vvEPA
United States
Environmental Protection
Agency
Office ol Research and
Development
Washington DC 20460
EPA600/R-98/162
November 1998
www.epa.gov/ncerqa
DRINKING WATER PROGRESS
REVIEW WORKSHOP FOR
THE 1995-1998 SCIENCE TO
ACHIEVE RESULTS
f. ,, *"•„# % C, -~& ^ fr - *, -v^vJ.^4 ^-^ *• * ^ .
(STAR) GRANTS
December 8-9,1998
Arlington, Virginia
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Table of Contents
Introduction vii
Section 1. Arsenic Grants
Arsenic Contribution From Dietary Sources 3
Edo D. Pellizzari, Reshan Fernando, James H. Raymer, Olujide Akiribo, C. Andrew Clayton
A Dose-Response and Susceptibility Investigation of Skin Keratoses and Hyperpigmentation Caused
by Arsenic in Drinking Water 4
Allan H. Smith, Reina Haque, D.N. Guha Mazumder, Binay K. De, 'Nilirna Ghosh, Soma Mitra,
David Kalman
Arsenic-Glutathione Interactions and Skin Cancer 6
Elizabeth T. Snow
Arsenicals, Glutathione Reductase and Cellular Redox Status 7
Miroslav Styblo
Carcinogenicity of Sodium Arsenite in p53+'~ Male Mouse on a Choline-Deficient Diet 9
Raymond Tice, Glenda Moser, Thomas Goldsworthy
Section 2. Microbial Pathogens Grants
Mechanisms of Inactivation of Viruses in Groundwater 13
Maria E. Alvarez, Suresh Pillai
Meaningful Detection of Known and Emerging Pathogens in Drinking Water 15
Gerard A. Cangelosi
Cryptosporidiumparvum Volunteer Study: Infectivity, Illness, and Immunity 16
Cynthia L. Chappell, Pablo C. Okhuysen, Herbert L. DuPont, Charles R. Sterling, Walter Jakubowski
Development of a Quantitative Cell Culture-Based Infectivity Assay for Cryptosporidium parvum
in Source and Finished Water 18
Ricardo De Leon, Paul A. Rochelle
Cultural and Enhanced RT-PCR Methods for Waterborne Caliciviruses 20
G. Shay Font
Virus Filtration and Water Reuse: From Microscale Phenomena to Field-Scale Observations 21
Stanley B. Grant
National Estimate of the Occurrence of Waterborne Disease 22
Deborah Levy
Studies of the Infectivity of Norwalk and Norwalk-Like Viruses 23
Christine L. Moe
PCR-Based Detection of Cytopathogenic and Noncytopathogenic Viruses in Water 25
Ian L. Pepper, Kelly A. Reynolds, Charles P. Gerba
Detection and Occurrence of Human Caliciviruses in Drinking Water 26
MarkD. Sobsey
111
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Table of Contents (continued)
Genomic Database for Cryptosporidium Species 28
Steve J. Upton, Christine C. Dykstra, Byron L. Blagburn
Understanding Risk Factors to Cryptosporidium parvum: Studies in Gnotobiotic Pigs 29
Lucy A. Ward, SukHan Cheung, Yunfei Wang, C.K. Nielsen
Section 3. Disinfection By-Products Grants
Development of Biomarkers, in Peripheral Blood, for Exposure to and Effects of'the Water
Disinfectant By-Products Haloacetonitroles 33
Ahmed E. Ahmed
Metabolic Fate of Halogenated Disinfection By-Products In Vivo and Their Relation to Biological Activity 34
LM. Ball
Evaluation of the Efficacy of a New Secondary Disinfectant Formulation By Using Hydrogen Peroxide
and Silver and the Formation of Disinfection By-Products Resulting From Interactions With
Conventional Disinfectants 35
Stuart Batterman
Integrated Approach for the Control of Cryptosporidium parvum Oocysts and Disinfection By-Products
in Drinking Water Treated With Ozone and Chloramines 37
Benito J. Marinas, Roger A. Minear
The Use of Ozonation and FBT for the Control of THM Precursors in Drinking Water 38
Susan J. Masten
Genotoxicity and Occurrence Assessment of Disinfection By-Product Mixtures in Drinking Water 39
Roger A. Minear, MichaelJ. Plewa
Molecular Weight Separation and HPLC/MS/MS Characterization of Previously Unidentified Drinking
Water Disinfection By-Products 41
Roger A. Minear, Sylvia Barrett
Direct Quantisation of Haloacetic Acids By Surface Enhanced Raman Scattering 42
Michael J. Natan
Health Risk of Trihalomethanes Found in Drinking Water: Carcinogenic Activity and Interactions 43
Michael A. Pereira
Assessment of Human Dietary Ingestion Exposures to Water Disinfection By-Products via Food 44
James H. Raymer, Gerald G. Akland, Edo D. Pellizzari, C. Andrew Clayton, Doris J. Smith
Analysis of Organic By-Products From the Use of Ozone/Chlorine and Ozone/Chloramines in Drinking
Water Treatment 45
David A. Reckhow
Overview of Disinfection By-Products Research and Preliminary ICR Findings 47
Susan D. Richardson
Physiologically Based Pharmacokinetic Modeling of Haloacid Mixtures in Rodents and Humans 48
Irvin R. Schultz
IV
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Table of Contents (continued)
Development of a New, Simple, Innovative Procedure for the Analysis of Bromate and Other Oxyhalides
at Sub-ppb Levels in Drinking Water 50
Howard Weinberg
Inhalation and Dermal Exposure to Disinfection By-Products of Chlorinated Drinking Water 51
Clifford P. Weisel, Jeffrey Laskin
Kinetic-Based Models for Bromate Formation in Natural Waters 52
Paul Westerhoff
Mechanistic-Based Disinfectant and Disinfectant By-Product Models 53
Paul Westerhoff, David Reckhow, Gary Amy, Zaid Chowdhury
Mechanisms and Kinetics of Chloramine Loss and By-Product Formation in the Presence of Reactive
Drinking Water Distribution System Constituents 55
Richard L. Valentine
Index
57
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Introduction
The mission of the United States Environmental Protection Agency (EPA) is to protect public health and
to safeguard and improve the natural environment—air, water, and land. Achieving this mission requires applying
sound science to assessing environmental problems and evaluating possible solutions. The National Center for
Environmental Research and Quality Assurance (NCERQA) 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 the EPA Office of Research and Development (ORD) is
Drinking Water. The Safe Drinking Water Act mandates that EPA identify and regulate drinking water contaminants
that may have any adverse health effects, and that are known or anticipated to occur in public water systems. EPA
regulations addressing requirements of the Act require disinfection of surface water and certain groundwater supplies.
Scientific evidence suggests that exposure to chemical by-products formed during the disinfection process may be
associated with adverse health effects. Reducing the amount of disinfectant or altering the disinfection process may
decrease by-product formation; however, these practices may increase the potential for microbial contamination.
EPA's challenge is to balance the health risks caused by exposure to microbial pathogens with the health risks caused
by exposure to disinfection by-products (DBFs). To meet this challenge, the Agency has developed comprehensive
research plans for microbial contaminants/disinfection by-products and for arsenic in drinking water. These plans
will form the basis for prioritizing research needs for the Agency's drinking water program.
Research described in this program review has been funded through EPA's Science to Achieve Results
(STAR) Program, which is managed by NCERQA. Grants were awarded through open, peer-reviewed competition
of proposals submitted in response to Requests for Application (RFA) for each of the years from 1995 to 1998. 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 microbial/disinfection by-products and
arsenic research plans. Some key questions are: What data are required to assess arsenic exposure in human
populations? What are the health risks caused by exposure to microbial pathogens, and what methods are needed to
measure occurrence of pathogens in drinking water? What are the health effects associated with exposure to DBFs,
and how can we better characterize the risk posed by exposure to multiple or complex mixtures of DBFs?
This program review will allow 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, William G. Stelz, at 202-564-6834 or stelz.william@epa.gov.
vn
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Section 1.
Arsenic Grants
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Arsenic Contribution From Dietary Sources
Edo D. Pellizzari, Reshan Fernando, James H. Raymer, Olujide Akinbo, and C. Andrew Clayton
Research Triangle Institute, Research Triangle Park, NC
This project's objective is to enhance the un-
derstanding of human exposures to arsenic (As), hi
speciated forms, especially with regard to the dietary
pathway. The contribution of diet to arsenic exposure
is an important piece of information to consider as
reductions hi the allowable drinking water concen-
trations are contemplated. This study is investigating
the distribution of toxic As species in composite food
samples from EPA Region 5 National Human Exposure
Assessment Study (NHEXAS) and food, water, and
urine samples from the NHEXAS Children's Study.
Data from these probability-based samples will generate
information about exposure to As species of all residents
of Region 5 and for children in two rural counties and
one urban county hi Minnesota.
The specific objectives of Phase I are: (1) to
refine, optimize, and validate a speciation method for
arseno-betaine (AsB), arsenocholhie (AsC), monomethyl
arsonic acid (MMA), dimethyl arsinic acid (DMA),
arsenite [As(III)], and arsenate [As(IV)] hi food (dup-
licate diets); and (2) to optimize and validate a spec-
iation method for As hi urine, and to set up a speciation
method for As hi drinking water. The objectives of
Phase II are to: (1) estimate the levels of speciated (and
total) As hi selected foods and estimate the effects of
food preparation methods on these levels, including the
cooking of foods hi water contaminated with As; (2)
identify food types associated with elevated levels of
speciated (and total) As based on food diaries and
analysis of samples to be collected during a mini-market
basket survey; (3) estimate the food exposure dis-
tributions for speciated forms of As, using measure-
ments on archived duplicate-diet food samples from the
NHEXAS Region 5 Study; (4) estimate the food and
drinking water exposure distributions, and urine dose
distributions, for speciated forms of As, using measure-
ments on archived samples from the NHEXAS Chil-
dren's Study; and (5) estimate correlations/associations
among urine/food/drinking water levels of speciated As,
using measurements on archived samples from the
NHEXAS Children's-Study.
Speciation is accomplished using ion chromato-
graphy hi conjunction with inductively coupled plasma/
mass spectrometry (IC-ICP-MS). Aspects of the re-
search include optimizing the separation and recovery of
As species from the matrix, preservation of chemical
species during extraction and analysis, and stability of
the As species during storage prior to extraction. Three
food extraction methods are being evaluated for their
ability to isolate As species from duplicate diet samples.
These methods are: (1) MeOH-Water-CHCl3, (2)
MeOH-Water, and (3) enzymatic digestion hi water
(Trypsin-(NH4)2CO3). A mass balance approach is
being used to evaluate the extraction methods for As
species. To challenge and evaluate the performance of
the extraction schemes, two standard duplicate diet
composite samples were prepared, one high (49%) and
one low (6%) in calories of fat, each composite con-
taining a food known to have As present. In addition,
high- and low^fat duplicate diet composite samples were
prepared, but without (or very low) As-containing foods.
The latter composites will be used to examine storage-
stability of As species and as quality control samples
during the analysis of NHEXAS duplicate diet samples.
Cooking experiments began to examine the
impact of cooking on As concentrations and species hi
foods and to determine whether As may be transferred
to foods eaten when foods are cooked hi water con-
taminated with As. In these experiments, the uncooked
food is being analyzed for total As. If any As is
measured, it is speciated. Then the food (e.g., shrimp,
tuna, chicken) is subjected to broiling, grilling, frying,
etc., to determine whether the species of As are
transformed from one form to another. Foods also are
being cooked in water containing a range of As
'concentrations typically found hi the environment. The
cooked food and residual water are being analyzed to
permit mass balance. The conversions of As com-
pounds during boiling and hi the absence of foods also
will be evaluated to improve understanding of the
system.
Using an anion-exchange separation procedure
and detection by ICP-MS, baseline resolution of AsB,
AsC, As(III), DMA, MMA, and As(IV) was achieved.
The chromatographic conditions to achieve this separa-
tion consisted of a Hamilton PRP-X100 Anion exchange
column and gradient elution with mobile phase A
consisting of 5mM Na2CO3/NaHCO3 (aq), pH7:MeOH,
94:6, v/v and Mobile phase B containing 25mM
NazCOj/NaHCO;, (aq), pH 7:MeOH, 94:6, v/v, at a
flow rate of ImL mhi"1. Measurement of levels as low
as 25 pg for each species (on column) was achieved.
The three methods were used to extract As
species from 2g of each composite food fortified with
As-containing foods, the low-fat and high-fat food
composites. The major component hi each sample was
AsB. Also, the intensity of the AsB peak hi ah* three
extracts were comparable. The preliminary findings are
that extraction efficiencies of these three methods appear
similar with these duplicate diet samples.
The remaining research includes completing the
method development and validation for As species hi
food, optimizing the As species method for urine,
analyzing duplicate diet samples, performing cooking
experiments, determining the fate of As in the foods,
and data analyses.
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A Dose-Response and Susceptibility Investigation of Skin
Keratoses and Hvperpigmentation Caused by Arsenic in Drinking Water
Allan II. Smith, Reina Hague
University of California, Berkeley, CA
D.N. Guha Mazumder, Binay K. De, Nilima Ghosh, Soma Mitra
Institute of Post Graduate Medical Education and Research, Calcutta, India
David Kalman
University of Washington, Seattle, WA
Keratoses and hyperpigmentation are hallmark
dermal signs of arsenic (As) toxicity. Keratoses are hard
raised lesions that appear on the palms and soles.
Hyperpigmentation is marked by raindrop-shaped
diffuse dark spots on the trunk and limbs. The first
detailed assessment of the dose-response relationship of
arsenic-induced keratoses and hyperpigmentation is
under way. This project's objective is to determine if
susceptibility varies by As methylation capability and
nutritional factors such as methionine and cysteine. As
methylation will. be assessed by urinary assays.
Nutritional status will be determined by blood
measurements of key macronutrients and micronutrients
as well as by analysis of a dietary questionnaire.
A case-control study has commenced that takes
advantage of the largest population-based survey con-
ducted in an As-affected area of West Bengal, India (see
Figure 1). The landmark cross-sectional survey, con-
ducted between 1995 and 1996, included more than
7,000 participants, 400 of whom were found to have
As-induced skin lesions. Approximately 280 of the 400
individuals with skin lesions were exposed to drinking
water containing less than 500 mg/L of inorganic As.
These 280 individuals comprise the case group for the
present investigation. The control group consists of
lesion-free individuals randomly selected from the cross-
sectional survey database matched on age and sex.
Data obtained from personal interviews and
chemical analyses of drinking water samples will be
used to assess As exposure. The interview consists of
questions about lifetime residential history, water
sources at work, and fluid consumption. The clinical
exam involves various dermatologic, neurologic, res-
piratory, and hepatic endpoints. A dietary question-
naire supplemented with results of blood assays will be
used to ascertain the participants' nutritional status.
