see Table 1). In general, chlorodibromoacetic
and tribromoacetic acids are not detected following
cooking, consistent with the rapid loss of these com-
pounds during boiling. The soaking of lettuce also
resulted in uptake of HAAs (1.8-7.8% of the total
available). Results for spaghetti indicated that as much
as 15 percent of the available HAAs are adsorbed/
absorbed during cooking in spiked water. Given the
large volumes of water used to cook pasta, the masses
absorbed could be much higher than for other types of
food.
In addition, the data obtained to measure uptake
following rinsing with spiked water indicate that
additional HAAs—up to 10 percent—are taken up from
the rinsing water. This was true whether the pasta was
cooked in reagent water or in spiked water. It also was
clear that the HAAs that typically show low recovery
following cooking (bromodichloroacetic acid, chloro-
dibromacetic acid, and tribromoacetic acid) can become
available to contribute to exposure following contact of
the pasta with fresh, DBF-containing water.
Initial evaluation of a method to extract bromate
from food (spaghetti) showed that material extracted
from pasta interfered with measurement of the recovery
of bromate. A specific and sensitive method for
bromate based on ion chromatographic separation/
postcolumn reaction method developed for water was
adapted for this work. Analysis of water recovered from
the cooking of spaghetti showed that dilution was
needed to affect detection of the bromate; whether this
was due to adsorption of bromate onto suspended/
dissolved solids or a suppression of the chromato-
graphic detection was not clear. Bromate was detected
in reagent water following its use to cook pasta, which
indicated its presence in the pasta. Initial extractions of
homogenized, cooked pasta indicated fewer interfer-
ences than with the recovered water, but that method's
precision was not very good. Further method work will
be conducted during the next year.
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Table 1. Percent uptake of haloacetic acids into foods during cooking
Percent Uptake (%RSD)
_, , Concentration c J —
Compound . ... , , . Green Pinto i,
1 in Water (ppb) Carrots „ OR Lettuce"
Beans Beans'"
Chloroacetic acid
Bromoacetic acid
Dichloroacetic acid
Trichloroacetic acid
Bromochloroacetic acid
Dibromoacetic acid
Bromodichloroacetic acid
Chlorodibromoacetic acid
Tribromoacetic acid
500
100
150
50
100
50
100
100
100
NRb
57C (3.8)
64C (24)
24 (294)
40C (9.7)
5T (8.0)
26C (43)
ND
ND
17C(38)
21 (50)
48C (39)
33 (92)
35C(8)
60C(18)
2.2(182)
ND
ND
62d(3.1)
NDe
85d(3.4)
INTf
37d (5.0)
51d(16.6)
5.3d (87)
ND
ND
2.5d(42)
4.5d(9.1)
3.9d(19)
3.1d(75)
4.2d(5.4)
7.2" (6.7)
1.8d(40)
6.5C(5.5)
7.8d(12)
Chicken8
15C(3.2)
ND
llc(22)
14C(121)
7.1C(45)
llc(23)
NR
ND
ND
a Uptake = [(HAA mass in food cooked in spiked water - mass in food cooked in reagent water) x 100] - total HAA mass in cooking water,
N=3
b NR = Not reported A decrease in concentration was measured following cooking in spiked water
c Significant at p < 0 05.
d p value not calculated because n = 1 for control case, a substantial increase was noted for these compounds relative to the food cooked in
reagent water
c ND = Not detected
f INT = Interferent. Very high trichloroacetic acid concentration in the blank made estimation of the uptake difficult.
8 Pinto beans soaked in reagent water and cooked in water spiked with HAAs, chicken soaked and cooked in spiked water
h Lettuce soaked for 5 minutes in water containing HAAs
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Bioavailabilitv of Haloacetates in Human Subjects
Irvin R. Schultz , Robert Shangraw ', and Torka Poet
1 Battelle, Pacific Northwest Division, Rich/and, WA; 2Oregon Health Sciences University, Portland, OR
The objective of this research project is to char-
acterize the absorption, disposition, and oral bioavail-
ability of chlorinated and brominated haloacetates in
human volunteers after consumption of drinking water
containing a natural mixture of these compounds. It is
hypothesized that accurate assessment of the oral bio-
availability of haloacetates can be achieved by the
simultaneous administration of an oral dose of I2C-
labeled haloacetate with an intravenous dose of LlC-
labeled haloacetate. It is hypothesized that measurable
plasma levels of dichloroacetate, bromochloroacetate,
and dibromoacetate can be detected from the debromi-
nation of bromodichloroacetate, dibromochloroacetate,
and tribromoacetate.
This research will directly test the hypothesis that
prolonged exposure to low concentrations of dihalo-
acetates reduces their metabolism and increases their
systemic bioavailability in humans. These experimental
results will be used to validate a physiologically based
pharmacokinetic (PBPK) model for haloacetates in hu-
mans that is based on in vitro metabolism parameters
obtained with human tissue homogenates (see Figures 1
and 2). Dichloroacetate (2 mg haloacetate/Kg) in 1 pint
of water will be given to volunteers. After 5 minutes,
'JC-labeled dichloroacetate will be given by intraven-
ous injection (via a catheter placed in the arm).
A similar experiment will be performed using
mixtures of chlorinated and brominated haloacetates in
rhesus monkeys. In a second experiment, volunteers
will consume 1 pint of tap water previously verified to
contain the seven haloacetates of interest. For all exper-
iments, serial blood samples will be removed using the
intravenous catheter, and the blood plasma will be
analyzed simultaneously for both the ljC haloacetates
and I2C haloacetates (using gas chromatography-mass
spectrometry or liquid chromatography-MS/MS tech-
niques). The area-under-the-curve ratio for the oral and
intravenous doses will be determined to estimate the
oral bioavailability.
This project will provide critical data needed to
make accurate and reliable exposure estimates of halo-
acetates to humans consuming municipal drinking
water supplies. In addition, this project will identify the
consequences of low-level exposure to haloacetates on
their subsequent metabolism and disposition. This in-
formation is needed to assess whether individuals who
consume water containing high levels of byproducts
experience greater than predicted exposure due to de-
creased elimination of haloacetates. This project also
will allow for the direct testing of physiologically based
pharmacokinetic model predictions of haloacetate dosi-
metry in humans.
10
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
^ 1.2 •
o
a.
o, 1.0 -
•| 0.8
E
S 0.6 H
o>
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S 0.2
03
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200
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4X0 600
pMDCA
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800
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0,050 -i
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0,040 -
0.035 -
~- 0,030 -
C 0.025 -
CD
Is 0020 -
^ 0.015 -
~ 0 010 -
" 0005 -
0000 -
K5
*
x = 0.058 «w = 60 1
= 0.065 Kg, = 112
_j j j j. | i | j j -j
0 14 28 42 56 70 84 88 112126140
\M BDCA
Figure 1. Left graph DCA metabolism in human liver S9 Right graph- BDCA metabolism (total, solid squares) and DCA formation (open
circles) in human liver microsomes Formation of DCA accounts for most of the consumption of BDCA Solid lines on both graphs
are the nonlinear least squares fit of the observed data to the Michaelis-Menten equation
100 -a
100 -
O
50 mg/kg
*«io -
ft
1 -j
1 mg/kg
1 mg/kg
i » •• ?
, | | i | . | , | .
01 2345878
Time (hours)
0123456
Time (hours)
Figure 2. Left graphs. PBPK model predictions of DCA plasma profiles in humans after 1 and 50 mg/kg oral doses. Observed data (circles) is
from Curry et al (1991) Biopharm Drug Dispos 12 375-390 Right graphs: PBPK model predictions of BDCA and DCA plasma
profiles in humans after 1 and 5 mg/kg oral doses of BDCA
The Office of Research and Development's National Center for Environmental Research
11
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2001 STAR Drinking Water Progress Review Workshop
Physiologically Based Pharmacokinetic
Modeling of Haloacid Mixtures in Rodents and Humans
Irvin R. Schultz, Robert D. Stenner, and Richard J. Bull
Battelle, Pacific Northwest Division, Rich/and, WA
The goals of this research project are: (1) char-
acterization of the comparative toxicokinetics and me-
tabolism of chloro, bromo, and mixed chlorobromo
haloacids (HAs) in rodents; and (2) development of a
physiologically based pharmacokinetic (PBPK) model
that can predict the tissue distribution and elimination
of HAs during chronic oral exposure in mice, rats, and
humans.
Prior experiments established that di- and tri-HAs
have distinct pharmacokinetic properties, and that prior
exposure to di-HAs can alter their metabolism. Current
studies have focused on characterizing the kinetics of
di- and tri-HAs at lower doses and as a mixture of HAs.
In vivo experiments used mice and rats given either
intravenous or gavage doses of an HA or HA mixture
and the blood plasma concentration-time profile and
urinary excretion characterized in individual animals.
Both control (naive) and animals pretreated with a di-
HA for 7 days at various drinking water concentrations
were used to measure the effects of prolonged HA
exposure on the disposition. In vitro metabolism exper-
iments of tri-HAs used mouse and rat microsomes.
These experiments were performed under varying
oxygen tensions ranging from zero (pure N2 atmo-
sphere), 2 percent O2, and normoxic (normal atmo-
sphere).
Di-HA elimination by naive rats was so rapid that
only higher doses (1-20 mg/kg for the intravenous and
5-20 mg/kg for the gavage) achieved blood plasma
concentrations above the analytical limit of detection.
The oral bioavailability was approximately 30 percent
in nai've rats at the 5 and 20 mg/kg doses. Oral bio-
availability increased to 39 percent and 82 percent at 5
and 20 mg/kg doses, respectively, in HA-pretreated
animals. The oral absorption plasma profile of di-HAs
was complex and exhibited secondary peaks several
hours after dosing. This phenomenon appeared to be
enhanced in mixtures of HAs. The metabolism of di-
HAs is known to be mediated by the enzyme gluta-
thione-S-transferase Zeta (GSTzeta).
Pretreatment of rats with a di-HA greatly dimin-
ished the GSTzeta activity and metabolism of all di-
HAs. This effect on metabolism could be observed after
pretreatment with drinking water levels as low as 1
mg/Kg (see Figure 1). Microsomal metabolism of tri-
HAs proceeded by reductive debromination forming a
di-HA free radical intermediate, which was stimulated
under a reducing environment. The Vmax for the loss of
parent trihaloacetate was 4-5 times higher under nitro-
gen headspace than under atmospheric conditions. In-
trinsic metabolic clearance was of the order Tri-
Bromoacetate > Chlorodibromoacetate»Bromodichlo-
roacetate.
Eadie-Hofstee plots for the consumption of the par-
ent tri-HA appeared linear, suggesting that a single
P450 enzyme is responsible. Carbon monoxide and
diphenylene-iodonium (a specific P450 reductase inhib-
itor) blocked the metabolism. However, inhibitors of
specific P450 proteins (CYP 2E1, 2D6, and 3A4) failed
to block metabolism significantly.
These results indicate that low-level exposure to
di-HAs decreases metabolism, causing an increase in
the oral bioavailability. The delayed absorption of di-
HAs may allow greater levels to reach the colorectal
region than previously thought. These results will be
incorporated into the working PBPK model for HAs to
predict tissue dosimetry during low exposure rates of
mixtures of HAs.
12
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
c
o
O
3?
100 -i
90 -
80 -
70 -
60 -
50 -
40 -
30 -
20 -
10 -
1 w
0 -
0 001 0 01 0
ill
i
05
nrU
$
0 2
I
i
2
BCA (g/L)
xs s
n T i
•i
m
0 01 0 05 02
DBA
T
i
gfclT
i§B
2 001 02 2
DCA (g/L)
Figure 1. GSTzeta activity in liver cytosol prepared from rats treated for 7 days with a di-HA at various drinking water concentrations
(mean ± SD)
The Office of Research and Development's National Center for Environmental Research
13
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2001 STAR Drinking Water Progress Review Workshop
Inhalation and Dermal Exposure
to Disinfection Byproducts of Chlorinated Drinking Water
Clifford P. Weisel and Jeffrey Luskin
Enviromental and Occupational Health Sciences Institute, Piscataway, NJ
Inhalation and dermal exposure to haloacetic acids,
haloketone, haloacetonitrile, and chlorohydrate from
showering and bathing are being determined. The ex-
posures will be determined from: (1) concentration of
the DBFs or their metabolites in urine or breath follow-
ing known exposures; (2) estimation of dermal trans-
port coefficients using in vitro measurements across
excised skin; and (3) size and number distribution of
residential water aerosols generated during showering
and disinfection byproduct (DBF) concentration.
The dermal absorption during both in vivo and in
vitro studies was minimal for chloroacetic acid, di-
chloroacetic acid, bromoacetic acid, trichloroacetic
acid, bromochloroacetic acid, and dibromochloroacetic
acid. Only trichloroacetic acid had measurable penetra-
tion through the skin in vitro at pH 7, with a Kp value
of 0.00039 cm2/hr at 37°C. The haloacetic acids were
either only slightly above the background levels in the
urine (total excess excretion 1-2 \ig for dichloroacetic
acid) or not detectable in the urine (dibromoacetic acid)
following a bath using concentrations of 200 [ig/L. Low
or nonmeasurable levels of the haloacetic acids in urine
after bathing could be due to rapid metabolism of the
compound, although a portion of even rapidly meta-
bolized compounds are expected to be excreted prior to
being metabolized after being dermally absorbed.
The in vitro and in vivo results strongly suggest
that dermal absorption of haloacetic acids from water at
pH of approximately 7 is insignificant. Dichloropropan-
one and trichloropropanone had Kp values of 0.019 and
0.0081 cm2/hr, respectively, at 21°C.
The in vitro measurements were made under
steady-state conditions of high concentrations (g/L), run
for 20-50 hours. Chloroacetonitrile rapidly adsorbed
across the skin within the in vitro studies, made under
nonsteady state (|J.g/L concentrations), with a peak flux
of 0.01 u.g/hr/cm2 within minutes of the exposure,
followed by a decline after several hours.
For water concentrations of 20 |0,g/L, a 30-minute
bath would provide a dose of 1-3 (ig, or 5-15 percent
of that obtained from ingestion of 1 L of water. The in
vivo studies also documented penetration through the
skin, although a majority of the compounds are meta-
bolized rapidly, making measurement of the DBF itself
difficult. Chlorohydrate was observed to dermally pene-
trate the skin based on measurement of its metabolite
trichloroacetic acid.
Aerosols of 0.5-2.0 microns were produced by
showers using different showerheads and temperatures.
The aerosol size distribution was characterized by p =
3.2 x 10"Jd""4, where p is the percentage of aerosols
within a certain size range, and d is the diameter of the
aerosol.
For water spiked with 25 |ig/L of haloketones and
200 |ig/L of haloacetic acids, the upper limits of what is
expected in a water distribution system, the airborne
particulate levels near the shower stream were 10 u,g/mj
for the haloacetic acids and 100 ng/nr5 for the halo-
ketones.
The expected inhalation contribution to the total
dose from these aerosols is calculated to be 1 percent or
less compared to an ingestion of 1 L of water, Jt is
expected that the haloketones also will have a vapor
component that will contribute more to the inhalation
dose.
These results provide evidence that dermal and in-
halation exposures are not important routes for halo-
acetic acid, but should be considered for haloaceto-
nitrile, chlorohydrate, and haloketone (see Table 1).
The data necessary for drinking water exposure
models, including dermal uptake, particle size, and par-
ticle number distributions are being generated. In vivo
studies need to be completed. Additional work also is
needed to develop biomarkers that adequately can mea-
sure exposure to these DBFs in field and controlled
studies.
14
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Table I. Importance of exposure route to total dose relative to ingestion
Compound Class Dermal
Haloacetic Acid
Haloketone
Haloacetonitrile
Chlorohyrate
No
Yes
Probable
Yes
Inhalation- Aerosol Inhalation-Vapors
No
No
No
No
No
Probable
Probable
Probable
The Office of Research and Development's National Center for Environmental Research
15
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2001 STAR Drinking Water Progress Review Workshop
NHEERL Research
on Carcinogenic Contaminants in Drinking Water
Douglas C. Wolf
Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, Office of
Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC
Water research in the Environmental Carcino-
genesis Division focuses on improved understanding of
the mechanisms of mutagenesis and Carcinogenesis of
water contaminants for incorporation into human cancer
risk assessment models. The program uses cellular,
animal, and computer models for assessing responses to
individual and mixtures of water disinfection by-
products (DBFs), arsenic, and chemicals on the U.S.
Environmental Protection Agency's (EPA) chemical
contaminant list. A significant component of the re-
search effort is directed toward a better understanding
of mechanisms underlying tumor development to in-
form the cancer risk assessment process. As examples,
research on potassium bromate (KbrO3), dichloroacetic
acid (DCA), arsenicals, and a defined mixture of four
water DBPs will be discussed.
Ozone has been proposed for water disinfection
because it is more efficient in killing microbes than
chlorine and results in much lower levels of trihalo-
methanes than chlorination. Ozone leads to formation
of hypobromous acid in surface waters with high
bromine content and forms brominated organic by-
products and bromate. The carcinogenicity and chronic
toxicity of KBrO3 was studied in mice and rats. KBrO3
is carcinogenic in the rat kidney, thyroid, and meso-
thelium and is a renal carcinogen in the male mouse.
These data were used to predict the human health risk
associated with exposure to bromate in drinking water.
DCA is the main component of the haloacetic
acids, the second most prevalent group of DBPs after
trihalomethanes. DCA is a rodent liver carcinogen that
results in tumor development after 2 years, even when
mice are exposed to DCA for only 10 weeks. DCA has
been shown to depress apoptosis in hepatocytes and is a
weak inducer of DNA mutations, as it has been weakly
positive in both in vitro and in vivo mutagenicity as-
says, including the Big-Blue Mouse*. These and other
data are being used to develop a biologically based
dose-response model for DCA hepatocarcinogenesis in
the mouse. Arsenic is a known human carcinogen and
has been shown to promote cancer in the rat urinary
bladder, kidney, liver, and thyroid as well as in the
mouse lung. It has been generally thought that arsenite
is the carcinogenic form, and that methylation is the
detoxification and excretion pathway. However, recent
evidence shows that methylation of arsenic also may be
a toxification pathway. Arsenic may act as both a geno-
toxic and nongenotoxic carcinogen, thereby acting as an
initiator and/or a tumor promoter. Additional research is
examining the interaction between arsenic and DNA
methylation, as well as the carcinogenic activity of di-
methylarsenic acid and the contribution free radicals
may have in arsenic-induced cancer. Current default
risk assessments for chemical mixtures assume addi-
tivity of carcinogenic effects, but this may under- or
over-represent the actual biological response.
A rodent model of hereditary renal cancer was used
to evaluate the carcinogenicity of a mixture of KBrO3,
3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone
(MX), chloroform, and bromodichloromethane. Treat-
ment with the mixture did not produce more neoplasms
than the individual compounds, suggesting less than an
additive response for carcinogenicity. Research within
the Environmental Carcinogenesis Division not only
attempts to develop methods for detecting environment-
al carcinogens, but also tries to define the mechanisms
of Carcinogenesis with the intent of resolving issues,
assumptions, and uncertainties in cancer risk assess-
ment for water contaminants. This abstract does not re-
flect EPA policy.
16
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Aluminum Toxicokinetics: Oral
Absorption From Drinking Water and Brain Retention
Robert A. Yokel , Patrick J. McNamara1, and David Elmore
College of Pharmacy and Graduate Center for Toxicology, University of Kentucky, Lexington, KY; 'Purdue Rare
Isotope Measurement Laboratory, Purdue University, Lafayette, IN
Aluminum (Al) is a neurotoxicant that plays a role
in dialysis encephalopathy and similar conditions. Al-
though highly controversial, some epidemiological
studies suggest a positive association between drinking
water Al concentration and the incidence of Alzhei-
mer's disease. It has been suggested that Al is better
absorbed from water than foods. The overall objective
of this study is to determine if Al in drinking water
significantly contributes to brain Al accumulation.
