645R97102
 1997
               a/?c/ Environmental Effects Research Laboratory
              ROGRAM UPDATES
               .       .                   .
            AND ENVIRONMENTAL EFFECTS RESEARCH
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                    August 1997

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This document contains a series of progress reports that describe a subset of the research
programs administered by NHEERL. Reports are provided for the following topics:

             * Contaminated Sediments
             + Tropospheric Ozone
             +• Endocrine Disrupters
             +• Drinking Water
             + Air Toxics
             + Indoor Air
             + Global Climate Change
             + Pesticides in the Diets of Infants and Children

Over the course of the next year, we anticipate additional reports describing the remainder
of NHEERL's research  programs.   The  reports are  intended  to  be client-friendly
communications that convey,  in non-technical  terms, the direction and  progress of
NHEERL research on high-priority environmental issues. The titles of the reports stem from
ORD's research planning categories (referred to as "subcomponents" in the FY96-97
budget).

The  reports are designed to capture critical features of NHEERL's  research in each
program area. Contained in the reports are 3 principal sections.

•     The first section presents a summary of the overall research program.  This section
      addresses the regulatory and programmatic issues associated with the program
      (including a discussion of statutes germane to the research), defines the program
      goal, discusses scientific uncertainties and research needs relevant to the program,
      and outlines NHEERL's strategy for realizing research objectives.

•     The second  section,  called program  highlights,  features  key research
      accomplishments of the fiscal year.

•     The third section provides more detailed descriptions of the research activities
      within each program area. Discussions are organized to reflect NHEERL's research
      strategy  (in  particular,  its  explicit  use  of the risk  assessment  paradigm).
      Accordingly, the headings that describe the research within each program relate to
      elements of risk assessment.  The descriptions also offer a chronological context
      to the  research.   Recent and on-going research  efforts  are chronicled, and
      anticipated research directions are projected, rendering a snapshot of the program
      at a given point in time.

We believe that the material presented here conveys the contributions made by NHEERL
in strengthening the scientific foundation for environmental decision-making and trust that
these reports  will be informative  and useful in understanding NHEERL research.
                                                O.S. Environmental Protection Agtmfr
                                                   'on 5 Library (PH2J)
                                                                        I2th

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 National Health and Environmental Effects Research Laboratory
    ECOSYSTEMS PROTECTION:
    CONTAMINATED SEDIMENTS

HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH
          PROGRESS REPORT

              August 1997

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                         CONTENTS
Introduction	3

Summary of the Contaminated Sediments
     Health and Environmental Effects Research Program  	4

FY95-96 Highlights	6

Contaminated Sediments Problem Formulation Research Program	7
               Toxicity Assessment Methods
               Ecological Hazard Characterization

Contaminated Sediments Determinants of Effect Research Program	11
               Mechanistic Research
               Models

FY95-96 Bibliography 	14
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996

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                        INTRODUCTION
The purpose of this report is to communicate results from the Contaminated Sediments
Research Program from EPA's National Health and Environmental Effects Research
Laboratory (NHEERL).
The report contains

      •    a summary of the NHEERL Contaminated Sediments Research Program,
           which includes an explanation of the regulatory and programmatic context
           of the program, the program's overall goal, the rationale for research in this
           area, and the research strategy

      •    a section which highlights recent key findings (FY95-96 Program Highlights)

      •    a more detailed  description of the NHEERL Contaminated  Sediments
           Research  Program, by program area, including a summary of research
           accomplishments and anticipated progress for the near future
The format of this report is still evolving, and we welcome feedback.  Readers with
comments or requests for further information are encouraged to contact:

Jennifer Orme-Zavaleta
National Health and Environmental Effects Research Laboratory (MD-51A)
U.S. EPA
Research Triangle Park, N.C. 27711

Phone: (919) 541-3558 or FAX: (919) 541-0642
E-mail: ormezavaleta.jennifer@epamail.epa.gov
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996

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             CONTAMINATED SEDIMENTS
         RESEARCH  PROGRAM  SUMMARY
Numerous environmental statutes authorize
EPA to address the health and ecological
risks   associated   with   contaminated
sediments. Legislation includes the National
Environmental Policy Act; the Clean Air Act;
the Coastal  Zone Management Act;  the
Federal Insecticide, Fungicide, and Rodenti-
cide Act; the Toxic Substances Control Act;
the  Marine  Protection,  Research,  and
Sanctuaries Act; the Clean Water Act; the
Great Lakes Water Quality Agreement; the
Comprehensive   Emergency   Response,
Compensation,  and Liability Act; the Great
Lakes Program Act; and the Water Resources
Development  Act.    EPA research on
contaminated sediments is conducted by the
laboratories of  its Office of Research and
Development (ORD); of fundamental concern
to ORD is the relationship between sediment
contaminants and the viability and sustain-
ability of  benthic (bottom-dwelling)  eco-
systems.  NHEERL, which is responsible for
effects-based research within ORD, supports
the  Contaminated  Sediments  Research
Program by developing methods and models
for evaluating  sediment toxicity  and by
predicting the effects of sediment contami-
nants on various  components of aquatic
ecosystems.   This document summarizes
NHEERL's research program and highlights
some of its recent accomplishments.

PROGJRAM^GOAL ••^•2i£& ."•. -   '
To determine the nature and magnitude of the
toxic  effects  associated with  contaminated
sediments.
 RATIONALE
 According to a recent evaluation of data from
 the  National  Sediment  Quality  Survey,
sediment contamination may pose serious
risks to  aquatic life, wildlife, and  human
health. Many potentially toxic and persistent
contaminants  are   found  in   sediments,
including metals (such as mercury and lead),
organic  chemicals   (such as  PCBs and
polycyclic aromatic hydrocarbons, or PAHs),
and various pesticides.  These contaminants
may adversely  affect  sediment biota and
aquatic communities,  and  they may bio-
magnify  in  the  food chain,  eventually
impacting wildlife or human health.  There is
a critical need for methods and models to
detect and predict sediment toxicity and to
assess the impact of contaminated sediments
on   benthic   organisms   and   aquatic
ecosystems.
To ensure that the Agency is equipped with
scientific and technical data relevant to the
formulation of sound environmental  policy,
ORD operates a research program founded
on the principles of risk assessment.  In the
area of ecological research, ORD's program
follows the  framework for ecological  risk
assessment developed by EPA in 1992 (Fig.
1).  The framework is conceptually similar to
the human health risk assessment paradigm
set forth by the  National  Academy  of
Sciences. The steps of the process include
problem formulation, analysis  (characteriza-
tion  of  exposure  and  effects), and  risk
characterization.  ORD conducts  research
that can be used  in each  of these  risk
assessment steps.  NHEERL supports the
Agency's  risk  assessment   efforts   by
developing test methods, predictive models,
and scientific data that strengthen regulatory
and policy decisions.

In  the area  of contaminated  sediments,
NHEERL's objective is to evaluate the impact
.NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996

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of contaminated sediments on the health and
sustainabilityof benthic species, communities,
and ecosystems.  Two categories of research
have been established to meet this objective.
In the first category, PROBLEM FORMULA-
TION, we are developing methods to detect
and   describe   the   ecological  hazards
associated with contaminated sediments. Our
methods  include   Toxicity  Identification
Evaluation (TIE) techniques for identifying
hazardous contaminants in sediment; toxicity
tests for characterizing potential acute and
chronic effects; and hazard assessments for
determining the sources, causes, and extent
of sediment contamination in selected areas.
Secondly, we are analyzing DETERMINANTS
OF EFFECT.  This research involves the
study of factors that control  and/or modify
sediment toxicity, such as bioavailability. Our
studies  of  bioavailability are  helping  us
understand the basis for sediment toxicity,
which enables us to more accurately define
safe levels of contaminants or mixtures  of
contaminants in sediment.   Models are
another  way  in   which   we   examine
determinants of effect. Through the develop-
ment  of   models   that   can   estimate
bioaccumulation and toxicity, we hope  to
reduce some of the uncertainties associated
with  risk  assessments of  contaminated
sediments.
FIGURE 1.  Elements of ecological risk assessment.
                         Problem
                         Formulation
                    Exposure  '   Effects
                    Analysis   f   Analysis
                        Risk
                        Characterization
                          Risk
                          Management
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996

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NHEERL CONTAMINATED SEDIMENTS RESEARCH
             FY95-96 PROGRAM HIGHLIGHTS
The goal of this research is to improve our ability to characterize the nature of the hazard
associated with contaminated sediments.

    >  We demonstrated for the first time that Toxicity Identification Evaluation (TIE)
       methods can be performed on marine sediment pore  waters; these methods
       showed that PCBs were the cause of acute toxicity in New Bedford Harbor, MA,
       sediments.

    >  We completed a major Technical Guidance Document describing marine methods
       for TIEs.
 The goal of this research is to better understand the environmental processes that
 influence sediment toxicity in order to more accurately predict adverse effects.

    >  Investigators in our Atlantic, Western, and Mid-Continent Ecology Divisions
       produced a series of reports appearing in a  special issue of Environmental
       Toxicology and Chemistry that provide the scientific basis for an equilibrium
       partitioning-based approach for predicting metal bioavailability and toxicity in
       sediments.

    » In conjunction with the Office of Water, Sediment Quality Criteria (SQC) documents
       were proposed for two pesticides (endrin and dieldrin) and mixtures of three PAHs
       (acenaphthene, fluoranthene, and phenanthrene).

    *•  We showed that the toxicity of some sediment contaminants (e.g., fluoranthene)
       increases in the presence of sunlight, and we developed a technique to identify
       these phototoxic chemicals in contaminated sediments.
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996

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               CONTAMINATED SEDIMENTS
 PROBLEM FORMULATION  RESEARCH  PROGRAM
 In the context  of  EPA's ecological  risk
 assessment  framework,  the  process  of
 problem formulation includes  the  identifi-
 cation  of assessment endpoints  and the
 relationship between these endpoints and
 stressors.  NHEERL contributes to both of
 these elements and defines its problem
 formulation  research  as  research  to
 demonstrate a causal connection between
 exposure and effect.
 What  toxic  effects  are  associated with
 exposure to  contaminated sediments, and
 what contaminants are responsible for the
 toxicity?
 Exposure to sediment contaminants may
 adversely affect benthic  biota and other
 aquatic communities.  Persistent contami-
 nants can bioaccumulate and enter the food
 chain,  where they  have  the  potential  to
 impact wildlife or human health. Methods
 are needed to detect toxic contaminants in
 sediments,  to  describe  their  effects on
 benthic organisms and communities, and to
 predict their  long-lasting impact to aquatic
 ecosystems.
 PROGRAM DESCRIPTION
 NHEERL has instituted two lines of research
 to characterize the toxicity of contaminated
 sediments. In the first area, we are develop-
 ing and validating TOXICITY ASSESSMENT
 METHODS to identify the contaminants
 responsible for sediment  toxicity  and to
 describe the effects that may result from
 exposures  to  contaminated  sediments.
 These methods enable us to establish causal
 relationships between specific contaminants
 and  toxic response.  Another focus of our
 sediments  research  is  ECOLOGICAL
 HAZARD CHARACTERIZATION, in which
we  are conducting field investigations to
assess  the  impact  of   contaminated
sediments on benthic ecosystems. Sediment
samples are being  collected  at  various
sampling stations and analyzed for temporal
and spatial  fluctuations in  chemistry and
toxicity.  These  data are then  related to
changes in population dynamics and benthic
community  composition.   This type of
analysis permits an initial hazard assessment
to be made and provides  a  means for
predicting the effects of sediment contami-
nants  on various components of  aquatic
ecosystems.
TOXICITY ASSESSMENT METHODS

Identifying the cause of sediment toxicity.
One way  to  identify and  localize toxic
contaminants in sediment is through the use
of Toxicity Identification Evaluation (TIE)
methodologies. TIE procedures employ a
toxicity-based chemical fractionation scheme
that segregates and identifies contaminants
responsible for acute toxicity.  Since 1992,
scientists in our Atlantic Ecology Division
(AED) have been developing and validating
TIE procedures for the marine environment.
During  that  time,  we  have  published
numerous  scientific  papers and  technical
support    documents   describing   the
application  of  TIE  methodologies  to
contaminated  sediments.   Considerable
progress was made in this area in FY96. For
the first time, we developed TIE techniques
that enabled us to assess marine sediment
pore waters.  Using  these  methods, we
analyzed contaminated  sediments in New
Bedford  Harbor, MA, and discovered that
PCBs were the cause of acute toxicity  in
these sediments. Our marine TIE methods
were recently released by EPA in the form of
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996
                                   7

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a Technical Guidance Document.