Urinary assays will be used to determine As methylation
efficiency.
The interviews and sample collection commenced
hi June 1998. The field team reached their target rate of
five interviews per week. To date, more than 100 par-
ticipants have .been seen. Approximately 50 urine and
blood samples have been transported to the United
States for analyses.
By building on an existing study, the present
investigation was specifically designed to identify the
shape of the dose-response curve for arsenic and skin
lesions, and to detect a possible threshold. This project
also will identify potential susceptibility factors, and
thereby reduce the uncertainty in generalizing risk to
other populations. Because keratoses and hyperpigmen-
tation are the most common and earliest occurring
endpoints of chronic ingestion, and because evidence
suggests that these skin lesions are biomarkers of sub-
sequent cancer risks, this project will significantly con-
tribute to a fuller understanding of the long-term health
effects of consuming water containing low levels of As.
Interviewing and sample collection will continue
in India, and data entry and analyses of urine and blood
samples will continue hi the United States. Approxi-
mately 400 individuals will be recruited by December
1999 (200 cases and 200 controls).
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KERATOSES
50-99
100-149 150-199 200-349 350-499
As Level in Ttabewell Drinking Water (ug/1)
10.7
500-799 >800
25.0 T
<50
HYPERPIGMENTATION
100-149 150-199 200-349 350-499
Arsenic Level in Tubewell Drinking Water (ug/1)
22.7
500-799 >800
Figure 1. Prevalence of keratoses and hyperpigmentation per 100 for females and males in West Bengal, India, 1995-1996 Cross
Sectional Survey (Adapted from Guha Mazumder et al., InternationalJournal of Epidemiology, 1998).
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Arsenic-Glutathione Interactions and Skin Cancer
Elizabeth T. Snow
Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, NY
This project's objective is to test the hypothesis
that arsenic (As)-induced cancer is the result of changes
in cellular redox control mediated by altered glutathione
(GSH) levels by exploring the effect of As on gluta-
thione-regulating enzymes in human keratinocytes in
vitro, and in mice in vivo. It is expected that exposure to
physiologically relevant, low doses of As will result hi
the activation of enzymes such as p21-ras, glutathione S-
transferase (GST), glutathione reductase (GR), y-glut-
amyl-cysteine synthetase (y-GCS), and glutathione per-
oxidase (GPx) due to changes in cellular phosphoryla-
tion and/or redox status, and will thereby potentiate the
induction of cellular stress responses. Subsequent to an
oxidative stress response in certain cell types, such as
keratinocytes, As can induce hyperproliferation and in-
hibit DNA repair.
This project's approach is three-fold: (1) Cultured
human keratinocytes will be treated with low doses of
As, and the relative activity of several key enzymes,
GST, y-GCS, GR, GPx, andp21-ras, will be examined.
(2) The activity of these enzymes will be assayed both
hi purified form and in extracts from untreated and As-
treated cells. The role of GSH and As(GS)x complexes
in mediating these responses will be examined by
measuring cellular GSH and GSSG levels, by assessing
the formation of As(GS)x complexes, and by modulating
GSH levels. (3) The role of GSH hi As carcinogenesis
will be evaluated by examining the effect of varied GSH
levels on the rate of papilloma induction hi a mouse skin
tumorigenesis model using normal mice and mice that
overexpress human GPx.
25Or
Preliminary findings have shown that physio-
logically relevant concentrations of As, in a variety of
forms, do not directly inhibit GSH metabolizing en-
zymes. However, low concentrations of inorganic As
can cause significant changes hi cellular GSH levels and
hi the relative activity of a variety of GSH metabolizing
enzymes, either in cultured human keratinocytes (see
Figure 1) or hi the epidermis of mice exposed via their
drinking water.
These results show that even low, relatively
nontoxic concentrations of As can directly modulate
cellular redox levels which, hi turn, may alter cellular
signalling and other aspects of intermediary metabolism
and thereby contribute to the carcinogenic process.
However, contrary to earlier hypotheses, this project's
findings indicate that these changes are not brought
about by the direct inhibition of GSH-related enzymes
by inorganic As or As metabolites. Most enzymes are
quite insensitive to physiological concentrations of As.
Inhibition is only seen at high As concentrations or with
complexes found at very low concentrations hi skin
cells. It is proposed that As produces alterations hi the
activity of these redox-regulating enzymes by initiating
changes hi the regulation of a variety of stress-response
genes. The means whereby As initiates these changes is
not yet established.
The next step is to evaluate the molecular reg-
ulation of these enzymes hi cultured human keratino-
cytes and to continue ongoing carcinogenesis experi-
ments to evaluate the role of redox control processes hi
the production of skin cancer.
5 7.5 10 12.5
Arsenic(lll)
Figure 1. Increased GSH, y-GCS activity, and cystine uptake in human keratinocytes after 24 hours As(HI).
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Arsenicals, Glutathione
Reductase and Cellular Redox Status
Miroslav Styblo
Department of Pediatrics, University of North Carolina, Chapel Hill, NC
This project's goal is to examine two related
aspects of the mode of action of inorganic arsenic (iAs)
as a carcinogen and toxin, including: (1) interactions
between metabolites of iAs and glutathione reductase
(GR), a key enzyme in redox metabolism of glutathione
(GSH); and (2) effects of the As-GR interactions on the
ratio of GSH to glutathione disulfide (GSSG), an
indicator of cellular redox status.
This project integrates studies at three levels of
biological complexity: purified enzyme, cultured cells,
and intact animals. The experimental approach includes:
(1) examination of the interactions of iAs and its meta-
bolites with GR purified from human erythrocytes; (2)
examination of the effects of arsenicals on GR activity,
GSH/GSSG ratio and intracellular peroxides in cultured
human cells as compared with patterns of arsenic
metabolism hi cells; and (3) examination of the in vivo
effects of arsenicals on GR activity and GSH/GSSG
ratio in tissues of mice.
During the first year of the project, metabolic
patterns for arsenicals have been examined in human
cell lines to be used in future experiments. These cell
lines have been derived from tissues that are known to
be a maui site for metabolism of iAs (liver) or targets
for its carcinogenic effects (skin and urinary bladder).
Primary hepatocytes isolated from rats have been used
as a positive metabolic control.
Among the cell lines examined, rat primary
hepatocytes had the greatest capacity for methylation of
arsenite (iAs111). The methylation capacity was sig-
nificantly lower in the human cell lines (primary
hepatocytes > epidermal keratinocytes > SV-40 trans-
formed urinary bladder [Urotsa] cells). In primary rat
hepatocytes, dimethylarsenic (DMAs) was the major
metabolite exported from cells to culture media. The
total methylation yield increased hi the range of 0.1 to
4 mM iAs1". At 10 to 20 mM iAs1", inhibition of meth-
ylation occurred. In cell lines with low methylation
capacity, iAs and/or monomethylarsenic (MAs) accum-
ulated hi the cells, suggesting that complete methylation
(dimethylation) is a precondition for clearance of As
from cells. Cytotoxicities of iAs and its metabolites also
have been examined. Trivalent methylated arsenicals
(MAs"1 and DMAs"1) were more acutely toxic than iAs1"
for all cell lines examined (see Table 1). GSH (2.5
mM) completely protected cells against toxicity of
triValent arsenicals. Addition of 2 ^M sodium selenite
reversibly inhibited methylation and increased retention
of iAs1" in cells. Presence of GSH hi culture media
dramatically enhanced the inhibitory effects of selenite
(see Figure 1). Selenite potentiated the cytotoxicity of
iAs1", MAs111, and DMAs1" and interfered with the
protective effect of GSH (see Figure 2).
These results indicate that: (1) the methylation of
iAs hi cultured cells yields potentially toxic trivalent
methylated metabolites; (2) the high methylation capa-
city did not protect cells from cytotoxic effects of
trivalent arsenicals; and (3) the methylation capacity is
concentration dependent and affected by the presence of
GSH and selenite.
The effects of iAs and its methylated metabolites on
GR activity and GSH/GSSG ratios in animal and human
cell lines are currently being studied. In parallel, the
relationship between As exposure and induction of
oxidative stress in cultured cells is being examined.
Interactions between arsenicals and GR isolated from
human erythrocytes will be examined during the second
year of the project followed by in vivo studies in mice.
MTTAssav
Cell Line
Methylation Capacity
(pmol iAsnl/106 cells/hr)
Estimated IC50
iAs1" MAsmO
Human Keratinocytes 0.1
1.4
0.3
'Values ()jM) for Arsenicals
MAsnlI2 DMAs"1!
Rat Hepatocytes 370
Human Keratinocytes 0.1
Urotsa ND
>5 2.6
»5 3.3
»5 1.2
2.5
2.3
1.6
2.6
»5
3.5
Neutral Red Assay
0.7
0.4
ICso is defined as a concentration of an arsenical that results in 50% decrease in viability of cells
over 24-hr incubation period.
Table 1. Methylation capacities and susceptibility of cells to toxic effects of trivalent arsenicals.
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-GSH
+GSH
o
E
.0.
CO
W
Q
40
30
20
10
0
0/C
4
(MM)
Figure 1. Inhibition of methylation of 0.1 u,M lAs"1 in primary rat hepatocytes by selenite in the
presence or absence of GSH (2.5mM). Total amounts of methylated metabolites (MAs
and DMAs) in cell culture (cells and medium) after 8-hour incubation.
100'
O
•S «M
_CONTROL_
CONTROL-t-Se'
-Ss
lAs»(10) MAsra(1) DMAs'" (10)
Arsenical (uM)
o
O
•5
Z%t CONTROL
r1
+Se
0.4 0.4 10 10
Concentration of MAs"1 (pM)
-GSH
+GSH
Figure 2. Effects of selenite on acute toxicity of trivalent arsenicals in primary rat hepatocytes: (a) effects of 2 |iM selenite or
cytotoxicity of iAs™ (10 uM) MAsm(l uM), and DMAs"1 (10 uM); (b) protective effects of 2.5 mM GSH against
cytotoxicity of MAs"1 in the presense and in the absence of 2 uM selenite. Cytotoxicity was examined after 24-hour
incubation using MTT assay.
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Carcinogenicity of Sodium Arsenite in
p53+/~ Male Mouse on a Choline-Deficient Diet
Raymond Tice, Glenda Moser, and Thomas Goldsworthy
Integrated Laboratory Systems, Research Triangle Park, NC
Arsenic is a multisite human carcinogen. There
are no known experimental model systems in which
arsenite is tumorigenic. This project's objectives are to:
(1) evaluate the carcinogenic ability of sodium arsenite
(AS) in drinking water as a function of hepatic methyl
donor status by comparing tumorigenicity in hetero-
zygous p53+'~ mice on a choline-deficient (CD) diet as
compared with mice on a choline-sufficient (CS) diet;
(2) determine the cocarcinogenic ability of sodium
arsenite with the skin carcinogen 4-vinyl- 1-cyclo-hexene
diepoxide (VCD) or the bladder carcinogen 2-meth-
oxy-5-methylaniline (p-cresidine); (3) extend acute
genotoxicity data; and (4) correlate hepatic methylation
activity to nutritional status and tumorigenicity.
A 28-day dose-finding study was done in male
C57B1/6 mice on CD and CS diets. The tumor study
was done in male p53+/~ mice on CD and CS diets. In
the 28-day study, groups of five mice 8 weeks of age
were exposed to 0.005, 0.01, or 0.02 percent AS in
drinking water. There were no treatment-related deaths,
clinical observations, or significant histopathological
changes in liver or skin after AS exposure. Body
weight gain was decreased hi mice on CD and CS diets
exposed to 0.02 percent AS. There were no differences
in water, AS, or food consumption hi mice on the CD
diet as compared with mice on the CS diet. As a
function of water consumption and concentration of As,
there was a dose-dependent, but not linear, increase in
AS consumption and decrease hi AS consumption with
time of AS exposure. Based on the results from the
28-day study, beginning at 8 weeks of age, groups of at
least 20 male p53+l~ mice on a CD and CS diet were
exposed to 0.005 percent AS with or without concurrent
VCD- or /j-cresidine exposure. At 6 months of treat-
ment, all mice except VCD-exposed were euthanized.
Mortality was less than 5 percent hi all groups, and
there were no consistent clinical findings associated with
treatment. Although there was no difference hi body
weight gain between mice on the CD and CS diets, there
was a decrease hi body weight gain in p-cresidine and
AS/p-cresidirie-exposed mice. Water consumption was
decreased hi arsenic-exposed mice, while food con-
sumption was decreased in ^-cresidine-exposed mice.
The only gross findings at necropsy were an increased
thickening of bladder and an increase in bladder tumors
hi p-cresidine-exposed mice on a CD diet. No necropsy
data is available on VCD-exposed mice because they
will be followed for an additional 4 months.
These data suggest 0.005 percent AS hi the drink-
ing water for 6 months did not increase the incidence of
gross tumors in.p53*'~ mice on a CD or CS diet. p53*'~
mice on a CD diet are more susceptible to ^-cresidine-
induced bladder tumors than^55+/ mice on a CS diet.
Histolopathological evaluations will be done on
hematoxylin-eosin-stained liver, skin, bladder, feet, and
lung to detect microscopic changes. Total arsenic bur-
den of the fur and arsenical speciation of urine, blood,
liver, skin, lung, and kidney will be determined to
detect differences hi tissue deposition of arsenic hi mice
on CD and CS diets. Micronuclei quantitation and
single-cell gel electrophoresis will be used to detect
DMA damage in blood, brain, liver, lung, kidney, testis,
skin, bladder, and epididymis of mice exposed to ar-
senic for 6 months under different nutritional conditions.
This information will be used to link exposure-
dose-responses for arsenite toxicity and carcinogenicity.
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Section 2.
Microbial Pathogens Grants
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Mechanisms of Inactivation of Viruses in Groundwater
Maria E. Alvarez
El Paso Community College, El Paso, TX
Suresh Pillai
Texas A&M University Research Center, El Paso, TX
Extensive information exists on the inactivation
kinetics of a variety of viruses in surface and ground-
water. However, little is known about the molecular
mechanisms of viral inactivation. The possibility that
viruses may become reactivated when environmental
conditions change has not been studied in detail. An
understanding of the structural and compositional
changes of viral particles as inactivation proceeds is
critical to fully establish the quality of water supplies
and the efficiency of disinfection protocols.
This project's objectives are to: (1) determine the
mechanisms of inactivation of MS-2 bacteriophage and
poliovirus in groundwater, and (2) determine whether
the viral particles that have entered the reversibly in-
activated state could become infectious, and to identify
the environmental factors that could promote or retard
the process.