There were four aims of the study. The first aim
was to determine the absolute bioavailability of Al from
drinking water.
The second aim was to assess whether water hard-
ness and/or food in the stomach affect oral Al bioavail-
ability. Repeated measurements of both 26A1 and 27A1 in
rat plasma were obtained after concurrent administra-
tion of a single stomach feeding of 26A1 in water and
continuous 27A1 intravenous infusion. The stomach
feeding was given in the absence ("soft" water) or pres-
ence of calcium and magnesium carbonate ("hard"
water) and in the absence or presence of stomach
contents. Al bioavailability was calculated from the
area under the Al concentration x time curve for 26A1
compared to the equivalent term for 27A1.
The third aim was to determine the fraction of Al
that enters the brain from blood by quantitation of 26A1
in the rat brain after its systemic administration.
The fourth aim was to determine the rate of brain
Al elimination. Two approaches were utilized. Rat
brain 26A1 was quantitated at multiple times after sys-
temic 26A1 dosing. Additionally, some rats received
repeated injections of the Al chelator desferrioxamine
to determine whether it affected the brain 26A1 half-life.
Approximately 0.3 percent of the 26A1 was absorbed
independent of whether it was given in "soft" or "hard"
water and independent of the absence or presence of
stomach contents.
After intravenous 26A1 administration, approxi-
mately 5 x 10"' percent of the dose appeared in each
gram of brain. The half-life of brain Al elimination was
approximately 150 and 85 days in the absence and pre-
sence of repeated desferrioxamine injections, respec-
tively. Al shows a prolonged residence in the brain. As
the human lifespan is about 30 times that of the rat, the
150-day half-life in rat brain translates to a human brain
Al half-life of approximately 12 years. Brain Al concen-
tration resulting from continuous Al intake can be pre-
dicted from the amount of Al that enters the brain and
the half-life of Al in the brain.
For this prediction, 0.3 percent absorption of the 8
mg Al consumed daily in drinking water and food,
entry into the brain of 5 x 10"J percent of the absorbed
Al, and a 12-year brain Al half-life were assumed. The
results suggest that brain Al in the average 60 year-old
human should be approximately tenfold more than is
reported. One explanation for this discrepancy is that
0.3 percent is an overestimate of oral Al bioavailability
from food. Water provides only about 1 percent of the
typical daily Al consumption, whereas food provides
about 95 percent.
If Al bioavailability from food is approximately
0.03 percent, the prediction would match observed
human brain Al concentrations. Drinking water then
would provide approximately 10 percent of the daily
absorbed Al that might contribute to brain Al accumu-
lation. The Al concentration in drinking water then
might impact human brain Al accumulation. Deter-
mination of the oral bioavailability of Al from food is
required to clarify the ability of Al in drinking water to
significantly contribute to brain Al.
The Office of Research and Development's National Center for Environmental Research
17
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Section 2.
Chemical Contaminants: Arsenic
-------
2001 STAR Drinking Water Progress Review Workshop
Overview of EPA's Arsenic
in Drinking Water Regulation
IreneS. Dooley
Office of Ground Water and Drinking Water, U.S. Environmental Protection Agency, Washington, DC
The goals and objectives of this project are to
propose arsenic regulation by January 1, 2000, issue a
final rule by June 22, 2001, and reevaluate at least
every 6 years. The Safe Drinking Water Act directed
the U.S. Environmental Protection Agency (EPA) to
revise its 1975 Drinking Water Standard (the Maximum
Contaminant Level [MCL]) and list affordable tech-
nologies for small systems. EPA may adopt an MCL
"that maximizes health risk reduction benefits at a cost
that is justified by the benefits."
The Act, as amended in 1996, charged the Agency
to develop a research plan by February 2, 1997, to
address the uncertainty in assessing health risks from
low levels of arsenic to support the rulemaking, and
conduct the research in consultation with the National
Academy of Sciences (NAS), other federal agencies,
and interested public and private entities.
In December 1996, EPA submitted its draft re-
search plan for peer review and issued the final plan in
February 1998. The plan identified the short-term re-
search (available before January 2000) that would sup-
port regulation development and long-term research to
reassess the arsenic MCL, as specified by the statute. In
1996, EPA asked the NAS to review arsenic health
effects research and EPA's risk characterization from
arsenic exposure. The NAS report, issued in March
1999, concluded that the Taiwanese studies provide the
best data available for quantifying risks. The report
stated that EPA's MCL of 50 ppb is not adequately
protective and should be lowered as soon as possible.
In the preamble to EPA's June 22, 2000, proposed
rule, the Agency explained how it used NAS' bladder
cancer risk analysis to prepare EPA's initial risk assess-
ment (see Figure I). In the proposal, EPA identified
sources of scientific uncertainty and requested comment
on MCL options ranging from 3-20 ppb. Although the
possible mechanisms for arsenic-induced cancer are
associated with sublinear dose-responses, available data
do not meet EPA's criteria for departing from a linear
extrapolation.
Studies published in 2000 show that trivalent
organic forms of arsenic are more toxic than inorganic
arsenic. Current studies do not identify infants and
children as being at greater risk.
In the January 22, 2001, final rule, EPA's lower
bound risk estimates accounted for the arsenic in Tai-
wanese food and from food preparation in Taiwan.
More than 2,900 of the 3,000 community water
systems projected to require treatment serve fewer than
10,000 people. The MCL of 10 ppb will avoid 37-56
bladder and lung cancers per year, and cost households
$38-$327 per year in systems serving under 10,000
people.
Although ecological studies identify health ef-
fects, uncertainties about exposure, differences in nutri-
tion, selenium, and linear extrapolation of data affect
the risk assessment. Additional mode of action studies,
animal studies, population studies, and studies of in-
fants and children could improve future risk assess-
ments.
The Office of Research and Development's National Center for Environmental Research
21
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2001 STAR Drinking Water Progress Review Workshop
Peer-Reviewed
Research
.>.•'
/ »EPA Research
] •IndustryStudies
f
Risk
Assessment
Hazard
Identification
Risk
Management
International
I R esearch,! D OSe-ReSpOIl SB / MC LG '!>
Needs*
Assessment
MCL
Treatment and
E xpo sii re
Assessment
ization^/
• Small System \
• Technologies l
• Test Methods
• C ost-Benefrts of /
Options /
• Occurrence,
Number of
Systems ^
* Arsenic Research Plan (www epa gov/ORDAVebPlus/final/arsenic pdf)
* 1996 Cancer Assessment Guidelines (61 FR 17960)
Figure 1. Role of research and studies in standards development.
22
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Arsenic-Glutathione Interactions and Skin Cancer
Catherine B. Klein ' and Elizabeth T. Snow 2
'Department of Environmental Medicine, New York University School of Medicine, Tuxedo, NY;2 Center for
Cellular and Molecular Biology, Deakin University, Victoria, Australia
The goal of this research project is to test the hypo-
thesis that arsenic-induced cancer could result from
changes in cellular redox control mediated by gluta-
thione (GSH). Specific aims were designed to: (1) ex-
amine the in vitro effects of arsenic on the activities of
GSH-metabolizing enzymes glutathione S-transferase-
pi (GST-Tt), glutathione reductase (GR), and glutathi-
one peroxidase (GPx); (2) assess GSH-dependent redox
status, production of reactive oxygen species (ROS),
GSH/GSSG levels, and the activities of GR and GPx in
low-dose inorganic arsenite (As"1) treated cultured hu-
man keratinocytes; and (3) evaluate the role of GSH in
arsenic-induced carcinogenesis by examining the effect
of varied GSH levels on the rate of skin papilloma
induction in normal and GPx-overexpressing mice.
The approach taken by this project was to explore
the effects of arsenic on glutathione-regulating enzymes
in human skin keratinocytes in vitro, and in mice in
vivo. It was predicted that exposure to physiologically
relevant, low doses of As could activate the gluta-
thione-related enzymes due to changes in cellular phos-
phorylation and/or redox status. If this occurred, it
could either potentiate or ameliorate the induction of
cellular stress responses, and perhaps skin carcinogene-
sis. This research has shown that physiologically rele-
vant concentrations of arsenic (less than 100 uM) do
not directly inhibit GSH-metabolizing enzymes. Direct
enzyme inhibition is only seen at high arsenic con-
centrations. Low concentrations of As"1 do, however,
cause significant changes in cellular GSH levels and in
the relative activity and gene expression of a variety of
redox-active enzymes in cultured human keratinocytes
or fibroblasts (see Figure 1).
These results show that low, relatively nontoxic
concentrations of arsenic can directly modulate cellular
redox activity that, in turn, may alter cellular signaling,
thereby contributing to the carcinogenic process.
In experiments designed to evaluate the hypothe-
sis that arsenic acts as a progressor or copromoter in the
production of skin cancer, this research has confirmed
that As does not act as a copromoter with 7,12-di-
methylbenz[a]anthracene (DMBA) and 12-O-tetrade-
canoyl phorbol-13-acetate (TPA) in a standard skin
carcinogenesis protocol. This is consistent with the lack
of As-induced carcinogenesis in other animal models,
and again shows that the biological response of humans
to As is quite different from that of rodents. In another
mouse experiment using a higher dose of TPA, the
significant level of papilloma formation in the DMBA/
TPA-treated animals is reduced in the arsenic-exposed
animals.
During the last year of this project, additional
animal experiments with the GPx-overproducing trans-
genie mice will be performed. GPx is one of the few
identified genes that is downregulated by a low dose of
As"1, and these animals also are highly sensitive to
DMBA/TPA-induced skin cancer. They should pro-
vide an interesting additional test of As as a copromoter
or progressor.
Frozen samples of skin and other tissues from the
first three animal experiments have been stored, and
other samples have been preserved in paraffin blocks.
These samples will be used to investigate tissue arsenic
levels and the levels of GSH-related proteins and
mRNA in the skin, bladder, and other tissues from the
As-treated and control animals.
The Office of Research and Development's National Center for Environmental Research
23
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2001 STAR Drinking Water Progress Review Workshop
0 5 10 15 20 25 30
As111 \M
Figure 1. mRNA levels in cells exposed to As for 24 hours
24
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
A Dose-Response and Susceptibility Investigation of Skin
Keratoses and Hyperpigmentation Caused by Arsenic in Drinking Water
Allan H. Smith', Reina Haque', Joyce Chung', Lee Moore', D.N. Guha Mazumder2, Binay K. De 2,
Nilima Ghosh2, Soma Mitra2, and David Kalman3
'University of California, Berkeley, CA;'Institute of Post Graduate Medical Education and Research, Calcutta,
India; ~ University of Washington, Seattle, WA
The first detailed assessment of the dose-response
relationship of arsenic-induced keratoses and hyper-
pigmentation at low doses has been completed. Key ob-
jectives of the study also included determining whether
susceptibility varies by arsenic methylation capability
and nutritional factors such as methionine and cysteine.
Arsenic methylation was assessed by urinary assays.
Nutritional status was determined by blood measure-
ments of key macronutrients and micronutrients as well
as by analysis of a dietary questionnaire.
Recently, a case-control study was completed that
took advantage of the largest population-based survey
conducted of a district in West Bengal, India, with ele-
vated inorganic arsenic levels in its drinking water
supplies. The source of the arsenic is geologic.
Potential participants for the case-control study
were identified from the 1995 cross-sectional survey for
further medical examination and detailed arsenic expo-
sure assessment. The cross-sectional survey included
more than 7,000 participants, and measured the resi-
dents' current arsenic concentration in their tubewell
water supplies. All individuals identified with keratoses
and/or hyperpigmentation in the survey, and exposed to
<500 ug/L of arsenic, comprised the case group for the
present investigation. The control group consisted of
lesion-free individuals randomly selected from the
cross-sectional survey database matched on age and
sex.
The information used in the earlier survey of ar-
senic in drinking water was expanded to include infor-
mation from all current and past water sources used in
households and work sites. Data obtained from personal
interviews and chemical analyses of drinking water
samples were used to assess arsenic exposure. The in-
terviews consisted of questions about lifetime residen-
tial history, water sources at home and work, and fluid
consumption. The clinical exam involved various derm-
atologic, neurologic, and respiratory endpoints. A num-
ber of participants suspected to have an arsenic-induced
skin lesion were photographed. A dietary questionnaire
supplemented with results of blood assays will be used
to ascertain the participants' nutritional status. Urinary
assays will be used to determine arsenic methylation
efficiency.
The interviews and sample collection started in
June 1998, and ended in December 1999. Participation
was excellent (93% in cases and 97% in controls). Ulti-
mately, 192 cases and 213 controls participated. Key
preliminary results on the dose-response relation in-
clude: (1) strong dose-response trends with peak and
average exposures were found based on known years of
exposure (test for trend with peak and average expo-
sures, /><0.0001); (2) these trends remained after adjust-
ing for sex, age, smoking status, socioeconomic fac-
tors, and body mass index; (3) the proportion of cases
confirmed as definite or probable by photograph was
high (87%)—an implication of this finding is that
keratoses and hyperpigmentation are the best biomark-
ers of long-term effect; and (4) some cases who were
initially thought to have consumed very low arsenic
levels actually were exposed to higher concentrations in
past decades.
For the first time, the dose-response relation of skin
lesions and arsenic ingestion at low doses was charac-
terized using a detailed exposure assessment. Also, it
was the first study to confirm cases by photograph.
Interviewing and sample collection have been com-
pleted for 405 total participants. Data entry and editing
have been completed, and scientific reports are being
prepared for publication regarding the dose-response
relation, arsenic methylation capability, and nutritional
factors.
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Trivalent Methylated Arsenicals: Novel
Biomarkers of Arsenic Toxicity in Humans
Miroslav Styblo', Luz M. Del Razo 2, Shan Lin s, William R. Cullen 4, and David J. Thomas5
'Departments of Pediatrics and Nutrition, University of North Carolina, Chapel Hill, NC; 'CINVESTA V-IPN,
Mexico City, Mexico; 3 University of North Carolina, Chapel Hill, NC; 4 University of British Columbia, Vancouver,
BC, Canada; ^ National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection
Agency, Research Triangle Park, NC
The major goal of this project is to study the effects
of inorganic arsenic on a major cellular redox enzyme,
glutathione reductase (GR), and on the redox status of
cells exposed to arsenic. To better understand the role
of metabolism in the toxicity of arsenic, the correlation
between effects induced by arsenic and the patterns of
its metabolic conversions are systematically examined
in live cells. Cells from tissues that metabolize arsenic
(liver) and tissues that are targets for carcinogenic
effects of arsenic (skin, lung, and bladder) are used in
the study. A summary of results obtained during the 3
years (1997-2000) of the study follows.
An inorganic form of trivalent arsenic, arsenite
(iAs111), is toxic for most cell types examined only at
relatively high concentrations. It is a weak inhibitor of
GR and of another major oxidoreductase, thioredoxin
reductase (TR), in cultured cells. iAs111 is effectively
metabolized to methylarsenic (MA) and dimethylarsen-
ic (DMA) species in primary human hepatocytes. How-
ever, the capacity of human hepatocytes to methylate
iAs"1 varies significantly among individuals. Human
epidermal keratinocytes and human bronchial epithelial
cells are much less effective methylators of iAs1" than
are hepatocytes. Epithelial cells derived from the hu-
man bladder do not methylate iAs1". There is no ap-
parent correlation between the capacity of cells to
methylate iAs and their susceptibility to acute toxicity
of iAs111.
Trivalent methylated metabolites of iAs"1, methyl-
arsonous acid (MAsm) and dimethylarsinous acid
(DMAs"1), are significantly more toxic for most cell
types than is iAs"1. In addition, MAs"' is a more potent
inhibitor of GR and TR than is iAs1" by an order of
magnitude. An acute exposure of cells to MAs111 results
in a significant reduction of intracellular levels of a
major cellular antioxidant, glutathione (GSH). Penta-
valent methylated metabolites of iAs"1, methylarsonic
acid (MAsv), and dimethylarsinic acid (DMAsv) do not
inhibit either GR or TR and are not cytotoxic. In cells
exposed to iAs1", inhibition of TR activity strongly
correlates with intracellular MAs, suggesting that the
trivalent form of MAs1" is a product of the metabolism
of iAs1" in cells.
Treatments with antioxidants (catalase, N-acetyl-
cysteine, or glutathione-ethyl ester) partially protect
cultured cells against the toxicity of iAs1", MAs1", or
DMAs"1, indicating that generation of reactive oxygen
species may be, in part, responsible for cytotoxiciry of
trivalent arsenicals. On the other hand, a concurrent ex-
posure to selenite increases cytotoxicity of all three
species.
Using an optimized hydride generation atomic
absorption spectrometric technique, it was found that
trivalent methylated arsenicals, MAs"1 and DMAs"1, are
indeed products of the methylation of iAs'" in human
hepatic (HepG2) cells (see Figure 1). Both MAs1" and
DMAs"1 also were found in the urine of individuals
chronically exposed to iAs from drinking water (see
Table 1). The amounts of MAs1" and DMAs1" posi-
tively correlated with total urinary arsenic and thus,
with the extent of the exposure to iAs.
In conclusion, trivalent methylated metabolites,
MAs111 and DMAs"1, are more potent cytotoxins and
enzyme inhibitors than is iAs1". Trivalent methylated
arsenicals are natural products of the metabolism of iAs
in humans, suggesting that methylation of iAs is not
necessarily a detoxification mechanism. In fact, these
species may significantly contribute to the adverse ef-
fects associated with the exposure to iAs. Because of
their pronounced biological effects, MAs111 and DMAs1"
may become sensitive biomarkers of arsenic toxicity in
humans.
26
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
100 -
co 80 -
o
-I—>
LI—
o
60 -
40
20 -
0 -
IAs"
iAsv
MAs"1
MAsv
DMAs"1
DMAsv
0.1 1 10
Concentration of iAsm (pM)
Figure 1. Arsenic metabolites in human hepatocellular carcinoma (HepG2) cells exposed to various concentrations of lAs"1 for 24 hours
Metabolites in the whole culture (medium + cells) are shown (n=2)
Table 1. Arsenic metabolites m urine (ng/mL) collected from residents of Zimapan region, Mexico.
r Age/Sex iAs"1 \ iAsv ^MAs1" ~~| MAsv F DMAs1" F DMAsv Total As 1
39/f
19/m
21/m
12/m
20/f
11/f
8.1
25.2
59.4
104.2
14.7
11.2
4.5
16.8
72.1
122.8
10.1
12.2
Nd
2.3
5.2
12.3
1.2
1.0
7.1
37.7
119.8
276.7
19.3
11.2
Nd
18.2
59.8
114.3
5.7
18.3
18.6
40.1
408.2
467.2
90.5
52.7
38.3
140.3
724.5
1097.5
141.5
106.6
The Office of Research and Development's National Center for Environmental Research
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Section 3.
Chemical Contaminants
Formation of DBFs
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2001 STAR Drinking Water Progress Review Workshop
Use of Differential Spectroscopy
To Study DBF Formation Reactions
Mark M. Benjamin and Gregory V. Korshin
Department of Civil and Environmental Engineering, University of Washington, Seattle, WA
The major goal of this study is to ascertain the
intrinsic mechanism of formation of halogenated dis-
infection byproducts (DBFs). The novelty of the em-
ployed approach is twofold. First, it is designed to ex-
plore the nature of intermediates formed prior to the
release of individual controlled DBFs (notably, trihalo-
methanes, haloacetic acids, chloral hydrate). Second,
the approach makes use of in situ techniques (such as
differential absorbance spectroscopy [DAS] and stop-
ped-flow spectrophotometry [SFS]) capable of probing
the system with adequate temporary resolution. These
techniques were applied to track and quantify the in-
corporation of halogens into representative organic sub-
strates, which were exemplified by natural organic mat-
ter (NOM) and model compounds (e.g., 3,5-dihydro-
benzoic acid, [DHBA] and resorcinol). The latter type
of species represents halogen attack sites in NOM.
