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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                                             - ~**m

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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.
                                                   52

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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.
                                                   53

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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.
                                  54

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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.
                                                 55

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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
                                               57

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