We also are conducting research to field
validate  TIE   methods  for   freshwater
sediments. Scientists in our Mid-Continent
Ecology Division (MED) used these methods
during FY96 to demonstrate differences in
the cause of toxicity in  surficial and deep
river sediments.   This finding could prove
important   when   determining   possible
sediment  remediation  strategies.   The
development of TIE procedures for use in
solid-phase sediment testing is technically
more challenging. Progress was made in
this area during FY96 using metal chelating
agents, zeolite,  and organic sorbents  to
differentiate among sources of acute toxicity.

TIE procedures were coupled with analyses
of quantitative structure-activity relationships
(QSAR) during FY96 to identify phototoxic
chemicals in sediments contaminated  by
mixtures of  polycyclic hydrocarbons. The
detection of these compounds is significant
given our findings that some contaminants in
sediments can be photoactivated, resulting in
increased sediment toxicity (see discussion
under  DETERMINANTS  OF  EFFECT,
Mechanistic Research).

Sediment toxicity tests. The development
and  evaluation  of  toxicity   tests  that
characterize  acute  and  chronic  effects
caused by contaminated sediment is another
important component of this program area.
During FY95-96, our evaluations of acute
toxicity tests led to a better understanding of
the behavior of  test endpoints and helped
bound  our  confidence  in  predictions  of
ecological effect. Several examples can be
cited. An evaluation of the relative sensitivi-
ties  of  several species of  amphipods
provided information valuable for conducting
interspecies extrapolations, an assessment
of  the  statistical   performance  of  the
amphipod mortality test provided a point of
departure   for   determining  ecologically
relevant  reponse  from  a  population
perspective, a study of the relative sensitivity
of   freshwater   invertebrates   to    10
contaminants helped validate current toxicity
test methods, and an examination of the
effects of storage duration and temperature
on the acute toxicity of whole sediments led
to recommendations  for sediment storage
conditions.

In addition  to the above-mentioned acute
toxicity  tests,  we also  developed   and
standardized several  chronic toxicity  tests
during FY95-96. A method for evaluating the
chronic toxicity of contaminated freshwater
sediments was  developed which uses an
aquatic midge to assess reproductive effects
in invertebrates,  and  a bioassay to detect
potential toxic and teratogenic effects caused
by contaminated estuarine sediments was
designed that uses grass shrimp embryos as
the test species. The latter assay has shown
exceptional performance in tests of individual
contaminants as  well as whole  sediment
samples and pore waters.  Currently, we are
using  the  assay at  a Superfund  site in
Charleston Harbor, SC (in collaboration with
the  National Oceanic  and  Atmospheric
Administration, or NOAA) to assess possible
reproductive and  developmental effects.
Initial results indicate delayed spawning at
impacted sites, but no  terata have  been
observed.

Most species recommended for sediment
toxicity testing are not indigenous to the area
of concern, and their  relevance in sediment
hazard assessments for that  region is
unknown.    For  this  reason,  we  are
conducting  research  to  examine  the
sensitivity   of    selected   species  to
contaminants  found  in  Gulf of  Mexico
sediments. The objective of the project is to
determine whether indigenous species are
more  appropriate  for hazard  assessment
than  conventional test species,  and  thus
better predictors  of  impact.    In  FY95,
scientists in our Gulf Ecology Division (GED)
began evaluating a variety of test species
common to southeastern estuaries, including
amphipods, bivalves, and  aquatic plants.
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996
                                      8

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These species are being examined for their
reponse to whole sediment and pore water
exposures.  During  FY96 we reported that
the most  sensitive  test organism was an
indigenous  species of  mollusc;  it  was
considerably more sensitive than standard
test species (such as the amphipod) used in
sediment toxicity tests. We also successfully
cultured aquatic plants in the laboratory, and
in F97 we intend to  expose these  plants to
contaminated  sediments   to  determine
relative  species  sensitivities  and  toxic
endpoints.  Our objective in future years is to
continue developing  toxicity test methods for
organisms  not  represented  by  existing
methodology. These studies will focus on
more subtle  responses, such as  behavior
(e.g., burrowing activity, avoidance).

ECOLOGICAL HAZARD CHARACTERIZA-
TION

Field studies. This  research is designed to
assess the  overall condition  of  benthic
ecosystems by correlating data on sediment
chemistry,    toxicity,    and   community
composition. During FY96, scientists in our
Western    Ecology    Division   (WED)
investigated the ecological effects of DDT
contamination   on  the    macrobenthic
community in sediments at a Superfiind site
in Richmond Harbor, CA.  Our  chemical
measurements  revealed  that  sediment
concentrations of DDT were as high as
100,000 times background.  Our analyses of
community   composition   showed   that
taxonomic composition and density changed
along the DDT pollution gradient; specifically,
as DDT increased, amphipod density and the
Infaunal  Index  decreased.   The  dose-
response relationships  determined  in this
study have permitted accurate predictions of
DDT effects on macrobenthic communities.

Comprehensive assessments of sediment
quality are underway in GED.  These multi-
year studies are designed to determine the
sources, causes, and extent of toxic impacts
in   Gulf   sediments   using  a   hazard
assessment approach. Our analytical tools
include acute and chronic sediment toxicity
tests, field biomonitoring, chemical analyses
of sediments (including  measurements of
heavy metals, pesticides, and  organics),
determinations of biocontaminant levels in
sediment species  (tissue  analyses), and
assessments   of  benthic   community
dynamics.   Areas  of study  include  the
northern  Gulf of Mexico  region and  South
Florida.   Sampling stations were set up at
various locations beginning in FY94. Water,
sediment, and biota samples were collected
and  analyzed  in  FY95,  and  from this
information  we  were  able  to produce
baseline information on contaminant levels.
Our data revealed considerable differences
in the  concentrations  of contaminants in
sediments collected from the same location
at different times, indicating that the ability of
sediment assessment guidelines to predict
toxic effects was dependent on when the
samples were collected. In contrast, results
from the acute and chronic toxicity tests were
consistent over time, suggesting that toxicity
tests  may  be  a more  reliable index  of
sediment quality than chemical analyses. In
FY97 and  beyond, we  will  evaluate  the
efficacy  of selected sediment  criteria  to
protect benthic organisms at the population,
community, and ecosystem levels. Sensitive
species will be identified to determine their
effectiveness as bioindicators  of  habitat
quality, and a protocol for conducting hazard
assessments of contaminated sediments will
be developed.

Effects    on   microbia!   community
dynamics.  GED is studying the structure/
function  of communities  of  sedimentary
microorganisms   in an   effort  to  better
understand  the  effects  of   sediment
contaminants on microbial ecology. During
FY96,  we  used  molecular techniques to
resolve the community structure of bacteria
in a sediment contaminated with mercury.
We  showed that the  bacteria could  be
stratified into distinct populations according
to sediment depth. Our results suggest that
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996

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microorganisms  form  distinct communities
whose localization in the sediment may be
influenced  by contaminants.   A specific
group of bacteria was identified in the layers
of sediment exhibiting high rates of mercury
methylation; it is likely that these bacteria are
responsible  for   the  transformation  of
mercury.   During  FY97, we  will  begin to
examine   the   effects   of   sediment
contaminants  on  bacterial  communities
associated with the rhizosphere (root zone)
of submerged aquatic plants. This research
will help determine  whether contaminants
alter  the   microbial  ecology   of   the
rhizosphere, leading to plant stress and die-
off.  In future years, we  plan  to  identify
sensitive communities that could serve as
indicators of pollutant stress.
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996
                                    10

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                   CONTAMINATED SEDIMENTS
  DETERMINANTS OF EFFECT RESEARCH PROGRAM
 NHEERL defines determinants of effect
 research  as  research  to  identify  and
 describe the  basis for the health and/or
 environmental effects caused by exposure to
 environmental   stressors  or  chemical
 contaminants.
ISSUE
 What factors affect the toxicity of sediment
 contaminants, and how can we account for
 these factors when establishing safe levels
 of contaminants in sediments?
 The toxicity of sediments may be influenced
 by  any number  of  physical  or chemical
 determinants (such as pH) and by biological
 processes    (such    as    contaminant
 bioaccumulation).  These factors affect risk
 and must be  considered  when defining
 acceptable thresholds for sediment contami-
 nation. In many cases, the mechanisms by
 which these factors regulate toxicity are not
 clearly understood. Such information would
 help explain the basis for toxicity, leading to
 more  scientifically  defensible  sediment
 quality criteria, and would  assist efforts to
 improve models that estimate toxicity.
 The primary objective of this  research
 program is to develop methods and models
 that advance  our understanding of  the
 environmental    processes    influencing
 sediment toxicity. Such information confers
 biological plausibility to toxicity estimates and
 places  us  in a  better position to  define
 realistic   benchmarks   for    sediment
 contamination. Two research areas form our
 program.  MECHANISTIC RESEARCH is
 being conducted to examine the factors that
 control  or  modify  sediment  toxicity.   A
 significant  research effort is  devoted  to
 analyzing   the  biological  availability  of
sediment contaminants.   A mechanistic
understanding of bioavailability is essential to
accurately predict contaminant uptake~and
hence    toxicity--from    concentrations
measured in the environment. We also are
developing MODELS that will enable us to
predict the uptake and bioaccumulation of
sediment contaminants.  These mathema-
tical models enhance our ability to predict
toxicity, which, in turn, strengthens ecological
risk assessments.
MECHANISTIC RESEARCH

Bioavailability.     Research   on   the
bioavailability of sediment contaminants has
been   a  fundamental   component  of
NHEERL's CONTAMINATED SEDIMENTS
RESEARCH  PROGRAM  for  10 years.
During  that  period,  we have  produced
numerous scientific papers, technical support
documents, and  Sediment Quality Criteria
(SQC)  documents that define acceptable
thresholds for sediment contaminants.  Of
particular concern are metals and organic
compounds.

METALS:     NHEERL   scientists   are
responsible for major advances over the last
five years in the  use of the equilibrium
partitioning (EqP) approach for assessing the
ecological risk of metals in sediments.  Our
studies have shown conclusively that acid
volatile sulfide (AVS) is a controlling factor in
the partitioning of metals  between sediment
and water: AVS binds  to metals,  effectively
sequestering  them   so  they  are  not
bioavailable. Consequently, AVS concentra-
tions can be used to predict bioavailability
and toxicity.   In 1994, we presented the
technical basis for using AVS in predictions
of bioavailability to the EPA Science Advisory
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996
                                  11

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Board. Their review concluded that the EqP
approach using AVS analyses was a more
reliable and predictable tool than any other
available.

Since 1994, we have continued to define the
state-of-the-science in the area of the EqP
approach.  In the December 1996 issue of
Environmental Toxicology and Chemistry, a
series of  16 research reports was published
by investigators  in AED,  WED,  and MED.
These papers describe  critical research
supporting the concept of an EqP approach,
and they provide the scientific basis for using
EqP to  predict  metal bioavailability and
toxicity in  sediments.   Results from this
important area of research, involving both
laboratory and field studies, are being used
to assist in efforts to derive sediment quality
criteria (SQC)  for metals.  During FY97, in
conjunction with the Office of Water, we
intend to  publish  SQC documents  for
cadmium, copper, lead,  nickel, and zinc.
Plans  for   future  research  include  an
evaluation  of  seasonal  variations in AVS
ratios  and  their relationship  to trends in
sediment toxicity.

ORGANICS: NHEERL research has led to
advances in the use of EqP for setting sound
SQC for non-ionic organic contaminants
found  in sediments.   Our studies have
resulted in  improved methods for predicting
the   biological   availability  of  organic
compounds, such as PCBs and pesticides, in
sediments  and sediment pore waters.  For
example, in FY95, we developed an EqP
technique  that  involves the isolation  of
colloidal and freely dissolved PCBs in marine
sediments,  and  in  FY96,  we  used  this
methodology to determine the distribution of
PCBs in New Bedford Harbor  sediments.
This project demonstrated the importance of
the   colloidal   phase  when  evaluating
contaminated marine sediments.