Several major findings have been made. It was
determined that the differential spectra of NOM record-
ed in the conventional mode are dissimilar to those of
the model compounds. Nonetheless, a secondary com-
ponent whose features were comparable to those found
for the model aromatic compounds has been detected.
The secondary differential spectra appear to be associ-
ated with the formation of chlorinated aromatic inter-
mediates that form prior to cleavage of small DBFs
from larger NOM precursors. The disappearance of the
secondary component of the differential spectra coin-
cided with the onset of release of individual DBFs such
as di- and trichloroacetic acids. Time-resolved experi-
ments indicated that the intensity of the features corres-
ponding to the formation of halogenated aromatic units
incorporated into NOM molecules is much higher in the
SFS mode than it was in the conventional regime.
Halogenation of the model compounds in the SFS mode
showed that each step of the halogen incorporation
consists of two main phases. The first rapid phase
corresponds to the formation of a charge-transfer com-
plex, while the slow second phase corresponds to the
actual incorporation of the halogen into the aromatic
ring. In the case of the model compounds, the charge-
transfer complexes exhibit distinct spectroscopic pro-
perties, which so far have not been observed for NOM.
The reason for this is being explored. It has been sug-
gested that either the rate of halogen interaction with
NOM is higher than that with model compounds, or the
stereochemistry of NOM does not allow the charge-
transfer complexes to form. Thus, the pathway of NOM
halogenation is distinct from that of the model com-
pounds.
Despite the need to ascertain the existence of the
transient complexes for halogenated NOM, the data of
DAS unambiguously indicate that the release of all
individual DBFs ranging from trihalomethanes to halo-
acetonitriles is associated with the breakdown of the
halogenated intermediates.
Experiments in which these intermediates were
subjected to hydrolysis, oxidation, or thermal treatment
demonstrated that the formation of haloacetic acids in-
volves oxidative destruction, while trihalomethanes are
released following their hydrolytical breakdown. These
data provide important insights into the identity, forma-
tion, and breakdown of the halogenated intermediates
during chlorination of NOM. The results also support
novel technological approaches pertinent to the control
and prediction of DBF concentrations in drinking water.
Currently, studies of the kinetics and mechanisms of
halogenation of model compounds (e.g., flavonoids) are
being conducted, as is exploration of the nature of se-
quential halogen incorporation into the reactive sites.
Eventually, it is planned to use the data to create a con-
sistent mechanistic model of DBF formation.
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Brominated DBF Formation and Speciation Based
on the Specific UV Absorbance Distribution of Natural Waters
James E. Kilduff' and Tanju Karanfil2
'Department of Energy and Environment, Rensselaer Polytechnic Institute, Troy, NY; 2 Department of
Environmental Engineering and Science, Clemson University, Clemson, SC
Understanding the characteristics of natural waters that
influence disinfection byproduct formation and
treatability is critical for providing safe water and for
meeting current drinking water regulations (e.g., the
disinfection/disinfection byproducts [D/DBP] rule). Un-
derstanding natural organic matter reactivity is of pri-
mary importance. Although sophisticated fractionation
and characterization of organic mater in natural waters
yields important information, one bulk water parameter,
the specific ultraviolet absorbance (SUVA), has proved
to be a useful and robust predictor of both reactivity
with oxidants and treatability. SUVA determination of
a water sample yields a single aggregate value that re-
presents the response of a distribution of chromophores
within a single natural organic matter (NOM) molecule
and among different NOM molecules. Similarly, reac-
tivity of bulk water represents the combined reactivity
of many different molecules and molecular moities. The
objective of this research is to examine how specific
UV absorbance, and more importantly, how the distri-
bution of SUVA in a source water, influences the
formation and speciation of brominated DBFs. Such
information will be useful for optimizing treatment
goals, understanding the effects of treatment processes,
and devising strategies to comply with the D/DBP rule.
Two high-SUVA waters (Charleston and Myrtle
Beach) and two low-SUVA waters (Troy and Water-
ford) were fractionated using four physicochemical
separation processes (i.e., carbon and XAD resin ad-
sorption, coagulation, and ultrafiltration). For each wa-
ter, approximately 50-60 fractions were obtained, each
having different SUVA values. The fractions were
chlorinated according to the uniform formation condi-
tion protocol. The formation of trihalomethane (THM),
nine species of haloacetic acids (HAA9), haloaceto-
nitriles, chloropicrin, chloral hydrate, and cyanogen
chloride were quantified. The relation between the for-
mation and speciation of DBFs and the SUVA of each
fraction was examined. Experimental procedures are
presented in detail elsewhere (Kitis et al., 2000a,b).
Preliminary results showed that for the three phy-
sicochemical separation processes (GAC adsorption,
XAD-8 [batch and column] adsorption, and alum co-
agulation), each fractionated dissolved organic matter
(DOM) in natural water samples by preferentially re-
moving higher SUVA components. Similar trends were
observed for all water samples tested. The SUVA of
DOM remaining in solution was plotted versus adsorb-
ent (GAC or XAD-8) or alum dose applied. The re-
sulting profiles indicated that by increasing the adsorb-
ent or coagulant dose in small increments, it is possible
to incrementally fractionate a natural water and to
probe the hypothetical SUVA distribution of the sample
from high to low values (see Figure 1). However, each
process showed a different fractionation endpoint, indi-
cating that low- or non-UV absorbing components of
DOM are removed to different extents by each pro-
cess. As expected, ultrafiltration separated DOM com-
ponents in solution based primarily on molecular size
differences; no preferential removal of higher SUVA
components was observed because there was not a
strong correlation between SUVA and molecular size.
The results of chlorination experiments and DBF
yields obtained thus far clearly indicate that for each
water tested, there are strong correlations between the
SUVA values of DOM fractions and their THM and
HAA9 yields, independent of how the fractions were
obtained (see Figure 2). The apparent single trend for
each DBF provides evidence that the observed behavior
represents the intrinsic reactivity profile of DOMs to
DBF formation in a (single) natural water. Also, it was
found that low SUVA components of DOM are more
efficient at incorporating bromine. The reactivity pro-
file concept (i.e., understanding how reactivity is cor-
related to SUVA) will allow water utilities to optimize
the degree of treatment required to comply with D/DBP
regulations. The reactive components that require re-
moval, and the degree of treatment necessary to accom-
plish this removal, may be directly obtained from the
relationship between SUVA removal and the degree of
treatment (e.g., alum dose).
Future work will evaluate whether there is any
impact of commonly used isolation processes (e.g., re-
verse osmosis, XAD adsorption) on the DBF reactivity
of isolates obtained from natural waters. The observed
DBF reactivity profile concept will be further tested for
waters having a range of hydrophobicity, measured by
the relative proportion of hydrophobic and hydrophilic
DOM components. Using physicochemical fractiona-
tion, DOM components with different SUVA values
will be obtained. The impact of bromine incorporation
by these fractions will be examined. Additional physi-
cochemical characterization of the fractions will be
attempted to provide insight to the differences in the
bromine incorporation by different DOM components.
The formation and speciation kinetics of brominated
DBFs will be investigated as a function of SUVA,
water chemistry, and reaction conditions.
32
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
References
Kitis M, Karanfi! T, Kilduff JE, Wigton A. The reac- Kitis M, Karanfil T, Kilduff JE, Wigton A. Probing
tivity of natural organic matter to disinfection the reactivity of natural organic matter to disinfection
byprod-ucts formation and its relation to specific byproduct formation using different separation pro-
ultraviolet absorbance. Proceedings of 2000 Annual cesses. Proceedings of 1st World Water Congress of
Conference, American Water Works Association, International Water Association, Paris, France (June
Denver, CO (June 2000a). 2000b).
The Office of Research and Development's National Center for Environmental Research 33
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2001 STAR Drinking Water Progress Review Workshop
Hypothetical SUVA Distribution
->
Lower MW hydrophilic
acids, hydrocarbons, etc. ^_
3+4 5
SUVAMean=SUVAMeasured
Fulvic acids
Humic acids
Typical Drinking Water Sources
Figure 1. A hypothetical SUVA distribution in a natural water
Increasing
Adsorbent or
7 8
SUVA254
O)
I
O
O
Q
100
80
60
40
20
0.0
1.0 2.0 3.0 4.0
SUVA280 (L/mg DOC-m)
O F400GAC
O XAD-8 batch
A XAD-8 column
A Alum
Coagulation
• Ultrafiltration
— Raw Water
— Linear (All
fractions)
5.0
6.0
Figure
2. THMs reactivity profile of the natural organic matter fractions obtained from Myrtle Beach water. The solid line
represents a linear fit to all experimental data.
34
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Molecular Weight Separation
and HPLC/MS/MS Characterization of Previously
Unidentified Drinking Water Disinfection Byproducts
Roger A. Minear' and Sylvia Barrett2
'Department of Civil Engineering, University of Illinois at Urbana-Champaign, Urbana, IL; 2Metropolitan Water
District of Southern California, Los Angeles, CA
New approaches are being examined for better
characterizing disinfection byproduct (DBF) molecular
weight profiles by using tandem mass spectrometry
(MS/MS) techniques. The study will include an ex-
amination of the differences in DBFs that result from
different water disinfection processes. The underlying
hypothesis of this work is that new approaches are
needed for this assessment and that tandem mass spec-
trometry, coupled with prior separations, offers promise
in that regard.
A prerequisite to making such procedures mean-
ingful is the development of preseparation procedures
that will simplify the mass spectral data. The MS/MS
system has the potential for assessment of molecular
weight in the first stage, followed by generation of
specific chemical structural data on selected mass peaks
in the distributions via measurement of related frag-
ments from the selected ion. The MS/MS system,
hence, has its own separation capabilities.
The work is directed at enhancing these capabilities
for complex DBF mixtures with preselection by mol-
ecular-size separations using ultra filtration (UF) mem-
branes and size exclusion chromatography (SEC).
These preselected molecular-size fractions then would
be followed by other high-performance liquid chro-
matographic (HPLC) techniques. Information has been
developed on determining levels of halogenated natural
organic matter (NOM), required to obtain measurable
mass spectra in both stages of the MS system, using
surrogate compounds and their mixtures and chlorin-
ated NOM, represented by Suwannee River Fulvic Acid
(SRFA) and a hydrophilic NOM. Studies have been
conducted on two instruments, Micromass AutoSpe-OA
TOP mass spectrometer at the Metropolitan Water
District of Southern California, and a TSQ 7000 ESI
quadrapole ion trap mass spectrometer at Kyoto Uni-
versity in Japan. From these studies, information has
been found on optimal solvent composition, flow rates,
and scan rates for quality spectra. Using individual
known compounds, their mixtures, and chlorinated
SRFA, variables for the second stage MS were exam-
ined as to the balance between obtaining spectral
fingerprints and demonstrating the presence of chlorine
in the mass selected.
From the complexity of bulk sample spectra, it is
apparent that preseparation of the DBF samples will be
essential. To that end, chlorination of SRFA and Su-
wannee River Humic Acid (SRHA) have been con-
ducted using chlorine 36 labeling to obtain molecular
size distribution of the chlorinated products on size
separation columns (see Figure 1). To date, this has
been a 25 x 200 mm BIAX column (Chrom, Germany)
packed with Toyopearl HW 508 resin (Japan), with a
nominal fractionation range of 100-20,000 g/mol.
It is apparent that chlorine is distributed across the
entire molecular size range, SRHA has chlorine at high-
er molecular size than SRFA (as expected), and radio-
activity also shows up at longer retention times than for
inorganic chloride. This is not surprising, as work with
known compounds has demonstrated that several low
molecular weight chlorinated compounds eluted after
chloride, including haloacetic acids. Longer chlorine
contact times show a slight shift to smaller molecular
size distributions (1 hour versus 24 hours versus 5
days).
The levels of sample concentration required to ob-
tain MS data have been defined; however, the prelim-
inary results demonstrate that separations to reduce the
complexity of the first stage spectra are going to be
essential to reduce the complexity of the second stage
MS.
The use of chlorine 36 affords the mechanism for
evaluation of the separation process by greatly facil-
itating data acquisition from SEC and other HPLC
columns. Traditional total organic halogen measure-
ments proved to be tedious and impractical.
The following are projected activities for the im-
mediate future: (1) additional HPLC columns will be
evaluated for separation of the components prior to
MS/MS examination—Protein Pak SEC and CIS col-
umns will be used; (2) solid-phase extraction will be
explored as a preconcentration step prior to mass spec-
trometry analysis; and (3) using a mixture of standards,
high resolution electrospray ionization will be inves-
tigated, which may provide accurate mass determin-
ation at the first stage (Am = 0.1 for m/z 500 at 5,000
resolution) and better definition of parent ions for
MS/MS.
The Office of Research and Development's National Center for Environmental Research
35
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2001 STAR Prinking Water Progress Review Workshop
1 .OE+04
•g- 8.0E+03
2;
•
2; 6.0E+03
4.0E+03
< 2.0E+03
O.OE+00
-SRFA
SRHA
20 40 60 80
Retention time (min)
100
120
Figure I. Comparison of SEC-CI36 chlorinated SRFA and SRHA samples.
36
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Membrane Introduction Mass Spectrometry Studies
of Halogenated Cyano Byproduct Formation in Drinking Water
Terese M. Olson
Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, MI
Increasing recognition is being given to the im-
portance of nonhumic precursors of disinfection by-
products (DBFs) in drinking water. This especially is an
issue in water supplies with low humic matter content
and as more effective removal of humic matter is
achieved. Proteins, peptides, and amino acids have been
implicated as important precursors of halosubstituted
nitriles and cyanogen halides under these conditions.
Efforts to reduce the use of chlorine disinfectants also
have increased the significance of brominated cyano
byproducts when bromide concentrations are elevated.
Colorado River Water (CRW), a source of drinking
water for more than 20 million people, represents a
water supply where these conditions collide; it is both
low in humic matter and contains moderately high bro-
mide concentrations. The proposed research seeks to:
(1) determine which amino acid precursor compounds
represent the most important source of these DBFs and
the structural features that contribute to their reactivity,
(2) characterize the kinetics and formation mechanism
of chlorinated and brominated cyanosubstituted DBFs,
and (3) model the formation of halogenated cyano com-
pounds in CRW. Initial phases of the project involve a
set of characterization and screening experiments that
will provide the basis for selecting a subset of amino
acids and peptides for later mechanistic study. The ami-
no acid composition of CRW samples currently is being
characterized. Relative reactivities of free and combin-
ed amino acids in CRW with chlorine and chloramine
will be determined, and the formation potentials of
haloacetonitriles (HANs) and cyanogen halides (CNX)
due to the chlorination, chloramination, and bromina-
tion of model amino acids will be examined.
Upon selecting a short list of amino acid and pep-
tide precursors, the kinetics of amino acid halogenation
will be studied by applying a new online technique
known as membrane introduction mass spectrometry
(MIMS). The method relies on the selective membrane
separation of volatile species directly from aqueous sol-
ution into the vacuum of a mass spectrometer ion trap
(see Figure 1). This "pervaporation" approach offers
important advantages for the kinetic study of volatile
byproduct formation, such as cyanogen halides and
nitriles, as extraction and reaction quenching steps are
avoided.
Based on the proposed kinetic studies of cyano
byproduct formation, a model for HAN and CNX
formation in CRW will be formulated. Simulation re-
sults will be compared with actual CRW chlorination
and chloramination experiments.
The findings of this research will help water
suppliers to assess the precursor sources of HAN and
CNX, gain insight into possible control strategies (e.g.,
controlling algal activity, adjusting solution chemistry),
and assess the risk of disinfection strategies. The re-
search also will help to establish optimal approaches for
applying MIMS techniques to disinfection byproduct
analysis.
The Office of Research and Development's National Center for Environmental Research
37
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2001 STAR Drinking Water Progress Review Workshop
—
******2f*'liUii "^.r— *"*^^
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o solvent
\^_ _^s
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2001 STAR Drinking Water Progress Review Workshop
Mechanisms and Kinetics of Chloramine
Loss and Byproduct Formation in the Presence
of Reactive Drinking Water Distribution System Constituents
Richard L. Valentine, Junghoon Choi, and Steve Duirk
Universitv of Iowa, Iowa City, IA
This project seeks to enhance understanding of the
influence of reactive substances in drinking water on:
(1) the fate of monochloramine and the nature of in-
organic reaction products, (2) the kinetics of mono-
chloramine loss, and (3) the formation of selected or-
ganic disinfection byproducts (DBPs). Results also will
be used to develop and extend mechanistic chloramine
reaction models to include the effects of these reactive
substances.
The approach has been to investigate reactions in
batch reactors containing laboratory prepared model
waters. These waters contained a variety of reactive
organic and inorganic components. Natural organic
matter (NOM) includes both extracted humic material
(HM) and nitrogenous compounds (NCs) such as di-
methylamine. Monochloramine reacts slowly with
humic-type organic matter. Most of this loss can be
attributed to relatively simple reduction reactions, not
substitution to form organic DBPs. The loss is charac-
terized by a biphasic reaction. Modeling results indicate
that a one-site reaction model is not adequate to de-
scribe monochloramine loss in the presence of HM. The
reactivity of monochloramine correlates with the UV
absorbance. After resolving several monochloramine
loss pathways occurring in the presence of humic
NOM, the decrease in UV absorbance with time was
observed to correlate with the amount of monochlor-
amine reacting with HM. The stability of both nitrite
and monochloramine in their mixtures successfully was
modeled by consideration of its oxidation to nitrate by
monochloramine via a direct reaction.
Haloacetic acid (HAA) formation was correlated
with monochloramine reactivity with HM. Several
HAAs also were reactive with iron oxides. The fastest
and most extensive decay was observed for monobro-
moacetic acid. Dichloroacetic acid was the most stable.
Several brominated trihalomethanes were reactive in
the presence of ferrous hydroxide. Monochloramine re-
acts with dimethylamine to form dimethylnitrosamine
(NDMA), a potent carcinogen. A mechanism is pro-
posed in which monochloramine reacts with dimethyl-
amine to form hydrazine, which in turn is oxidized by
additional monochloramine to NDMA. A reaction mo-
del, which considers several important reactions, ap-
pears reasonably capable of predicting NDMA forma-
tion in this simple system (see Figure 1).
Reaction of monochloramine with humic type
NOM may be an important loss pathway, which also
results in the formation of several DBPs, especially
HAAs. Reaction of some HAAs with pipe deposit ma-
terial may be an important loss pathway in distribution
systems.
The stability of nitrite in the presence of mono-
chloramine indicates that they may coexist in drinking
water. It is believed that formation of NDMA (and
other nitrosamines) by reaction of monochloramine
with appropriate precursor substances may be important
in chlorinated and chloraminated water and wastewater.
The hypothesized mechanism is in contrast to classical
nitrosation reaction, which requires the presence of ni-
trite. NDMA should therefore be considered a "new"
disinfectant byproduct.
Future work will focus on improving the model for
monochloramine loss and HAA formation in the pre-
sence of NOM and nitrite. A special emphasis will be
on improving the understanding of the newly proposed
NDMA formation mechanism, and its potential signifi-
cance in chlorinated and chloraminated waters.
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2001 STAR Drinking Water Progress Review Workshop
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
DMA, mM
Figure 1. Formation of dimethylnitrosamme (NDMA) by reaction of monochloramine with dimethylamine (DMA). Reaction time 24 hours
Monochlorammeconcentration 0.1 mM Temperature' 25°C. Air: saturated 1 mM bicarbonate buffer. pH: adjusted to 7 0± 0.1.
40
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2001 STAR Drinking Water Progress Review Workshop
Development of a New, Simple, Innovative
Procedure for the Analysis of Bromate and Other
Oxy-Halides at Sub-ppb Levels in Drinking Water
Howard S. Weinberg and Carrie Delcontyn
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC
This project developed a relatively simple method-
ology to provide the tools for assessing exposure to
bromate in drinking water at the 10~6 cancer risk level
of 0.05 ug/L that has hitherto been impeded by the lack
of sensitivity of existing methodologies. This new ana-
lytical methodology provides the U.S. Environmental
Protection Agency and the water monitoring commun-
ity with the ability, using existing analytical equipment
with simple add-on accessories, to monitor bromate,
chlorite, and iodate in drinking water in a linear range
from 0.05-100 u.g/L without being impacted by the
presence of higher levels of anions.