Microcosm studies were performed on ground-
water collected from the Rio Grande floodplain. Viral
concentration was determined by the plaque assay
method. Microcosm samples showing complete inacti-
vation of seeded MS-2 phage at 27°C when incubated at
4°C showed up to three logs of viral reactivation after
the temperature shift (see Figure 1). No reactivation
was detected when poliovirus was studied under similar
conditions, Indicating that the reactivation phenomenon
may be unique to the bacteriophage system or that the
sensitivity of the plaque assay for poliovirus does not
allow detection of reactivated viruses. Structural analy-
ses of viral particles as inactivation proceeds hi ground-
water show the presence of virus particles with different
sedimentation coefficients. These results support the
hypothesis that virus inactivation proceeds in a stepwise
process, which involves the rearrangement of viral
components that may eventually lead to the ejection of
the viral genome from the capsid. Because groundwater
components interfere with protein analyses of
inactivated viruses, subsequent studies were conducted
hi buffered systems using chlorine as the inactivating
agent. Viruses with sedimentation coefficients inter-
mediate between intact and empty capsids were observed
when MS-2 bacteriophage and polioviruses were
inactivated with chlorine in the presence of Mg++ ions.
The protein components of viruses inactivated by
chlorine showed no difference hi protein components, as
compared with intact viruses, when analyzed by SDS-
PAGE and chromatofocusing. Studies also were con-
ducted on the stability of RNA in groundwater. The re-
sults indicate that naked viral RNA can persist for
extended periods of time in groundwater in the presence
of indigenous heterotrophic microbial populations.
Viral RNA does not appear to be the primary target for
viral inactivation in groundwater. Whether RNA
associated with viral capsids behave in a different
manner has yet to be determined. The results also
indicate that reverse transcriptase-polymerase chain
reaction (RT-PCR) analyses of groundwater samples
could be used to detect viral RNA sequences not
contained within capsids. The data obtained thus far
indicate that viral inactivation hi groundwater is due to
minor rearrangements of the capsid protein that may
interfere with the adsorption or penetration of the virus
or viral genome into the host cell.
Future studies are aimed at increasing the
sensitivity of the assay to detect minor changes hi viral
structure as inactivation proceeds.
13
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MS2 Bacteriophage
Poliovirus
024
8 9
Days
Days
Figure 1. Inactivation of viruses in groundwater at 27°C.
14
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Meaningful Detection of Known
and Emerging Pathogens in Drinking Water
Gerard A. Cangelosi
University of Washington and Seattle Biomedical Research Institute, Seattle, WA
Providers of safe drinking water must balance the
conflicting needs of controlling microbial contamination
and minimizing health risks associated with disinfection
by-products. Essential to this endeavor are micro-
biological monitoring methods that are practical,
meaningful, and adaptable to newly discovered or
emerging pathogens. Many such pathogens are difficult
or impossible to cultivate in laboratory media, and
polymerase chain reaction (PCR) detection of their
genetic material hi water can be of uncertain sig-
nificance because of the detection of dead cells or their
remnants. The project's goal is to overcome these
problems by using standard molecular methods to detect
two nonstandard nucleic acid analytes, rRNA precursors
(pre-rRNA) and bromodeoxyuridine-labeled DNA
(BrdU-DNA) (see Figure 1). Both of these analytes can
be detected in species-specific fashion, and both are
diagnostic of cells capable of nucleic acid synthesis and
growth (i.e., viable cells). Preliminary data have
demonstrated the potential utility of pre-rRNA and
BrdU-DNA assays in clinical diagnostic laboratories.
The assays will be modified for water supply analysis
and studied to answer questions regarding the survival
in drinking water of two bacterial pathogens, Myco-
bacterium avium and Helicobacterpylori.
Bacterial model systems (Escherichia coli, M.
avium, and/or H. pylori) and water samples from the
Seattle Department of Public Utilities will be used to test
the feasibility and utility of measuring bacterial pre-
rRNA and BrdU-DNA in water supplies. The assays
will be used to study the starvation kinetics of M. avium
in drinking water and the resistance of this pathogen to
disinfection. Also, it will be determined whether the
assays can distinguish replicative (viable) from non-
replicative forms of H. pylori hi water.
The assays are designed to overcome the most
significant challenge associated with genotypic de-
tection of microorganisms in environmental samples,
namely the false-positive detection of residual nucleic
acid from dead cells or contaminants. If successful,
these methods can help drinking water providers to
know where, when, and how to control emerging
pathogens in'the water supply. These tools also will
yield long-awaited answers on the role, if any, of
drinking water in the transmission of M. avium and
H. pylori.
Total (mature)
Pre-iRNA rKNA
Dividing
cells
Non-dividing
(stationary
phase) cells
Death-
phase cells
Figure 1. Slot blot detection of pre-RNA and mature rRNA pools in dividing,
stationary-phase, and dying cells of Mycobacterium smegmatis.
15
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Cryptosporidium parvum Volunteer
Study: Infectivity. Illness, and Immunity
Cynthia L. Chappell, Pablo C. Okhuysen, and Herbert L. DuPont
University of Texas, School of Public Health and Medical School, Houston, TX
Charles R. Sterling
University of Arizona, Tucson, AZ
Walter Jakubowski
National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH
Cryptosporidium parvum causes diarrheal disease
in both immunocompetent and immunocompromised
individuals. Until recently, information concerning this
pathogen in humans has come from outbreak situations,
case studies, and infections in travelers. In 1993, a
study of Cryptosporidium infectivity and natural history
of the infection in healthy adult volunteers was instituted
at the University of Texas Health Science Center in
Houston. This project's goal is to establish the infec-
tivity of diverse C. parvum isolates in healthy adults and
to evaluate the effect of preexisting serum antibody on
infectivity. These studies have generated information
that can be used in risk assessment of waterborne
transmission.
Volunteers from 18 to 55 years of age are
enrolled only after they have undergone a complete
history and physical examination as well as a battery of
tests to ensure that they are in excellent health.
Importantly, all volunteers are proven HIV-negative
with normal T-cell subsets and no immunodeficiencies.
After challenge, volunteers are monitored daily for the
first 14 days and three times per week for an additional
4 weeks. Active surveillance of volunteers' households
and/or other close contacts for diarrheal illness is
maintained throughout the study period. When diarrhea
occurs in the subjects or their contacts, a complete
enteric workup is performed. No secondary trans-
missions have been documented to date. All C. parvum
oocysts are used within 6 weeks of calf production and
are tested for viability by excystation rate and mouse
infectivity. All of the isolates used in these studies
belong to the genotype 2 (animal) subgroup of C.
parvum as defined by multi-locus analysis. Fecal oocyst
excretion is detected using the direct immuno-
fluorescence assay (Merifluor kit, Meridian Diag-
nostics).
To date, three geographically diverse isolates
have been studied for their infectivity hi volunteers who
had no evidence by ELISA of previous exposure.
Challenge doses ranging from 10 to 1 million oocysts
have been given, and infectivity has been documented
by the presence of fecal oocysts and/or a diarrheal
illness characteristic of cryptosporidiosis. The dose
necessary to cause infection in 50 percent of the
volunteers (ID50) varied with isolate and ranged from
approximately 10 to 1,100 oocysts (see Figure 1).
Interestingly, the isolate with the lowest ID50 also was
the most virulent when assessed for illness attack rate
(86 percent vs. 50-55 percent). However, the onset,
duration, and severity of illness did not differ among
infected individuals.
Protective immunity was studied in 17 volunteers
who had high levels of serum IgG before challenge. In
this group, both infection and illness were correlated
with dosage levels exceeding 5,000 oocysts. Indeed, the
ID50 was significantly increased to approximately 1800
oocysts, a 20-fold increase over the ID50 in serologically
negative individuals. Subjects receiving dosage levels
similar to those that might be associated with waterborne
exposure were protected from infection and illness. Of
those who did become infected and/or ill, fewer
volunteers shed detectable oocysts. The data reveal that
significant variability in infectivity and virulence occurs
among C. parvum genotype 2 oocysts. Individuals with
serum antibodies to C. parvum are less susceptible to
subsequent exposure, especially with low numbers of
oocysts.
Future studies will be designed to compare
infectivity and virulence of genotype 1 oocysts and to
examine the crossprotection between genotype 1 and
genotype 2 C. parvum. Also, studies on the infectivity
of nonparvum species of Cryptosporidium are planned.
16
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Cumulative % infected
110
100
90
80
70
60
50
40
30
20
J pj t
/ / /
/ / / ~ —
/ / /
/ /
/ f f
/ *
/ f
/ 1
rf •
-»-TAMU
012345
Challenge dose (log)
Figure 1. JD50's for various isolates in volunteers. Reference of method: Reed and Muench, AmJ Hyg 27:493, 1938.
17
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Development of a Quantitative Cell Culture-Based
Infectivity Assay for Cryptosporidium parvum in Source and Finished Water
Ricardo De Leon and Paul A. Rochelle
Metropolitan Water District of Southern California, La Verne, CA
This project's goal is to develop a quantitative cell
culture infectivity assay and a rapid molecular screening
assay for Cryptosporidium parvum in finished and
source water concentrates. Specific objectives include:
(1) optimization of cell culture for infection with low
levels of oocysts; (2) development of a molecular
detection assay targeting messenger RNA from a C.
parvum-spscific heat shock protein gene; (3) develop-
ment of a quantitative in situ nucleic acid-based assay
for detecting infections; (4) development of sample
cleanup procedures that are compatible with cell culture
and recovery of infectious oocysts; and (5) evaluation
and validation of the method with environmental
samples.
The general approach involved recovery and
purification of C. parvum oocysts from environmental
water samples using a combination of filtration, centri-
fugation, and oocyst purification, and inoculation of
these oocysts onto monolayers of human cells grown on
adapted microscope slides. Following incubation, infec-
tious foci are detected using in situ hybridization. While
developing the in situ hybridization detection method, an
intermediate detection method was employed. This in-
volved detection of C. parvum-speciRc gene transcripts
by reverse transcriptase-polymerase chain reaction (RT-
PCR) on messenger RNA extracted from infected cell
cultures (see Figures la and Ib).
Cell culture growth conditions were optimized to
permit detection of infection with low levels of oocysts,
and PCR primers were used for the specific detection of
C. parvum. Using RT-PCR, infections were detected in
human cells inoculated with £ 10 oocysts. A variety of
immunomagnetic separation (IMS) methods were
developed and evaluated as sample cleanup procedures.
Oocyst recoveries of up to 100 percent were obtained,
and it was demonstrated that IMS did not affect oocyst
infectivity. Considerable effort was expended on col-
laboration with the U.S. Environmental Protection
Agency in the development and validation of Draft
Method 1622, which is a new procedure for the
recovery and purification of Cryptosporidium oocysts
from environmental water samples. A protocol was
developed that allowed the in vitro infectivity assay to
be performed directly on oocysts recovered using this
new procedure. The results clearly demonstrated that
oocysts recovered using Draft Method 1622 retained
their infectivity.
The results demonstrated that in vitro cell culture
combined with molecular detection methods provides a
practical method for determining the infectivity of
waterborne C. parvum. The overall method of oocyst
recovery, cell infection, and molecular detection of
infections is robust, sensitive, and specific for C.
parvum. Specificity is an important consideration be-
cause there are a variety of species of Cryptosporidium
that may be found in water but only C. parvum is
recognized as a human pathogen. In addition, it is
essential that methods used to recover and purify
oocysts from environmental samples do not adversely
affect the infectivity of oocysts. Consequently, the
demonstration that oocysts recovered using Draft
Method 1622 retain their infectivity is an important
finding.
Future research will include continued develop-
ment of the quantitative aspects of the cell culture
infectivity assay with the primary focus on colorimetric
in situ hybridization, comparison of different cell lines
for their ability to support low levels of infection, and
evaluation of the final method with environmental water
samples.
Immunomagnetic
separation
Primary
detection
method
Intermediate
detection
method
Figure la. Outline of approach.
18
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1234567
Control:
Present in all
human cells
C. parvum
specific
1. Molecular size standards
2. Untreated oocysts
3. Oocysts recovered by immunomagnetic separation
4. Oocysts recovered using USEPA Draft Method 1622
5. Cells inoculated with sample recovered from an unspiked
environmental water concentrate
6. Cells inoculated with inactivated oocysts
7. Uninfected cells
Figure Ib. Detection of infectious Cryptosporidium parvum by reverse transcriptase-polymerase chain reaction
on messenger RNA extracted from infected cells.
19
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Cultural and Enhanced RT-PCR
Methods for Waterborne Caliciviruses
G. Shay Font
National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH
The Biohazard Assessment Research Branch of
the U.S. Environmental Protection Agency's National
Exposure Research Laboratory is developing reverse
transcriptase-polymerase chain reaction (RT-PCR)
detection techniques for Norwalk and other calici-
viruses. This project's goal has been to develop univer-
sal RT-PCR procedures for processing water environ-
mental samples for these nonculturable human enteric
viruses. Human caliciviruses are divided into two major
structural groups, each with many distinct members.
Norwalk virus and other small round structured viruses
such as Snow Mountain, Southampton, Hawaii,
Taunton, and Bristol agents belong to one structural
group; the Sapporo-like human caliciviruses belong to
a second group. Members of the second group are
more closely related genetically to animal caliciviruses
than are members of the first group and have a classic
"Star of David" morphology like the animal calicivi-
ruses.
Norwalk virus can be directly amplified using
RT-PCR. However, RT-PCR is subject to inhibition
from environmental inhibitors concentrated along with
viruses during the processing procedure. These inhibi-
tors affect both the reverse transcriptase enzyme that
produces the DNA copy of an RNA virus genome and
the DNA polymerase that amplify the DNA copy during
PCR. To date, most procedures that have been devel-
oped to remove inhibitors have not worked with all
water sample types, or sample volumes have been too
small to be used for exposure assessments.
This project's objective is the detection of calici-
viruses using cultural methods and RT-PCR. Powerful
molecular and immunological screening methods for
virus replication hi cell lines have been developed.
These methods are being used to develop a cultural
method for caliciviruses. A method to directly detect
caliciviruses in environmental samples also has been
developed. The approach removes organic and inor-
ganic inhibitors by combining virus purification and
concentration methods with chemical and solvent
treatments. It results in an 800-fold concentration of the
portion of the sample used for molecular assays, which
greatly Increases the amount of sample assayed per RT-
PCR reaction. The approach has been successfully eval-
uated on more than 300 field samples from different
geographical regions; 1 percent of the samples was
positive for Norwalk virus.
No information is available on the levels of
caliciviruses in environmental waters. This information
is important because viral gastroenteritis is a common
illness in the United States. Although this disease can
be caused by several virus types, it is estimated that
Norwalk virus is responsible for 20-40 percent of the
outbreaks of illness hi adults and in children more than
2 years of age.
Primer sets to detect additional caliciviruses are
currently being developed. After their development
and initial testing, they will be incorporated into the
method and the final method will be field evaluated
again.