Our   research   has   resulted  in   the
development  of  SQC  documents   for
numerous  organic contaminants.  In FY96,
we helped produce SQC documents for the
pesticides dieldrin and endrin and mixtures
of three PAHs: acenaphthene,  phenan-
threne, and fluoranthene.

Biological   processes  involved   in  the
bioavailability of organic contaminants, such
as  uptake and assimilation  by  benthic
organisms, are  also  important considera-
tions in assessments of ecological risk.  In
FY95 we found that infaunal and epibenthic
organisms accumulate similar concentrations
of non-ionic organic chemicals regardless of
feeding habit. This has important implica-
tions because it demonstrates that  SQC
must  be  derived to protect all  benthic
species, not just infaunal deposit-feeding
species.  We also observed that metabolic
alterations of PCBs by marine organisms
resulted in changes in the pattern  and
abundance  of   PCB  congeners  in  the
organisms (as  well  as in  the water and
sediment).    During   FY96,  we  found
similarities in the uptake of nonplanar and
coplanar PCB congeners in blue  mussels.
This finding  opens  up the  possibility  of
substituting the  more easily measured and
less costly nonplanar PCB analytes for the
more difficult and costly coplanar compounds
in PCB risk assessments.  In future years,
we  plan  to address  contaminant  bio-
availability by constructing bacterial indicator
strains  that  respond  to  pollutants   by
producing light that can be readily measured.

Water quality  characteristics.   During
FY95,  we conducted extensive research to
determine the influence of water chemistry
(pH and hardness) on the acute toxicity of
ammonia, a common sediment contaminant.
The   studies  were   conducted   using
freshwater benthic  invertebrates  (amphi-
pods).  Our results showed that, unlike
responses  previously  reported  for  other
organisms,  ammonia  toxicity  was  not
affected by pH in soft water.

In FY96, we demonstrated that humic acids
and  total  organic  carbon  affect  pH-
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996
                                   12

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dependent sorption  of contaminants  on
estuarine sediments, and these interactions,
in turn, affect toxicity.

Ultraviolet (UV) radiation.  During FY95-96,
scientists in MED and WED showed that the
toxicity of some sediment pollutants (specifi-
cally, PAHs)  increased in the presence of
sunlight. Toxicity was demonstrated in both
freshwater and marine invertebrates, and it
increased in direct proportion to the intensity
and energy of the light.  Our data suggest
that sediment toxicity tests conducted on
chemicals prone  to photoactivation may
underestimate potential  toxicity  by several
orders  of  magnitude.    Consequently,
predictions   of  sediment  toxicity   (and
subsequent estimates of risk) should take
into consideration  the enhanced effects of
phytotoxic contaminants.   These findings
have verified predictions made by our QSAR
models.   During  FY96,  we  expanded
research in this area by devising a technique
whereby phototoxic chemicals in sediments
could be identified through a combination of
chemical fractionation and QSAR modeling
(previously  discussed  under  TOXICITY
ASSESSMENT METHODS).

MODELS

During   FY95-96,   scientists  in  WED
developed  a  bioaccumulation  model  of
pollutant   uptake  for  neutral   organic
compounds. This model, which is applicable
to organisms that ingest sediment particles,
differs  from the  established  method for
calculating Biota-Sediment Bioaccumulation
Factor (BSAF) values in that it estimates the
internal dose to the organism and assumes
that equilibrium between tissue residues and
gut contents is the most important factor
governing bioaccumulation. We applied this
new model in predictions  of  PCB  bio-
accumulation and  found that our BSAFs
were less variable across sediment types
than those predicted by the standard model,
and they  were in better agreement  with
established BSAF maximal values.

In FY95,  we  developed and tested an
innovative method for predicting the toxicity
of a mixture of PAHs in sediments.  Called
the "£PAH Model," it predicts the probability
of survivorship of sensitive species exposed
to mixtures of PAHs. Comparisons of model
predictions with observed toxicity in field-
collected   PAH-contaminated  sediments
showed an 86% correspondence in results.
This research enhances our ability to predict
interactions  of multiple  contaminants  in
sediment, leading to more realistic estimates
of risk.
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996
                                    13

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                       FY95-96 BIBLIOGRAPHY
Ankley GT (1996) Laboratory versus field measurement endpoints: a contaminated sediment
    perspective, in: Risk Assessment Considerations for Contaminated Sediments, SETAC
    Pellston Series.

Ankley GT (1996) Evaluation of metal/acid volatile sulfide relationships in the prediction of metal
    bioaccumulation by benthic macroinvertebrates, Environ. Toxicol. Chem. 15:2138-2146.

Ankley GT, SA Collyard, PD Monson, PA Kosian (1994) Influence of ultraviolet light on the toxicity
    of sediments contaminated with polycyclic aromatic hydrocarbons, Environ. Toxicol. Chem.
    13:1791-1796.

Ankley GT, MK Schubauer-Berigan (1994) Comparison of the techniques for the isolation of pore
    water for sediment toxicity testing, Arch. Environ. Contamin. 27:507-512.

Ankley GT, MK Schubauer-Berigan (1995) Background and overview of current sediment toxicity
    identification evaluation procedures, J. Aquat. Ecosystem Health 4:133-149.

Ankley GT, MK Schubauer-Berigan, P Monson (1995) Influence of pH and hardness on toxicity of
    ammonia to the amphipod Hyalella azteca, Can. J. Fish Aqat. Sci. 52:2078-2083.

Ankley GT, DM  DiToro, DJ Hansen, WJ Berry (1996) Assessing the ecological risk of metals in
    sediments (Editorial), Environ. Toxicol.  Chem. 15:2053-2055.

Ankley GT, DM DiToro, DJ Hansen, WJ Berry (1996) Technical basis and proposal for deriving
    sediment quality criteria for metals, Environ. Toxicol. Chem. 15:2056-2066.

Ankley GT, RJ Erickson, GL Phipps, VR Marttson, PA Kosian, BR Sheedy, J Cox (1995) Effects
    of light intensity on the photoxicity of fluoranthene to a benthic macroinvertebrate, Environ. Sci.
    Technol. 29:2828-2833.

Ankley GT, K Liber, DJ Call, T Markee, T Canfield, CG Ingersoll,  LA Carnahan (1996) A field
    investigation of the relationship between zinc and acid volatile sulfide concentrations in
    freshwater sediments, J. Aquatic Ecosyst. Health, in press.

Ankley GT,  OG Mekenyan, P Kosian, DR Mount, P Monson, DJ Call (1996)  Identification of
    phototoxic  polycyclic hydrocarbons in sediments through sample fractionation  and QSAR
    analysis, SAP QSAR Environ. Res., in press.

Ankley GT, MK Schubauer-Berigan, J Dierkes (1996) Application of toxicity identification evaluation
    techniques to pore water from Buffalo River sediments, J. Gt. Lakes Res., in press.

Benoit DA, PK Sibley, PS Juenemann, GT Ankley (1996) Chironomus tentans life cycle test: design
    and evaluation for use in assessing toxicity of contaminated sediments, Environ. Toxicol.
    Chem., in press.

Bergen BJ, WG Nelson, RJ Pruell (1996) Comparison of nonplanar and coplanar PCB congener
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996
14

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     partitioning in seawater and bioaccumulation in blue mussels (Mytilus edulis), Environ. Toxicol.
     C/7em.15:1517-1523.

 Berry WJ, DJ Hansen, JD Mahony, DL Robson, DM DiToro, BP Shipley, B Rogers, JM Corbin, WS
     Boothman (1996) Predicting the toxicity of metal-spiked laboratory sediments using acid-
     volatile sulfide and interstitial water normalizations, Environ. Toxicol. Chem. 15:2067-2079.

 Boese BL, M Winsor,  H Lee, S Echols, J  Pelletier, R Randall (1995) PCB  congeners and
     hexachlorobenzene biota-sediment bioaccumulation factors for Macoman asuta exposed to
     sediments with different total organic carbon contents, Environ. Toxicol. Chem., 14:303-310.

 Boese BL, JO Lamberson, RC Swartz, RJ Ozretich (1996) Photoinduced toxicity of fluoranthene
     to seven marine benthic crustaceans, Arch. Environ. Contamin.  Toxicol., in  press.

 Boese  BL, H Lee, DT Specht,  J Pelletier,  R  Randall  (1996)  Evaluation  of  PCB  and
     hexachlorobenzene biota-sediment bioaccumulation factors based on ingested sediment in
     a deposit-feeding clam, Environ. Toxicol. Chem., 15:1584-1589.

 Burgess RM, GE Morrison (1995) Clams as a species for sublethal sediment toxicity studies,
     Quintessence, 1:48.

 Burgess RM, WA Brown, RA McKinney, JG Quinn (1996) Enrichment of marine sediment colloids
     with polychlorinated biphenyls (PCBs): Effects of PCB solubility and chlorination, Environ. Sci.
     Technol, 30:1923-1932.

 Burgess RM, KT Ho, GE Morrison, G Champman, PL Denton (1996) Marine Toxicity Identification
     Evaluation (TIE) Phase I Guidance Document, EPA/600/R-96/054.

 Burkhard LP, BR Sheedy (1995) Evaluation of screening procedures for bioconcentratable organic
     chemicals in effluents and sediments, Environ.  Toxicol. Chem., 14:697-711.

 Burton G, TJ Norberg-King, CG Ingersoll, GT Ankley,  PV Winger, J Kubitz, J Lazorchak, ME Smith,
     E Greer, F Dwyer, DJ Call, KE Day, P Kennedy, M Stinson (1996) Interlaboratory study of
     precision: Hyalella azteca, Chironomus tentans freshwater sediment toxicity assays, Environ.
     Toxicol. Chem.,  15:1335-1343.

 Cotter AM, P Kosian, RA Hoke, GT Ankley (1996) Extraction and analysis of low levels of DDT,
     DDE and ODD in small volumes of sediment pore water, Chemosphere, 33:1341-1354.

 Devereaux R, MR Winfrey, J Winfrey, DA Stahl (1996) Depth profile of sulfate-reducing bacterial
     ribosomal RNA and mercury methylation in an estuarine sediment, Microbiol. Ecol., 20:23-31.

 DeWitt T,  R Swartz, D Hansen, D McGovern, W Berry (1996) Bioavailability and chronic toxicity of
     cadmium in sediment to the estuarine amphipod Leptocheirus plumulosus, Environ. Toxicol.
     ChemA 5:2102-2112.

 DiToro D, CS Zarba, DJ Hansen, WJ Berry, RC Swartz, CE Cowan, SP Pavlou, HE Allen, NA
     Thomas,  PR Paquin (1995)  Equilibrium partitioning in setting sediment quality criteria,
     Quintessence-Excellence in Environ. Contamin. Toxicol. 1:45.
NHEERL CONTAMINA TED SEDIMENTS ANNUAL REPORT 1996                            15

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Ferraro SP, FA Cole (1996) Effects of £DDT sediment contamination on macrobenthic community
    structure and composition in San Francisco Bay, Mar. Biol., in press.

Gundersen J. WG Maclntyre, RC Hale (1996) pH-dependent sorption of chlorinated guaicols on
    estuarine sediments: the effects of humic acids and TOC, Environ. Sci. Technol., in press.

Hansen DJ, WJ Berry, J Mahony, DM DiToro, GT Ankley, ZQ Ma (1996) Predicting the toxicity of
    metal-contaminated field sediments using interstitial concentrations of metals and acid-volatile
    sulfide normalizations, Environ. Toxicol. Chem. 15:2080-2094.

Hansen DJ, JD Mahony, WJ Berry, S Benyi, J Corbin, SD Pratt, DM DiToro, MB Able (1996)
    Chronic effects of cadmium in sediments on colonization by benthic marine organisms: an
    evaluation of the role of interstitial cadmium and acid-volatile sulfide in biological availability,
    Environ. Toxicol. Chem. 15:2126-2137.

Hassan S, AW Garrison, HE Allen, D DiToro, GT Ankley (1996) Estimation of partition coefficients
    of five trace metals in  sandy sediments and application to the sediment quality criteria,
    Environ. Toxicol. Chem. 15:2198-2208.

Ho K, R McKiney, A Kuhn, M Pelletier, R Burgess (1997) Identification of acute toxicity in New
    Bedford Harbor sediments, Environ. Toxicol. Chem.,  16:551-558.