The postcolumn reaction converts bromate, chlor-
ite, iodate, and to a lesser extent chlorate, eluting from
an ion chromatographic column into the highly sensi-
tive chromophore, the tribromide ion, which can be de-
tected at levels as low as 0.05 ug/L in drinking water
(see Table 1). This method was evolved with the ex-
press purpose of providing a tool for meeting the ob-
jective of evaluating bromate occurrence at the 10"6
cancer risk level resulting primarily from the use of
ozone in drinking water treatment.
Coincidentally, the sensitivity of this method pro-
vided proof of the presence of bromate resulting from
the use of hypochlorite as the agent of disinfection. De-
pending on the dosage and number of points of hy-
pochlorite addition during treatment, the levels of bro-
mate resulting from its usage are indicated in the range
0.1-3 ug/L.
Among 20 plants surveyed using this technique,
the average level of bromate contributed by the use of
hypochlorite was 0.94 ug/L. A mass balance between
the levels of bromate in the hypochlorite feedstocks and
finished water proved beyond doubt the source of the
added contaminant. The results do suggest that the level
of contamination of bromate in hypochlorite varies
across the country with those plants sampled in Region
5 exhibiting the highest levels of contamination. With
discussions towards future regulation of bromate in
drinking water required to balance risk with the cost of
alternative treatment, this finding seriously is impeding
attempts to regulate bromate closer to 0.05 ng/L from
its current regulated level of 10 ug/L in the United
States.
Table 1. Statistics of analysis.
„ . , i PQL ,, Standard ! RSD MDL
Detection Analvte , ,. . N . . ;
! (ug L) Deviation | Percent (ug/L) |
UV-PCR
Conductivity
Iodate
Chlorite
Bromate
Chlorate
Bromide
Chlorate
0.06
0.10
0.05
70.0
10.0
10.0
7
6
7
7
7
7
0.01
0.02
0.004
8.5
1.5
1.0
12
18
7.8
12.2
15.4
10.1
0.04
0.08
0.01
31.5
5.6
3.7
PQL = Practical quantitation limit.
RSD = Relative standard deviation.
MDL = Method detection limit.
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2001 STAR Drinking Water Progress Review Workshop
Formation and Stability
of Ozonation Byproducts in Drinking Water
Howard S. Welnberg, Alice Harris, and Shikha Bhatnagar
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC
Generally, it is perceived that disinfection byprod-
ucts (DBFs) produced by ozonation represent less of a
health hazard than those produced by the chlorine-
containing disinfectants, at least by the use of short-
term bioassays. Also, it has been noted that the types of
oxidation byproducts produced by ozonation of natural
waters are in many cases the same as compounds pro-
duced by natural oxidation processes in streams, lakes,
and reservoirs. The implication is that naturally occur-
ring compounds will be safer than "unnatural" com-
pounds such as trihalomethanes (THMs) and haloacetic
acids (HAAs) produced by chlorination. However, there
are fallacies associated with each of these arguments.
Short-term bioassays are not at a stage of de-
velopment to use in relative risk assessments. Also, the
preconcentration methods used to obtain extracts for
bioassays may not efficiently trap polar byproducts
from ozonation of natural waters. Natural waters them-
selves often are mutagenic, so the argument that ozone
produces "natural" organics is not encouraging.
With less than 50 percent of the assimilable organic
carbon generated by ozonation remaining unidentified,
this project is investigating new methodologies for target-
ing unidentified byproducts. These together with refined
existing techniques will be employed to study the impact
of water quality parameters on the formation and stability
of these compounds in distributed drinking waters.
Stage one of this study focused on evaluating
methodologies for accurately targeting carbonyl-contain-
ing compounds tentatively identified in a variety of lit-
erature results combining field-, pilot-, and laboratory-
scale ozonations. The species targeted in this initial ap-
proach include the following compounds: CrC12 mono-
saturated aldehydes, glyoxal, methyl glyoxal, dimethyl
glyoxal, trans-2-hexenal, formic, acetic, and oxalic acids,
and the three mixed functional pyruvic, glyoxylic, and
ketomalonic acids.
Previous attempts to quantify these byproducts in
aquatic matrices involving derivatization techniques may
have suffered from inadequate recoveries. This conclu-
sion was arrived at by synthesizing the relevant oximes
and esters and comparing derivatization efficiency in the
aquatic medium alongside the pure derivatized standard.
Purification of the derivatized standards involved utilizing
a stepwise thin layer chromatography with the isolated
product assayed by proton nuclear magnetic resonance.
Stage two of the project involved the development
of new analytical methods for determining three distinct
groups of proposed ozonation byproducts that hitherto
have not been identified in ozonated waters. These in-
clude organic peroxides, epoxides, and multifunctional
carbonyl-containing organic byproducts. In the absence
of many commercial standards for these compounds,
these efforts have focused on evolving strategies for
maximizing isolation from the aquatic matrix and en-
hancing detection for low molecular weight repre-
sentative compounds from each of these classes.
The peroxides are separated on a C-18 column by
high-performance liquid chromatography (HPLC). A
postcolumn reaction then converts the individual spe-
cies into hydroxyl radicals, which then react with para-
hydroxyphenylacetic acid at pH 11 to produce a fluor-
escent dimer whose detector response is directly pro-
portional to the concentration of organic peroxide in the
original sample. Current detection limits are in the
range of 10-30 u.g/L, but these probably can be lowered
by an order of magnitude using a preconcentration
approach. Epoxides are extracted from water after
derivatization with difluoroaniline, but the kinetics of
the latter reaction are slow and are impacting a realistic
minimum quantitation limit.
Carbonyl-containing compounds that have not been
amenable to derivatization with pentafluorobenzylhy-
droxylam successfully are isolated from water using
2,4-dinitrophenylhydrazine, which produces a finger-
print diode-array spectrum following resolution of indi-
vidual components by HPLC. Online preconcentration
permits detection at the 10 nanomolar level, and the
technique is compatible with electrospray mass spectro-
metric detection, which will be used to identify new
byproducts. Laboratory-generated ozonated surface wa-
ters are being used to assess these methods using a
semibatch operation to generate the byproducts (see
Figure 1).
42
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2001 STAR Drinking Water Progress Review Workshop
Air Ozonizer
NaOH Trap
5-Wav Valve
To measure gas
inflow absorbance
10L
To measure
^ gas outflow
absorbance
A
A
Kl
Figure 1. Scheme of the approach used in the laboratory for generating ozonated waters
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2001 STAR Drinking Water Progress Review Workshop
Kinetic-Based Models
for Bromate Formation in Natural Waters
Paul Westerhoff
Department of Civil and Environmental Engineering, Arizona State University, Tempe, AZ
Ozone (O3) is an effective disinfectant, but it can
form byproducts (e.g., bromate). Bromate forms via ox-
idation of naturally occurring bromide through a series
of steps involving Oj and hydroxyl radicals (HO).
There is a need to develop tools to understand and pre-
dict bromate (BrO3~) formation while still achieving
high levels of microbial disinfection. The central hypo-
thesis is that a kinetic-based understanding of natural
organic matter (NOM) reactions with HO and aqueous
bromine (HOBr/OBr~) over a range of temperatures is
necessary to develop mechanistic-based models for
bromate formation in bulk waters.
The objectives of this project are to: (1) develop a
comprehensive database of BrO3~, O3, and HO radical
concentrations; (2) determine rates of reaction between
HOBr and OBr~ and NOM; (3) calibrate and verify a
BrO3~ formation mechanistic-based model that includes
NOM; (4) simulate BrO3~ control measures necessary
to meet proposed and future maximum contaminant
levels; and (5) link the numerical BrO3~ formation
model with hydraulic and concentration x time disin-
fection models.
Approximately one-half of the database including
oxidant concentrations (O3 and HO) and bromate data
has been completed for ultrapure water and Colorado
River Water. R^ values have been calculated that
represent the ratio of O3 to HO concentrations. A
correlation between RC, and dissolved organic carbon
(DOC), alkalinity, pH, and temperature has been de-
veloped from literature values.
After testing several experimental methods for
determination of reaction rates between NOM and halo-
gens (e.g., aqueous bromine), a colorimetric approach
(ABTS) was adopted. A large experimental matrix of
reaction rate constants have been determined for the
reaction of HOBr and OBr~ with unozonated and ozon-
ated NOM (see Figure 1). Although not directly related
to bromate formation, companion experiments using
HOC1 and OC1~ were simultaneously undertaken to
better understand the reaction mechanisms and provide
insight into organohalogen formation.
As an example of representative results, the second
order reaction between HOBr and CAP-NF for HOBr
was 31 M'V and only 1.4 M'V for HOC1. Hence,
HOBr reacts faster with NOM than HOC1, but the rate
of reaction is quite low given the rates of reaction
between important oxidants (ozone and HO radicals)
and bromine species during the formation of bromate.
The presence of NOM is likely to exert a larger effect
on the amount of oxidant present than affect the inter-
mediate bromine species during bromate formation.
A mechanist model has been developed to predict
bromate formation as a function of oxidant pathways
and key water quality factors (pH, ammonia concen-
tration, alkalinity). The model handles the presence of
DOC as a site-specific rate constant value.
44
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2001 STAR Drinking Water Progress Review Workshop
2.5
1.5
o
c
o
U
0.5
00*
0 1 .
02 |
03 ,
04 ;
05 '
-06 :
A "J
O
• o
B
0 Blank
•0 24 uM
A0 6 uM
°1 2uM
A 1 8 uM
O
2
o
o
A
O
A
O
A
O
B
^
O
0
•
A
0
A
30 60 90
Reaction Time (sec)
120
"*~~ SR-RO:Br,
-*— CAP-NF:Br2
*•'- SR-RO:C12
^-'CAP-NFrCl,
20 40 60 80 100 120
Reaction Time (min for HOC1, sec for HOBr)
140
Figure 1. Consumption of HOBr and HOC1 by a reverse osmosis isolate (SR-RO) from the Suwannee River or nanofiltration
isolate (CAP-NF) from the Colorado River (pH = 5, DOC = 50 (iM) The inset figure represents analysis for
estimation of pseudo first-order rate constant for HOBr consumption by CAP-NF
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Mechanistic-Based Disinfectant
and Disinfectant Byproduct Models
Paul Westerhoff', David Reckhow2, Gary Amy3, and Zaid Chowdhury4
'Department of Civil and Environmental Engineering, Arizona State University, Tempe, AZ;2 University of
Massachusetts, Amherst, MA;} University of Colorado, Boulder, CO; 4Malcom Prinie, Inc., White Plains, NY
The water industry faces new challenges in under-
standing and controlling disinfection byproduct (DBF)
formation as health concerns demonstrate a need for
more stringent regulatory DBF requirements. Mechan-
istic tools for understanding and predicting the rate and
extent of DBF formation are required to facilitate the
evaluation of DBF control alternatives.
This research project involves the development of a
mechanistic-based numerical model for chlorine decay
and regulated DBF (trihalomethane [THM] and halo-
acetic acid [HAA]) formation derived from (free) chlor-
ination; the model framework will allow future modi-
fications for other DBFs and chloramination.
Predicted chlorine residual and DBF results will be
compared against predictions from several other quasi-
mechanistic models. It is anticipated that a significant
improvement in prediction accuracy over existing em-
pirical models will occur. The central modeling hypo-
thesis is that a two-site reaction mechanism can be used
to predict disinfectant decay in the presence of natural
organic matter (NOM). It assumes that NOM contains
both slow and fast disinfectant-reacting and DBF-
forming sites. NOM site densities and concentrations
are related to the concentration, size, structure, and
functionality of NOM. The project is approximately 90
percent completed. Raw waters from the Colorado
River (AZ), Lake Houston (TX), and Harwoods Mill
(VA) were collected and subjected to laboratory treat-
ment by the following processes: filtration only plus
coagulation, ozonation, chemical softening, activated
carbon sorption, and ultrafiltration followed by filtra-
tion. A total of 18 samples were produced and subse-
quently subjected to chlorination.
The amount and characteristics of the dissolved
organic carbon were altered by these processes, and
were quantified by UV absorbance, fluorescence, mole-
cular weight, and hydrophobicity. Upon chlorination,
the kinetic consumption of chlorine residual and DBF
formation was monitored under different water treat-
ment conditions (pH, temperature, chlorine dose, spiked
bromide) over the timeframe of a few minutes to days.
Figure 1 represents typical THM species pro-
duction (symbols), shown for raw Colorado River
water. DBF production was simulated using a C+-
language model that simultaneously solved differential
equations (lines in Figure 1). A good correlation
between experimental and predicted values has been
obtained for chlorine residual, THM species, and di-
halogenated HAAs.
The model has been encoded into Version 2 of the
U.S. Environmental Protection Agency's Water Treat-
ment Plant Simulation Model, which has the option of
using either the mechanistic model developed herein, or
the existing empirical models, for predicting DBF for-
mation in water treatment plants and distribution sys-
tems.
46
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2001 STAR Drinking Water Progress Review Workshop
_ 2 x
20
40
time (h)
60
80
Figure 1. Kinetic production of THM species observed in laboratory batch tests (symbols) and simulated by the mechanistic
model (lines) upon chlonnation (38 nM) of raw Colorado River water (T= 15°C, pH=7.5, Br= 1.18 uM)
The Office of Research and Development's National Center for Environmental Research
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Section 4.
Drinking Water Treatment
Studies
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2001 STAR Drinking Water Progress Review Workshop
Evaluation of the Efficacy of a New Secondary
Disinfectant Formulation Using Hydrogen Peroxide
and Silver and the Formulation of Disinfection Byproducts
Resulting From Interactions With Conventional Disinfectants
Stuart A. Batterman ', Khali! H. Money', Shuqin Wang', Lianzhong Zhang', James Warila',
Ovadia Lev2, HillelShuval2, Badri Fattal2, and An-Tsun Huang1
'Department of Environmental Health Sciences, School of Public Health. University of Michigan, Ann Arbor, MI;
'Division of Environmental Science, Graduate School of Applied Science and Technology:, Hebrew University,
Jerusalem, Israel
The objectives of this research project address two
critical issues associated with the use of a new second-
ary disinfectant formulation utilizing hydrogen perox-
ide (H2O2) and silver (Ag+): (1) the efficacy of the for-
mulation to provide long-term residual disinfection,
including the control of coliform bacteria, bacterial re-
growth, and slime/biofilm control; and (2) the identifi-
cation and quantification of disinfection byproducts
(DBFs) that may result from interactions with conven-
tional chlorine- and oxidant-based disinfectants. The re-
search encompasses laboratory studies and demonstra-
tions to evaluate the effectiveness of the alternative dis-
infectant in a range of source waters and utility system
characteristics. The secondary disinfectant is one of the
few nonchlorine-based disinfectants that can provide
long-term residual disinfection in drinking water sys-
tems. By combining two or more disinfection agents, it
may be possible to lower concentrations of each com-
ponent, reduce exposures, minimize the formation of
toxic and undesirable DBFs, and thus minimize health
risks associated with disinfection.
The approach to this research includes the follow-
ing: (1) laboratory evaluation of microbial disinfection
efficacy, including optimal formulation of the second-
ary disinfectant and optimal doses of primary and sec-
ondary disinfectants; (2) laboratory evaluation of DBF
formation resulting from interactions with various pri-
mary disinfectants; and (3) demonstration of the disin-
fectant's effect on biofilm formation and removal to
provide "real world" results. These components are de-
signed to provide a comprehensive evaluation of the
microbial disinfection efficiency and DBF formation
potential of the new disinfectant.
Preliminary findings indicate that the addition of
the secondary disinfectant following the use of chlorine
as a primary disinfectant produces very dramatic reduc-
tions in DBF formation (e.g., trihalomethanes [THMs]
and haloacetic acids [HAAs]), as demonstrated in Fig-
ure 1 using local groundwater. This results from the
reduction of chlorine to chloride by H2O2, which halts
further reaction of chlorine with dissolved organic mat-
ter and other DBF precursors. When used with ozone,
H2O2 quenches formation of THMs and also reduces, to
an extent, the formation of inorganic byproducts (e.g.,
bromate), as demonstrated in Figure 2. These reduc-
tions result from several reactions that have been in-
vestigated in both empirical and mechanistic studies
(e.g., Figure 3 shows the kinetics of bromate formation
in a model system). The reduction in DBFs resulting
from interactions among primary and secondary disin-
fectants applies to a wide range of temperatures, pH,
bromide concentrations, and dissolved organic carbon
levels. Sequential additions of chlorine, ozone, or other
primary disinfectant (possibly with ammonia) and
H2O2/Agf as a secondary disinfectant may provide op-
timal performance. Aldehyde formation from reactions
between H2O2 and ozone are being investigated.
The inactivation performance of the combined disin-
fectant, its individual components, and a commercially
available stabilized formulation of H2O2 and Ag+ have
been evaluated for several bacteria and viruses. Labora-
tory studies indicate that the combined disinfectant ex-
hibits a synergistic action on the viability of E. coli;
however, no increased virucidal action was observed.
The biocidal action generally increased with higher
temperature and pH, and decreased in secondary and
tertiary effluents. The H2O2 component induced a wide
array of stress responses, and bacteria deficient in the
. ability to activate central cellular stress responses were
hypersensitive to both H2O2 and Ag+.
These studies suggest that the combined disinfec-
tant may be appropriate for use as a long-term second-
ary residual disinfectant for relatively high-quality
water. However, further experiments examining biofilm
prevention showed that the bacteria surviving after 48
hours of disinfection had high catalase activity, hinting
that the combined disinfectant may have limited effec-
tiveness in continuous operation. Figures 4a and 4b
show that biofilm prevention effectivity is largely due
to H2O2, and that inactivation activity is higher at the
first stages of biofilm formation and less effective at
later ones.
Widespread use of the combined disinfectant, if
practical, might result in potential for uptake in fish and
humans. An ecological model was constructed to simu-
late partitioning between water and sediment, uptake by
algae, invertebrates, and fish (trout and carp) as well as
risks to humans from fish consumption. Monte-Carlo
The Office of Research and Development's National Center for Environmental Research
51
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2001 STAR Drinking Water Progress Review Workshop
simulations were used to represent the uncertainty and
variability of input parameters. The modeling effort
used a variety of scenarios, including "worst case" con-
ditions in which receiving waters provided small
amounts of dilution and subsistence fishers consumed
large amounts of high trophic-level feeders. The results
suggest that risks are minimal under all likely scenarios.
Information is being developed regarding long-
term disinfection efficacy in different source waters and
environmental and utility conditions. Effects on DBFs
of the primary disinfectants and any new byproduct
formation are quantified, as are optimum dosages and
pathogen inactivation. These results will be compared
to the disinfection efficacy and DBF formation of con-
ventional disinfectants. The research results will be
suitable for use in exposure and risk assessment pur-
poses to support future policies and decisions regarding
disinfection approaches.
Laboratory tests on the DBF formation potential
are being concluded with an investigation of aldehyde
and other species resulting from interactions with H2O2
and ozone. The inactivation performance evaluation
studies for poor water quality conditions (i.e., high total
organic carbon-level water) are underway.
p p b
H202/Ag
Figure 1. Reduction in THMs and HAAs as a result of the addition of a secondary disinfectant following the addition of chlorine as a
primary disinfectant in laboratory tests. Mixed ground and surface water in the local city supply was used The graph reflects
DBFs resulting after 24 hours. The bars in the foreground reflect the secondary disinfectant, the bars in the rear reflect Cl alone.
The basic water parameters were: Br" = 0.081 mg/L, TOC = 3 1 mg/L, initial Cl = 5 mg/L, residual Cl = 1 85 mg/L, pH = 6.85
(buffered) TTHM=total THMs, THAAs=total HAAs.