20
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Vims Filtration and Water Reuse: From
Microscale Phenomena to Field-Scale Observations
Stanley B. Grant
Department of Civil and Environmental Engineering, University of California, Irvine, CA
Many communities in the United States rely on
groundwater to supply their drinking water needs. To
maintain a steady-state groundwater supply, water dis-
tricts increasingly use urban runoff and/or reclaimed
sewage to recharge then" groundwater aquifers. An
important human health concern associated with this
practice is the possibility that human enteric viruses
present in the recharge water may be transported
through the subsurface to production wells, enter water
distribution systems, and cause outbreaks of gastro-
intestinal disease. This project's goal is to increase un-
derstanding of the biogeochemical features of recharge
basins that most influence the removal of viruses from
recharge water by physicochemical filtration.
The viruses that have been studied thus far
include several that infect bacteria (bacteriophage) and
a recombinant analog of the important human pathogen,
Norwalk virus. Each of these viruses has its own uni-
que electrostatic "signature" (as determined by capillary
microelectrophoresis), and the electrostatic signature of
each vims strongly influences its removal from water by
physicochemical filtration.
These results have important implications relative
to: (1) the suitability of bacteriophage as indicators for
viruses of genuine human health concern (like Norwalk
virus), and (2) the optimal siting and operation of re-
charge basins employing recycled sewage and/or urban
runoff.
21
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Deborah Levy
Centers for Disease Control
and Prevention
National Estimate of the Occurrence of
Waterborne Disease
(Abstract to be provided)
22
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Studies of the Infectivity
of Norwalk and Norwalk-Like Viruses
Christine L. Moe
Deptartment of Epidemiology, University of North Carolina, Chapel Hill, NC
This project's objective is to develop an under-
standing of the risks associated with exposure to
waterborne human caliciviruses as a function of dose
and host susceptibility factors. This study will deter-
mine the infectious dose of two important human
caliciviruses (HuCVs), a prototype Genogroup I virus
(Norwalk virus [(NV]), and a prototype Genogroup II
virus (Snow Mountain Agent [SMA]), which are recog-
nized as major waterborne pathogens. The specific ob-
jectives are to: (1) identify the dose range of NV and
SMA (ID10, ID50 and IDg,,) hi human volunteers with
various levels of preexisting antibodies; (2) examine the
immune response (serum and secretory antibodies) and
determine the characteristics of volunteers who are
susceptible to infection; and (3) evaluate the fit of
several mathematical models of dose-infectivity to the
data.
Two double-blinded human challenge studies are
proposed to determine the dose-infectivity relationships
for NV and SMA. These studies will build on a pilot
NV dose-ranging study previously supported by the
U.S. Environmental Protection Agency. The NV Low
Dose Study, with 40 subjects, will focus on infectivity
hi the critical low-dose region of the dose-response
curve and will provide more accurate information on the
risks of NV infection associated with low levels of virus
typical of the concentrations in water. The SMA Dose-
Ranging Study, with 45 subjects, will be conducted in
three rounds. Each round will have 15 subjects ran-
domized to one of three doses that approximate the ID10,
ID50, and ID^. In both studies, subjects will be moni-
tored for gastrointestinal symptoms for 5 days and will
return for Day 8, 14, and 21 followup visits. Stool
specimens will be assayed for NV or SMA RNA by
reverse transcription-polymerase chain reaction (RT-
PCR). NV and SMA serum antibodies and secretory
antibodies will be measured by enzyme immunoassay.
Infection will be defined as excretion of NV or SMA or
seroconversion.
The outcomes of interest in these studies are both
symptomatic and asymptomatic NV and SMA infection.
Symptomatic infection is of concern because of the
disease burden on the population, the effect on absen-
teeism and the impact on the health care system. In
terms of public health protection, asymptomatic in-
fection also is of concern because of the potential for
secondary transmission and the consequences of these
infections for the immunocompromised population,
including infants and the elderly. Several mathematical
models of dose-infectivity (probit, log-linear, and beta-
Poisson) will be evaluated.
The results of the pilot dose-ranging study
indicated that antibody status of the subject prior to
dosing was a significant predictor of infection. Crude
analyses (that did not control for host factors) did not
indicate a simple dose-infectivity relationship. The best
fit to the data was with a two-population beta-Poisson
model that modeled the dose-infectivity relationship for
subjects with preexisting NV IgG separately from those
without preexisting NV IgG (see Figure 1). Subjects
with preexisting NV IgG were infected at lower doses,
and infection was related to dose. Subjects without pre-
existing NV IgG tended to be younger and were pro-
bably a mix of susceptible individuals who had not been
previously exposed to Norwalk virus and individuals
who were resistant to Norwalk virus infection. The data
indicated that the latter case was more common because
most of these individuals did not become infected.
Preliminary estimates of risk of enteric virus in-
fection and illness from drinking water using this new
NV dose-response data are higher than previous esti-
mates of annual risk of waterborne viral disease based
on rotavirus dose-response data and using similar
assumptions and models. Different dose-infectivity
models yielded different risk estimates, and the range of
these estimates reflects the uncertainty associated with
infectivity at. low doses. Our preliminary results are
consistent with observations that NV is highly in-
fectious hi water; ingestion of untreated water is
associated with significant risk of illness; and many
waterborne outbreaks of gastroenteritis are due to
human caliciyiruses.
Currently, the first 20 subjects in the NV Low
Dose Study are being dosed. This data will be used to
refine our models of the dose-response relationship
observed in the pilot dose-ranging study. The infectivity
of NV and SMA hi humans will be defined in terms of
RT-PCR detectable units because this is the only available
method that can measure HuCVs in both clinical and
environmental samples. This study will provide impor-
tant date on the relationship between viral dose and
susceptibility to infection (as measured by preexisting
serum antibody levels and other host factors), clinical
symptoms, and immune response. The results of these
studies will help to estimate the risk of NV and SMA
infection and gastroenteritis associated with exposure to
contaminated water and establish safe exposure limits for
HuCVs hi water to reduce waterborne disease.
23
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Q Antibody - positive
O Antibody - negative
Antibody Pos
' Antibody Neg
»•• •**""""""
Two-population beta-Poisson model
Figure 1. Norwalk virus dose response.
24
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PCR-Based Detection of Cytopathogenic
and Noncytopathogenic Viruses in Water
Ian L. Pepper, Kelly A. Reynolds, and Charles P. Gerba
University of Arizona, Tucson, AZ
This study focused on a new concept for virus
detection utilizing a biological amplification step
followed by an enzymatic amplification step. This is
known as integrated cell culture (ICC) polymerase
chain reaction (PCR) or ICC/PCR. This strategy
combines both cultural and molecular techniques for
rapid detection of viable human viruses, in large
equivalent volume concentrates, without the limitations
of toxicity or inhibition. The combined methodology
provides the first reliable method for practical analysis
and direct monitoring of environmental samples for
viral pathogens posing a significant threat to public
health. ICC/PCR allows for detection of infectious
viruses in days compared to the weeks necessary with
cell culture alone.
ICC/PCR was able to detect 2.8 plaque-forming
units (pfu) of poliovirus in 24 hours compared with 72
hours with conventional cell culture alone. Likewise,
ICC/PCR detected 1 pfu/flaskof the noncytopathogenic
virus, HAV, in 3 days compared with 14 days for cell
culture. Noncytopathogenic strains of rotavirus also
could be detected with ICC/PCR. This new meth-
odology was able to detect low concentrations of polio-
virus, that were not detected by cell culture until a third
passage had been undertaken, approaching 6 weeks of
incubation. Therefore, ICC/PCR was able to overcome
the potential for false negative results. ICC/PCR
detection of virus in environmental samples, including
sewage concentrates, required 24 hours as compared
with 10 days with conventional cell culture.
The ICC/PCR methodology allows for detection
of infectious viruses in hours to days compared to the
days to weeks necessary with cell culture alone, over-
coming the major flaws of cell culture and direct PCR
methodologies. Another advantage to ICC/PCR is that
no new method has been created, but rather two well
used techniques are now being combined in a new way,
enhancing the known strengths, while eliminating the
known weaknesses of each method.
What does this new technology mean for en-
vironmental health, service, and regulatory agencies in
the future? It demonstrates that a reliable method now
exists for routine analysis of viruses in the environ-
ment. Previously, bacterial indicator organisms have
been used to determine water quality with respect to
fecal contamination and potential public health impacts.
These organisms do not correlate well with the presence
of viruses, but a rapid, reliable method has not been
available for direct virus testing, until the advent of
ICC/PCR. With improved, viable virus detection
sensitivity and reduced assay times (ICC/PCR requires
24-72 hours versus 5-14 days, or more, with 'con-
ventional cell culture), ICC/PCR is the future for
effective environmental virus monitoring. Even with
samples that are suitable for direct PCR amplification
monitoiring, having low inhibitory compounds and
sufficiently high levels of target organisms, subsequent
use of ICC/PCR would be useful to evaluate the viable
nature of the target, with minimal cost and time
involvement.
25
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Detection and Occurrence
of Human Caliciviruses in Drinkini
MarkD. Sobsey
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC
This project's objectives are to: (1) develop
improved methods to recover, concentrate, and purify
human caliciviruses (HuCVs) from water; (2) develop
new and improved molecular methods using reverse
transcriptase-polymerase chain reaction (RT-PCR) and
oligoprobing (OP; gene probe) to amplify and detect
these HuCVs; (3) apply these methods to the detection
of field HuCVs in environmental sewage and water
samples, thereby determining HUCV occurrence; and
(4) attempt to cultivate Norwalk virus (NV) in dif-
ferentiated intestinal and other cell cultures and develop
a practical HuCV infectivity assay system.
The following methods will be further optimized
and evaluated for HuCV concentration from water:
adsorption to and elution from electropositive, micro-
porous, Virosorb 1MDS filters, precipitation by
polyethylene glycol (PEG) and possibly other pre-
cipitating agents, immunocapture using polyclonal anti-
bodies (human serum immune globulin) bound to
paramagnetic beads, organic solvent (chloroform or
fluorocarbon) extraction, and molecular exclusion by gel
(Sephadex) chromatography and centrifugal ultrafil-
tration. Improved methods to amplify the nucleic acid
of HuCVs concentrated from water will use specific
primers and probes and conditions for RT-PCR
amplification and nucleic acid hybridization that will be
developed in this study. In general, RT-PCR amplifi-
cation and nonradioactive oligoprobe hybridization of
HuCVs will be done according to previously described
procedures, but will involve the selection and testing of
new and unproved RT and PCR primers for amplifi-
cation of genomic targets and new and improved
nonradioactive oligoprobes for hybridization detection
of the resulting amplicons. The methods developed in
this study for concentration, purification, RT-PCR
amplification and oligoprobe hybridization detection of
HuCVs will be applied to field samples of water.
Initially, HuCVs in water samples implicated in HuCV
gastroenteritis will be detected. Later, HuCV detection
will be applied to geographically representative surface
and groundwaters of the United States. Some will be
fecally contaminated from identifiable sources of sewage
or other human fecal waste and others will be from
relatively uncontaminated or "pristine" sources that are
drinking waters or the protected raw (surface or ground)
sources of drinking water supplies. Attempts will be
made to cultivate Norwalk virus in primary cell and
organ cultures, diploid cell strains, and continuous cell
lines.
This project will develop new methods to detect
human caliciviruses and other enteric viruses hi water
that will be immediately applied to field samples of
water as soon as they are developed. The research will
produce one or more detailed protocols or stepwise
procedures, including specification of the needed ma-
terials and full descriptions of methods, used to recover
HuCVs as well as other enteric viruses in water and then
detect them by RT-PCR amplification and oligoprobe
hybridization. This project also may provide a method
for propagation and infectivity assay of Norwalk virus
and possibly other HuCVs.
Human caliciviruses are important sources of
waterborne disease hi the United States and elsewhere
(see Figure 1). To better assess, prevent, and control
the health risks from these viruses, unproved methods
are needed for their detection in water and wastewater.
26
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Figure 1. Electron photomicrograph of human caliciviruses.
27
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Genomic Database for Crvptosporidium Species
Steve J, Upton
Division of Biology, Kansas State University, Manhattan, KS
Christine C. Dykstra and Byron L. Btagburn
Auburn University, Auburn, AL
This project's objective is to acquire various iso-
lates and species of Cryptosporidium from the environ-
ment, and produce gDNA libraries to be deposited into
the American Type Culture Collection (ATCC). In this
project, oocysts are acquired from various sources,
purified from feces on CsCl gradients, and surface ster-
ilized using 10 percent Clorox bleach. Oocysts are then
washed three times using phosphate buffered saline and
centrifugation, and the DNA is extracted from each iso-
late. The DNA is digested with EcoRl and/or SauSa.
Fragments from the EcoRl digestions are ligated into
pBluescript II plasmid vector and the plasmids used to
transform competent Escherichia coli XLl-Blue cells.
Fragments cut with SauSa are ligated into a bacterio-
phage vector (LambdaZap express). Prior to submiss-
ion of the libraries to the ATCC, quality control checks
are performed. These include: (1) determination of the
percentage of recombinants; (2) determination of the
average insert size; and (3) probes for the presence of
hsp70, the 18s rRNA subunit, and/or actin inserts. To
date, enough isolates/species have been acquired so that
more than 20 independent libraries are either available
or soon will be (see Table 1).
The acquisition of additional isolates and species
is continuing. Once a large number of genomic libraries
representing a variety of strains and species of Crypto-
sporidium are available to researchers, investigators will
be able to sequence and compare any region of the ge-
nome desired. In addition, investigators will no longer
need to rely solely on the sequences submitted by mul-
tiple laboratories into the national databases to make
comparisons. This should allow for the development of
many different types of specific, rather than random,
primers for developing diagnostic tests.
Species
C. batteyi
C. meleagridis
C. baileyi
C.muris
C.parvum
C,parvum
Cparvum
C-parvum
C.parvum
Cparvum
C.parvum
C-parvum
C.parvum
C.parvum
C.parvum
C.parvum
C-parvum
C. parvum
C. serpentis
C.sp.%
C.sp.%
C.sp.%
C. sp. t
Origin
Alabama
California
Georgia
Virginia
Alabama
Colombia
Colorado
Florida
Kansas
New York
Spain
Massachusetts
Iowa
Iowa
Louisiana
Peru
Texas
Iowa
Kansas
Idaho
Idaho
California
Alabama
Isolate
AuCbl
Fresno
none yet
108735
AuCpl
Colombia
6304321
AuCp2
KSU-1
Cornell
Galicia
GCH1
Iowa(A)
Iowa®
Louisiana
Peru
TAMU
UCP
KSU-2
95-400
VS1742
VMTRC49
AuCml
ATCC No.