Hoke RA, GT Ankley, JF Peters (1995) Use of freshwater sediment quality database in evaluation
    of sediment  quality criteria based  on   equilibrium partitioning  and screening  level
    concentrations, Environ. Toxicol. Chem. 14:451-459.

Hoke  RA, PA Kosian, GT Ankley, AM Cotter, FM VanderMeiden.GL Phipps, EJ Durhan (1995)
    Check studies with Hyalella azteca and Chironomus tentans in support of the development
    of a sediment quality criterion for dieldrin, Environ. Toxicol. Chem. 14:435-450.

Hoke  RA, GT  Ankley, PA Kosian, AM Cotter, FM VanderMeiden.GL Phipps, CW  West, J Cox
    (1996) Equilibrium partitioning as the basis for an integrated laboratory and field assessment
    of the impacts of DDT, DDE, and DDD in sediments, Ecotoxicol., in press.

Ingersoll CG, GT Ankley, DA Benoit, EL Brunson, GA Burton, FJ Dwyer, RA Hoke, PF Landrum,
    TK Norberg-King, PV Winger (1995) Toxicity and bioaccumulation of sediment-associated
    contaminants using freshwater invertebrates: a review of methods and applications, Environ.
    Toxicol. Chem. 14:1885-1894.

Ingersoll CG, GT Ankley, R Baudo, A Burton, W Lick, S Louma, D MacDonald, T Reynoldson, K
    Solomon, R Swartz, W  Warren-Hicks (1996) Methodological uncertainty in conducting risk
    assessment  with contaminated sediments,  in: Risk  Assessment  Considerations  for
    Contaminated Sediments, SETAC Pellston Series, Chapter 17.

Kosian PA, RA Hoke, GT Ankley, FM VanderMeiden (1995) Determination of dieldrin binding to
    dissolved organic material in sediment pore water using a reverse phase technique, Environ.
    Toxicol. Chem. 14:445-450.

Lake  JL,  R McKinney, CA  Lake, FA Osterman, J Heltshe (1995) Comparison of patterns of
    polychiorinated biphenyl congeners in water, sediment, and indigenous organisms from New
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996
16

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     Bedford Harbor, MA, Arch. Environ. Contamin. Toxicol. 29:207-220.

 Leonard EN, VR Mattson, GT Ankley (1995) Horizon-specific oxidation of acid volatile sulfide in
     relation to the toxicity of cadmium spiked into a freshwater sediment, Arch. Environ. Contamin.
     Toxicol. 28:78-84.

 Leonard EN, GT Ankley, RA Hoke (1996) Evaluation of metals in marine and freshwater surficial
     sediments from the Environmental Monitoring and Assessment Program relative to proposed
     sediment quality criteria for metals, Environ. Toxicol. Chem. 15:2221-2232.

 Leonard EN, AM Cotter, GT Ankley (1996) Modified diffusion method for analysis of acid volatile
     sulfides and simulaneously extracted metals in freshwater sediment, Environ. Toxicol. Chem.
     15:1479-1481.

 Liber K, DJ Call, T Markee, KL Schmude, MD Balcer, FW Whiteman, GT Ankley (1996) Effects of
     acid volatile sulfide on zinc bioavailability and toxicity to benthic macroinvertebrates:  a spiked
     field experiment, Environ. Toxicol. Chem. 15:2113-2125.

 Liber K, DJ Call, TD Dawson, FW Whiteman, TM Dillon (1996) Effects of Chirononus tentans larval
     growth retardation on adult emergence and ovipositing success: implications for interpreting
     freshwater sediment bioassays, Hydrobiologia 323:155-167.

 Masunaga S,  S Susarla, JL Gunderson, Y Yonezawa (1996) Pathway and rate of chlorophenol
     transformation in anaerobic sediment, Environ. Sci. Technol. 30:1253-1260.

 Mekeyan OG,  GT Sankley, GD Veith,  DJ Call (1995) QSARs for photoinduced toxicity of  aromatic
     hydrocarbons, SAR QSAR Environ. Res. 4:139-145.

 Monson PD, GT Ankley, PA Kosian (1995) Phytotoxic response of Lumbriculus  variegatus to
     sediments contaminated by polycyclic aromatic hydrocarbons, Environ. Toxicol. Chem.
     14:891-894.

 Mount DR (1996) Use of metal chelating agents to differentiate among sources of acute aquatic
     toxicity, Environ. Toxicol.  Chem., in press.

 Oremland, RS, LG  Miller, P Dowdle, T  Connell,  T Barkay (1995) Methylmercury oxidative
     degradation potentials in contaminated and pristine sediments of the Carson River,  Nevada,
     Appl. Environ. Microbiol. 61 -.2745-2753.

 Pesch CE, DJ Hansen, WS Boothman, WJ Berry, JD Mahony (1995) The role of acid- volatile
     sulfide in  determining bioavailability of cadmium and nickel from contaminated sediments:
     experiments with Neanthes arenaceodentata, Environ. Toxicol. Chem. 14:129-141.

 Peterson GS,  GT Ankley, EN  Leonard (1996) Effect of bioturbation on metal-sulfide oxidation in
     surficial freshwater sediments, Environ. Toxicol. Chem. 15:1147-2155.

 Phipps GL, VR Mattson, GT Ankley  (1995)  Relative sensitivity of three freshwater benthic
     invertebrates to ten contaminants, Arch. Environ.  Contam. ToxicoL 28:281-286.

 SchlekatCE, KJ Scott, RC Swartz, B Albrecht, L Antrim, K Doe, S Douglas, J Ferretti, DJ Hansen,


NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996                            1 7

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    DW Moore, C Mueller, A Tang (1995) Intel-laboratory comparison of a 10-day sediment toxicity
    test method using Ampelisca abdita, Eohaustorius estuarius and Leptocheirus plumulosus,
    Environ. Toxicol. Chem. 14:2163-2174.

Schubauer-Berigan MK, PD Monson, CW West, GT Ankley (1995) Influence of pH on the toxicity
    of ammonia to Chironomus tentans and Lumbriculus variegatus,  Environ. Toxicol. Chem.
    14:713-718.

Schuytema GS, AV Nebeker, MA Cairns (1995) A comparison of recirculating, static, and elutrite
    aquatic sediment bioassay procedures, Bull. Environ. Contamin. Toxicol. 56:742-749.

Schuytema GS, AV  Nebeker, TW  Stutzman (1996) Salinity tolerance of Daphnia magna and
    potential use for estuarine sediment toxicity tests, Arch. Environ. Contamin. Toxicol., in press.

Sibley PK, GT Ankley, AM Cotter,  EN Leonard (1996) Predicting chronic toxicity of sediments
    spiked with zinc: and evaluation of the acid volatile sulfide model using a life cycle test with
    the midge (Chironomus tentans), Environ. Toxicol. Chem., in press.

Sibley PK, DA Benoit, GT Ankley (1996) The significance of growth in Chironomus tentans
    sediment toxicity tests: relationship to reproduction  and demographic endpoints, Environ.
    Toxicol. Chem., in press.

Solomon K, G  Ankley, R Bauso, A Burton, C Ingersoll, W Lick,  S Luoma, D MacDonald, T
    Reynoldson, R  Swartz, W Warren-Hicks (1996) Methodological uncertainty in  conducting
    sediment  ecolgical  risk  assessments with  contaminated sediments, in:  Ecological  Risk
    Assessments of Contaminated Sediments, GT Biddinger, T Dillon, C Ingersoll (Eds.), SETAC
    Special Publication Series, in press.

Swartz R, D Schults, R Ozretich, J Lamberson, F Cole, T DeWitt, M Redmond, S Ferraro (1995)
    £PAH: A model to predict the toxicity of polynuclear aromatic hydrocarbon mixtures in field-
    collected sediments, Environ. Toxicol. Chem. 14:1977-1987.

Swartz RC, DM DiToro (1996) Sediments as complex mixtures: an overview of methods to assess
    ecotoxicological significance, in: Ecological Hisk Assessments of Contaminated Sediments,
    GT Biddinger, T Dillon, C Ingersoll (Eds.), SETAC Special Publication Series, in press.

Thursby  G, Heltshe, JK Scott (1996) A statistical performance assessment of the 10-day solid-
    phase toxicity test using the marine amphipod Ampelisca abdita, Environ. Toxicol. Chem., in
    press.

Tracey G, D Hansen, (1996) Use of biota-sediment accumulation factors to assess similarity of
    non-ionic organic chemical exposure to benthically-coupled organisms of differing trophic
    mode, Arch. Environ. Contamin. Toxicol. 30:467-475.

Veith GD, OG Mekenyan, GT Ankley, DJ Call (1995) A QSAR analysis of substituent effects on the
    photoinduced acute toxicity of PAHs, Chemosphere 30:2129-2142.

Weber DE, CL McKenney, MA MacGregor, DM Celestial (1996) Use of artificial sediments in a
    comparative toxicity study with larvae and postlarvae of  the grass shrimp, Palaemonetes
    pugio, Environ. Pollut. 93:129-133.
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996
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Whiteman FW, GT Ankley, DJ Call, PM Cook, D Rau (1996) Evaluation of interstitial water and a
    route of exposure for ammonia in sediment tests with benthic macroinvertebrates, Environ.
    Toxicol. Chem. 15:794-801.
NHEERL CONTAMINATED SEDIMENTS ANNUAL REPORT 1996
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 National Health and Environmental Effects Research Laboratory
    CRITERIA AIR POLLUTANTS:
      TROPOSPHERIC OZONE

HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH
            ANNUAL REPORT

              JULY, 1997

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                         CONTENTS
Introduction	3

Summary of the Tropospheric Ozone
     Health and Environmental Effects Research Program  	4

FY95-96 Highlights	6

Tropospheric Ozone Epidemiology Research Program	8
           Acute and Chronic Effects
           Sensitive Subpopulations
                Children
                Asthmatics
                Other Variables

Tropospheric Ozone Determinants of Effect Research Program  	11
           Dosimetry
           Mechanisms of Toxicity
           Risk Models

Tropospheric Ozone Ecological Effects Research Program	15
           Effects of Ozone on Trees
           Effects of Ozone on Forests
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996

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                       INTRODUCTION
BTOROSE
The purpose of this report is to communicate results from the Tropospheric  Ozone
Research Program of EPA's National Health and Environmental Effects Research
Laboratory (NHEERL).
The report contains
           a summary of the  NHEERL Tropospheric  Ozone Research Program,
           including an explanation of its regulatory and programmatic context, the
           overall program goal,  the rationale for the  program, and the research
           strategy

           a section which highlights recent key findings (FY95-96 Program Highlights)

           a more detailed description of the NHEERL Tropospheric Ozone Research
           Program,  by  program   area,   including  a  summary  of   research
           accomplishments and anticipated progress for the near future
COMMENTS WELGOMEli    "••.-.  •-,-;;?;.-     ; -t:^- W::f;\    ,  •

The format of this report is still evolving, and we welcome feedback.  Readers with
comments or requests for further information are encouraged to contact:

John Vandenberg, Assistant Laboratory Director
National Health and Environmental Effects Research Laboratory (MD-51A)
U.S. EPA
Research Triangle Park, N.C. 27711

Phone: (919) 541-4527 or FAX: (919) 541-0642
E-mail: vandenberg.john@epamail.epa.gov
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996

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                  TROPOSPHERIC  OZONE
         RESEARCH PROGRAM SUMMARY
EPA is required by the Clean Air Act (CAA) to
prescribe  National  Ambient  Air  Quality
Standards (NAAQS) that protect public health
and the environment from the harmful effects
of tropospheric ozone (O3).  The standards
must be reviewed  on a regular basis and
revised as appropriate. In July 1997, the EPA
promulgated new NAAQS for ozone in which
the  level of the standard was reduced from
0.12 ppm to 0.08 ppm, and the averaging time
increased from 1 hour to 8 hours.  Scientific
support for revision of the standards and their
implementation is provided by the Agency's
Office of Research and Development (ORD).
NHEERL,  a  research  arm   of  ORD,
investigates the effects of O3 on human health
and ecosystem vitality.  The objective of
NHEERL's research is to reduce uncertainties
in  ozone  risk  assessment,   thereby
strengthening  the  scientific foundation  for
future O3 NAAQS decisions and attainment
strategies. NHEERL  studies were critical to
the  decision to  establish  a new primary
standard (human health based) and to  the
decision to retain with confidence the existent
secondary standard  (welfare based). This
document summarizes NHEERL's research
program and highlights some of its recent
accomplishments.
To develop scientificaly sound and defensible
data on the health and ecological effects of O3
for use in criteria development.