52
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2001 STAR Drinking Water Progress Review Workshop
250
200
g 100
50
0
D without S/H
E3vvitliS/H
50
~ 40
!30
| 20
10
0
03(mg/L)
D
10
20
Time(min)
* without S/H
D with S/H
D
30
40
Figure 2. BrO-, formation with and without additions of Ag'/HiO?
(S/H) at various initial O; concentrations [Br~] = 600
ug/L, [DOC] = 30 mg/L. pH = 7 0. T = 30 C. t = 30 mm
Figure 3. Time trend of BrO-,' formation with and without
AgVH,O, (S/H) addition [O5] = 60 mg/L, [Br'] = 600
ug/L. [DOC] = 30 mg/L, pH = 7 0; T = 25. Ag+/H,O, (30
Hg/L/30 mg/L) added 1 mm after ozonation started
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
CFU/ml, with AgNO,
CFU/c m with AgNO,
-HO— CFU/ml, with H2O2
CFU/c m with H2O2
Q CFU/ml, with H2O2+AgNO3
CFU/c m with H2Oz+AgNO3
5O 1OO ISO
Time (hours)
200
A CFU/ml, with AgNO3
CFU/c m with AgNO,
—Q— CFU/ml, with H2O2
CFU/c m with H2O2
f • I • • • •'• I
50 60 70 80 90 100 110 120
Time (hours)
Figure 4a. Effect of AgNO; (30 ppb) and H2O2 (30 ppm) combined addition after 48 hours of biofilm formation In this case, silver
ions reduced biofilm bacteria by a half order of magnitude for short time (approx. 5 hours), then bacterial counts returned
to previous values A much sharper reduction was observed with free living and biofilm bacteria under the activity of H2C>2.
For 20 hours, biofilm count decreased by four orders of magnitude, while free living bacteria continued up to 95 hours to
drop by four orders of magnitude
Figure 4b. Effect of AgNOi (30 ppb) and H2C>2 (30 ppm) on free living and adsorbed bacteria (biofilm) on galvanized iron coupons in
tap water. Free living bacteria were reduced by 3.5 logs during 24 hours under combined AgNCb and HjOi Biofilm
bacteria were reduced under similar conditions by 2 logs for the same period of time. In both cases, bacterial numbers did
not change and remained stable up to 100 hours (termination time of the experiment)
54
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2001 STAR Drinking Water Progress Review Workshop
Evaluation of Ozone Byproduct Formation
Under Ozone Dose, Temperature, and pH Variation
Michael S. Elovitz', Jehng-Jyun Yao 2, and Dick J. Miltner'
1 Treatment Technology Evaluation Branch, Water Supply and Water Resources Division,
U.S. Environmental Protection Agency, Cincinnati, OH; 'Oak Ridge Institute for Science and Education
Postdoctoral Fellow, Oak Ridge, TN
This project was one part of a comprehensive re-
search plan (Research Plan for Microbial Pathogens
and Disinfection By-Products in Drinking Water) estab-
lished by the U.S. Environmental Protection Agency's
Office of Research and Development in 1997. The
principal goal of this study was to study the effects of
ozone dose, temperature, and pH on the formation of
ozonation byproducts (OBPs) as well as ozone's benefi-
cial use for decreasing ultraviolet (UV) absorbing com-
ponents and the precursors of chlorinated disinfection
byproducts.
A main objective of this project was to examine
bromate (BrC>3~) formation resulting from low to mod-
erate bromide levels (< 200 ppb) and higher than nor-
mal ozone dosages in accord with anticipated high
ozone CT requirement for inactivation of Qyptospo-
ridium. In addition, at the time the Research Plan was
written, nonhalogenated organic OBPs still were receiv-
ing considerable scrutiny; therefore, another objective
was to include the formation of aldehydes, carboxy-
lates, and ketoacids in the analyses.
Finally, another objective was to examine the abil-
ity of ozonation to eliminate reactive sites within the
dissolved organic matter that would otherwise react
with chlorine to form chlorinated DPBs. Assessment
was made by evaluating the loss of UV-absorbance and
performing chlorination experiments and analyzing for
trihalomethanes, haloacetic acids, and total organic ha-
lide formation. A natural water (East Fork Lake, OH)
with a final dissolved organic carbon (DOC) concen-
tration of 2 mg/L was used for the study. The water was
spiked with bromide to either 100 ppb or 200 ppb.
Ozonation experiments were conducted in both a 1 -
L batch reactor and in a 2-L (14-minute theoretical hy-
draulic residence time) flow-through reactor, which
consisted of a counter-current bubble column followed
by a second reaction column and equipped with 10
sampling ports along the flow path.
The main experimental matrix included: (1) five O3
doses
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2001 STAR Drinking Water Progress Review Workshop
80
70
60
50
Q.
30
_ V. ,,
2Q ^ -O •Batch-2.6mg/L
-~ O * Batch-3 8mg/L
— O OColumn-2.6mg/L
10 Q 1Column-3.2mg/_L
0 5 10 15 20 25 30
O3-exposure(/-CT) [mg/Lxmin]
Figure 1. Bromate formation as a function of CVexposure and O,-CT in the batch (solid symbols) and column (open symbols) reactors,
respectively Circles represent a low Ch dose (2.6 mg/L) and squares a high O:, dose (3.2 or 3 8 mg/L), 22° C, pH 7.5, 2 mg/L
DOC.
56 The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Integrated Approach for the Control
of Cryptosporidium parvum Oocysts and Disinfection
Byproducts in Drinking Water Treated With Ozone and Chloramines
Benito J. Marinas, Roger A. Minear, Jaehong Kim, Hongxia Lei, Jason L. Rennecker,
Amy M. Driedger, and Benito Corona-Vasquez
Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, Urbana, IL
The overall goal of this project is the development
of process design recommendations for the simulta-
neous control of Cryptosporidium parvum oocysts and
disinfection byproducts (DBFs) during ozone/chlora-
mines sequential disinfection of natural waters. Be-
cause the main objective of the study is to develop an
integral control strategy, the scope of work focuses on a
limited number of selected DBFs (bromate, formalde-
hyde, and cyanogen halides) associated with the ozone/
chloramines sequential disinfection process.
Experiments have been performed to investigate
the kinetics of C. parvum oocyst inactivation resulting
from sequential application of ozone and monochlor-
amine. Experiments with single disinfectants resulted in
inactivation curves characterized by the presence of an
initial lag phase, during which little inactivation occur-
red followed by pseudo-first order inactivation kinetics.
The rate of C. parvum oocyst inactivation with mono-
chloramine was enhanced when this chemical was used
as secondary disinfectant. Ozone pretreatment resulted
in the removal of the lag phase during the secondary
inactivation with monochloramine. Furthermore, the
rate of secondary monochloramine inactivation was
faster than that observed for postlag-phase primary dis-
infection. This synergistic effect was more pronounced
at lower temperatures.
Experimental results revealed that the CT (product
of disinfectant concentration and contact time) required
to achieve a certain level of inactivation was unique. No
pH dependence was observed for primary inactivation
with ozone in the pH range of 6-10, or primary and
secondary inactivation with monochloramine at pH val-
ues of 8 and 10. The mechanism of cyanogen bromide
(BrCN) formation during O3/NH2C1 sequential disin-
fection also is under investigation. BrCN can be formed
through the reaction between HCHO and NH2Br, the
latter compound produced from the reaction between
NH2C1 and Br~ ion. Based on the reaction pathway re-
ported for cyanogen chloride, potential intermediates in
the formation of BrCN include JV-bromoaminometh-
anol, N-bromodimethanolamine, iV-bromomethanimine,
and cyanide. The competing decomposition of NH2Br
involves many reactions with the overall rate affected
by pH, [NH3]/[HOBr] ratio, initial NH2Br concentra-
tion, and temperature.
An integrated model has been developed to simul-
taneously predict bromate formation and C. parvum
oocyst inactivation during ozone treatment of natural
waters. The model consists of elementary chemical
reactions that are responsible for ozone decomposition
and bromate formation as well as a delayed Chick-
Watson kinetic expression for ozone disinfection of C.
parvum oocysts. This model has been evaluated experi-
mentally with a laboratory-scale batch reactor, and a
laboratory-scale flow-through ozone bubble-diffuser
contactor using synthetic solutions. In addition, semi-
empirical expressions taking into account the effect of
natural organic matter on relevant chemical and dis-
infection reactions have been incorporated into the
model. The kinetic expressions were combined with
empirical mass transfer correlations, and the axial dis-
persion model to simulate the gas transfer and hydro-
dynamic characteristics in pilot- and full-scale ozone
bubble-diffuser contactors. Figure 1 shows an example
of the integrated model application to simulate the full-
scale ozone contactor located at the Los Angeles Aque-
duct Filtration Plant.
Future research plans include the performance of
additional experiments to assess the role of natural
organic matter in the formation of bromate at tempera-
tures in the range of 5-25° C. The kinetic information
obtained from laboratory-scale experiments will be in-
corporated into the computer model. Model evaluation
with full-scale ozone contactors will be attempted using
data available either in the literature or from water util-
ities.
Future efforts also will focus on the quantitative
study of the fast equilibrium reactions and subsequent
slower reactions between monobromamine and formal-
dehyde. Experiments also will be performed with var-
ious preozonated natural waters dosed with NH2C1 and
Br.
The Office of Research and Development's National Center for Environmental Research
57
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2001 STAR Drinking Water Progress Review Workshop
A
D F G H
(a)
Liquid Phase
Ozone
Conccntratio
Profile
(c)
C. parvum
Inactivation
— 100% capacity
75% capacity
— 50% capacity
— 25% capacity
0.1 0.2 0.3 0.4 0.5 0.6 0.7
Normalized Cumulative Volume
0.8 0.9
1.0
Figure 1. Simulation of dissolved ozone concentration, bromate formation, and C. parvum mactivation at the ADWP plant ozone
contactor (water flow rate = 85 MOD; gas flow rate = 17.8 kscfh; temperature = 9° C, pH = 8.2; Br~ = 33 Ug/L; DOC = 1.9
mg/L; ozone input capacity = 25-100%)
58
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Pilot Studies of an Ozonation/FBT Process
for the Control of DBPs in Drinking Water
Susan J. Masten, Kyung-Hyuk Lee, Kuan-Chung Chen, and Alexander A. Yavich
Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI
A combined ozonation/biological fluidized bed
treatment (FBI) for the removal of trihalomethane
(THM) and other disinfection byproduct (DBF) pre-
cursors from drinking water is being investigated. This
project also aims at developing design criteria for the
proposed ozonation/FBT system. The goals of this pro-
ject are being accomplished through bench-scale and
pilot-scale studies using Lake Erie water collected at
the Monroe Water Filtration Plant (Monroe, Ml) and
Lake Lansing water (Haslett, Ml). Huron River water
has a total organic carbon (TOC) concentration of 6-8
mg/L and is typical of rivers across the United States.
Although Lake Lansing water does not provide source
water to any treatment plant, it has been selected be-
cause of its high TOC concentration (9-11 mg/L). Fu-
ture work will involve other source waters.
The effects of ozonation reaction pathways on the
formation of both DBPs and biodegradable organic
carbon (BDOC) were investigated using a bench-scale
ozonation system. The study showed that the produc-
tion of OH radicals relative to ozone dose adjusted for
alkalinity was the greatest in water with the lowest
TOC concentration (Lake Erie water) and the lowest in
water having highest TOC concentration (Lake Lansing
water). This suggests that organic matter in selected
source waters acted more as a scavenger rather than as a
promoter of radical reactions. Direct ozone reactions
appear to favor the production of BDOC, whereas ra-
dical reactions result in the removal of organic carbon.
In biodegradation studies involving a bench-scale
biodegradation system, several parameters were identi-
fied that described the kinetics of the removal of organ-
ic matter during biodegradation. These included: (1) the
minimum empty bed contact time (EBCTmm), which
represented the minimum EBCT required to remove
rapidly BDOC ("fast" BDOC); (2) BDOC5,OW, which
represented the amount of BDOC that at least remained
after biodegradation at EBCTmm; and (3) Rmax, which
was defined as the maximum rate of the biodegradation
of "fast" BDOC. It was found that essentially all or-
ganic matter in Lake Erie water was refractory (i.e., not
subject to biodegradation). Huron River water con-
tained approximately 20 percent of potentially bio-
degradable organic matter, whereas nearly one-half of
the organic matter in Lake Lansing could be bio-
degraded. The ozonation of Lake Erie water at doses of
up to 3 mg/mg C did not result in the production of
biodegradable organic carbon. Ozonation of Huron Ri-
ver water resulted in an increase of BDOC concentra-
tion from 1.2 mg/L in raw water to 2.8 mg/L in water
ozonated at a dose of 1 mg/mg C. However, the con-
centration of BDOCS|OW also was increased after ozona-
tion.
Unlike Huron River water, for which no significant
changes in biodegradation kinetics were observed at
doses greater than 0.5 mg/mg C, the biodegradation
parameters for ozonated Lake Lansing water were
affected by ozone doses to a much greater extent.
Ozonation of Lake Lansing water at a dose of 0.75
mg/mg C resulted in an increase in BDOC concen-
tration from 5.04 to 6.06 mg/L. Ozonation at ozone
doses of 1.5 and 3 mg/mg C resulted in the formation of
additional 0.8 and 1.33 mg/L BDOC, respectively. An
increase in ozone dose resulted in an increase in Rmax
and a decrease in EBCTmm. The most striking dif-
ference between Huron River water and Lake Lansing
water was observed with respect to "slow" BDOC. The
concentration of BDOCsiow in Lake Lansing water
decreased with an increase in ozone dose compared to
Huron River water, in which BDOCS|OW increased at a
dose of 0.5 mg/mg C and leveled off at higher ozone
dosages.
A pilot-scale study of the ozonation/FBT system
has been initiated. The 1-gpm pilot-scale system has
been installed at the Monroe Water Treatment Plant in
Monroe, MI. Although all necessary kinetic data and
operating information have been generated, bench-scale
studies with selected source waters will continue.
These studies will be directed towards further optimiz-
ing parameters for ozonation and biodegradation to in-
crease the efficiency of the combined ozonation/FBT
system.
The Office of Research and Development's National Center for Environmental Research
59
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Section 5.
Microbial Contaminants
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2001 STAR Drinking Water Progress Review Workshop
Prevalence and Distribution of Genotypes
of Cryptosporidium parvum in United States Feedlot Cattle
EdwardR. Aftviil1, C. Elmi2, W.P. Epperson3, D.M. Grotelueschen4, J. Kirkpatrick*, B. Hoar6,
W.M. Sischo7, L.y. Carpenter*, D. Brewster9, W. Riggs10
'Department of Population Health and Reproduction, School of Veterinary Medicine, University of California,
Davis, CA; 'U.S. Department of Agriculture, Fresno, CA; 'South Dakota State University, Brookings, SD;
4 University of Nebraska, Lincoln, NE; ^Oklahoma State University, Stillwater, OK; ^California Department of
Health Sen-ices, Davis, CA; 7 University of California, Davis, CA;*U.S. Department of Agriculture, Olympia, WA;
9 U.S. Department of Agriculture, Lakewood, CO; "'U.S. Department of Agriculture, Austin, TX
The overall goal of the proposed research project is
to establish the prevalence and distribution of geno-
types of Cryptosporidium parvum in western and cen-
tral U.S. feedlot cattle. In addition, the fecal concen-
tration of oocysts from each genotype will be quantified
to estimate oocyst loading rates for this source of patho-
gen. A secondary objective is to determine manage-
ment and animal risk factors that are associated with
cattle shedding one or more genotypes of C. parvum.
The original geographic focus for the multistate cross-
sectional survey of feedlot cattle was California, Ne-
braska, Washington, South Dakota, and Colorado. Two
additional states with large feedlot cattle populations
have been added—Texas and Oklahoma—for an over-
all enrollment of seven states located throughout the
central and western United States. As of December 31,
2000, approximately 240 cattle per feedlot have been
sampled from feedlots located in six of the seven en-
rolled states, resulting in 1,440 tested samples. Using a
direct immunofluorescent assay that reliably can detect
600 or more oocysts per gram of fecal material, four
infected cattle have been identified out of the 1,440
tested. To confirm that the diagnostic method is not
falsely classifying fecal samples as negative, 10 ran-
domly chosen samples per feedlot are being tested us-
ing immunomagnetic separation of oocysts, followed
by the standard direct immunofluorescent assay.
This research project has shown previously that
this combined method can, on average, detect as few as
one oocyst per gram of bovine fecal material. Of the 60
samples tested to date, none contained detectable levels
of C. parvum oocysts. Using a polymerase chain re-
action-restriction fragment length polymorphism (PCR-
RFLP) technique developed by the Centers for Disease
Control and Prevention that targets the 18S small-
subunit rRNA gene locus, three of these four presump-
tive C. parvum isolates have been confirmed as C. par-
vum, displaying the typical bovine PCR-RFLP pattern.
DNA sequencing of the amplicon is in progress to
improve the discriminatory power of this genotyping
method and to detect minor polymorphisms in the ge-
nome. Preliminary inferences from the first year of re-
search are that feedlot cattle located throughout central
and western United States are not serving as an envi-
ronmental source of C. parvum. It should be stressed
that there still are more than 4,500 cattle to examine, so
valid and precise conclusions should not be formed
until the cross-sectional survey is completed. In parti-
cular, the survey data will be statically modeled to de-
termine the oocyst loading rate from this cohort of adult
cattle. It is this parameter, the oocysts loading rate, that
is the primary parameter of public health interest and an
ideal parameter for risk assessment applications, not the
overall prevalence of fecal shedding.
The Office of Research and Development's National Center for Environmental Research
63
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2001 STAR Drinking Water Progress Review Workshop
Development of Detection and Viability Methods
for Waterborne Microsporidia Species Known To Infect Humans
David A. Battigelli', MM. Marshall2, and M. Borchardt3
'Wisconsin State Laboratory of Hygiene, Department of Preventive Medicine, University of Wisconsin, Madison,
Wl; 'Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ; 3Marshfield Medical
Research Foundation, Marshfield, Wl
The proposed study targets the development of a
strategy for the recovery and identification of the hu-
man microsporidia from natural waters, and it is divid-
ed into five major components: (1) sample collection,
(2) sample concentration, (3) sample processing, (4) di-
agnostic assay, and (5) method validation in natural
waters. The study design emphasizes the application of
several biotechnologies that, when combined, offer the
potential for the development of a highly sensitive and
specific environmental monitoring tool.
The techniques proposed in the study (continuous
flow centrifugation [CFC], flow cytomerry/cell sorting,
DNA amplification, and oligoprobe hybridization) were
selected in part due to their successful application for
the development of recovery methods for Cryptospo-
ridium and Giardia. In addition, the proposed strategy
offers the potential to detect low levels of microsporidia
while simultaneously providing strain-specific identifi-
cation. Continuous infections of model microsporidia
(E. intestinalis, E. hellem, and E. cuniculi) were estab-
lished in rabbit kidney cells, and a preliminary viability
assay has been evaluated using coverslips according to
a 24-well plate format. Sensitivity characterization sug-
gests that fewer than 100 viable spores can be detected
per coverslip in this format. A variety of monoclonal
and polyclonal antisera have been produced and charac-
terized for their binding characteristics against model
microsporidia. Both primary and secondary labeling
methods have been evaluated. Preliminary results indi-
cate optimal fluorescence with fluorescein isothiocya-
nate-conjugated polyclonal antisera—improvements to
indirect labeling methods with monoclonal antisera are
underway. Spores labeled with fluorescent antibodies
have been successfully sorted using automated cytome-
try technology.
Evaluation of sample collection methods during the
first 3 months has focused on assessing the efficiency
of filter/elution and CFC. Seeded challenge studies
using reticulated foam filter units to capture spores in-
dicate that recover}' efficiencies as high as 60 percent
may be achieved with idealized waters (reverse osmosis
grade). Preliminary recovery experiments using CFC
indicate that recovery efficiencies as high as 90 percent
are possible.