*
*
87666
*
*
*
*
87439
*
*
87665
87667
87668
*
*
*
*
87664
*
*
*
*
Enzyme
SauSa
EcoRl
SauSa
EcoRl
SauSa
EcoRl
EcoRl
SauSa
EcoRl
EcoRl
EcoRl
EcoRl
EcoRl
EcoRl
EcoRl
SauSa
SauSa
EcoRl
EcoRl
EcoRl
EcoRl
EcoRl
SauSa
Hosl
Original Host
chicken
chicken
turkey
rock hyrax
calf
calf
human
calf
calf
human
calf
human
calf
calf
calf
human
human
calf
corn snake
beef steer
dairy cow
dairy cow
dairy cow
t Data
Last Passage Host
chicken
chickenf
turkeyt
mouse
calf
calf
N/P
calf
calf
N/P
calf
calf
calf
calf
calf
calf
calf
calf
corn snake
N/P
N/P
N/P
N/P
*ATCC number not yet acquired.
tCurrently under propagation.
JBovine abomasal Cryptosporidium sp.
N/P not passaged.
sometimes termed C. muris-like.
Table 1. Isolates and species of Ciytosporidium associated with EPA grant #R825148-01-0.
28
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Understanding Risk Factors to
Crvvtosvoridium parvum: Studies in GnotoMotic Pigs
Lucy A. Ward, Suk Han Cheung, Yunfei Wang, C.K. Nielsen
The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, OH
This project's goal is to increase knowledge and
understanding of risks associated with cryptosporidiosis.
A gnotobiotic pig model is being used to pursue the
following objectives: (1) assess and compare the patho-
genesis (infectivity and virulence) of Cryptosporidium
parvum in neonatal versus older gnotobiotic pigs; (2)
evaluate the susceptibility and clinical responses of
immunosuppressed gnotobiotic pigs to C. parvum; and
(3) characterize the humoral (B cell), cellular (T cell),
and cytokine immune responses in gnotobiotic pigs with
cryptosporidiosis.
Detailed comparative pathogenesis and immunity
studies are being conducted in gnotobiotic pigs of
different ages and immune status following inoculation
with the human C. parvum isolate, GCH1 (from Saul
Tzipori, Tufts University School of Veterinary Medi-
cine, Grafton, MA) and a second C. parvum isolate
(designated OH or Ohio) obtained from an Ohio Agri-
cultural Research and Development Center (OARDC)
laboratory worker in 1997. The median lethal dose,
infectious dose, and diarrhea! dose for these two C.
parvum isolates in neonatal gnotobiotic pigs have been
established. As few as five GCH1 oocysts infected
neonatal gnotobiotic pigs with a duration of shedding of
greater than 10 days with little diarrhea. The median
diarrheal doses were 730 and 6,900 oocysts for GCH1
and OH, respectively, resulting in moderate diarrheal
disease of 5 to 10 day duration. Doses of > 5 x 107
GCH1 oocysts were lethal with onset of oocyst shedding
and diarrheal disease observed by 24 hours post-
inoculation. In contrast, although less than 50 OH
oocysts infected neonatal pigs, doses of ;> 108 OH were
not lethal. It is assumed that environmental isolates
vary in viability, infectivity, and virulence, although
little is known of this aspect.
The results demonstrated that the GCH1 isolate
was more virulent (approximately 10 times) in terms of
infectious, diarrheal, and lethal doses compared with the
OH isolate in gnotobiotic pigs. The median diarrheal
doses are significantly lower than the suckling mouse
model (104 to 105 oocysts) and more consistent with
human and nonhuman primate studies. Further, pre-
liminary studies suggest that the infectivity of either of
the C. parvum isolates is not affected by the age of the
host, although host age appears to influence the severity
of subsequent disease. In summary, the findings sug-
gest that the onset of oocyst shedding, severity of
diarrheal disease, number of oocysts shed, and duration
of shedding is dose- and age-dependent and notable
differences exist in terms of infectivity and virulence
between the GCH1 and OH C. parvum isolates in neo-
natal gnotobiotic pigs.
Future studies will further establish the sig-
nificance of age and immune status as determinants of
risk to cryptosporidiosis. These studies also will include
basic immuhological studies to assess the role of the
host's immune response and the risk to crypto-
sporidiosis.
29
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Section 3.
Disinfection By-Products Grants
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Development of Biomarkers,
in Peripheral Blood, for Exposure to
and Effects of the Water Disinfectant Bv-Products Haloacetonitroles
Ahmed E. Ahmed
Department of Pathology, University of Texas Medical Branch, Galveston, TX
Drinking waters are contaminated with a mixture
of halogenated hydrocarbons that are disinfection by-
products. Among those are haloacetonitriles (HAN).
This project's goal is to develop unique biomarkers, in
peripheral blood, for HAN exposure and cellular injury
that may result from HAN-induced alkylative or
oxidative damage to cellular molecules.
The water disinfectant by-product dichloro-
acetonitrile (DCAN) is a mutagen that is known to in-
duce DNA strand breaks in cultured human lympho-
blasts. The effect of DC AN in cultured mouse peritoneal
macrophages (MPMs, RAW 264.7) was explored.
MPMs were exposed to various concentrations (100
/tM- 400 fM.) of DCAN for 4 hours. The phagocytic
activation of MPMs was characterized by the production
of reactive oxygen intermediates (ROI) and secretion of
TNF-a. The ratios of intracellular reduced glutathione
(GSH) and oxidised glutathione (GSSG) were assessed
and used as an indicator of oxidative stress. Electro-
phoretic detection of DNA degradation and light micro-
scopy were used for the characterization of DCAN-
induced apoptosis. Lactate dehydrogenase (LDH) leak-
age and trypan blue exclusion were used as markers of
the necrotic effects of DCAN.
Following treatment with DCAN, GSSG was
increased (135% of control, P<0.05). DCAN activa-
tion of MPMs was observed by elevated levels of ROI
(190-250% of control, P<0.05) and increased secretion
of TNF-a (450% of control, P<0.05). Electrophoresis
of DNA of treated MPMs indicated a dose-dependent
increase in the degradation of genomic DNA. This in-
formation, combined with morphological studies, in-
dicated that exposure of MPMs to lower levels of
DCAN (100 /*M and 200 /*M) incites apoptic cell death.
At 400 fiM DCAN, cellular necrosis was observed as
indicated by increased LDH leakage and decreased
viability (45% of control, P<0.05).
These studies suggest that the dose-dependent
DCAN-induced apoptosis or necrosis in MPMs is due to
the disturbance in GSH/GSSG ratio and initiation of
ROI-mediated oxidative damage mechanisms. These stu-
dies should provide a basis for the development of bio-
markers for regulatory guidelines and policies governing
the tolerancelevels for chronic human exposure to HAN.
Future steps will focus on the characterization and
quantitative determination of alkylative (DNA and
hemoglobin adducts) and oxidative damage to cellular
macromolecules.
33
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Metabolic Fate of Halogenated Disinfection
Bv-Products In Vivo and Their Relation to Biological Activity
L.M. Ball
University of North Carolina, Chapel Hill, NC
Halogenated by-products of drinking water disin-
fection are of concern because of their widespread
human consumption and the current uncertainty over
their health effects. Haloacids as a class are the most
abundant of the halogenated disinfection by-products.
Many of these compounds are suspect carcinogens and,
in addition, may form macromolecular adducts that
could be useful as biomarkers of exposure to disin-
fectants. This project's goal is to elucidate mechanisms
of biological activity and disposition of haloacids.
Two specific approaches will be followed: one
directed towards DBFs that are highly abundant,
although less potent biologically, and the other directed
towards DBFs that appear to be highly biologically ac-
tive, though present at low levels. The first approach
involves the identification and quantitation of macro-
molecular adducts formed by the haloacetic acids, chlor-
oacetic acid, dichloroacetic acid, from their brominated
analogs—bromoacetic and dibromoacetic acid—and from
the mixed haloacetic acid, bromochloroacetic acid.
Structural identification and quantitation of DNA
adducts will be approached by chromatographic and
mass spectrometric techniques. Quantities of adducts
sufficient for detailed structural elucidation are often
difficult or impossible to obtain by direct modification
of DNA; hence, direct chemical synthesis will be under-
taken to provide standards for investigation of adduct
formation in vivo. The second approach will involve
investigation of the metabolism and disposition of MX,
to identify the chemical nature and mechanism of forma-
tion of metabolites of the potent bacterial mutagen (Z)-
2-chloro-3-(dichloromethyl)-4-oxobutenoic acid, more
commonly known in its ring-closed form as 3-chloro-
4-(dichloromethyl)-5-hydroxy-2(5H)-furanone or MX
(for Mutagen "X")- Structural identification of MX
metabolites, and their tissue distribution in the rat, will
be carried out to gain an understanding of the nature of
the active species present at potential target organs,
and the levels and duration of internal exposure.
Synthesis and characterization of putative DNA
adducts from haloacetic acids is in progress. Gaining
knowledge of the chemical structures of macromolecular
adducts formed will provide information on routes of
genotoxic activity. In addition, this information consti-
tutes the foundation for development of assays to
measure rates of adduct formation and loss, and the
development of biomarkers of exposure and effect.
Future steps involve the comparison of techniques
for sensitivity and specificity in detecting putative
adducts in biological matrices.
34
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Evaluation of the Efficacy of a New Secondary Disinfectant Formulation
By Using Hydrogen Peroxide and Silver and the Formation of Disinfection
Bv-Prodncts Resulting From Interactions With Conventional Disinfectants
Stuart Batterman
School of Public Health, University of Michigan, Ann Arbor, MI
This project's objectives are to address two
critical issues associated with the use of a new secondary
disinfectant formulation using hydrogen peroxide and
silver. The issues are: (1) the efficacy of the formu-
lation to provide long-term residual disinfection, in-
cluding the control of coliform bacteria, bacterial
regrowth and slime/biofilm control; and (2) the
identification and quantification of disinfection by-
products (DBFs) resulting from interactions with con-
ventional chlorine-based and oxidant-based disinfec-
tants. The project encompasses laboratory studies and
field demonstrations to evaluate the efficacy of the
proposed alternative disinfectant in a range of source
waters and utility system characteristics. Secondary
objectives include investigations related to taste and
odor. The proposed secondary disinfectant is one of the
few nonchlorine based disinfectants that can provide
long-term residual disinfection in drinking water
systems. By combining two or more disinfection
agents, it is possible to lower concentrations of each
component, reduce exposures, minimize DBF forma-
tion, and thus minimize the health risks associated with
disinfection.
The approach consists of three major compo-
nents: (1) laboratory evaluation of microbial disinfec-
tion efficacy, including optimal doses of primary and
secondary disinfectants; (2) laboratory evaluation of
DBF formation potential resulting from interactions with
various primary disinfectants; and (3) field demonstra-
tion of the disinfectant to provide "real world" results.
Inactivation performance for hydrogen peroxide
ranged between 0.18 and 0.65 logs for 5 and 30 ppm,
respectively, and between 0.54 and 2.87 logs for silver at
5 and 30 ppb, respectively. The combined disinfectant was
more potent than each of its components alone. This
synergistic effect may be attributed to combined stress
conditions, exerted by a combination of two disinfectants,
rather than to the formation of highly active species that are
known to evolve during the reaction of hydrogen peroxide
and multiple valence transition metal ions [such as
Fe(H)/Fe(ffl) and Cu(I)/Cu(H)]. (Results are based on
several model wild-type indicator organisms, E. coli - B,
E. coli 4100/MC, and E. coli - K12, and 1 hour contact
time.) The addition of the secondary disinfectant drama-
tically reduced DBF levels when using chlorine as a
primary disinfectant (see Figure 1). These reductions
averaged 72+9 percent for trihalomethanes (THM) and 67
±11 percent for haloacetic acids (HAA) across three types
of water and two chlorination levels. (Experiments were
performed at 25°C, pH = 7, 10 min contact time for
chlorine, secondary disinfectant at 30 mg/L for H2O2 and
30 Mmg/L for Ag+, and DPBs determinations after 24 hr.)
This reduction in by-product formation is most likely due
to the reaction of Cl~ by H2O2. Followup mechanistic
studies will be conducted to characterize this process.
A screening level health and ecological risk as-
sessment evaluated risks due to the silver that would be
discharged from wastewater treatment systems if the new
disinfectant formulation was adopted. The assessment uses
a probabilistic three trophic-level (freshwater algae, aquatic
invertebrates, and trout/carp) simulation model to simulate
the bioaccumulation of silver in freshwater aquatic species.
Each level considers uptake from water, food or sediment,
with first-order uptake and elimination kinetics, typical
feeding rates, and metal assimilation. Most of the model
parameters, derived from the literature, were considered
lognormally distributed. Silver discharges were simulated
in three scenarios: river (high dilution, low turbidity),
clear-stream (low dilution, low turbidity), and turbid stream
(low dilution, high turbidity). Although some model inputs
are uncertain; many predictions were comparable to field
results. Although the highest (^O"1 percentile) silver
concentrations in the clear-stream scenario may pose a
developmental risk to some invertebrates, a chronic risk to
early survival and development of trout,, and a chronic risk
of argyria to subsistence fishers, predictions under most
conditions show that the bioaccumulation of sEver is
unlikely to result in toxic effects to aquatic wildlife and
humans consuming fish.
Results of this research regarding residual
disinfection efficacy, disinfection by-products, optimum
dosages, and risks of the new disinfectant will be compared
with that of conventional disinfectants. This information is
significant in exposure and risk assessments of pathogens
and DBFs that support future policies and decisions
regarding the most appropriate disinfection approach.
Ongoing and planned future activities include: (1) further
laboratory tests on disinfection efficacy of the new
formulation, including viral inactivation studies; (2) further
tests on DPB formation potential using the new
formulation with other primary disinfectants, such as
chlorine dioxide; (3) a comparative analysis of candidate
disinfectants under a range of conditions; and (4) and full-
scale field tests.
35
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ppb
O
H2O2/Ag
Flgure 1. Reduction in THMs and HAAs as a result of addition of secondary disinfectant. Mixed ground and surface water used in
the local city supply. DBFs resulting after 24 hours. Foreground - with secondary disinfectant; rear - Cl alone. Basic water
parameters: Br- - 0.081mg/L, TOC = 3.1mg/L. Initial Cl = 5mg/L; residual Cl = 1.85mg/L, pH = 6.85 (buffered). TTHM =
total THMs; THAAs = total HAAs.
36
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Integrated Approach for the Control of
Ctyptosporidium parvum Oocysts and Disinfection
Bv-Products in Drinking Water Treated With Ozone and Chloramines
Benito J. Marinas and Roger A. Minear
Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, Urbana, IL
This project's goal is to develop process design
recommendations for the simultaneous control of
Cryptosporidium parvum (C. parvum) oocysts and
disinfection by-products (DBFs) in natural waters treated
with ozone and chloramines. Because the main ob-
jective of the study is to develop an integral control
strategy, the scope of work focuses on a limited number
of selected DBFs (bromate, formaldehyde, and cyan-
ogen halides) associated with the ozone/chloramines
disinfection strategy.