RpfONALE
Ozone,  a major component of smog, is a
reactive gas that can irritate the lungs and
damage   agricultural  crops  and  forest
ecosystems. While the acute health effects of
O3  are fairly  well documented  (e.g., its
tendency to exacerbate asthma), very little is
known  about  the  effects  of chronic  O3
exposure on humans or the role of ozone in
respiratory disease.   From an ecological
standpoint,   there   remain   unresolved
questions regarding ozone's impact on tree
growth  and  its contribution  to  reported
declines in forest productivity.  For EPA to
promulgate  a  standard  that  adequately
protects  both  human  health  and  the
environment, critical  areas  of  uncertainty
surrounding O3 toxicity must be resolved.


to ensure that the Agency is equipped with
scientific and technical data relevant to the
formulation of sound environmental policy,
ORD operates a research program founded
on principles of risk assessment. In the area
of  health  effects,  the  risk  assessment
paradigm  of  the  National  Academy  of
Sciences  (NAS)  provides  the  research
context.   This  paradigm  consists  of 4
fundamental  steps (hazard identification,
dose-response   assessment,    exposure
assessment, and risk characterization) that
support risk management decisions. In  the
area of ecological  effects, ORD's research
program follows the framework for ecological
risk assessment developed by EPA in 1992,
consisting of problem formulation, analysis
(characterization of exposure and effects),
and risk  characterization.    NHEERL's
research in tropospheric ozone  emphasizes
two areas of risk assessment:

    +  hazard  identification (or  problem
formulation), in  which  we  are developing
methods that  enable  us  to  detect and
characterize human  health and ecological
hazards associated with O3, and
    +  the assessment of dose-response, in
which  we are studying the  events that  link
exposure and effects; these data form the
 NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996

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basis for predictive models used to quantify
risk.

In the area of health effects, the objective of
our program is to elucidate the role of O3 in
the  development   and  exacerbation   of
respiratory disease.    Two   fundamental
questions need to  be  resolved:  1)  Do
exposures to O3 lead to an Increase in acute
respiratory illness in sensitive populations?
and 2) Do long-term exposures lead to the
development of chronic lung disease, such as
asthma or chronic obstructive lung disease?
To   answer  these   questions,   we   are
performing epidemiology studies and clinical
research  in  humans,  and   lexicological
assessments of O3 morbidity and mortality in
experimental  animals.   In   the  area  of
EPIDEMIOLOGY, we are characterizing the
acute and chronic effects associated  with
ambient exposures to O3 in urban environ-
ments, and we are identifying susceptible
subpopulations  who  may be  especially
vulnerable to those effects.  We are studying
DETERMINANTS  OF  EFFECT  to better
understand the biological basis for injury and
disease.    Included  is research  on  O3
dosimetry and mechanisms of toxicity, the
results of which are used to help us develop
biologically plausible   models of  human
response to O3. In the area of ECOLOGICAL
EFFECTS, we are examining the effects of O3
on trees, including  direct effects on growth
and indirect effects below ground  (on the
roots  and soil).  These effects  are being
evaluated in the context of individual tree
characteristics, changing exposure dynamics,
and multiple stressors.  We are using these
experimental data to construct risk models
that can  estimate large-scale  forest effects
over time.
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996

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NHEERL TROPOSPHERIC OZONE RESEARCH
         FY95-96 PROGRAM HIGHLIGHTS
       In July 1997, the EPA promulgated new National Ambient Air Quality Standards
       for Tropospheric Ozone to protect public health and welfare. Our research results
       were included in the Air Quality Criteria Document for Ozone and Related
       Photochemical Oxidants, which we helped to develop. This document provided
       the scientific basis for decisions on the primary and secondary NAAQS for O3
       decisions with multi-billion dollar implications for public health and the economy.
 The goal of this research is to characterize the human health effects associated with O3 exposures
 and to identify subpopulations who may be especially susceptible to O3.

     *  Collaborating with epidemiologists from Harvard University and Mexico City, we
       demonstrated that exposure to O3 is associated with decrements in lung function
       in children. A similar response was demonstrated in controlled clinical studies at
       our exposure facility in Chapel Hill, NC, suggesting that data from clinical studies
       can be used to predict O3 response in children.

     *  We demonstrated for the first time that asthmatics are more susceptible to O3
       than healthy individuals.  Furthermore, we showed that asthmatics experience a
       different kind of inflammatory response (eosinophil-driven) than non-asthmatics
       (neutrophil-driven), indicating that there may be objective  ways to measure
       differences in O3 sensitivity between individuals.

     *•  In cooperation with researchers at Loma Linda  University,  we found that O3
       exposure is significantly related to the development of asthma  in males but not in
       females.

     *  We found that age plays an important role in O3 sensitivity: responsiveness to O3
       is greatest in young adults and decreases with age.
  DETERMINANTS OF EFFECT (pg 11)                         3
  The goal of this research is to describe the dose distribution of O3 in the lungs and the mode of
  action for O3 toxicity.

     *• We formulated an artificial lung lining fluid that simulates human lung lining fluid;
       it is being used to improve dosimetry estimates  and to elucidate  the toxic
       mechanisms of O3 damage.
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996

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    >  We showed that O3 induces lung injury and inflammation as early as one hour
       following exposure. Some inflammatory mediators are elevated at this early time
       point, while others do not appear until 18 hours later, suggesting mediators have
       different kinetics of appearance in the lung.

    >  We demonstrated that a common anti-inflammatory drug (Ibuprofen*) reduced
       decrements in lung function caused by O3 but failed to reduce underlying lung
       injury and inflammation. This is the first study to show that O3-induced changes
       in lung function can be dissociated from lung damage and inflammation in
       humans.

    *•  Our findings in O3-exposed mice and in human lung cells suggest that Vitamin C
       confers a protective anti-oxidant effect against 03. In other studies in Guinea pigs,
       we showed that a combination of Vitamin C deficiency,  O3 exposure, and
       infectious challenge results  in hypersusceptibility.

    *•  We developed a cross-species, biologically based dose-response model for ozone
       toxicity based on concentration x time  (CxT) relationships that assists criteria
       development.

    *   We  constructed a mathematical model capable of accurately predicting the
       proportion of individuals expected to respond adversely to O3 as a function of O3
       dose and concentration.
                                 ~fi1PP/M&N;i(rihH*iV-i;;*'
 	 			     asllsSESlk!:!^^
 The goal of this research is to quantify the effects of acute and chronic O3 exposures on trees and
 forests.

    >•  Ozone  was shown to have profound effects on the rhizosphere (the root/soil
       complex)  of tree seedlings, making them more susceptible  to nutrient  and
       moisture stress and affecting carbon movement and sequestration. These results
       suggest that O3 may magnify the effects of global climate change.

    *•  In a study of 11 important tree species, we demonstrated that ponderosa pine,
       black cherry, and aspen are the most sensitive species to O3, while douglas fir,
       Virginia pine, and red maple are relatively insensitive.

    *•  We showed that plants are more apt to take up O3 during the daylight hours and
       that there is a greater effect on plant growth during episodic exposures to higher
       concentrations of O3. These results were used in the NAAQS review process to
       support decisions on setting a secondary NAAQS for O3.

    * Mention of trade names is not an endorsement or recommendation for use.
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996                               7

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                TROPOSPHERIC  OZONE
    EPIDEMIOLOGY RESEARCH PROGRAM
NHEERL defines epidemiology research
as research to identify and describe the
human health  risks posed by exposure to
environmental contaminants.
ISSUE
Does ambient exposure to O3 lead to an
increase in acute and/or chronic respiratory
effects?   Do  individuals  differ in their
responsiveness to O3?
Millions of individuals are exposed to levels
of tropospheric O3 that exceed the national
ambient air quality standard.  Although a
number of health effects (e.g., respiratory
ailments, asthma) have been documented or
are suspected to occur as a result of these
exposures, very little is  known about the
magnitude of the public health burden or the
long-term effects of exposure to high levels
of O3. The issue is complicated by suscepti-
bility.  Some individuals may be at greater
risk than the general population  to the
adverse effects  of air  pollutants,  and
protecting these individuals is an  explicit
requirement of many environmental laws. It
is important to identify  and  investigate
sensitive subpopulations in order to support
decisions on the form  and level  of the
National Ambient Air Quality Standard for
tropospheric ozone.
PROGRAM DESCRIPTION

The purpose of the Epidemiology Research
Program is to characterize the public health
burden  associated   with  ambient  O3
exposure.  The program is supported in large
part by  the Ozone Epidemiology Initiative,
which is designed to relate acute and chronic
O3 exposures with adverse health outcomes,
such as asthma or chronic obstructive lung
disease.  The  Initiative  received first-year
funding in FY92, and many of the projects
are now coming to fruition.  Among our
investigative tools are questionnaires, health
databases, measurements of lung function,
and autopsy samples (lung tissue).  One
important facet  of  the  research is the
identification of ACUTE AND CHRONIC
HEALTH EFFECTS caused by O3. Another
focus of our research is the identification and
characterization  of  SENSITIVE  SUB-
POPULATIONS, such  as asthmatics and
children, who may be more susceptible than
the general population to the adverse effects
of O3. Our findings are leading to a more
confident evaluation of the need for revisions
to the NAAQS for O3.
ACUTE AND CHRONIC HEALTH EFFECTS

NHEERL, through  an FY92  Cooperative
Agreement with Loma Linda University, is
studying the respiratory health of California
Seventh  Day Adventists  (non-smokers)
exposed  to  ambient  O3.   The  project,
originally initiated  in  the 1960s,  is  a
prospective epidemiology study. Respiratory
health questionnaires  were administered
periodically over a 15-year period to a cohort
of approximately 3000 individuals.  Ozone
exposures were estimated from measure-
ments taken at various monitoring stations,
and these estimates were then related to the
respiratory health findings.  A subset of
participants underwent lung function testing
in FY93, which was intended to provide a
physiological basis for the findings from the
questionnaire. By FY96, we were able to
report that O3 was significantly related to the
development of asthma in males, but not in
females.  We have  postulated that  the
gender  difference may  be the  result of
different exposure patterns; for example,
males spend more time outdoors in summer
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996
                                   8

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than females. As a corollary to this study, we
are examining the relationship between long-
term exposure to O3 and mortality.  During
FY96, data  collection  on  mortality was
completed, and we will begin analysis of the
data in FY97.

In  collaboration with  the University  of
California  at  San  Francisco,  we  are
determining the  relationship between short-
term  O3  exposure  and  morbidity and
mortality. We are using the California Kaiser
Permanente  database,  which  offers  a
population of 10 million from which to draw
information.  A  pilot study was conducted
during FY92, in which data on medical care
(e.g., hospital admissions,  physician visits,
emergency room visits) for acute respiratory
conditions were obtained.  A final  assess-
ment of the pilot study was made in FY95,
and a three-year Cooperative Agreement
was executed to determine the relationship
between  O3 exposure  and medical care
usage.  During FY96, air pollution (O3) data
were collected and assembled. An  analysis
of mortality data is now underway. In FY97,
hospitalization  data will  be collected and
analyzed, and we will begin to plot trends in
hospital admissions over time, relating these
trends with O3 levels.

SENSITIVE SUBPOPULATIONS

Children.  Scientists in our Human Studies
Division   (HSD)  are   collaborating  with
investigators from  Harvard University and
researchers in Mexico City on a series of
studies designed to document the effects of
O3 on respiratory disease in children. Pilot
studies were conducted during FY91-92 in
Mexico City, an area of high O3 concentra-
tions.  Two schools were selected for the
study: one located  in an area of  high O3
levels and one in  an area of moderate
exposure levels. Pulmonary function tests
were administered to schoolchildren from
both regions. Upon analysis of the data, we
reported in FY95 that exposure to  O3 was
associated with  decrements in lung function
in the children. The magnitude of response
was similar to the responses we observed in
young adults exposed to equivalent levels of
O3 in clinical studies conducted by HSD.
These results suggest that clinical data can
be used to predict the responses of children
living in areas of high ambient levels of O3.