Initial studies indicate that the viability of model
microsporidia spores can be measured using cell cul-
ture-based techniques, and that an enumerative assay
based on epifluorescence microscopy is possible. Con-
tinued improvements to these diagnostic procedures
may make it possible to produce a finished method for
the assessment of the fate, ecology, and distribution of
infectious microsporidia in natural waters, and will as-
sist in the effort to collect data for regulatory discus-
sions on the contaminant candidate list of microorgan-
isms.
The balance of the first year's effort will focus on
the development of in situ labeling methods to improve
the sensitivity and specificity of microsporidia detection
using flow cytometry. The research also will focus on
improving the sensitivity of the viability assay and will
continue to optimize the immunological labeling proce-
dures.
64
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Meaningful Detection of Mycobacterium avium Complex
in Drinking Water; Are Disinfectant-Resistant Morphotypes Virulent?
Gerard Cangelosi, Christine Palermo, and Shawn Faske
Seattle Biomedical Research Institute and Department ofPathobiology, University of Washington, Seattle, WA
The bacterial pathogen Mycobacterium avium-
intracellulare (MAC) is resistant to chlorine and diffi-
cult to eliminate from water supplies. The public health
significance of MAC in drinking water is not well
understood, and correlations between the occurrence of
MAC in water and the incidence of MAC disease are
not always perfect.
One source of variation is the unique propensity of
the pathogen to segregate into "colony type" variants,
also known as morphotypes, with differing character-
istics of virulence, resistance to antimicrobial agents,
and ability to survive in the environment. Typically,
any MAC cell found in water is assumed to be a threat
to public health, even though some MAC morphotypes
are orders of magnitude more infectious than others.
This research is aimed at identifying virulent morpho-
types and the circumstances under which they come in-
to contact with humans.
Four morphotypic variants of MAC were examin-
ed for virulence (ability to cause disease), sliding mo-
tility (ability to spread over solid surfaces), and sensi-
tivity to chlorine. The morphotypes, distinguishable by
colony opacity and ability to bind to Congo red, were:
red-opaque (RO), white-opaque (WO), red-transparent
(RT), and white-transparent (WT).
Virulence was assessed by using a cultured human
blood cell model of disease, a mouse model of disease,
and analysis of very fresh clinical isolates. Sliding mo-
tility was measured on soft agar plates. Chlorine sensi-
tivity was measured in side-by-side and combined ex-
periments, using hypochlorite as the chlorine source.
WT was the only form that survived and grew within
cultured human cells and mouse organs, suggesting that
the other forms were weakly virulent or avirulent. Con-
sistent with this, white was the predominant morpho-
type in 25 of the clinical isolates examined. RT and RO
variants were capable of sliding motility, whereas WT
and WO variants were not. RT cells were markedly
more resistant to chlorine than the other three morpho-
types.
In the isolates that were studied, only one morpho-
type, RT, was resistant to chlorine. This form was not
virulent. Other forms, including the virulent WT form,
were more easily killed by chlorine (see Figure 1). If
this pattern applies to all MAC strains, it would suggest
that virulent MAC might be easier to eliminate from
drinking water than previously believed. This is an ex-
ample of how colony morphotype can be a factor in
determining the human health consequences of MAC in
drinking water.
The role that morphotype plays in other character-
istics important to the survival of MAC in water sup-
plies will be examined, including the abilities to form
biofilms and to grow on filters. The morphotypic char-
acteristics and virulence properties of isolates taken di-
rectly from various public water supplies without exten-
sive passage in the laboratory (which inevitably results
in morphotypic switching) will be determined. If water
distribution environments favor specific morphotypes,
then the infectivity of those morphotypes must be taken
into account when gauging their public health signifi-
cance.
The Office of Research and Development's National Center for Environmental Research
65
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2001 STAR Drinking Water Progress Review Workshop
1E6
1E5
D)
f 1E4
1E3
RT (attenuated)
\ WT (virulent)
0(0) 2(1.3) 4(3) 6(4)
ppm NaOCI (free CI-)
8(6)
Figure 1. Killing of RT and WT variants of M avium strain HMCO2 by sodium hypochlonte
66
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Detection of Emerging Microbial Contaminants
in Source and Finished Drinking Water With DNA Microarrays
Darrell P. Chandler', Ricardo DeLeon2, and Timothy M. Straub'
1 Battelle Memorial Institute, Pacific Northwest Division, Richland, WA; Metropolitan Water District of Southern
California, LaVerne, CA
DNA microarrays have not been applied to envi-
ronmental samples (in general), and commercial array
companies are focused solely on the high-throughput
genotyping and drug discovery markets. With the po-
tential to detect multiple genera of organisms and abil-
ity to incorporate live/dead discrimination via mRNA
analysis within a single water sample, microarray tech-
nology may provide a significant technological advance
in the pathogen monitoring of drinking water supplies.
The objective of this research project is to develop
DNA microarrays for the detection of Ciyptosporidium
parvum and Helicobacter pylori and evaluate their sen-
sitivity, specificity, and quantification ability relative to
standard techniques for the detection of these pathogens
in source and finished water. Genotyping arrays have
been constructed for the vacA and cagA genes of H.
pylori (36 oligonucleotide probes), and the hsp70 gene
of C. parvum (68 oligonucleotide probes). Biotin-label-
ed polymerase chain reaction products are generated
from genomic DNA and hybridized to their respective
arrays (Cryptosporidium or Helicobacter). The signal
from hybridized probes is generated using a strepavi-
din-alkaline phosphatase, ELF® chemiluminescent sub-
strate system (Molecular Probes, Eugene, OR). The hy-
bridized probes are visualized via ultraviolet transillu-
mination using a BioRad Fluor-S imager.
Preliminary results from the H. pylori array dem-
onstrate that reproducible single-base mismatch dis-
crimination within vacA and cagA amplification prod-
ucts is possible. C. parvum hsp70 genes also have been
successfully labeled, amplified, and detected on a pro-
totype microarray.
This research project now is poised to investigate
microarray sensitivity and matrix effects in an environ-
mental context, and to assess the specificity of the mi-
croarrays on a collection of Helicobacter and Crypto-
sporidium (human versus nonhuman) isolates. Work
during the next year will focus on: (1) optimizing hy-
bridization conditions for the Cryptosporidium array to
differentiate between human and non-human sources of
C. parvum, (2) processing environmental water samples
containing C. parvum to determine matrix effects on the
sensitivity and specificity of the arrays, and (3) expand-
ing the H. pylori arrays to detect additional bacterial
pathogens (such as E. coli O157:H7) in source and fin-
ished water.
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Infectivity and Virulence of Cryptosporidium
Genotype 1/H Oocysts in Healthy Adult Volunteers
Cynthia L. Chappell', Pablo C. Okhuysen ', Saul Tzipori 2, and Giovanni Widmer 2
University of Texas-Houston Health Science Center, Houston, TX; Tufts University School of Veterinary Medicine,
North Crafton. MA
Cryptosporidium parvum is comprised of a genet-
ically heterogeneous population, which can be divided
into isolates that are transmitted among humans (geno-
type 1) and those transmitted between human and ani-
mal hosts (genotype 2). Experimental infection with
genotype 2 oocysts has previously revealed significant
variability in infectivity (ID50 range between 9-1,042
oocysts) and clinical outcome (asymptomatic to diar-
rheal illness). Further, a comparison of serologically
negative versus serologically positive individuals show-
ed a 20-fold increase in the ID,o in those persons with
preexisting serum IgG. These antibody-positive individ-
uals excreted much fewer oocysts than the antibody-
negative individuals, suggesting that secondary infec-
tions would be less likely to occur with this population.
Although the presensitized volunteers typically were
resistant to low-level exposures, high challenge doses
(>5,000 oocysts) resulted in infection and diarrheal ill-
ness in a number of the volunteers. Indeed, some ill-
ness measures indicated that the diarrhea in those with
preexisting antibody was more severe than in serolog-
ically negative persons.
The overall goal of the present study is to generate
dose-response curves in healthy volunteers using geno-
type 1 oocysts and to compare the resulting infectivity,
clinical outcomes, and immune responses to previous
genotype 2 results. Two different genotype 1 isolates
have been proposed for testing. The specific objectives
of the study are to: (1) establish the infectious dose
(ID50), clinical outcomes, and intensity of infection for
two Cryptosporidium genotype 1 isolates in seronega-
tive individuals; (2) investigate the antibody and cellu-
lar responses in volunteers to genotype 1 isolates; and
(3) examine isolates for subtle genetic differences and
determine the stability of DNA markers prior to and
after passage in pigs and humans.
Year 1 activities have included: (1) identifying two
genotype 1 isolates from HIV-negative donors that can
be amplified in gnotobiotic (GNB) pigs; (2) establish-
ing the genetic stability of the isolate in multiple pig
passages; and (3) testing the first of several challenge
doses in four healthy volunteers. The genotype 1 isolate
used to date was obtained from an HIV-negative donor
who developed a relatively mild, symptomatic illness
with C. parvum. Genetic polymorphism studies re-
vealed a banding pattern typical of genotype 1 isolates.
Multiple passages were carried out in GNB pigs to ex-
amine the "fingerprint" of the amplified oocysts at each
passage. All passages showed a stable pattern with no
detectable changes in the mobility of PCR products of
any of the loci studied.
After oocyst purification and safety testing for ad-
ventitious agents, 100 genotype 1 oocysts were ingested
by each of four serologically negative volunteers. Vol-
unteers were monitored daily for 14 days and three
times per week for a total of 6 weeks. All four vol-
unteers showed either clinical or parasitological evi-
dence of infection, suggesting that the ID50 for this
isolate (TU502) may be similar to the genotype 2
isolate, TAMU (ID50 = 9 oocysts). A diarrheal illness
was noted in three of the volunteers, while the fourth
volunteer had unformed stools and gastrointestinal
symptoms, but did not meet criteria for diarrhea (see
Table 1). Two of the four volunteers shed oocysts that
were detectable by direct fluorescence assay and en-
zyme-linked immunosorbent assay. Peripheral blood
mononuclear cells and serum samples, as well as saliva
samples, have been collected at five time points (pre-
and postchallenge) from volunteers, but evaluation of
immune responses has not been completed. Continuing
studies will test additional volunteers at 100 oocysts
and other groups at lower dosages.
68
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2001 STAR Drinking Water Progress Review Workshop
Table 1. Outcome of Ciypto^pondtum parvitm (genotype 1) challenge in serologically negative.
healthy adult volunteers Each volunteer received 100 oocvsts of the TU502 isolate
Volunteer „. , ,
Diarrhea"
Number
132
133
134
135
+
+
+
-
GI
Symptomst
-
+
+
+
Oocysts
Detected
+
-
-
+
* Diarrhea is defined as the passage of unformed stools fitting any of the following
criteria along with at least one additional gastrointestinal symptom (1) > 3 in 8 hours,
(2) > 4 in 24 hours, or (3) >200 grams per day
t GI symptoms are defined as the presence of two or more gastrointestinal symptoms
with one or more concurrent unformed stools
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2001 STAR Drinking Water Progress Review Workshop
Mycobacterium avium Complex in Drinking
Water; Detection, Distribution, and Routes of Exposure
Timothy E. Ford, Anand Patel, and Yu-Rong Chu
Harvard School of Public Health, Boston, MA
Organisms of the Mycobacterium avium complex
(MAC) are an increasingly prevalent cause of clinical
disease and are known to be widespread in drinking
water supplies. However, there are significant gaps in
current knowledge of MAC. At present, there are no
validated methods for the detection of MAC in bio-
films, a likely treatment-resistant reservoir for micro-
organisms in water distribution systems. Also, com-
monly used techniques in isolating MAC from water
are relatively insensitive due to aggressive decontamin-
ation steps.
Objectives of the research project are to: (1) devel-
op improved methods for detecting MAC in biofilms,
(2) explore and implement more sensitive techniques
for the detection of MAC in drinking water samples,
and (3) determine the prevalence of MAC in municipal
drinking water distribution systems and at sites of end-
user exposure (drinking water, hot water, and toilet tank
water).
MAC in biofilms will be quantified using powerful
image analysis equipment and software. A microscope
coupled with a digital camera will provide high-resolu-
tion images of the specimens. Florescence in situ hy-
bridization, using highly specific labeled DNA probes
and antibodies, will allow for the direct labeling of
MAC on histological sections of biofilms. These bio-
films will be obtained from cartridges (see Figure 1)
that form part of a bypass apparatus (see Figure 2) in-
tegrated into the hot water recirculating system of a
suitable large building. Also, it is proposed to inves-
tigate a novel method for selectively culturing MAC on
paraffin wax slides. MAC's unique ability to utilize the
wax as its sole source of carbon will be investigated,
and modifications to the paraffin recipe to increase
specificity will be explored.
Water and biofilms will be systematically sampled
in the reservoirs, distribution systems, and end-user
sites (commercial, institutional, and residential build-
ings) of four geographically separate communities in
eastern Massachusetts. Samples will be collected over
both space and time to investigate both physical and
seasonal factors. Within end-user buildings, liquid and
biofilm samples will be obtained from drinking water,
hot water, and toilet tank water. Data on the presence of
MAC will be correlated with water quality parameters.
To implement a bypass system, a suitable building
water distribution system that is colonized by MAC
first had to be identified. Water samples from 20 build-
ings were processed to selectively isolate MAC, and
several have shown to be positive to varying degrees.
In the interim, artificial MAC-Pseudomonas aeroginosa
biofilms are being grown to develop and test the detec-
tion and image analysis methods.
This research should significantly improve the abil-
ity to detect MAC in environmental samples and pro-
vide information on the factors influencing the distri-
bution of MAC in municipal water systems. Successful
completion of this research will provide information on
optimal methodologies for assessing MAC in drinking
water. Designs for the bypass system have been final-
ized, and the construction and implementation stage
will proceed in the coming 2-3 months. Also, in the
next phase, collection of biofilm and water samples
from the target list of municipal drinking water dis-
tribution systems and end-user exposure sites will be-
gin.
70
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Coupon Stack Insert
Reducer
Figure 1. A three-dimensional cutout view of the Biofilm Cartridge that integrates into a bypass apparatus of a building hot water re-
circulating system Biofilms will form on the flat surfaces of the coupons in the insert Several inserts may reside in the car-
tridge—these may be slid in and out by unscrewing the reducer
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2001 STAR Drinking Water Progress Review Workshop
Hot Water From
Recirculating
System
Clamp
Hot Water Back
to Recirculating
System
Pump
Flowmeter
On/Off Valve
I
Tri-CIover or Range
sanitary fitting
Figure 2. Schematic diagram of the bypass apparatus with three biofilm cartridges The diagram shows the positions of the pumps, flow-
meters, valves, and various other fittings that connect the system together
72
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NERL Microbial Program
With Emphasis on Protozoan Methods
H.D. Alan Lindquist
National Exposure Research Laboratory, Office of Research and Development, U.S. Environmental Protection
Agencv, Cincinnati, OH
The National Exposure Research Laboratory's fa-
cility in Cincinnati is engaged in a variety of microbio-
logical research projects that include studies on bac-
teria, viruses, fungi, and protozoa. One of these was a
protozoology project to determine the best way to eval-
uate 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 com-
pare these alternative methods, there is no way to select
a method or battery of methods that will address the
basic question of the underlying distribution of C. par-
vum in the environment.
Procedures for evaluating methods have inherent
variability, and thus, it is difficult to determine whether
the method, or the procedures for evaluating the meth-
od, 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 ex-
ample of an evaluation of four methods using this tech-
nique is presented in Figure 1.
Ail spiked
sari-pies
Figure 1. Presumptive recoveries.
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Studies of the Infectivity
of Norwalk and Norwalk-Like Viruses
Christine L. Moe1, Lisa Lindesmith', Ralph S. Baric1, Paul Stewart2, William Heizer3, and Jeffrey A. Frelinger4
'Department of Epidemiolog)', 2Department of Biostatistics,3 Department of Medicine, and 4 Department of
Microbiology and Immunology, University of North Carolina, Chapel Hill, NC
The objective of this research is to develop the under-
standing of the risks associated with exposure to water-
borne caliciviruses as a function of dose and host suscep-
tibility factors. Specifically, this research will determine
the infectious dose of three important human caliciviruses
(HuCVs), a prototype Genogroup 1 virus (Norwalk virus
[NV]). and two prototype Genogroup II viruses (Snow
Mountain Agent [SMA] and Hawaii virus [HV]). that are
recognized as major waterborne pathogens. The specific
objectives are to: (1) identify the dose range of NV, SMA,
and HV (ID!0, ID50, and IDW) in human volunteers with
various levels of preexisting antibodies; (2) examine the
immune response (serum and secretory antibodies) and
determine the characteristics of volunteers that are sus-
ceptible to infection; and (3) evaluate the fit of several
mathematical models of dose-infectivity to these data.
Double-blinded human challenge studies are being
conducted to determine the dose-infectivity relationships
for NV, SMA, and HV. These studies build on a previous
pilot NV dose-ranging study also supported by the U.S.
Environmental Protection Agency (EPA). The NV study
focuses on infectivity in the critical low-dose region of the
dose-response curve and provides more accurate infor-
mation on the risks of N V infection associated with low
levels of virus typical of the concentrations in water. The
SMA and HV dose-ranging studies will examine a range
of doses that approximate the IDio, ID50, and ID90. In all
studies, subjects are monitored for gastrointestinal symp-
toms for 5 days, and return for Day 8, 14, and 21 fol-
lowup visits. Stool specimens are assayed for NV, SMA,
and HV RNA by reverse transcription-polymerase chain
reaction (RT-PCR). NV, SMA, and HV serum antibodies
and secretory antibodies are measured by enzyme immu-
noassay. Infection is defined as excretion of NV, SMA, or
HV or seroconversion.
The outcomes of interest in these studies are sympto-
matic and asymptomatic NV, SMA, and HV infection.
Symptomatic infection is of concern because of the dis-
ease burden on the population, the effect on absenteeism,
and the impact on the health care system. In terms of
public health protection, asymptomatic infection also is of
concern because of the potential for secondary trans-
mission and the consequences of these infections for the
immunocompromised population, including infants and
the elderly.
Several mathematical models of dose-infectivity will
be evaluated. A total of 76 subjects were challenged with
NV, and 25 became infected. NV doses ranged from 1 x
10"1 to 1 x 107PCR detectable units (PDU). Seventy-two
percent of the infected subjects had gastrointestinal symp-
toms. Asymptomatic infections occurred more frequently
at low doses. Most subjects shed virus for at least 8 days
postchallenge, and several continued to shed virus for 18—
23 days postchallenge. A simple dose-infectivity relation-
ship was not observed. The presence of anti-NV serum
IgG in prechallenge sera was a significant predictor of
infection, and subjects who were anti-NV IgG positive
had consistently higher infection rates at all doses.
Assays for anti-NV salivary antibodies were devel-
oped and used to examine the salivary immune response.
Ninety-one percent of challenged subjects developed an
anti-NV salivary IgA response. However, the timing of
this response was different for subjects who developed in-
fection compared to subjects who did not develop infec-
tion, and suggests a mechanism of protective immunity to
NV infection. The role of T-cell mediated immunity in
NV infection currently is being evaluated. A two-popu-
lation beta-Poisson model provided the best fit to the
dose-infectivity data. Preliminary risk assessment using
this NV dose-infectivity data indicates that the risk of
waterborne NV is higher than previous EPA estimates
based on rotavirus infectivity data.
SMA and HV inocula were extensively safety tested
to ensure the absence of other pathogens and toxins. To
date, two subjects have been challenged with the SMA
inoculum (105 PDU), and both developed symptomatic
infections. Seroconversion could not be detected by re-
combinant NV antigen, so SMA excreted by these sub-
jects was cloned and inserted into a Venezuelan Equine
Encephalitis virus vector. This expression system then
was used to make recombinant SMA capsid protein that
self-assembled into particles. This recombinant protein
will be used to examine the immune response in subjects
challenged with SMA and HV, and to assess the preex-
posure immune status of the subjects in all of the chal-
lenge studies.