This project includes experimental tasks designed
for the simultaneous study of C. parvum oocyst in-
activation and selected DBF formation/decomposition in
natural waters treated with ozone and chloramines using
both batch and flow-through reactors. An integrated
predictive model will be developed and calibrated with
these experimental results. The model will be used to
determine optimum process design, and verified hi full-
scale systems using fluorescent-dyed polystyrene micro-
spheres as nonbiological surrogate indicators for C.
parvum oocyst inactivation.
Tasks recently initiated include setting up ex-
perimental and analytical systems, and formulation of a
modeling approach.
It is anticipated that this research project will result
in a better and more integral understanding of the kinetics
of DBF formation/decomposition and C. parvum oocyst
inactivalion in water supply systems using ozone and
chloramines as primary and secondary disinfectants.
A predictive model incorporating this information
will be developed for the overall optimization of both C.
parvum and DBF formation control.
37
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The Use of Ozonation and FBT for the
Control of THM Precursors in Drinking Water
Susan J. Masten
Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI
A biological fluidized bed treatment system (FBT)
in combination with ozonation was used for the control
of trihalomethane (THM) precursors in Huron River
water. The average annual levels of total organic
carbon (TOC), trihalomethane formation potential
(THMFP), and turbidity were 6.5 mg/L, 420 mg/L, and
3.3 MTU, respectively. The major water quality para-
meters that were monitored included turbidity, pH,
alkalinity, TOC, biodegradable organic carbon (BDOC),
ultraviolet absorption measured at 254 nm (UV-254),
THMFP, apparent molecular weight (AMW) distri-
bution, and humic and nonhumic fractions of natural
organic matter (NOM).
Ozone dose, hydraulic retention time, and the
concentration of dissolved ozone controlled both the
destruction of organic carbon and the production of low-
molecular weight organic compounds and biodegradable
organic matter during ozonation of Huron River water.
The study showed that biodegradable organic matter in
Huron River water consisted of rapidly and slowly
biodegrading fractions ("fast" and "slow" BDOC),
which agreed with findings of other researchers who
used different source waters. The biodegradability of
water and biodegradation efficiency were characterized
by the maximum biodegradation rate of "fast" BDOC
(Rmax), the minimum empty bed contact time (EBCT)
that was required to eliminate "fast" BDOC (EBCTmin),
and by the minimum concentration of "slow" BDOC
that remained in the water after biodegradation at
EBCTmin (BDOCmin). The study showed that FBT was
more efficient than biofiltration hi terms of NOM
removal and EBCT.
Among the processes investigated (single-pass
ozonation/FBT, ozonation/FBT with recycle, single-pass
FBT/ozonation with biofiltration, recirculating FBT/
ozonation with biofiltration), the recirculating FBT/
ozonation process followed by biofiltration was most
efficient in terms of the removal of NOM relative to
ozone consumption and biodegradation rate. At an
ozone dose of 1 mg/mg C and total EBCT of 40-50
minutes, the removal of NOM was comparable with that
achieved at the Ann Arbor Water Treatment Plant,
which uses lime softening, flocculation/sedimentation,
ozonation and granular activated carbon (GAC) filtra-
tion to treat Huron River water. For a design capacity
of 1 million gallons per day, the cost of treatment by the
FBT/ozonation process followed by biofiltration was
estimated to be at least 40 percent lower man that of a
conventional flocculation/sedimentation process with
ozonation and GAC filtration.
38
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Genotoxicity and Occurrence Assessment of
Disinfection Bv-Product Mixtures in Drinking Water
Roger A. Minear and Michael J. Plewa
Department of Civil Engineering, University of Illinois at Urbana-Champaign, Urbana, IL
This project addresses the questions of whether
the single-cell gel electrophoresis (SCO COMET) assay
using transgenic mammalian cells, rather than bacterial
genotoxicity tests, can predict human risks from
drinking water disinfection by-products (DBFs) and if it
is sufficiently responsive to evaluate DBFs produced
from different disinfection processes and under different
conditions. The study is founded on the premise that
mammalian cells may be more representative of direct
cytotoxic and genotoxic effects from DBFs on humans
than the "traditional" bacterial assays.
This project's objectives are to: (1) calibrate
Salmonella typhimurium and transgenic mammalian cells
genotoxicity assays using regulated DBFs; (2) compare
the relative genotoxicities of chlorinated versus bro-
minated DBFs; (3) compare the relative genotoxicities
of chlorination by-products (CBPs) versus brominated
ozonation by-products (OBPs); (4) compare the relative
genotoxicities of DBFs derived from singular versus
combination (sequential) use of ozonation and chlori-
nation; and (5) provide a DBF occurrence database for
extrapolating genotoxicity results to current disinfection
practice. Representative DBFs will be produced from
organic matter isolated from a series of representative
source waters used for drinking water supplies using
both chlorination and ozonation in laboratory reactors
under a range of disinfection conditions. Selective
conditions will allow differential evaluation of bro-
minated DBFs via ozonation of bromide containing
waters and also provide information on the relation-
ship of toxicity to DBF molecular weight. Bulk DBFs
will be analyzed for toxicity and mutation induction in
S. typhimurum using a suspension test n S9 (mam-
malian microsomal metabolic activation).
The same DBFs will be analyzed with transgenic
Chinese hamster lung cells n S9 using the SCO
COMET method to detect direct genomic DNA
damage. The cytotoxicity of each sample will be
determined using a microtiter well technique. The
relative genetic damage per unit mass of each DBF
sample will be calculated and rank ordered. The level
of correlation of the DBF-induced genetic damage will
be correlated both qualitatively and quantitatively with
the Salmonella mutation assay and the mammalian cell
SCO COMET assay. The relative genotoxic risk for
specific DBFs or DBF samples will be determined.
The project team has developed and calibrated a
rapid, semiautomated, microplate-based cytotoxicity
assay for S. typhimurium. This method provided a mea-
surement of relative cytotoxicity for each DBF. The
DBF standards have been evaluated and rank-ordered
(see Figure 1). A novel microplate cytotoxicity assay
also was developed for mammalian cells. Reverse
mutation at the hisG46 and hisD3052 target genes was
analyzed for each DBF with and without mammalian
microsomal metabolic activation. This assay is being
calibrated with DBF standards. DBFs from each of the
four disinfection processes on the same natural organic
matter (NOM) source were compared, and the effect of
bromide on the total quantities of DBFs and their
distribution was examined. DBFs analyzed using the
(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine(PFBHA)
gas chromatographic method included nonhalogenated
aldehydes. In addition, procedures developed in our
laboratories were used to determine the distribution of
total organic halogen (TOX) between chlorine- and
bromine-containing species and to evaluate the fraction
of the total organic chlorine and total organic bromine
that was accounted for by known and measurable
compounds. The DBF standards were assayed with S.
typhimurium tester strains under suspension conditions
for both cytotoxicity (repression of growth relative to
the negative control) and for mutation induction at the
hisD3052 and hisG46 gene targets. This is the first tune
that these agents have been analyzed in such a quantita-
tive manner. Cytotoxicity is usually ignored when
conducting Salmonella plate incorporation tests. By
using our novel microplate cytotoxicity method, the
highest concentration of DBF to be the %C1/2 value was
established. Concentrations then declined for the con-
centration response mutagenicity experiments. These
data demonstrate the relationship between cytotoxicity
and mutagenicity of the DBF standards.
For future Salmonella cytotoxicity and muta-
genicity studies, the DBF mixture generated from
known water sources will be analyzed and compared to
the results from our DBF standards. Future research
will focus on defining the methods for analysis of the
DBF standards using SCO electrophoresis in mammalian
cells. In addition, a genotoxic potency for these agents
in these different assays will be determined, and their
rank order and relative sensitivity will be compared.
39
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E
8 100
in
Q
O
1 75
I
50
25
r
MX
BromoawHcAdd
Bromofbrm
DibrociMMCetlcAcId
Trlbromoacdlc Acid
Chlcrofbrm
Ethind
DlmethylsuKoxldo
10-3 10-2 10-1 10° 101
Test Agent Concentration (mM)
Figure 1. Comparative cytotoxicity of water DBFs measured as a repression of the growth rate as compared with the concurrent negative control
in S. typhimuri strain TA100.
40
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Molecular Weight Separation and HPLC/MS/MS Characterization
of Previously Unidentified Drinking Water Disinfection Bv-Prodiicts
Roger A. Minear
Department of Civil Engineering, University of Illinois at Urbana-Champaign, Urbana, IL
Sylvia Barrett
Metropolitan Water District of Southern California, Los Angeles, CA
This project's goal is to develop new approaches
for better characterizing disinfection by-product (DBF)
molecular weight profiles by using tandem mass spec-
trometry (MS/MS) techniques. The project will include
examination of the differences in DBFs that result from
different water disinfection processes. The underlying
hypothesis is that new approaches are needed for this
assessment, and tandem mass spectrometry, coupled
with prior separations, offers promise in that regard.
A prerequisite to making such procedures
meaningful is the development of preseparation pro-
cedures that will simplify the mass spectral data. The
MS/MS system affords easy assessment of molecular
weight in the first stage followed by generation of
specific chemical structural data on the mass selected
species distributions via measurement of related frag-
ments from the selected ion. The MS/MS system,
hence, has its own separation capabilities. This project
is directed at enhancing these capabilities for complex
DBF mixtures with preselection by molecular size sep-
arations using ultrafiltration (UP) membranes and size
exclusion chromatography (SEC). These preselected
molecular size fractions then would be followed by other
high performance liquid chromatographic (HPLC)
techniques.
It is expected that more than molecular weight
information will be developed from the planned ex-
periments. The use of several disinfection processes on
the same samples also will provide additional insight as
to how DBFs differ as a result of variations in the
disinfection process. The specific HPLC separation
processes, such as size exclusion chromatography and
reverse phase chromatography, are expected to provide
additional clues regarding the chemical characteristics of
unidentified DBFs. The research will provide infor-
mation on the molecular distribution of the DBFs col-
lectively and specific information on the halogenated
components distribution.
IQiere are no findings to date; however, given
current expectations that DBFs with molecular weights
exceeding 5,000 Daltons will be of little toxicological
significance, the expected results from the proposed
research will provide a means for assessing the potential
differences in disinfection processes for generating
greater or lesser contributions to the lower molecular
weight DBF fractions. This information affords an op-
portunity to improve risk assessment using the mol-
ecular weight criterion and better information of the
location of the halogens in the less than 5,000 Dalton
components of DBFs.
41
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Direct Quantitation of Haloacetic
Acids Bv Surface Enhanced Raman Scattering
MichaelJ. Natan
Department of Chemistry, The Pennsylvania State University, University Park, PA
There are five reasons why the surface enhanced
Raman scattering (SERS) is potentially a powerful tool
for environmental analysis. First, Raman spectra com-
prise fingerprint-like collections of molecular vibrations
that can uniquely identify low molecuar weight species.
Second, water is a weak Raman scatterer, and is thus the
preferred solvent for Raman measurements. Third, SERS,
the process whereby molecules in close proximity to
suitably roughened noble metal surfaces experience
enormous increases in Raman cross section, has been
shown to have single-molecule sensitivity. Fourth,
Raman is a scattering process, and is particularly well-
suited to analysis of solid interfaces. Finally, Raman
instrumentation has been advanced to the stage where
briefcase-sized instruments are commercially available.
This project has focused on two areas. The first is the
development of SERS detection for capillary electro-
phoresis (CE). Known for its high separation efficiency,
short separation time, and ease of sample handling, CE is
rapidly becoming a powerful separation technique used in
an extremely broad range of analytes. However, most
detection methods that have been used (e.g., fluore-
scence) require postcolumn derivatization with fluorescent
reporters. A method in which the CE eluate can be
directly deposited onto a SERS substrate that is rastered
underneath the tip has been developed. In this way,
separated compounds are deposited at different (known)
positions. The path of the capillary is then retraced with
a Raman epi-illumination probe, allowing the Raman
spectra of the unlabelled analytes to be acquired.
The second area concerns the design, fabrication,
and testing of new, highly enhancing three-layer SERS
substrates. The three-layer substrates comprise a layer
of colloidal gold, a layer of chemically deposited silver
(Ag), and a layer of evaporated Ag. These substrates
have been shown to be highly reproducible from both
intra- and intersubstrate perspectives, and have been
shown to allow subpicogram detection of pesticides.
42
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Health Risk of Trihalomethanes Found in
Drinking Water: Carcinogenic Activity and Interactions
Michael A. Pereira
Department of Pathology, Medical College of Ohio, Toledo, OH
This project's objective is to evaluate the
mechanisms through which the trihalomethanes (THMs)
and chlorinated acetic acids (dichloroacetic acid, DCA,
and trichloroacetic acid, TCA) cause liver cancer in
B6C3F1 mice and, for the THMs, colon cancer in F344
rats. In mouse liver, the carcinogenic activity of the
THM, DCA, and TCA has been proposed to result from
a nongenotoxic mechanism involving their ability to in-
duce cell proliferation. One of the mechanisms con-
trolling the expression of the mRNA for the immediate-
early protooncogenes associated with cell proliferation
is DNA methylation. Methylation of the 5-position in
cytosine is a normal occurrence in DNA and controls
the synthesis of mRNA, including the expression of the
mRNA protooncogenes. Hence, decreased methylation
of their genes is expected to increase their expression.
The THMs, DCA, TCA, and trichloroethylene (another
environmental contaminant and a parent compound of
DCA and TCA) have been determined to decrease the
level of 5-methylcytosine in liver DNA and in DCA-
and TCA-induced liver tumors. Upon termination of
exposure, liver rumors promoted by DCA regressed and
the level of DNA methylation returned to control level,
while those promoted by TCA continued to progress and
maintained the reduced level of DNA methylation.
Furthermore, chloroform administered hi the drinking
water did not affect DNA methylation corresponding to
its lack of carcinogenic activity when administered by
this route. Because chloroform followed by DCA and
TCA are the most prevalent chlorine disinfection by-
products, a study is being conducted to determine the
interaction of chloroform with the two chlorinated acetic
acids when administered hi the drinking water. The
study will determine whether chloroform affects the po-
tency of the tumor-promoting activity of the chlorinated
acetic acids. , It is predicted that chloroform will de-
crease the carcinogenic activity of DCA and TCA, sug-
gesting that their common occurrence hi drinking water
would be less hazardous than exposure to them alone.
In the NTP bioassay hi rats, bromodichloro-
methane and bromoform administered by gavage hi corn
oil were carcinogenic hi the colon. Four THMs ad-
ministered hi drinking water were evaluated for then-
ability to induce and/or promote azoxymethane (AOM)
induced aberrant crypt foci (ACF) hi the colon of F344
rats. ACF are putative precancerous lesions that appear
to predict an increased risk of colon cancer.