The  relationship  between   ambient  O3
exposures and absenteeism from school due
to respiratory-related causes was examined
in these same Mexico City school districts
during FY93-94.  Our findings, reported in
FY96, indicated  that children in the  "high
ozone" region suffered acute  respiratory
illness   severe  enough   to  result   in
absenteeism. Currently, we are examining
the effect of O3 on  lung  growth in these
schoolchildren and on emergency room visits
for respiratory complaints.  Although results
are still being analyzed, preliminary findings
suggest that high O3  levels (and paniculate
matter exposure) account for a significant
increase   in  emergency  room   visits  for
pneumonia. These findings  support those
from   another   EPA-sponsored   study
conducted in 1992 in the  U.S.,  which also
showed an increase in pneumonia-related
emergency  visits   associated   with  O3
exposures.

Through a Cooperative Agreement with the
University  of Southern California  (USC),
NHEERL and the California Air  Resources
Board  are  co-funding  an  epidemiologic
investigation designed to identify the health
effects of long-term  exposure to O3 (and
other pollutants) in children. The  project was
initiated in FY92 and involves approximately
4000 children  living in  twelve Southern
California communities.  During  Phase I of
the  study (FY92-94),  communities  were
selected  based on air pollution character-
istics and population demographics. During
Phase II  (FY93-96), children were enrolled
from each  of  the 12  communities, and
baseline   data on  health status, medical
history, demographics, etc. were gathered.
Additional  children  were enrolled  during
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996

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Phase III (FY95-97)  to  compensate for
children who might withdraw from the study,
and questionnaires were updated during this
period. Pulmonary function tests are being
performed annually over a seven-year period
(1993 - 1999).  A longitudinal study is now
underway in which  the  children will be
followed  for up to seven  years to define
which pollutants and what concentrations are
associated with changes  in health status.
Respiratory  illness  surveillance  will be
achieved by monitoring absenteeism from
school and conducting telephone interviews
with parents.

Through  another Cooperative  Agreement
with  USC, we are comparing lung tissues
from young, accidental-death victims who
lived in an area of high-level O3 exposures
(Los   Angeles)   to  'lung   tissues   from
individuals who lived in an area of low-level
O3 exposure (Miami).  The autopsy samples
are being examined for evidence of disease
and damage. The presence of pathological
lung lesions, which may be a marker of early
chronic lung disease, is being used  as  a
measure of damage.  During FY94-96, the
lungs of  75  individuals in  each area were
collected and studied. Results are currently
being analyzed.

Asthmatics. The sensitivity of asthmatics to
O3 is a question that has produced conflicting
answers  in  the past, but  clinical studies
conducted during FY95-96 by HSD helped
clarify the issue.  Our scientists determined
that allergic asthmatics (dust-mite sensitive)
exposed to 0.1 ppm  O3 do not experience
decrements  in lung  function, nor do their
lower airways appear to be sensitized to a
subsequent allergen  challenge.  However,
higher levels  of O3 (0.16  ppm) did  cause
decrements in lung  function,  and at this
concentration, the asthmatics were indeed
more susceptible to  the effects of O3 than
normal, healthy individuals. We also showed
that  subsequent  challenge by  dust-mite
allergen  resulted  in  increased  airway
reactivity, suggesting that exposure to  O3
may  contribute  to   asthma  morbidity.
Bronchoscopies   were   performed    on
asthmatics exposed to O3, and lung fluids
were recovered for analysis.  Our findings
show that asthmatics experience a different
kind of inflammatory response (eosinophilic-
driven)  than  nonasthmatics  (neutrophil-
driven), indicating a biological  difference in
the way asthmatics respond to O3.  These
results  support   the   contention  that
asthmatics are more susceptible to O3 than
healthy individuals, both in  terms of direct
and indirect response (i.e., when they come
in contact with an  allergen  following  O3
exposure). Our findings also offer biological
plausibility for epidemiology findings that
indicate a relationship between ambient O3
exposure and asthma attacks, emergency
room visits, etc.

Other  Variables.    In  clinical  research
conducted during FY93-94, we  measured
spirometric responses to O3  and demon-
strated that neither gender nor race affected
responsiveness at low concentrations  for
exposures of  moderate duration.  During
FY95, we further demonstrated that neither
socioeconomic status nor hormonal status
(i.e.,  stage  of  menstrual  cycle)  altered
pulmonary response  to O3.   In  FY96,  we
reported that there was no  evidence of an
effect of body size  on  response to  O3.
However, we did find that age was a factor in
O3 sensitivity: responsiveness  was greatest
in  young   adults  and  decreased  with
increasing age.

These  findings  in  humans  have  been
corroborated by our studies in rats conducted
during FY96,  which  suggest  that younger
rats are more sensitive to O3 exposure than
older animals. This age factor is important in
light of the  Clean Air  Act  requirement to
protect the most sensitive individuals in the
population.
NHEERL TROPOSPHER1C OZONE ANNUAL REPORT 1996
                                    10

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                      TROPOSPHERIC OZONE
  DETERMINANTS OF EFFECT RESEARCH PROGRAM
NHEERL defines determinants of effect
research  as  research  to  identify  and
describe the bases for the effects caused by
exposure  to  environmental  stressors or
chemical contaminants.
How does O3 produce its toxic effects?
The relationship between O3 exposure, dose
to  the  target tissue,  and effect  (lung
injury/disease) has not been  adequately
explained.  Accurate  estimations of target
dose,  which  are  based  on  regional
deposition and cellular uptake, are important
because the extent of lung damage depends
on where and how much O3 is deposited in
the lung.   In  addition, it is important  to
understand the mechanisms by which O3
produces  its toxic effects  because such
information would help explain the biological
basis for disease initiation and progression,
thereby leading  to  a more  biologically
defensible standard for €3.
 PROGRAM DESCRIPTION
The primary emphasis of this  research
program is to improve our ability to estimate
O3 dose and effect in target tissues.  Our
DOSIMETRY research focuses on obtaining
accurate estimates of dose to specific target
tissues in humans. While clinical studies are
the preferable and prevailing approach for
examining O3 dosimetry, we also  conduct
laboratory studies (in vitro and in vivo) to
describe   cellular  uptake  and   regional
deposition.  These  data are then extra-
polated to  humans.  In addition, we  are
conducting  studies   to   elucidate   the
biochemical MECHANISMS by which O3
causes lung damage  and inflammation.
Understanding how O3 damages lung cells is
critical for developing biologically plausible
risk models and  for explaining  individual
variations in susceptibility to O3.  Using the
information we generate on dosimetry and
mechanisms of toxicity, we are developing
RISK MODELS to predict human response
toO3.
DOSIMETRY

Historically,O3 dosimetry has been difficult to
study because  there  have  been  few
approaches for tracing the deposition of this
rapidly decaying chemical once it comes in
contact with biological tissue. Scientists in
our Experimental Toxicology Division have
advanced  the  science  in  this area  by
developing  a  system that  uses a non-
radiolabelled isotope (18O)  to  trace  O3
deposition and estimate target dose. During
FY94-95, we used this method to compare
the dose of O3 that reaches the respiratory
tract of  humans  and laboratory animals
(rats).  Following exposure to 18O-labelled
ozone, we collected lung cells and fluids by
bronchoalveolar lavage (BAL), measured the
amount of recovered 18O, and calculated O3
dose. Effects measurements (molecular and
cellular responses) were made on both sets
of fluids to determine whether there was a
correspondence between dose and effect.
We found that human cells incorporated 4 to
5 times  the O3 dose of rat cells, and that
effects were more  marked in humans.  In
FY96, we  used 18O-labelled O3 to test the
effect of ventilatory parameters (tidal volume
and respiratory rate)  on site-specific dose.
We  found that  changes  in  ventilation
produce only small variations in site-specific
doses in rodents.    However,  mucociliary
clearance does affect site-specific dose. Our
findings are enabling us to more accurately
estimate cellular O3 dose and response, to
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996
                                  11

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analyze species differences in dosimetry,
and to examine the influence of other factors
on dose distribution.

Chemical reactions that take place in the thin
mucus layer of the  lung  may mediate the
dose (and effect) of inhaled air pollutants.
For this reason, we formulated an artificial
lung lining fluid in FY94 that duplicates the
contents of human lung fluid. In FY95, we
measured the incorporation  of O3 into this
fluid;  results were  comparable to those
obtained with broncheoalveolar and nasal
lavage fluids collected from rodents. These
findings indicate that our test system may
represent a simple, but effective, approach
for studying O3 dosimetry. In the future, this
method  will   be   used   to   elucidate
mechanisms of toxic effect.

MECHANISMS OF TOXICITY

In an  effort to better understand how O3
causes lung damage and inflammation, we
are  analyzing  the  mode   of  action  for
mediators   of   cellular   response   and
inflammation  (e.g.,   prostaglandins   and
cytokines).   In FY95, scientists in  HSD
showed that lung epithelial cells  are the
primary target  of O3.  Ozone causes the
epithelial cells to increase  production  of
prostaglandins.   The  release  of these
prostaglandins  sets up a cascade of events
leading to  lung function decrements and
inflammation.   During FY96, we examined
some  possible biochemical  pathways that
might  help  explain  the   link  between
prostaglandin production and lung damage/
inflammation.  We found that O3 increases
prostaglandin   production   by   inhibiting
arachidonic acid esterification. This leads to
increased availability of arachidonic acid in
the lung, which metabolizes to produce the
prostaglandins that mediate  inflammation.

We also  are examining the mechanisms
involved in cytokine mediation of O3-induced
response. During FY95, we used a sampling
technique called nasal lavage to examine O3-
induced cytokines in humans.  We demon-
strated that asthmatic children have elevated
baseline levels of inflammatory cytokines in
their upper respiratory passages, which may
be related to their increased sensitivity to
inhaled compounds. In FY96, we reported
that the cytokine interleukin-6 is an essential
mediator of cellular adaptive response to O3.

Additional research by scientists in HSD has
suggested  that  different  mediators  of
inflammation  have  different  kinetics  of
appearance. We examined lung fluid for the
presence  of inflammatory mediators  at
different time points following O3 exposure,
and we examined the speed with which lung
injury and inflammation was induced. We
demonstrated . that   lung   injury   and
inflammation  occur within one hour of O3
exposure, and that while some inflammatory
mediators were elevated during this period,
others did not appear until 18 hours later.
This  study  will  enhance  our  ability  to
correlate  mediators   and  inflammatory
response with rapid changes in lung function.

We are exploring the  possibility that there
may be ways to protect individuals from the
harmful effects of O3. We have attempted to
determine whether an over-the-counter anti-
inflammatory drug (Ibuprofen) can  protect
individuals from the effects of O3 by reducing
or eliminating lung inflammation.  In  FY96,
we reported that while  Ibuprofen  blunts
decrements in lung function caused by O3, it
does not  reduce  underlying lung injury or
inflammation.   However,  levels  of  some
inflammatory mediators were lowered  by
Ibuprofen, which may help us understand the
mechanism  by  which  O3 reduces lung
function.    This   is  the   first   study  to
demonstrate that O3-induced changes in lung
function  can  be  dissociated from lung
damage and inflammation in humans.

Finally,  we  are  conducting  research to
determine the mechanistic basis for adaptive
responses to O3.  Ozone, an oxidant,  is
known  to impart tolerance to  itself  with
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996
                                    12

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continued exposure, but the reasons for this
are unclear.  In FY96  we found that mice
repeatedly exposed to O3 exhibit an increase
in the anti-oxidant Vitamin C (ascorbic acid)
in their  lung lining  fluid, suggesting  that
adaptation  to O3  is associated with the
protective anti-oxidant effects of Vitamin C.
We  also have  shown that  anti-oxidants
confer protection to human lung cells treated
in vitro to oxidants. Our findings are currently
being examined more fully in human clinical
studies.

RISK MODELS

Models  capable  of  predicting  human
response to O3 are critical to  setting an O3
standard. Our scientists have made signifi-
cant advances in this  area by developing
mathematical and biologically-based models
from  observations made in  humans  and
experimental animals.  In FY95 scientists in
our   Experimental   Toxicology   Division
developed   a  biologically  based  dose-
response (BBDR)  model of O3 toxicity by
combining  spirometric  data obtained from
humans with information on lung permeability
in animals.  This model, based on concentra-
tion  X  time (C x  T) relationships,  has
demonstrated  remarkable  homology  of
response between humans and animals over
a range of exposure  concentrations  and
durations.