These findings indicate that NV is highly infec-
tious. The low infectious dose, mild illness or asymp-
tomatic infections, and prolonged shedding facilitate
waterborne and secondary transmission of this virus.
Humoral and mucosal immune responses are markers
of host susceptibility to NV infection. These challenge
studies measure HuCV infectivity in humans in terms
of RT-PCR detectable units because this is the only
avail-able method that can measure HuCVs in both
clinical samples and environmental samples. These
studies will provide the EPA with important data on the
relationship between viral dose and host susceptibility
74
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2001 STAR Drinking Water Progress Review Workshop
to infection, clinical symptoms, and immune response. to: (1) challenge a small number of subjects with HV
The results of these studies are valuable for estimating inoculum to determine whether it is infectious, (2) con-
trie risk of HuCV infection and gastroenteritis associ- duct dose-ranging studies with SMA and HV-inocula
ated with exposure to contaminated water and to estab- and compare their dose-infectivity relationships to NV,
lish safe exposure limits for HuCVs in water to reduce and (3) examine host immune response to SMA and
waterborne disease. The next steps of this research are HV challenge.
The Office of Research and Development's National Center for Environmental Research 75
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2001 STAR Drinking Water Progress Review Workshop
Development and Evaluation of Procedures
for Detection of Infectious Microsporidia in Source Waters
Paul A. Rochelle
Water Qualit)' Laboratoiy, Metropolitan Water District of Southern California, La Verne, CA
The microsporidia Enterocytozoon bieneusi and
Encephalitozoon spp. are considered as emerging path-
ogens that represent critical threats to public health with
potentially fatal consequences for immunodeficient in-
dividuals who become infected. Some microsporidia
can lead to disseminated infection affecting almost
every organ of the human body, and they also can infect
immunocompetent individuals. Because many animals
can carry microsporidia, it is possible that surface wa-
ters can be contaminated, and consequently, may serve
as a route of transmission to humans. However, very
little is known about the occurrence of microsporidia in
environmental water sources, and there is a critical need
to determine the role drinking water plays in the epide-
miology of this group of parasites. Moreover, there are
no routine methods for the detection of microsporidia in
water.
The objectives of the proposed research project are
to develop a method for detection of microsporidia in
environmental waters, determine the viability and infec-
tivity of detected spores, and use the methods to deter-
mine the occurrence of microsporidia in source waters
in the United States. The recovery and purification
methods that will be evaluated include filtration of
water samples using a variety of different filter formats
and porosities, immunomagnetic separation, and den-
sity gradient centrifugation (see Figure 1).
Optimized DNA amplification assays and micro-
scopic methods will be used for detection and identi-
fication. Viability will be assessed using a spore germi-
nation assay coupled with a nucleic acid stain, a fluor-
escent dye exclusion assay, and phase contrast micro-
scopy. Infectivity will be determined by inoculating
spores into cell cultures and detecting infections using
molecular and microscopic methods.
A variety of fluorescent agents have been evaluated
for detecting spores. Staining procedures using calco-
fluor were useful for enumerating purified spore sus-
pensions, but could not be used for detecting spores
recovered from natural water samples due to the high
level of staining of background material in the samples.
Nonfluorescent stains, such as a modified trichrome
method, only were practical for enumerating purified
spore preparations. A commercially available fluores-
cently labeled antibody to Encephalitozoon spp. also
was evaluated, but it demonstrated extensive nonspe-
cific binding to sample debris and other microorgan-
isms. Published amplification primers were evaluated,
and those reported to be specific for E. cuniculi and E.
hellem did not amplify DNA from other parasites.
However, primers reported to be specific for E. intest-
inalis also amplified DNA from E. hellem and E. cuni-
culi. A cell culture-based spore propagation method has
been developed for E. intestinalis using Madin-Darby
canine kidney cells and will be used as the basis for an
infectivity assay for spores detected in water.
The immediate future plans for the project include
evaluation of different filtration formats for recovery of
spores from environmental water samples. Custom
manufactured capsule filters with pore sizes of 0.1-0.8
fim will be assessed along with compressed foam filters
comprising stacks of 62-68 filter disks. The expected
outcome of this research will be optimized methods for
the detection of infectious microsporidia in environ-
mental water samples. Information will be obtained on
the prevalence of microsporidia in environmental wa-
ters, which will allow the water industry and public
health officials to determine whether drinking water
represents a significant route of transmission for these
parasites.
76
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2001 STAR Drinking Water Progress Review Workshop
Water sample
Capsule filtration
(Gelman)
Dual porosity
filtration
Compressed foam
disks (Filta-Max)
J *
Method demonstrating highest recovery efficiencies
1
Immunomagnetic separation
Gradient centrifugation
Dynal jBiomag MPG
Percoll-] "Nycodenzj | Ficoll
sucrose
Method demonstrating highest recovery efficiencies and retention of infactivity
Centrifugation
DNA extraction!
Microscopic
detection
Viability and
Infectivity
Polymerase chain reaction
Figure 1. Outline of the approach for the development of a detection procedure for waterborne microsporidia.
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2001 STAR Drinking Water Progress Review Workshop
Attachment and Inactivation
During Virus Transport in Groundwater
Joseph N. Ryan ', Menachem Elimelech2, and Ronald W. Harvey*
] University of Colorado, Boulder, CO: 2Yale University, New Haven, CT; *U.S. Geological Survey, Boulder. CO
The major objective of this research is to provide a
better understanding of the processes that govern virus
transport in groundwater. The specific goals are to in-
vestigate four key processes that are the source of much
of the uncertainty in predicting virus transport and to
develop a virus transport model that accounts for these
processes. The four key processes, outlined as hypo-
theses, are: (1) organic matter will enhance virus trans-
port in aquifers by adsorbing to positively charged grain
surfaces and occupying these favorable virus attach-
ment sites; (2) the reversibility of virus attachment to
aquifer sediments is controlled by heterogeneity of aq-
uifer grains and virus interactions with different mineral
and organic matter surfaces; (3) the inactivation of vi-
ruses in groundwater is accelerated by strong, irrevers-
ible attachment, but not by weak, reversible attachment;
and (4) the transport of viruses during long-term release
will be enhanced by blocking of favorable attachment
sites by attached viruses, but the extent of blocking will
be diminished by accelerated inactivation of attached
viruses.
The first task is to conduct virus attachment and
release experiments in pulse-injection flow-through col-
umns and a two-dimensional aquifer tank. These ex-
periments are examining the effect of organic matter on
virus attachment and release and the reversibility of
virus attachment using the bacteriophage PRD1, syn-
thesized ferric oxyhydroxide-coated quartz sand, vari-
ous surfactants, two natural organic matter samples, and
sewage-derived organic matter.
The second task is to determine the effect of attach-
ment on virus inactivation. These experiments were
conducted in static column experiments using the bac-
teriophages PRD1 and MS2, and ferric oxyhydroxide-
coated sand and groundwater from the U.S. Geological
Survey Cape Cod field site. The third task is to conduct
virus attachment and release experiments in continu-
ous-injection flow-through columns to test the dynam-
ics of virus attachment. These experiments are being
conducted in columns similar to those described in the
first task.
The fourth task is to develop a two-dimensional
virus transport model that incorporates physically and
geochemically heterogeneous porous media, irreversi-
ble and reversible virus attachment, deposition dynam-
ics, and surface inactivation.
Virus transport experiments in geochemically and
physically heterogeneous sands have been conducted in
flow-through columns and the two-dimensional aquifer
tank. Geochemical heterogeneity, in the form of ferric
oxyhydroxide coatings, dominates virus attachment in
the aquifer tank (see Figure 1) and in a field experiment
at the Cape Cod site. Virus transport results from col-
umn studies with porous media used to fill the aquifer
tank have been to calibrate the virus transport model.
Attachment of viruses to ferric oxyhydroxide-coated
quartz sand accelerates virus inactivation relative to in-
activation of viruses in the solution phase. The findings
of this research currently indicate that geochemical he-
terogeneity and attachment-enhanced inactivation must
be included in modeling and predictions of virus trans-
port. The remaining work will focus on the effects of
organic matter and deposition dynamics on virus trans-
port.
78
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2001 STAR Drinking Water Progress Review Workshop
u
1 0
08
°6
04
02
00
10 cm above
the heterogeneiH
10cm belcm
the heterogeneity
chemical
heterogeneity
Figure 1. Breakthrough curves of bactenophage PRD1 at a 6-m transport distance within, above, and below a "chemical heterogeneity" layer
in a two-dimensional aquifer tank The chemical heterogeneity layer is a partially ferric oxyhydroxide-coated sand The break-
through curves are presented as the concentration of PRD1 at a given time normalized by the maximum concentration of PRD1 The
concentration of PRO 1 was measured by radioassay of a P-32 label
The Office of Research and Development's National Center for Environmental Research
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2001 STAR Drinking Water Progress Review Workshop
Molecular Detection
of Vegetative and Coccoid Forms of H. pylori
Manoucher Shahamat', C. Povlick', J. Hind', F. Robb', K. Sowers', M. Levin', and B. Bradley 2
1 University of Maryland Biotechnology Institute, Baltimore, MD;2 Department of Biological Sciences, University of
Man'land Baltimore Count\>, Baltimore, MD
Helicobacter pylori is a major factor in the etiology
of peptic ulcer disease and is the predominant cause of
chronic gastritis, one of the major risk factors in the
development of gastric cancer. More than 50 percent of
the world's population may be infected with H. pylori.
H. pylori has two morphological forms. In fresh culture
and in the exponential phase of growth, most of the
cells are curved, helical, rods; as the culture ages, or is
environmentally stressed, the cells change to coccoid
form (VBNC). The VBNC cells cannot be cultured on
or in the media that support the growth of the vegetative
form, although they can multiply if they encounter the
appropriate environmental conditions. Coccoid H. pylo-
ri is suspected to be responsible for disease transmis-
sion or relapse of infections.
The basic goals of this project are to validate an
accurate and sensitive molecular and/or immunodiag-
nostic method for the rapid detection of H. pylori in
both the culturable and coccoid forms from environ-
mental samples. The ability to do this is crucial for pre-
venting underestimation of the number of bacteria in
samples, developing epidemiological evidence, and un-
derstanding the ecological and public health signifi-
cance.
Three laboratory approaches have been followed in
the development of H. py/on'-specific molecular and
immunodiagnostic probes: (1) the application of mono-
clonal antibody specific to H. pylori—the monoclonal
antibody must be capable of crossreacting with both
helical and coccoid cells and be specific to H. pylori;
(2) the evaluation and standardization of a polymerase
chain reaction (PCR) based technique with specific oli-
gonucleotide probes to detect vegetative and coccoid
forms of H. pylori; and (3) the analysis of the entire
protein output (proteome) of the organism for suites of
proteins specific to each form and unique to H. pylori
as a species. The monoclonal antibody has been applied
using the immunofluorescence assay method, and the
preliminary results using IgG are promising.
Work to identify variable regions that can be used
for the design of strain-specific primers for PCR or the
development of high throughput microarray screening
has started. Sequencing of amplified fragments derived
from eight strains has identified unique DNA in all
eight strains as well as consensual primer sites. Evi-
dence to date suggests that this variable region may
provide a unique sequence that can be used to discri-
minate between individual strains of H. pylori. A PCR
or microarray technique may be particularly suited to
rapid screening and also to the detection of the coccoid
form of//, pylori from environmental samples.
Proteomic analysis has started and several proto-
cols have been tested. Homogenization methods spe-
cific to H. pylori have been developed to yield the opti-
mum resolution of the proteins. The preliminary test
results are promising—early analysis indicates that the
2D-PAGE protein pattern can be used to detect H. py-
lori in a mixed culture as well as differentiate specific
strains. Further analysis will determine the protein ex-
pression signatures for morphological form.
80
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2001 STAR Drinking Water Progress Review Workshop
Detection and Occurrence
of Human Caliciviruses in Drinking Water
Mark D. Sobsey
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC
The general objectives of this research project are
to: (1) develop improved methods to recover, concen-
trate, and purify Norwalk-like caliciviruses (NLVs) of
both genogroups from water; (2) develop new and im-
proved reverse transcription-polymerase chain reaction
(RT-PCR) and oligonucleotide probe (OP) materials
and methods to amplify and detect the recovered, con-
centrated, and purified NLVs; and (3) further evaluate
these methods by applying them to the detection of
field NLVs in environmental sewage and water sam-
ples, and thereby determine occurrence of NLVs in
representative samples of sewage and raw and finished
waters from both surface and ground sources. Lysine,
glycine, and arginine eluants were evaluated at different
pH levels with and without added nonionic detergents
for efficient recoveries of Norwalk virus (NV) adsorbed
to electropositive microporous filters from seeded tap
water. These amino acid eluants were evaluated for in-
hibition of RT-PCR detection of NV compared to inhi-
bition by beef extract-containing eluants.
Seven primer sets, representing four approaches to
broad detection, were evaluated on a panel of eight
samples representing seven distinct clusters within both
genogroups, including Norwalk, Bristol, Hawaii, Desert
Shield, Toronto, Gwynedd, and Cruise Ship (see Table
1). To confirm the presence of the desired amplicon, a
subset of samples was dot-blot hybridized with a set of
four oligoprobes, previously demonstrated to confirm
NLV amplification.
Amino acid eluents efficiently eluted viruses ad-
sorbed to electropositive microporous filters. Eluants
were most compatible with RT-PCR at pH 9.5. At pH
8.5, glycine was the most compatible eluant. Eluants
were least compatible with RT-PCR at pH 7.0. Initially,
candidate detergents resulted in one log!0 decrease in
the detection limit by RT-PCR. Detergent concentra-
tions and NV detection limits were not consistently cor-
related. Inhibition of RT-PCR by detergents was over-
come by using them at low concentrations, and the de-
tergents performed best when used at a concentration of
0.01 percent.
All of the primer sets evaluated were able to pre-
sumptively detect virus in the majority of samples.
However, most of the primer sets also demonstrated
some nonspecific amplification demonstrated by smear-
ing or distinct bands of inappropriate size.
Although experiments to evaluate sensitivity to low
copy number are not complete at this time, YGDD/
GLPSG1 and YGDD/GLPSG2 primers seem to be the
most promising for heat-release RT-PCR. However, ini-
tial results suggest differences in primer performance
between heat-released and chemically extracted RNA.
The results of these studies suggest that NLVs can
efficiently be recovered from water by adsorption-
elution methods that are compatible with RT-PCR, and
that the recovered viruses can be detected efficiently by
RT-PCR and oligoprobe hybridization.
Additional experiments will be completed on the
detection of low copy number of NLVs in environmen-
tal samples. Additional experiments to optimize recov-
ery and RT-PCR compatibility of adsorbed NLV from
positively charged filters will be completed. Additional
experiments on the effect of RNA extraction on the per-
formance of candidate primer pairs will be evaluated.
Attempts will be made to propagate human NLVs in
cell cultures.
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2001 STAR Drinking Water Progress Review Workshop
Table 1. Detection of NLVs by RT-PCR with confirmation by dot-blot hybridization is summarized for seven primer sets.
Primer Set
Ando
GI/GII
Vinje
LeGuyader
Wright
Green
Jiang
Monroe
Gwynedd
C
C
+
+
C
-
+
Cruise
Ship
+
P
C
C
C
+
+
Desert^
Shield
C
-
P
C
C
P
+
Bristol 1
C
+
+
C
C
P
+
Hawaii
C
C
C
C
C
C
+
Norwalk
C
+
+
C
C
-
+
Bristol 2
C
C
-
C
C
C
+
Toronto
C
C
C
C
C
C
+
+ = Presumptive positive, visible band failed probe
- = Did not detect
C = Confirmed positive, visible band probed positive
P = Probed positive, no visible band
82
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2001 STAR Drinking Water Progress Review Workshop
Development and Evaluation of Methods
for the Concentration, Separation, and Detection
of Three Protozoa From Large Volumes of Water
Saul Tzipori, Udi Zuckerman, Michael Buckholt, Giovanni Widmer, and Abhineet Sheoran
Division of Infectious Disease, Tufts University School of Velerinaiy Medicine, North Grafton, MA
The first phase of this project includes two aims.
Aim 1 is the optimization and standardization of a port-
able continuous flow centrifuge (CFC) device. The
CFC was modified and built from a blood separator into
a compact portable unit for concentrating C. parvum
oocysts, Giardia cysts, and Microsporidium spores from
various volumes of water (10-1,000 L) and different
matrices. Flow rate ranged from 0.5-1.4 L/min, with a
maximum speed of 8,000 rpm (3,000 xg). After centri-
fugation, the residual volume was 250 mL, from which
oocysts and cysts were dislodged by injecting a concen-
trated 10 mL elution buffer. The centrifuged residue
was transferred for immunomagnetic separation, fol-
lowed by reaction with specific monoclonal antibodies
(MABs) and enumeration by fluorescence microscopy.
The entire procedure, including parasite enumeration,
takes approximately 2 hours. For C. parvum oocysts (90
oocysts) spiked into 10 L of filtered tap water, the aver-
age recovery (n = 12, flow rate 0.5, 0.75/ LPM) was
90.3 percent ± 49.8 percent. For the same dose spiked
into 50 L (n = 11, flow rate 0.5-1.0/LPM), recovery
was 82.7 percent ± 42 percent. For oocysts (250) spiked
in 10 L of secondary effluent (turbidity = 2.8^.6 NTU,
n = 5, flow rate = 0.75/LPM), the average recovery was
56.5 percent. For a spike dose of 100 oocysts (80 ± 40
oocysts) in 1,000 L of tap water (turbidity = 0.5 NTU, n =
43, flow rate = 0.75/LPM), the average recovery was
57.33 percent ± 42.85 percent. C. parvum presence was
confirmed by immunofluorescence microscopy and by
polymerase chain reaction.
For Giardia cysts (95 and 1,080 cysts) spiked into
50 L (same matrix, n = 12, flow rate = 0.75 and I/
LPM), the average recovery was 106.1 percent ± 24.4
percent. Aim two is the production of antibodies against
Microsporidia. Encephalitozoon intestinalis and E. cun-
iculi were grown in rabbit kidney cells (RK13). Mouse
anti-£. intestinalis MABs were obtained by immuniz-
ing BALB/c mice with E. intestinalis spores and sub-
sequent fusion of spleen cells with Ag 8.653 myeloma
cells. Hybridoma supernatants were tested by EL1SA on
E. intestinalis and E. cuniculi lysate-coated plates.
Positive supernatants also were tested on methanol-
fixed spores by indirect immunofluorescence and in
parallel by immunoblotting. Positive hybridomas were
cloned twice and isotyped. Two clones, MABs CE5 and
CG9, were derived following the screening of 1,000
wells from two fusion experiments. Both MABs react
with E. intestinalis as well as E. cuniculi by ELISA and
immunoblot. CG9 identifies proteins of about 48,000
and 44,000 daltons, whereas CE5 reacts with proteins
of about 70,000, 41,000, and 36,000 daltons. CG9 ex-
hibits strong surface staining of both E. intestinalis and
E. cuniculi by indirect immunofluorescence, suggesting
that a spore wall antigen located on the surface is de-
tected, whereas CE5 shows no reactivity with either
spore. Rabbit polyclonal antibodies also were raised by
immunization with E. intestinalis, which crossreacts
with E. cuniculi.
Currently, CG9 is being evaluated for its ability to
inhibit invasion by mature spores, and is being charac-
terized for its cross-reactivity with other bacteria, fungi,
and protozoa. Also, two additional fusions have been
performed to obtain E. intestinalis-specific MABs.
Enterocytozoon bieneusi spores for antibody pro-
duction were purified from the stool of infected
humans. E. bieneusi spores, detected by chromotrope
stain, were votexed in suspension and filtered sequen-
tially through four sieves of pore diameters 425, 280,
106, and 45 urn.
Density gradient centrifugation was performed
with various concentrations of Percoll and Nycodenz.
To eliminate or reduce bacterial and fungal load, the
30/37 percent band of Nycodenz was mixed with anti-
biotics. The sterile concentrate will be used to immu-
nize rabbits and mice to produce polyclonal and mono-
clonal antibodies.