None of the four THMs either induced ACF or
promoted AOM-induced ACF. Presently, the ability of
the THM to induce cell proliferation hi the colon of
F344 rats is being studied as a function of route of
administration (i.e., in drinking water or by gavage hi
corn oil). This study also attempts to determine whether
bromodichloromethane induced colon cancer by a
genotoxic mechanism. Rats are being administered bro-
modichloromethane to develop tumors that will be
evaluated for mutations hi the K-ras oncogene, other
oncogenes, and tumor suppresser genes. This study will
determine whether the mechanism of colon carcino-
genesis by bromodichloromethane is genotoxic and thus
suggest appropriate hazard assessment for its environ-
mental (drinking water) exposure.
43
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Assessment of Human Dietary Ingestion
Exposures to Water Disinfection By-Products via Food
James H. Raymer, Gerald G. Akland, Edo D. Pellizzari, C. Andrew Clayton, and Doris J. Smith
Research Triangle Institute, Research Triangle Park, NC
This project's objective is to estimate the
magnitude of exposure to disinfection by-products
(DBFs) in drinking water via their ingestion after uptake
into food during cooking. The tests have shown that
foods can become contaminated with chemicals in the
water used in the home during food preparation (e.g.,
cooking). The magnitude of this contamination process
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, chloropic-
rin, 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 exposures of
children are higher than for an adult living hi the same
household.
Analytical methods will be developed for
ozonation DBFs in foods and beverages. Controlled,
laboratory experiments will be conducted to determine
how selected DBFs, especially those produced during
ozonation, are adsorbed by food during the cooking
process. Other DBF methods for foods and beverages
for halogenated compounds (currently under develop-
ment) will be brought in as needed. The relation
between DBF concentrations hi water and in foods
following cooking hi contaminated water will be
modeled. This project will specifically address food
items commonly eaten by children, hi addition to food
items eaten by adults, so that estimates of ingestion of
the study compounds to this subgroup can be de-
termined. A field study will be conducted hi two cities
each having different factors hi water disinfection (e.g.,
secondary disinfection, bromide concentration, high
dissolved organic carbon) to test the validity of the
model for predicting potential exposures and to
estimate the human exposures to DBFs from food and
water.
The major benefit is that current risk assessments
assume that ingestion consists of adding the con-
centration levels of the specific compounds known to
exist in the drinking water (multiplied by the normal
assumption of volume of drinking water consumed) to
the levels of the specific compounds that exist hi the
food, as determined by the Food and Drug Ad-
ministration hi various surveys. However, this project
will attempt to verify that the estimates of exposure
related to ingestion actually underestimate the actual
exposures, and to estimate the amount of under-
estimation for the two population groups of interest,
children and adults, based on actual measurements of in-
home prepared foods and drinking water. Furthermore,
this research will provide new information on dietary
exposure to a specific set of compounds that is currently
not available. The risk assessment process will be
improved hi that a greater understanding of DBF ex-
posure via food will be obtained. Confirmation of the
significance of this exposure pathway for DBFs will lead
to more accurate risk assessments, and provide evidence
for judging the extent to which children might be
included hi the portion of the population that has higher
total exposures.
44
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Analysis of Organic By-Products From the Use of
Ozone/Chlorine and Ozone/Chloramines in Drinking Water Treatment
David A. Reckhow
Department of Civil and Environmental Engineering, University of Massachusetts, Amherst, MA
This project's goal is to test, develop, and refine
new and emerging analytical methods for nonvolatile
organic disinfection by-products (DBFs), and to identify
new ozonation and mixed ozonation/chloramination by-
products. Special emphasis will be placed on new aqu-
eous-phase derivatization agents. Because new deriva-
tization and extraction techniques will be used, it is
expected that some new DBFs will be discovered over
the course of performing this research. In addition, the
results from this study will fill in some of the gaps in
our knowledge of differences between-DBFs hi plant
effluents and DBFs at exit points in a distribution
system. This information will be valuable for deter-
mining research needs related to water quality in distri-
bution systems.
The methods studied in this research largely
employ the use of new or existing, but little-used,
derivatizing agents, especially those that can be used
directly in aqueous samples. Early phases of this re-
search are being conducted on pure solutions of known
disinfection by-products, and laboratory-treated model
waters. However, the work has quickly progressed to
field samples from several New England water treatment
facilities that use ozone as a primary disinfectant. The
main thrust of this project has been to apply some of the
new and emerging techniques for aqueous-phase deriv-
atization to "tag" nonvolatile DBFs and render them
more hydrophobic and/or more easily detected.
In some cases, the derivatized compounds are
subsequently transferred to an organic phase, where they
are analyzed directly by gas chromatography (GC) or
further derivatized prior to analysis. Where possible,
GC with electron capture detection and gas chromato-
graphy-mass spectrometry (GC/MS) with negative
chemical ionization are being used. For compounds
without strong electron affinities or for highly polar
derivatives, LC/MS with electrospray is being used.
A search for new DBFs is parallelling the search
for new analytical methods. A comprehensive resin ad-
sorption procedure is being employed that was devel-
oped in the geochemistry field. This is providing the
platform from which some of the new and emerging
analytical technologies for identifying new nonvolatile
DBFs can be used.
Progress to date covers three general areas: (1)
new DBF method development, (2) persistence of
known DBFs in drinking water distribution systems, and
(3) sample preparation with gross characterization of
natural organic matter (NOM). Work on chloroformate
derivatization with GC/MS has yielded good results with
a wide range of poly functional organic compounds.
Clean chromatograms and mass spectra have been
obtained for several dozen authentic standards using
hexylchloroformate as a derivatizing reagent. Linear
standard curves have been developed and extended
down to the high ppb range. Unfortunately, the project
has not yet been able to demonstrate the higher levels of
sensitivity with GC/MS that would be needed for direct
analysis of water samples (i.e., the low ppb or high ppt
range).
Plant sampling and DBF analysis have shown that
many aldehydes and ketoacids can persist well into
distribution systems, when biologically active filtration
is not practiced. There has been success in extracting
and isolating the more hydrophilic organic constituents
of treated drinking water. Gross characterizations show
this material to be relatively unreactive with chlorine
and low in hydroxyaromatic content (by pyrolysis
GC/MS with and without methylation).
Ozonation by-products (OBPs) are only partly
removed through treatment and distribution in
nonfiltration plants (see Figure 1). Some compounds
appear to be lost in transmission/distribution, which
suggests biodegradation. Others are more persistent and
survive largely unattenuated to the consumers' tap. This
suggests that specific ozone by-product analysis may be
a sensitive test for water systems or segments of water
systems that have uncontrolled biodegradation.
This project is continuing with methods
development for the aqueous-phase derivatization and
GC/MS approach. The focus is on improving method
sensitivity as well as on sample isolation and con-
centration techniques. In addition, work is in progress
on adapting the LC/MS methods for use with drinking
water matrices. The most promising method is one
developed for marine pore water that uses 2-nitro-
phenylhydrazine with phase transfer catalysts.
Water treatment plant sampling will continue (a
new set every, 1-2 weeks) independent of these method
development studies. Samples will be analyzed for
known by-products (e.g., aldehydes, ketoacids). As new
methods become available, they will be applied as well.
45
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Sampling date: 1 Oct 98
Formaldehyde
Glyoxal
Me-Glyoxal
Glyoxalic Acid
Pyruvic Acid
Figure 1. Following OBPs in a nonfiltration ozonation system.
46
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Overview of Disinfection By-Products
Research and Preliminary ICR Findings
Susan D. Richardson
National Exposure Research Laboratory, U.S. Environmental Protection Agency, Athens, GA
Chlorine, ozone, chlorine dioxide, and chlor-
amine are currently the major disinfectants being used
to disinfect drinking water. Following the discovery of
chloroform as a chlorination disinfection by-product
(DBF) in 1974, researchers began to identify disin-
fection by-products. Of the disinfectants, chlorination
by-products have been the most studied, and, as a result,
more than 300 chlorine DBFs have been reported in the
literature (Richardson, 1998). Fewer studies have been
carried out for the alternative disinfectants; however,
there is some information known about their by-
products.
The U.S. Environmental Protection Agency
(EPA) has been interested in identifying the previously
unidentified DBFs with the goal of determining if there
are any potentially harmful DBFs present. Researchers
at EPA's National Exposure Research Laboratory in
Athens, GA,'have been carrying out research to ac-
complish this: goal, with an emphasis on DBFs from
alternative disinfectants and on polar DBFs. Research-
ers at the EPA's National Exposure Research Labora-
tory in Cincinnati, OH, have been carrying out methods-
related research to improve the identification of polar
aldehydes, improve the extraction of polar compounds
(to enable the isolation and identification of polar
DBFs), and to lower the detection limits for bromate, an
animal carcinogen that will be regulated under Stage 2
of the DBF Rule. Preliminary Information Collection
Rule (ICR) data are now available. These data, col-
lected at treatment plants across the United States,
include quantitative occurrence data for aldehydes,
bromate, and cyanogen chloride.
References
Richardson, S.D. 1998. Drinking water disinfection by-products. The Encyclopedia of Environmental Analysis & Remediation,
Robert A. Meyers, editor, vol. 3, pp. 1398-1421, John Wiley & Sons, New York.
47
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Physiologically Based Pharmacokinetic
Modeling of Haloacid Mixtures in Rodents and Humans
Irvin R. Schultz
Batelle Memorial Institute, Pacific Northwest Division, Richland, WA
This project's goals are twofold: (1) characteri-
zation of the comparative pharmacokinetics of chloro,
bromo, and mixed chlorobromo haloacids (HAs) in
rodents, and (2) development of a physiologically based
pharmacokinetic (PBPK) model that can accurately
predict the tissue distribution and elimination of HAs
during chronic oral exposure in mice, rats, and humans
(see Figures la and Ib).
Groups of mice and rats were given either
intravenous injections or gavage doses of an HA and the
blood concentration-tune profile and urinary excretion
characterized in individual animals. Additional groups
of mice and rats were pretreated with a di-HA for 14
days at various drinking water concentrations, and the
subsequent effect's of these treatments on metabolism
were measured. These experiments used both in vivo
(mice: blood elimination after intravenous injection;
rats: CO2 formation after gavage dosing) and in vitro
methods to quantify metabolic activity. The blood
elimination and oral bioavailability of the HAs were
characterized using compartmental and statistical
moment pharmacokinetic methods. The in vitro
metabolism was measured in liver homogenates and
determination of the Michaelis-Menten constants (Vmax
and ATm) of HAs determined using nonlinear least-squares
regression methods. These data sets will be used to test
and validate a PBPK model for di- and tri-HAs in the rat
and mouse. Later, the PBPK model will be extended to
humans and used to predict tissue levels of haloacetates
during chronic, low dose exposure to HAs resulting
from drinking water consumption.
After the mice received intravenous dosing, their
blood concentrations of all HAs declined hi a mono- or
biexponential manner with a short distributive phase.
There was a remarkable similarity in the extent of extra-
vascular distribution among HAs, while pronounced
differences existed hi the rate of excretion. The most
dramatic structural feature that influenced the disposi-
tion of HAs was substitution of a halogen for a hy-
drogen. All di-HAs had elimination half-lives of less
than 2 hours hi contrast to the tri-HAs, where the blood
elimination half-life varied from 0.6 to 8 hours. The
urinary excretion of all di-HAs was low and accounted
for less than 3 percent of the dose hi contrast to the tri-
HAs where urinary excretion accounted for at least 30
percent of me dose. All di-HAs are more rapidly meta-
bolized than tri-HAs. Pretreatment with a specific di-
HA greatly diminished the in vitro metabolism of all the
di-HAs.
These results indicate that large differences exist
hi the excretion rate of HAs, which are apparently
attributable to differences hi both metabolism and uri-
nary elimination. The metabolism of di-HAs decreases
during prolonged administration, which suggests that
previous pharmacokinetic studies of HAs using control
animals may be biased because our results indicate that
elimination of di-HAs is reduced during chronic
exposures. Therefore, estimation of target organ con-
centrations of these compounds during chronic dosing
may be higher than previously thought.
This project will establish the linearity of HA
pharmacokinetics at low doses, and the threshold drin-
king water concentration that causes inhibition of
metabolism for the di-HAs. These results will be in-
corporated into the working PBPK model for HAs to
predict tissue dosimetry during low exposure rates.
48
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•
Inspired Air I—
IC02 T • A A. [L
1 (EAaledAir) Inspired Air L-
Di-HA treatment
Figure la. Schematic view of the HA PBPK model.
80
60 -
EQ 40-1
o
O
20 -
0 J
O- DCA Pretreated CD -Control
•,• PBPK Model Predictions
120
CD
80
< 40
O
Q
0 J
T I 1 i 1
0 2 4 6 8 10
Time (Hr)
0 5 10 15 20 25
Time (hr)
Figure Ib. Examples of model prediction in rats. Left panel: CO2 formation from oral doses of I4C-DCA. Right panel:
Liver concentrations of DCA after gavage dosing. Pretreated rats were exposed to DCA up to 14 days prior to
challenge doses. Observed data for DCA in liver is from Evans, O.B. Biochem Pharmacol 1982; 31(19):3124-
3126.
49
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Development of a New, Simple, Innovative Procedure for the Analysis
of Bromate and Other Oxyhalides at Sub-ppb Levels in Drinking Water
Howard Weinberg
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC
The oxyhalide disinfection by-product bromate is
most commonly associated with the ozonation of drink-
ing water containing bromide and has been identified as
a potential source of kidney tumors. However, accurate
exposure assessment studies at the low concentrations
typically found in drinking water have been hindered by
the absence of sensitive analytical procedures for
detection and quantitation. Health effects studies sug-
gest that bromate should be regulated at 0.5/tg/L or less
in drinking water. Accordingly, an analytical method
that can be used routinely is needed to quantify this
contaminant with great sensitivity and selectivity.
This project is employing a relatively unconven-
tional approach to the analysis of chemical components
of drinking water. Ion chromatographic (1C) separation
with no pretreatment followed by a postcolumn reaction
to produce tribromide (Br3~) from bromate is applied to
the analysis of a variety of aqueous samples. The tri-
bromide ion is detected by ultraviolet absorbance at 267
run. This method is very sensitive for bromate with a
limit of quantitation of 0.2jig/L and also is very
selective. Common anions typically separated by 1C
exhibit no interference, even at the levels normally
found in drinking water. The method also has been
optimized for similar sensitive quantitation for the
oxyhalides iodate and chlorite; with the use of recently
available higher capacity chromatographic columns,
even lower detection levels are predicted. The meth-
odology is being applied to a wide variety of drinking
waters throughout the United States, including those
disinfected by water utilities as well as those produced
commercially and bottled.