Also  during  FY95,  scientists  in  HSD
constructed a mathematical model  capable
of utilizing data on multiple variables related
to O3 exposure and response. Because the
model  is   dynamic,  different  exposure
scenarios and individual  factors (e.g.,  age,
changing patterns of exercise, body size)
may be  considered.   We  developed  new
statistical methods that permitted us to use
clinical"data collected since the 1980s at our
human  exposure  facility  in  Chapel  Hill.
These data, which represent lung  function
measurements  (decrements  in   FEV^
collected  from  485   individuals,  were
integrated  into  our  model to  predict the
proportion of individuals expected to respond
adversely to  O3 as  a  function  of O3
concentration and duration.  The predictions
of our model were in good  agreement with
observed human response.  Specifically, we
showed that exposure to 0.12 ppm O3 for 6.6
hours resulted in a 10% decrement in lung
function in 47% of individuals. Our results,
which have assisted criteria  development,
led us to conclude that O3-induced changes
in lung function (FEV^ can be accurately
described as a sigmoid-shaped function of
exposure rate and duration of exposure.

Our  studies   on  ozone   prior  to  FY94
suggested that O3  impairs host  defense
systems  in  the  lungs  of  experimental
animals.   Ozone, it was  shown,  affects
immune  defenses,  rendering the  animal
more susceptible to  lung infections caused
by  bacteria  (specifically,   Streptococcus
infection). We went on to show  that the
increase  in susceptibility was caused by  a
depression  of  the pulmonary macrophage
defense system, which allowed the Strepto-
coccus bacteria to form protective capsules,
thus increasing their virulence. During FY94,
we expanded this research effort to include
humans.  Using a combination of in vitro and
in vivo studies,  we compared pulmonary
macrophage activity in humans and mice.
Our    data   demonstrated   comparable
sensitivity between the species, suggesting
that humans, like mice, may be at increased
risk of bacterial infection when  exposed to
O3.    From our  data,  we  published  a
qualitative extrapolation model  in  FY95 to
predict response in humans;  a quantitative
model  is now  under development that will
enable the Air Program Office to make better
use of animal data in assessing human risk.

In a related study, we are examining the role
of diet in altered susceptibility. Epidemiology
evidence  has  suggested  a  relationship
between low consumption of dietary anti-
oxidants (particularly Vitamin C), exposure to
air pollution, and respiratory infection  and
asthma.  Furthermore, as discussed in the
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996
                                    13

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previous section, our own  laboratory data
suggest that Vitamin C confers a protective
effect against O3.   We are developing an
animal model to help explain this association.
During  FY95-96, we  induced  Vitamin  C
deficiency in Guinea pigs and exposed these
Guinea  pigs to O3 followed  by an infectious
challenge   to  Streptococcus   bacteria.
Preliminary   findings   indicate  that   a
combination of diet (Vitamin C deficiency), O3
exposure, and infectious challenge results in
hypersusceptibility.
We are expanding this research to examine
the  effects  of  O3  on  viral  infections.
Surprisingly, our in vitro models suggested in
FY96 that lung cells pre-exposed to O3 were
actually more resistant-rather than  less
resistant-to subsequent viral infection. This
may be due to the inflammatory mediators
induced by O3, which impair viral replication.
We plan to continue our studies in this area
to explain this phenomenon.
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                                    14

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                    TROPOSPHERIC OZONE
 ECOLOGICAL EFFECTS  RESEARCH  PROGRAM
 NHEERL   defines  ecological  effects
 research as research that leads to a better
 understanding   of  the   response  of
 ecosystems (and their component parts) to
 anthropogenic stressors.
 ISSUE
 What are the effects of O3 on trees and
 forests?
 Tropospheric ozone is the most widespread
 air pollutant affecting vegetation in the U.S.
 However, its effects  on trees and forest
 systems   are   not   clearly  understood.
 Although it is known that the primary, or more
 direct, site of O3 action is in the leaves (which
 affects plant growth), little is known about the
 indirect effects of O3 (those affecting the root
 system  and  soil biota).  Studies of plant
 growth generally have involved less complex
 levels of biological organization, such as
 seedlings or individual  tree species; the
 results from these studies are subsequently
 used to make inferences on the effect of O3
 on mature trees or  mixed forests.   To
 accurately characterize the risk of O3 to trees
 and forests, additional information is needed
 to describe the direct and indirect effects of
 O3 in the context of changing exposure
 dynamics and other environmental stressors,
 and models  are  needed that can simulate
 long-term growth effects in  forest systems.

 PROGRAM DESCRIPTION

 The purpose of this research is to quantify
 the effects of acute and chronic O3 exposure
 on tree  species at  the  single-tree  and
 community level, and to extrapolate those
 effects to large-scale forest responses. To
 accomplish   our  objective,  we   have
 developed a two-pronged approach that
 takes into consideration different levels of
 biological complexity.  We are studying the
EFFECTS OF OZONE ON TREES, including
the nature and extent of O3 damage above-
ground (direct effects)  and below-ground
(indirect effects).    Effects  are  being
evaluated in  the context of individual tree
characteristics,  such as  age,  size, and
species; changing exposure dynamics, such
as concentration, frequency, and duration of
exposure; and exposure to other stressors,
such as temperature changes or drought.
The experimental data are then used to
inform  the  modeling component  of  our
program, in which we  are predicting  the
EFFECTS OF OZONE ON FORESTS. Our
models  characterize forest  behavior by
combining physiological data from individual
trees  or species with  information on O3
exposure  and   regional  environmental
conditions.   Results from this  research
program are forming the biological basis for
the secondary standard for O3.
PROGRAM
EFFECTS OF OZONE ON TREES

Indirect Effects. This research is designed
to characterize below-ground responses of
trees to O3, which includes effects on root
physiology   and   growth,   rhizosphere
processes (activity in the root zone of the
soil), and soil biota.  In research conducted
during FY95-96 by scientists in our Western
Ecology Division  (WED), O3 was found to
have profound effects on the rhizosphere.  It
reduced root growth and mycorrhizal activity
(the  symbiotic  associations of fungi and
roots),  and it  altered  the  movement  of
carbohydrates in  plants. We have shown
that the consequences of such changes are
substantial.  Reduced root growth makes the
seedlings   more  susceptible  to  other
stressors,  such  as drought and nutrient
deficiencies. Changes in mycorrhizal activity
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996
                                  15

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affect carbon movement and sequestration,
yielding a less favorable carbon  balance.
These results suggest that O3 stress may
magnify the effects of global climate change.
By adversely affecting the rhizosphere, O3
impacts on forest ecosystems may be more
widespread than previously estimated based
on conventional measurements of foliage
damage.

During FY95-96, we found that when plants
are exposed to O3, root exudation increases,
which increases levels of soil organic matter,
providing    substrate    for    rhizosphere
organisms. Consistent with this observation,
we demonstrated that bacterial and  fungal
biomass also increase in the soil  of plants
exposed to  O3, and the respiration  of soil
organisms is altered. The extent to which O3
disrupts soil biota is of importance because
changes within the soil food web may affect
the  long-term  health  and  productivity  of
forested systems.  In  FY97, we will study
shifts in soil microbial populations caused by
O3 and the  impact of these shifts on soil
properties.

Direct Effects. This research area has two
major   components:  1.) quantifying  the
response of different tree  species   to
changing O3 concentrations, and 2) defining
the role of exposure dynamics, both temporal
and  seasonal, on biological response to O3.

1) Response of tree species: During FY94-
95, we demonstrated the effects of  O3 on the
growth  of  11  important  tree  species.
Scientists in WED grew seedlings in open-
top  chambers,  keeping variables such as
water  and   temperature  constant  while
varying ozone concentrations.  The experi-
ments  were conducted over a  two-year
period, and  various measures of  growth
response-such as gas exchange, water use,
and changes in biomass partitioning--were
made.  In FY96,  we  assembled  the data
geographically    using   a   Geographic
Information  System (GlS)-based approach.
From these data, we were able to estimate
biomass losses as a function of changing O3
exposures and to rank species according to
their sensitivity to O3.   We  found that
ponderosa pine, black cherry, and aspen are
the most sensitive to O3, while douglas  fir,
Virginia pine, and red maple are relatively
insensitive. These exposure-response data
are critical for predicting the potential risks of
O3  to individual species  and  to  different
geographic regions.

2) Exposure dynamics: Because exposures
to O3 may be episodic or chronic (depending
on  such  factors  as geographic region,
season, etc.), we are conducting research
that considers the effects of changes in  O3
concentration,   duration,  and  frequency.
Research conducted by WED prior to FY94
demonstrated that growth response in trees
and grasses differs according to O3 exposure.
regimen, even when the overall seasonal O3
concentration  remains  constant.   Ozone
uptake was shown  to be a  function  of
temporal distribution  and  frequency of  O3
occurrence.   These early  studies also
indicated that  changes in growth do not
always occur during the season of exposure,
but may be exhibited the following year. This
"carry-over" of response indicates that the
effects of O3 on growth may be cumulative.
More recently, we have associated this effect
with reduced storage  of carbohydrates.

During FY95, we found that plants are more
likely to take up O3 during the daylight hours.
We also showed that there is a greater effect
on plant growth with episodic exposures to
high concentrations of  O3.  These results
were recently used in the Criteria Document
to support setting a secondary NAAQS  for
O3. During FY96 we showed that because
O3 uptake  is  a function   of  temporal
distribution and frequency of O3 occurrence,
exposure in different regions of the country
can be expected to have differing effects on
plant response. In future studies, we plan to
identify the exposure components that  are
most influential in long-term,  multiple-year
exposures, we  will  examine how these
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996
                                    16

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exposure components affect the rate of O3
uptake, and  we  will  determine  how  this
information can be incorporated into an index
allowing extrapolation of effects both spatially
and temporally.

EFFECTS OF OZONE ON FORESTS

During FY95, spatial  analysis techniques
were   used   to  develop  a  method  for
characterizing the risk of tropospheric O3 to
forests  in  the U.S.   We  developed a
Geographic Information System (GlS)-based
framework that predicts forest behavior by
linking  exposure-response  data   with
information  on  O3  exposures, regional
environmental  conditions,  and species
distribution. Findings from our experimental
studies of the effects of O3 on tree species
are being incorporated  into a  series of
models that can simulate long-term growth
effects on a regional scale. Using a model
called TREGRO, we predicted the long-term
effects of O3 on tree growth based on our
experimental  data  that  describes  the
physiological reponse of leaves, seeds, and
seedlings. We then integrated the TREGRO
simulated growth rate  into a model called
ZELIG that simulates  the response of a
stand or community of trees to O3 over time.
This GIS-based approach  quantifies the
impact of current ozone air quality and has
been used in benefits analyses for the Office
of Air Quality Planning and Standards in the
review of the O3 NAAQS.  Due to reductions
in this program, FY96 was the final year to
study the impact of O3 on forest systems.
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996
                                   17

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 National Health and Environmental Effects Research Laboratory
    ENDOCRINE DISRUPTORS
HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH
             ANNUAL REPORT




             NOVEMBER, 1996

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                           CONTENTS
Introduction
Summary of the Endocrine Disrupters
      Health and Environmental Effects Research Program   	4

FY95-96 Highlights	6

Endocrine Disrupters Hazard Identification Research Program 	7
           Screening and Toxicity Tests
           Biomarkers

Endocrine Disrupters Predictive Models Research Program  	  12
           Mechanisms of Action
           Pharmacokinetic Studies
           Mixtures
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996

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                        INTRODUCTION
The purpose of this report is to communicate results from the Endocrine Disrupters
Research Program of EPA's National Health and Environmental Effects Research
Laboratory (NHEERL).
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The report contains

•     a summary of the NHEERL Endocrine Disrupters Research Program, including
      an explanation of its regulatory and programmatic context, the overall goal, the
      rationale for the program, and the research strategy

•     a section that highlights recent key  findings (FY95-96 Program Highlights)

•     a description of the NHEERL Endocrine Disrupters Research Program, by
      program  area,  including  a  summary of  research accomplishments and
      anticipated progress for the near future


COMMENTS WELCOME

The format of this report is still evolving, and we welcome feedback. Readers with
comments or requests for further information are encouraged to contact:

      Sue McMaster, Assistant Laboratory Director
      National Health and Environmental Effects Research Laboratory (MD-51A)
      U.S. EPA
      Research Triangle Park, N.C. 27711

      Phone:  (919) 541-3844 or FAX: (919) 541-1440
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996

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                   ENDOCRINE  DISRUPTORS
             RESEARCH PROGRAM  SUMMARY
REGULATORY AND  PROGRAMMTIC
Several pieces of legislation sanction EPA to
protect human health and the environment
from  the  potentially harmful effects  of
endocrine  disruptors. These laws include
the Toxic  Substances Control Act, which
mandates  EPA to evaluate the toxicity of
new and  existing chemicals;  the  Federal
Insecticide, Fungicide, and Rodenticide Act,
which regulates pesticides; and the newly
enacted Food Quality  Protection  Act  of
1996 and the  Safe Drinking Water Act
Amendments of  1996,  both of  which
authorize a screening program for endocrine
effects.  To assist the Agency in meeting its
requirements in these areas, EPA's Office of
Research and Development (ORD) maintains
an Endocrine Disruptors Research  Program
that assesses the risks posed by chemicals
affecting the endocrine system.   NHEERL
supports  this  program  by  developing
methods to identify endocrine disrupting
chemicals, by  evaluating  the  potential
effects of these chemicals on human health
and the environment, by producing models
that improve quantitative risk  assessment,
and by providing chemical-specific data on
contaminants of unknown toxicity.