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2001 STAR Drinking Water Progress Review Workshop
Risk Factors for Cryptosporidium parvum Infection and Disease
Lucy. A. Ward, Y. Wang, S. Pereira, and S. Clarke
Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State
University, Wooster, OH
The risk of transmitting the protozoan parasite
Cryptosporidium parvum to humans via drinking water
is multifactorial and includes not only the conditions
under which C. pan'iim is introduced and survives in
water, but also knowledge of the minimum infective
dose, strain virulence variability, and a variety of indi-
vidual host risk factors. A gnotobiotic (Gn) pig model is
being used to assess risk factors to cryptosporidiosis.
Comparative studies in Gn pigs using two C.
parvum genotype 2 (bovine) strains originating from
humans (GCH1 and Ohio) suggest that host age is a sig-
nificant determinant of risk to disease and death, but not
infection. These Type 2 strains also demonstrated a 10-
to 100-fold difference between their median diarrheal
and lethal doses, but not median infective dose for neo-
natal pigs. However, upon deriving single oocyst
"clones" of each strain, these differences were lost, with
the cloned strains demonstrating significantly lower, al-
beit identical median diarrheal, lethal, and infective
doses of less than five oocysts. In contrast to the par-
ental strains, the phenotypic behavior of these "clones"
has remained predictable and uniform between different
pools and among multiple animal passages. This find-
ing further suggests that the clones are more genetically
homogenous (or clonal) compared to each parental
strain.
Comparative morphologic and cytokine studies
have demonstrated a positive correlation between cyto-
kine mRNA levels and C. /wrrvww-induced pathology,
suggesting that the host immune response may be in-
volved in disease expression, at least for genotype 2
strains. Comparative analyses of the different cytokine
levels present in the intestine suggest a mixed Thl-Th2
cytokine response. Ongoing humoral and cellular im-
muneity studies suggest that blood lymphoproliferative
responses to C. parvum develop sooner than serum anti-
body responses following infection. In fact, serum hu-
moral responses do not develop until 3 weeks or more
postinfection and loosely correlate with final clearance
of the parasite from the intestinal epithelial surface.
Neonatal Gn pigs given 105-106 Type 2 oocysts
typically begin shedding oocysts 2-3 days pbstinocula-
tion and often shed intermittently or continuously up to
19 days postinfection. Hence, this apparent "delay" in
serum antibody production (until 3 or more weeks post-
infection) suggests that there may be a need for multi-
ple or prolonged exposure to C. parvum to facilitate
seroconversion, as has been suggested in studies of
adult human volunteers.
This research recently has propagated three geno-
type 1 (human) C. parvum strains and one C. meleagri-
dis strain (derived from an AIDS patient) in neonatal
Gn pigs, and now is in the process of deriving single
oocyst clones from each of these isolates. The mere
availability of several Type 1 and 2 C. parvum clones
as well as other non-C. parvum clones (such as C. mele-
agradis) for research purposes is an enormous advance-
ment.
Preliminary studies demonstrate a uniquely differ-
ent phenotypic behavior for the Type 1 strains com-
pared to the Type 2 strains in the Gn pig model. Further
studies will be conducted to determine whether differ-
ences also exist in the cytokine, antibody, and cellular
responses of Gn pigs to Type 1 versus Type 2 strains.
84
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2001 STAR Drinking Water Progress Review Workshop
Development of a Rapid, Quantitative Method for the
Detection of Infective Coxsackie and Echo Viruses in Drinking Water
Marylynn V. Yates, W. Chen, and A. Mulchandani
University of California, Riverside, CA
The objective of this research is to improve on the
current analytical methods for quantitative detection of
infective coxsackie and echo viruses in drinking water.
The specific objectives are to: (1) improve the sensi-
tivity and specificity of immunomagnetic separation
polymerase chain reaction (IMS-PCR) for infective
coxsackie and echo virus detection in water using poly-
clonal or monoclonal antibodies; (2) improve the effi-
ciency and quantitation capabilities of IMS-PCR using
molecular beacons; and (3) quantify the presence and
viability of coxsackie and echo viruses in concentrated
drinking water samples.
IMS-PCR will be combined with molecular bea-
cons to increase the sensitivity and specificity of anal-
ysis of environmental water samples for coxsackie and
echo viruses. The research approach includes; (1) opti-
mization of IMS-PCR with polyclonal or monoclonal
antibodies to the coxsackie and echo viruses; (2) devel-
opment of molecular beacon-based, real-time reverse
transcription PCR (RT-PCR) assays for the quantitative
detection of target viruses; (3) analysis of virus-seeded
water with the IMS-molecular beacon combination; and
(4) testing of protocols on seeded drinking water con-
centrates to enable an assessment of the sensitivity of
the method to inhibitors present in environmental wa-
ters. The specificity and quantitative capability of the
protocols will be assessed by comparing results to
quantitative cell culture analysis. To date, experiments
have been conducted in which the quantitation of polio-
virus I, echovirus 11, and coxsackie virus B6 are com-
pared using typical cell culture plaque assay on buffalo
green monkey (BGM) kidney cells, direct RT-PCR, and
IMS-RT-PCR. Results of these experiments indicate
close agreement between the number of viruses de-
tected using cell culture and IMS-RT-PCR (see Figure
1). The number of viruses detected using RT-PCR is
100 times higher than with the other two methods. This
finding has been consistent for all three viruses studied.
Primers to amplify a 149-base pair fragment of the con-
served noncoding region of echovirus 11 and coxsackie
virus B6 have been selected. Molecular beacons were
designed to recognize a 25-base pair region on the
amplicon. The selectivity and the specificity of the mo-
lecular beacon are being assessed. Detection and quan-
titation are carried out entirely in sealed tubes, enabling
fast and direct measurements in a semiautomated for-
mat.
The results to date suggest that the use of IMS-RT-
PCR allows for the detection of only infective virus
particles. However, quantitation of virus particles using
this method still is approximate. The use of the mole-
cular beacon to quantify particles in combination with
the IMS process to separate the infective particles
should provide a rapid method for the enumeration of
infective virus particles in water. After the methods
have been refined, experiments will be conducted to
assess the ability of the combined IMS-molecular bea-
con procedure to detect and quantify infective virus par-
ticles, using cell culture plaque assay results as a stan-
dard for comparison.
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2001 STAR Drinking Water Progress Review Workshop
Virus Enumeration Using Cultural
and Molecular Methods
pfu/mL
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
D Plaque Assay
BIMS-RT-PCR
DRT-PCR
Echo virus 11 Coxsackievirus P olio virus 1
B6
Figure 1. Virus enumeration using cultural and molecular methods
86
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Section 6.
Contaminant Sources
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2001 STAR Drinking Water Progress Review Workshop
Molecular Tracers of Contaminant
Sources to Surface Water Drinking Supplies
Laurel J. Standley, Louis A. Kaplan, and J. Denis Newbold
Stroud Water Research Center, Avondale, PA
The objective of this research project is to develop
a more quantitative method for apportioning the contri-
bution of contaminants from point source effluents and
nonpoint source runoff to surface waters that are drink-
ing water supplies (e.g., rivers and reservoirs). In pre-
liminary research, a suite of molecular tracers was de-
veloped for several potential contaminant sources that
included wastewater treatment plants, agricultural run-
off, urban/suburban runoff, and wildlife. Although ac-
curate, the molecular tracer method is not yet quanti-
tative. It is hypothesized that: (1) unique compounds
(i.e., molecular tracers) that are constituents of runoff or
effluent of contaminant sources reflect the contribution
of these sources to contaminant budgets in drinking
water supplies; (2) although the instream fate of con-
taminants and corresponding molecular tracers may dif-
fer, selection of more than one tracer can strengthen
quantification of contaminant sources; and (3) threshold
values for molecular tracers can be determined that are
predictive for unacceptable levels of contamination
(i.e., contaminant threshold values).
The approach is threefold: (1) concentrations and
relative proportions of molecular tracers will be deter-
mined for contaminant sources; (2) the distribution of
tracers in various riverine compartments (e.g., water
column, suspended particles, and sediments) will be
measured in receiving waters of study streams; and
(3) transport and transformation processes that control
the riverine fate of the molecular tracers will be investi-
gated.
Results from this research will provide essential
information regarding tracer occurrence and fate that is
needed to develop a quantitative method for appor-
tioning sources of contaminants in drinking water
supplies. By quantitatively addressing fate and trans-
port of the molecular tracers in streams and rivers, this
work will provide the framework for future modeling
efforts.
Beneficiaries of this work include the drinking wa-
ter industry and all users of water resources by targeting
remedial efforts where they will achieve the greatest
improvement.
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Appendix
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2001 STAR Drinking Water Progress Review Workshop
U.S. Environmental Protection Agency
National Center for Environmental Research
STAR Drinking Water Progress Review Meeting
February 22-23, 2001
Holiday Inn Silver Spring
8777 Georgia Avenue
Silver Spring, MD
Agenda
Thursday, February 22.2001
Lincoln Ballroom, 4th Floor
8:30-9:10 Opening Remarks
Dr. Peter W. Preuss, Director, National Center for Environmental Research, USEPA
Ms. Cynthia Dougherty, Director, Office of Ground Water and Drinking Water, USEPA
Ms. Cynthia L. Nolt-Helms, Program Manager, National Center for Environmental Research,
USEPA
9:10 - 9:30 Overview of Regulatory Context for Drinking Water Research
Ephraim King, Director, Standards and Risk Management Division, OGWDW, USEPA
9:30 - 9:45 Oven'iew ofORD^ Drinking Water Research Program
Fred Hauchman, National Drinking Water Program Manager, USEPA
Chemical Contaminants: Exposure, Effects, & Assessment
Moderator: Joyce Donohue, Office of Water, Office of Science and Technology, Health and Ecological Criteria
Division, USEPA
9:45 - 10:15 Genotoxicity and Occurrence Assessment of Disinfection Byproducts in Drinking Water
Michael J. Plewa, University of Illinois at Urbana-Champaign, 1997 Recipient
10:15-10:30 BREAK
10:30 - 11:00 Assessment of Human Dietary Ingestion Exposures to Water Disinfection Byproducts via Food
J.H. Raymer, Research Triangle Institute, NC, 1998 Recipient
11:00 - 11:30 The Toxicokinetics and Metabolism ofHaloacids in Rodents: Effect of Chronic Exposure and Co-
Administration
Irvin R. Schulz, Battelle Memorial Institute - Pacific Northwest Division, 1997 Recipient
11:30-1:00 LUNCH ON YOUR OWN
1:00 -1:30 Development of Biomarkers for Haloacetonitriles-Induced Cell Injury in Peripheral Blood
Ahmed E. Ahmed, University of Texas- Medical Branch at Galveston, 1997 Recipient
1:30 - 2:00 NHEERL Research on Carcinogenic Contaminants in Drinking Water
Douglas C. Wolf, National Health and Environmental Effects Research Laboratory, USEPA
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2001 STAR Drinking Water Progress Review Workshop
2:00 - 2:30 Aluminum Toxicokinetics: Oral Absorption From Drinking Water and Brain Retention
Robert A. Yokel, University of Kentucky and Purdue University, 1996 Recipient from Human
Health Solicitation
2:30-2:45 BREAK
2:45 - 3:15 Overview ofEPA^ Arsenic in Drinking Water Regulation
Irene Dooley, Office of Water, USEPA
3:15-3:45 Overview ofORDu Arsenic Research Activities
Herman J. Gibb, National Center for Environmental Assessment, USEPA
3:45 -4:15 Arsenic-Glutathione Interactions and Skin Cancer
Catherine B. Klein, New York University School of Medicine, 1997 Recipient from Arsenic
Solicitation
4:15-4:45 Trivalent Methylated Arsenicals: Novel Biomarkers of Arsenic Toxicity in Humans
Miroslav Styblo, University of North Carolina at Chapel Hill, 1997 Recipient from Arsenic-
Solicitation
4:45 - 6:00 POSTER SESSION
6:00 First Day of Meeting Adjourns
Friday, February 23.2001
Session A: Potomac Room on the Lobby Level
Moderator: Jennifer Mclain, Office of Water, Office of Ground Water and Drinking Water, Standards and Risk
Management Division, USEPA
Chemical Contaminants: Formation of DBFs
8:00-8:15 Introductions
8:15 - 8:45 Development of a New, Simple, Innovative Procedure for the Analysis of Bromate and Other Oxy-
Halides at Sub-ppb Levels in Drinking Water
Howard Weinberg, University of North Carolina at Chapel Hill, 1997 Recipient
8:45 - 9:15 Kinetic-Based Models for Bromate Formation in Natural Waters
Paul Westerhoff, Arizona State University, 1998 Recipient
9:15- 9:45 BrominatedDBF Formation and Speciation Based on the Specific UVAbsorbanceDistribution of
Natural Waters
Tanju Karanfil, Clemson University and James (Chip) E. Kilduff, Rensselaer Polytechnic Institute,
1999 Recipients
9:45 - 10:00 BREAK
10:00 - 10:30 Mechanistic-Based Disinfectant and Disinfectant Byproduct Models
Paul Westerhoff, Arizona State University, 1998 Recipient
10:30 - 11:00 Use of Differential Spectroscopy To Probe Reactions Between Natural Organic Matter and
Chlorinated Oxidants
Gregory Korshin and Mark M. Benjamin, University of Washington, 1998 Recipient from
Exploratory Research - Environmental Chemistry Solicitation
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2001 STAR Drinking Water Progress Review Workshop
11:00 - 11:30 Mechanisms and Kinetics of Chloramine Loss and Byproduct Formation in the Presence of
Reactive Drinking Water Distribution System Constituents
Richard L. Valentine, University of Iowa, 1998 Recipient
11:30 - 12:30 LUNCH ON YOUR OWN
12:30 - 1:00 Molecular Weight Separation and HPLC/MS/MS Characterization of Previously Unidentified
Drinking Water Disinfection Byproducts
Roger A. Minear, University of Illinois at Urbana-Champaign, 799$ Recipient
1:00 - 1:30 Formation and Stability ofOzonation Byproducts in Drinking Water
Howard Weinberg, University of North Carolina at Chapel Hill, 1998 Recipient
Drinking Water Treatment Studies
1:30 - 2:00 Evaluation of Ozone Byproduct Formation Under Ozone Dose, Temperature, and pH Variation
Michael S. Elovitz, National Risk Management Research Laboratory, USEPA
2:00 - 2:30 Pilot Studies of the Ozonation/FBT Process for the Control of Disinfection Byproducts
in Drinking Water
Susan J. Masten, Michigan State University, 7998 Recipient
2:30 - 3:00 Integrated Approach for the Control o/Cryptosporidium parvum Oocysts and Disinfection
Byproducts in Drinking Water Treated With Ozone and Chloramines
Benito J. Marinas, University of Illinois at Urbana-Champaign 1998 Recipient
3:00 Adjourn
Friday, February 23,2001
Session B: Lincoln Ballroom, 4th Floor
Moderator: Robin Oshiro, Office of Water, Office of Science and Technology, Health and Ecological Criteria
Division, USEPA
Microbial Contaminants
8:00-8:15 Introductions
8:15 - 9:00 General Overview of'USEPA/National Exposure Research Laboratory Microbial Activities With
Concentration on Protozoan Methods
Bruce Mintz, NERL/USEPA, and Alan Lindquist, NERL/USEPA
9:00 - 9:30 Mycobacterium avium Complex in Drinking Water: Detection, Distribution, and Routes of
Exposure
Timothy E. Ford, Harvard School of Public Health, 1999 Recipient
9:30 - 10:00 Meaningful Detection o/Mycobacterium avium Complex in Drinking Water: Are Disinfectant-
Resistant Morphotypes Virulent?
Gerald Cangelosi, University of Washington and Seattle Biomedical Research Institute, 1998
Recipient
10:00-10:15 BREAK
10:15-10:45 Detection of Emerging Microbial Contaminants in Source and Finished Drinking Water With
DNA Microarrays (also a poster)
Darrell P. Chandler, Battelle Memorial Institute - Pacific Northwest Division, 7999 Recipient
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2001 STAR Drinking Water Progress Review Workshop
10:45 - 11:15 Development and Evaluation of Methods for the Concentration, Separation, and Detection of
Three Protozoa From Large Volumes of Water
Saul Tzipori, Tufts University, 1999 Recipient
11:15-11:45 Development ofDetection and Viability Methods for Waterborne Micmspovidla Species Known To
Infect Humans
David A. Battigelli, Wisconsin State Laboratory of Hygiene and University of Wisconsin -
Madison, 1999 Recipient
11:45-1:00 LUNCH
1:00 - 1:30 Investigation of Secondary Transmission of Cryptosporidium parvum
Mary E. Brown, National Center for Environmental Assessment, USEPA
1:30 - 2:00 Infectivity and Virulence o/Cryptosporidium Genotype 1/HOocysts in Healthy Adult Volunteers
Cynthia L. Chappell, University of Texas Health Science Center, 7999 Recipient
2:00 - 2:30 Attachment andInactivation in Virus Transport in Ground Water
Joseph N. Ryan, University of Colorado at Boulder, 1998 Recipient from Exploratory Research-
Environmental Chemistry Solicitation
2:30 - 3:00 Studies of the Infectivity ofNorwalkandNorwalk-Like Viruses
Christine L. Moe, Rollins School of Public Health of Emory University, 1997 Recipient
3:00 - 3:30 Detection and Occurrence of Human Caliciviruses in Drinking Water
Mark D. Sobsey, University of North Carolina at Chapel Hill, 1998 Recipient
3:30 Adjourn
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2001 STAR Drinking Water Progress Review Workshop
Posters
Membrane Introduction Mass Spectrometry Studies ofHalogenated Cyano By-Product Formation in Drinking
Water
Terese M. Olson, University of Michigan, 7999 Recipient
Haloacid Kinetics in Humans and Rhesus Monkeys
Irvin Schultz, Battelle Memorial Institute - Pacific Northwest Division, 7999 Recipient
Detection of Emerging Microbial Contaminants in Source and Finished Drinking Water With DNA Microarrays
(also oral presentation by Darrell P. Chandler)
Timothy M. Straub, Battelle Memorial Institute - Pacific Northwest Division, 7999 Recipient
Prevalence and Distribution of Genotypes of Cryptosporidium parvum in Feedlot Cattle in the Western United
States
E. Robert Atwill, University of California - Davis, 7999 Recipient
Development of a Rapid, Quantitative Method for the Detection of Infective Coxsackie and Echo Viruses in Drinking
Water Marylynn V. Yates, University of California, 7999 Recipient
Molecular Detection of Vegetative and Coccoid Helicobacter pylori
Manoucher Shahamat, University of Maryland Biotechnology Institute, 7999 Recipient
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2001 STAR Drinking Water Progress Review Workshop
Index of Authors
Ahmed, A.E., 3
Atwill, E.R., 63
Ball, L.M., 5
Batterman, S.A., 51
Battigelli, D.A., 64
Benjamin, M.M., 31
Cangelosi, G., 65
Chandler, D.P., 67
Chappell, C.L., 68
Dooley, I.S., 21
Elovitz, M.S., 55
Ford, T.E., 70
Kilduff, J.E., 32
Klein, C.B., 23
Lindquist, H.D.A., 73
Marinas, B.J., 57
Masten, S.J., 59
Minear, R.A., 6, 35
Moe, C.L., 74
Olson, T.M., 37
Raymer,J.H.,8
Rochelle, P.A., 76
Ryan, J.N., 78
Schultz, I.R., 10, 12
Shahamat, M., 80
Smith, A.H., 25
Sobsey, M.D., 81
Standley, L.J., 89
Styblo, M., 26
Tzipori, S., 83
Valentine, R.L., 39
Ward, L.A., 84
Weinberg, H.S., 41, 42
Weisel, C.P., 14
Westerhoff, P., 44, 46
Wolf, D.C., 16
Yates, M.V., 85
Yokel, R.A., 17
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