A series of controlled experiments has been
performed that employ a synthetic aquatic matrix con-
taining a mixture of anions together with reconstituted
natural organic matter as a source of organic carbon, all
dissolved in deionized water. These studies were done
to determine the method's applicability to a wide range
of drinking waters varying hi total organic carbon
(TOC), natural bromide, and treatment. Ozonation of
this water at varying ozone to TOC doses, different
levels of alkalinity, pH, and bromide provided an
opportunity to investigate correlations between bromate
(at sub-ppb levels) and transferred ozone dose in these
waters (see Figure 1). Furthermore, the analytical me-
thod has been applied in practice to the determination of
bromate hi a pilot ozonation plant treating a surface
water with low-level bromide (< 50/ig/L) at a variety of
ozone to TOC doses. The results of quality-controlled
analysis of these samples indicate the applicability of
this analytical method to the determhiation of sub-ppb,
levels of bromate that are indeed likely to be found in
ozonated waters that contain low bromide.
The availability of this relatively simple meth-
odology will provide the tools for assessing exposure to
bromate in drinking water that has hitherto been im-
peded by the lack of sensitivity of existing method-
ologies. This analytical methodology will provide the
U.S. Environmental Protection Agency (EPA) and the
water quality monitoring community with the ability,
using existing analytical equipment with simple add-on
accessories, to fulfill the goals of the Information Col-
lection Rule and criteria for future drinking water regu-
lations by achieving the required sensitivity very easily
in oxyhalide analysis.
The next steps are to further simplify the meth-
odology, share split sampling with EPA Cincinnati to
compare and validate different methods, and to begin a
comprehensive survey of U.S. drinking waters contain-
ing low levels of bromide.
1.0
mA
-0.5
Bromate
.(0.185 |ig/L)
234
Time (minutes)
Figure 1. Ion chromatographic measurement of bormate in ozonated surface water at various ozone
to TOC doses (mg/mg) - TOC = 2.5 mg/L; 0 (upper), 0.25 (middle), and 0.5 (bottom).
50
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Inhalation and Dermal Exposure to
Disinfection By-Products of Chlorinated Drinking Water
Clifford P. Weisel and Jeffrey Luskin
Environmental and Occupation Health Sciences Institute, Piscataway, NJ
This project's goal is to determine the inhalation
and dermal exposure to disinfection by-products (DBFs)
of chlorination from the multiple water uses that occur
within a home.
The exposures will be determined by: (1) mea-
surements of the concentration of the DBFs or their
metabolites in urine or breath following known expo-
sures; (2) estimation of dermal transport coefficients
using in vitro measurements across excised skin under
controlled conditions; and (3) measurement of size and
number distribution of residential water aerosols gen-
erated by typical water uses that result in inhalation ex-
posures. During the first year, biomarker measurement
methodology is being optimized. The targeted DBFs are
haloacetic acids, haloketones, chloral hydrate, andhalo-
acetonitriles. Subjects will be exposed to the DBFs via
inhalation to volatile and nonvolatile emissions from
water and by dermal absorption by direct contact to
water in showers and baths. The DBFs will be added
directly to purified water that is used for showering or
bathing at the upper range of concentrations measured
hi drinking water systems. Tune series breath levels of
volatile DBFs will be measured to assess the uptake and
release rate of the DBFs in the body.
The methodology for haloacetic acids and halo-
ketones analyses hi urine has been optimized. A 5 mL
urine sample is acidified with concentrated sulfuric acid,
extracted twice with ethyl ether, centrifuged, the ether
evaporated, the halogenated acids and ketones methy-
lated at 60 °C for 1 hour with 10 percent sulfuric
acid/methanol mixture in methyl tertiary butyl ether
(MTBE), sodium sulfate is added and the MTBE is
analyzed by gas chromatography/electron capture de-
tection (GC/ECD). A similar method is used for the
water analysis, except that MTBE is used initially in
place of ethyl ether. The mean percent recoveries based
on replicate analyses of spiked samples were between 80
and 100 percent for all compounds except dibromoacetic
acid from urine, which was 67 percent. The detection
limits were all less than 0.2 ;ig/L from water and 0.5
Hg/L from urine, except bromochloroacetic acid from
urine, which had a value of 1.2 jtg/L. A coeluting peak
to methyl bromochloroacetate has been found in the
urine extracts. Changes to the chromatographic
conditions, including dual column chromatography, are
being considered to resolve this problem.
This project will provide data on the total DBF
exposure and dose for a series of compounds that have
not been previously evaluated. Fundamental data nec-
essary for drinking water exposure models from a
variety of water uses will be obtained, including
particle size and number distributions. The preliminary
results demonstrated that the proposed biomarker mea-
surement methodologies have the necessary sensitivity
to be used for the planned exposures at environmental
levels.
Human studies are to begin. Initially, ingestion
studies will be done to provide a baseline on the excre-
tion pattern for the DBFs when maximal metabolism
will occur (see Figure 1). The DBF spiking system for
the shov/er experiments and the dermal immersion
system are being developed.
o
&
£
§
!
&
-2000 -1000 0 1000
Elapsed Time (min)
2000
Figure 1. Temporal change in urinary excretion rates of TCAA during the ingestion exposure study.
Exposure occurred at zero minute; concentration of TCAA in water was 25.5
volume of water was 500 mL; and amount of TCAA ingested was 12.75 ftg.
51
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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 by-products (e.g., bromate). Bromate forms via
oxidation of naturally occurring bromide through a
series of steps (see Figure 1). There is a need to develop
tools to understand and predict bromate (BrO3~)
formation while still achieving high levels of microbial
disinfection. The central hypothesis is that a kinetic-
based understanding of natural organic matter (NOM)
reactions with hydroxyl (HO) radicals and aqueous
bromine (HOBr/OBr~) over a range of temperatures is
necessary to develop mechanistic-based models for
bromate formation in bulk waters.
This project's objectives include: (1) developing
a comprehensive database of BrO3~, O3, and HO radi-
cal concentrations; (2) determining rates of reaction bet-
ween HOBr and OBr~ and NOM; (3) calibrating and
verifying a BrO3~ formation mechanistic-based model
that includes NOM; (4) simulating BrO3~ control
measures necessary to meet proposed and future MCLs;
and (5) linking the numerical BrO3~ formation model
with hydraulic and CT disinfection models.
A mechanistic-based, numerical, kinetic BrO3~
formation program will be developed (see Figure 2).
The program links an oxidant module for predicting O3
and HO radical concentrations with a BrO3" formation
module. The model employs a set of bromide oxidation
reactions that have been previously developed by the
investigator, and calibrated against bromine and BrO3~
formation hi NOM-free water; NOM reactions now will
be incorporated. The oxidant module will be calibrated
against experimental O3 decay data (e.g., simple first-
order decay) and HO radical concentrations (calculated
from the disappearance of a HO radical probe com-
pound during ozonation). Predicted BrO3~ levels will be
calibrated and verified against an internal database that
accounts for synergistic effects of key parameters
(bromide, pH, ozone dose, temperature, inorganic car-
bon, and ammonia) on ozone decay, HQ radical con-
centrations, and BrO3~ formation, and an external U.S.
Environmental Protection Agency database.
The project will start on December 15, 1998, so
there are no preliminary findings to date.
f °3
!< .NOM ,
(Oxidation)
HC
Or
03
\ HO BrO/*
r BrO '
NOM
(Substitution)
;ano-bromine compounds
Figure 1. Pathways for bromate formation.
Data Input
DOC, Br, pH, NH3, Temp
O3 decay rate parameter
Oxidant Modular
O3 (+ NOM) -» nHO (+ products), k,
Bromate Formation Modular
Reactions Provided in Tatjle 1
Temperature Correction Relationship
(Hydraulic Module i
| CSTRs in Series :
I CT Calculation Module j
Predicted Bromate & Ozone Concentrations
Figure 2. Framework for proposed model.
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Mechanistic-Based Disinfectant and Disinfectant By-Product Models
Paul Westerhoff
Department of Civil and Environmental Engineering, Arizona State University, Tempe, AZ
David Reckhow
Department of Civil and Environmental Engineering, University of Massachusetts, Amherst, MA
Gary Amy .
University of Colorado, Boulder, CO
Zaid Chowdhury
Malcolm-Pimie, Inc., Phoenix, AZ
The water industry faces new challenges hi
understanding and controlling disinfection by-product
(DBF) formation as health concerns demonstrate a need
for more stringent regulatory DBF requirements.
Mechanistic tools for understanding and predicting the
rate and extent of DBF formation are required to
facilitate the evaluation of DBF control alternatives.
A mechanistic-based numerical model will be de-
veloped for chlorine decay and regulated trihalomethane
(THM) and haloacetic acid (HAA) formation derived
from (free) chlorination (see Figure 1); the model
framework will allow future modifications for other
DBFs and chloramination. Predicted chlorine residual
and DBF results will be compared against predictions
from several other quasimechanistic models. A signifi-
cant improvement in prediction accuracy over existing
empirical models is anticipated. Several modeling hy-
potheses are proposed as a basis for a mechanistic-based
model for disinfectant decay and DBF formation. The
central modeling hypothesis is that a two-site reaction
mechanism can be used to predict disinfectant decay hi
the presence of natural organic matter (NOM). It as-
sumes that NOM contains both slow and fast dis-
infectant-reacting and DBF-forming sites. NOM site
densities and concentrations are related to the con-
centration, size, structure, and functionality of NOM.
This project's model also will include fitted rate
constants that are a function of pH and temperature. A
series of distribution functions, based upon the predicted
ratios of free-bromine to free-chlorine, will be used to
estimate each of the four THM species (THM4) and
each of the nine HAA species (HAA9).
DBF experimental data from completed projects
conducted by the investigators and other researchers will
be integrated into a single unified database. Existing
empirical models and newly developed numerical
models will initially be calibrated with our unified data-
base. Additional experimental data will be collected be-
cause prior databases lack complete documentation of
NOM characteristics before and during disinfection
addition. Controlled laboratory disinfection and DBF
formation studies will be conducted using water col-
lected at several points through different water treatment
plants, including raw, coagulated, softened, and pre-
oxidized (ozone and/or chlorine dioxide) waters; thus,
the waters represent a wide range of water qualities and
NOM characteristics. Experiments will investigate the
effects of pH and temperature, and NOM, bromide, and
free-chlorine concentrations. DBF hydrolysis studies
also will be conducted.
The project will start on December 15, 1998, so
there are no preliminary findings to date.
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Organic DBP Formation j
Reaction Module
(OFRM) J
| Disinfectant Consumption |
Reaction Module
^ (DCRM) J
User-Interface
&
Numerical Solution Module
Inorganic Reaction Module
(IRM)
CI2(aq, + H20 -» HOCI+H++Cr pK=-3.3
HOCI^H* + OCr pK.=7.5
HOCI+Br-5- HOBr+OCr k=2950M'1s'1
HOBr<->H*+OBr'
DBP Stability Reaction Module
(DSRM)
DBP(x)+OH' -» DBP'(x) or X kH
Hydroly5ls
DBP(x)+{B} ->DBP'(x) or X kBiodegrad.
where "x" is halogen and "B" is biomass
Figure 1. Schematic illustration of computer program.
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Mechanisms and Kinetics of Cfaloramine
Loss and By-Product Formation in the Presence
of Reactive Drinking Water Distribution System Constituents
Richard' L. Valentine
Department of Civil and Environmental Engineering, University of Iowa, Iowa City, I A
The fate of chloramines in drinking water
distribution systems, the nature of the reactions and
by-products involved, as well as the factors that
influence these processes are largely unknown. Recent
exploratory work at the University of Iowa indicates
the potential importance of several ubiquitous reactive
distribution system components. These substances
include natural organic matter (NOM), reduced iron
and manganese, iron oxides, bromide, nitrite, and
oxygen. This project's goal is to enhance our under-
standing of the influence of these reactive substances
on: (1) the fate of monochloramine and the nature of
inorganic reaction products, (2) the kinetics of mono-
chloramine loss, and (3) the formation of selected
organic disinfection by-products (DBFs). Results also
will be used to (4) extend existing mechanistic chlora-
mine reaction models to include the effects of these
reactive substances.
This project's overall approach is to study chlora-
mine loss and DBF formation to provide a com-
prehensive picture of the fate of chloramines in the
presence of NOM, bromide, nitrite, complexed and
uncomplexed reduced iron and manganese, iron oxides,
and oxygen. Studies will be conducted using collected
and laboratory prepared water in batch reactors to assess
the influence of aqueous phase constituents. Reactions
involving whole pieces of authentic pipe deposit material
also will be studied in a recirculating tubular reactor to
better mimic the fluid flow in pipes. Constituent con-
centrations will be systematically varied as will be
monochloramine concentration, pH, and the Cl/N ratio.
Variations may include investigations of the effect of
prechlorination and preozonation of the waters. Mono-
chloramine concentrations will be measured over a
period of at least 5 days. Samples will be taken for an-
alysis of selected DBFs. Mass and redox balances will
be made to try to account for the oxidizing potential of
monochloramine. Results will be analyzed hi the context
of an existing mechanistic description of the reactions
responsible for the autodecomposition of monochlo-
ramine.
This project will enhance the understanding of the
management of risks associated with microbial patho-
gens caused by an inability to maintain an adequate
disinfectant residual, and to risks associated with the
formation of DBFs. Information gained will facilitate
improvements hi water quality and water plant op-
eration by providing water plant managers new informa-
tion on the fate and effects of chloramines. New models
will provide guidance hi chloramination practices and
assessment of the significance of chloramine loss and
DBF formation potential.
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Index
Note: The year of the grant award appears in parentheses following the Principal Investigator's name.
Arsenic
Grants
Pellizzari, E.D. (1997), 3
Smith, A.H. (1997), 4
Snow, E.T. (1997), 6
Styblo, M. (1997), 7
Tice, R. (1997), 9
Microbial Pathogens
Grants
Alvarez, M.E. (1995), 13
Cangelosi, G.A. (1998), 15
Chappell, C.L. (1995), 16
De Leon, R. (1996), 18
Fout, G.S. (current work for
EPA/NERL - Cincinnati), 20
Grant, S.B. (1995), 21
Levy, D. (current work for
CDC), 22
Moe, C.L. (1997), 23
Pepper, I.L. (1995), 2^5
Sobsey, M.D. (1995), 26
Upton, S.J. (1996), 28
Ward, L.A. (1997), 29
Disinfection By-Products
Grants
Ahmed, A.E. (1997), 33
Ball, L.M. (1997), 34
Batterman, S. (1996), 35
Marinas, B.J. (1998), 37
Masten, S.J. (1998), 38
Minear, R.A. (1997), 39, 41
Natan, M.J. (1996), 42
Pereira, M.A. (1996), 43
Raymer, J.H. (1998), 44
Reckhow, D.A. (1996), 45
Richardson, S.D. (current
work for EPA/NERL), 47
Schultz, I.R. (1997), 48
Weinberg, H. (1997), 50
Weisel, C.P. (1997), 51
Westerhoff, P. (1998), 52, 53
Valentine, R.L. (1998), 55
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