PROGRAM GOAL
To determine the nature and magnitude of
the health  and ecological effects associated
with exposure to endocrine disruptors.

RATIONALE
A growing  body  of scientific  evidence
suggests that domestic animals and wildlife
have  suffered adverse consequences from
exposures to environmental  chemicals that
disrupt    endocrine   function.    These
chemicals,  collectively  called endocrine
disrupting  chemicals (EDCs),  exert  their
toxicity by mimicking or interfering with the
actions of hormones. Most of the effects
associated with EDCs, such as reproductive
dysfunction and sexual abnormalities, have
been  observed  in wildlife  populations
receiving relatively high levels of exposure.
Whether similar, albeit more subtle, effects
are occurring in humans is unclear. Reports
of declining sperm production in humans
over the last four decades—as  well  as
increases in  rates of cancers that may have
an endocrine-related basis (breast, prostate,
testicular)--have led to speculation about
environmentally mediated endocrine disrup-
tion  in humans.    These  observations,
coupled with available data from laboratory
studies,  have generated  a  climate  of
concern surrounding the potential  long-term
consequences of  exposure to endocrine
disruptors.
              -•          .•    •*
            STRATEGY
To ensure that the Agency is equipped with
scientific and technical data relevant to the
formulation of sound environmental policy,
ORD operates a research program founded
on principles of risk assessment.  Research
in the area of health effects is guided by the
risk assessment paradigm of the National
Academy of Sciences (NAS), which outlines
4 steps in risk assessment: hazard identifi-
cation, dose-response assessment, exposure
assessment,   and   risk   characterization.
Research on ecological effects follows the
framework  for ecological risk assessment
developed  by EPA in 1992, comprised of
problem  formulation, analysis (characteri-
zation  of exposure and  effects), and risk
characterization.

NHEERL's  research programs  adhere to
these risk-based  strategies.  Emphasis is
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996

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placed on two areas of risk assessment:
       • hazard identification, or problem
formulation, in which research focuses on
the development and  utilization of methods
to  identify human  health and ecological
hazards, and
       • the  characterization  of  dose-
response, which  seeks to explain events
linking exposure to effects; these events
form the basis of the predictive models used
to quantify risk.

NHEERL  has  designed  its  Endocrine
Disrupters Research Program to address the
uncertainties surrounding  the health  and
ecological effects of endocrine disrupters.
The program  can be sectioned  into  two
categories.  In the first category, our efforts
are focused on HAZARD IDENTIFICATION.
We are developing test methods-including
screening  assays,  toxicity   tests,   and
biomarkers-to identify potential EDCs and
to  characterize  endocrine-mediated  toxic
effects.   Secondly,  we  are  constructing
PREDICTIVE MODELS that more accurately
estimate risks to humans and wildlife posed
by  endocrine disruptors.   To  support the
development of  these risk models, we are
conducting  research that describes the
uptake  and  metabolism  of  EDCs,  the
fundamental   processes   involved   in
endocrine regulation, and the toxicity of
mixtures of endocrine disrupting chemicals.
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996

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 NHEERL ENDOCRINE  DISRUPTORS RESEARCH
            FY95-96  PROGRAM  HIGHLIGHTS
   *• We organized and sponsored two international workshops involving all stakeholders that
   identified EDO-related research needs in areas of human health and ecological effects.

   > We helped draft the ORD Research Plan for Endocrine Disrupters, which provides strategic
   direction to Agency research efforts in endocrine disruption.
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The goal of this research is to develop and validate cost-effective test methods for identifying
human health and ecological hazards associated with endocrine disrupting chemicals.

    *•  We have uncovered significant new data that challenge the prevailing scientific
   opinion  regarding endocrine disruption.  In research that has received widespread
   national and international recognition, we found that in addition to chemicals that mimic
   estrogens, there are anti-androgenic chemicals present in the environment.

    *• We have identified a sperm protein in rodents, which is also common to humans, that
   is  highly correlated with fertility.  Because  this protein may represent a suitable
   biomarker for fertility, there is significant commercial interest, and it is the subject of
   a recent EPA patent application.

    *• A novel assay was developed to detect the effects of EDCs on early development in
   fish.  The approach permits correlations to be made between EDO exposure, P450
   enzyme induction, and transgenerational health effects.
PREDICTIVE MODELS IPS
The goals of this research are to better understand the factors that influence dose-response
relationships and to use this information to develop reliable risk models.

   *• We found that p,p'-DDE, the major metabolite of DDT, binds to the androgen receptor,
   thereby blocking the action of endogenous androgens in the body.  This important
   discovery represents a new mode of action for endocrine disrupters.

   *• We showed that the persistent hearing loss caused by developmental exposure of
   rodents to PCBs is associated with reduced levels of circulating thyroid hormones, which
   may help explain the endocrine-related mechanism of action for this neurotoxic effect.

   *•  Exposing rats  to  dioxin during development  was found to reduce their core
   temperature by decreasing the homeostatic set point, suggesting that fundamental
   metabolic processes may be altered by endocrine disruptors.
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996

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                    ENDOCRINE DISRUPTORS
 HAZARD IDENTIFICATION RESEARCH PROGRAM
NHEERL   defines  hazard  identification
research  as  research to characterize the
association between  environmental expo-
sure and adverse effect.
What chemicals interact with the endocrine
system? What effects are caused by these
chemicals?
Disruption of endocrine function provokes a
cascade of reactions involving many organ
systems.  Response  is  often  subtle and
complex, making it difficult to establish a
direct link between EDC exposure  and  an
adverse effect.  Existing toxicity tests for
endocrine disruption, such as those  used in
product  registration  or  for  monitoring
environmental samples, are limited  in their
ability to detect key reproductive, develop-
mental, and immunological effects.  New
and  improved tests are needed that are
capable of detecting  a broad range  of
endocrine-mediated responses.

PROGRAM DESCRIPTION

NHEERL's efforts to characterize and  predict
the hazards associated with exposure  to
endocrine disrupting chemicals are focused
on two research  areas. In the first area, we
are developing and validating SCREENING
AND TOXICITY TESTS for EDCs.  Short-
term in vitro and in vivo assays, as  well as
first  tier  computer  models,  are  being
developed to  screen  putative  EDCs for
endocrine disrupting activity and to evaluate
the types of toxic effect  that may  be
produced in humans and wildlife.   We are
concentrating  our efforts   on  endpoints
relevant  to   endocrine  disruption  (e.g.,
estrogenicity,   anti-androgenicity,   Ah-
receptor binding, and anti-thyroidal activity)
and on effects most likely to be produced
(such as  reproductive and developmental
toxicity).     We  also   are   developing
BIOMARKERS to serve as early indicators of
response  specific  to EDC  exposure.  At
present,  our  focus  is on biomarkers of
reproductive dysfunction in aquatic wildlife
and in humans.

PROGRAM PROGRESS

SCREENING AND TOXICITY TESTS

Structure-Activity   Relationships   (SAR).
NHEERL  is   using  a   combination  of
computational chemistry, molecular model-
ing, and toxicity test outcomes to study the
interactions  that  occur  between  key
structural features of a  chemical  and its
biological target.   During FY95, we made
significant progress in modeling quantitative
structure-activity  relationships (QSAR) for
compounds that have the ability to bind to
steroid hormone receptors.  Using advanced
computer techniques,   scientists  in our
Experimental Toxicology Division developed
a preliminary three-dimensional QSAR model
that predicts  the  endocrine  disrupting
potential   for  polychlorinated  hydroxy-
biphenyls.  Our  model,  which  estimates
estrogen  receptor binding  affinities,  was
among the first  to  offer  evidence of a
structural basis for estrogenic activity for
this class of compound.   During FY96, we
expanded our  efforts to  include a more
diverse set of chemicals and additional toxic
endpoints.   For  example,  using in vitro
androgen receptor (AR)  affinity data,  we
have developed a QSAR model based on AR
ligands.   Our goal is   to describe the
structural parameters that underlie binding
to the androgen receptor.
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996

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In other SAR studies, we are examining a
subclass    of    PCBs   (ortho-substituted
congeners) that do not  bind to the aryl-
hydrocarbon (Ah) receptor.  These chemi-
cals   have   been   reported   to   cause
neurological effects, and we are attempting
to link key  neurochemical  events with
structural  features  of these  chemicals.
Such a linkage could provide a strategy for
predicting the endocrine disruptive potency
of these  and other congeners.  Utilizing a
number of neurochemical measures from
cell culture systems, we determined during
FY95 that these  PCB  congeners  perturb
calcium homeostasis, a balance critical to
the  functional  integrity  of  the  nervous
system.    These  findings are important
because they suggest that a neurochemical
measure  may be used  in structure-activity
modeling for EDCs. We are now conducting
studies to further characterize this effect in
an effort to identify a receptor target for
neurotoxic endocrine disruptors.

Our  Mid-Continent  Ecology  Division  is
modeling  a mechanistically-based  QSAR
that addresses the conformational flexibility
of molecules.   During FY95-96, we used
results from  mammalian toxicity tests to
model the binding  of halogenated aromatic
compounds to  the  Ah  receptor and  the
binding of PCBs and related  compounds to
the estrogen receptor.   In" the future, we
will focus on modeling post-transcriptional
and  post-translational  events  using  data
from representative aquatic species; cross-
species  comparisons   will  be  made  to
determine  how  well  mammalian  tests
predict  perturbations   of receptor-based
processes in aquatic species.

Reproductive  and Developmental Toxicity
Tests.   During FY95, a landmark paper
published  by  scientists  in   NHEERL's
Reproductive Toxicology Division challenged
the prevailing scientific notion of endocrine
disruption. Our scientists found that some
environmental chemicals, including p,p'-DDE
(the major metabolite of DDT), are potent
anti-androgens  and  not  environmental
estrogens as previously thought.  Exposure
of rats to these chemicals demasculinized
male offspring such that they displayed--
among other effects—undescended  testes
and retained nipples. Exposure also delayed
the onset of puberty. Thes^e effects are
consistent with androgen receptor binding.
Combined with our observations of altered
sex differentiation in male rats exposed to a
fungicide whose metabolites inhibit binding
of the androgen response elements on DNA,
it  became apparent  that anti-androgens
exist in the environment and that they can
cause developmental effects. We recently
demonstrated  that  these chemicals also
bind to the human androgen receptor. With
the publication of these important findings,
the  scientific   community   immediately
enlarged its research focus to include anti-
androgens in its assessments of  endocrine
disruption.

In FY95,  we  reported that a number of
adverse  reproductive effects,  such  as
delayed  puberty,  abnormalities of  the
vagina, and  difficulties in mating, occur in
the female offspring of rats and hamsters
exposed to an endocrine  disrupter (dioxin)
during pregnancy. Male offspring exhibited
reduced accessory sex gland weight and
reduced sperm  counts.    Upon   further
analysis,  we  have  found  evidence  to
suggest that the primary target for damage
in the males is the epididymis. We currently
are pursuing this hypothesis to help explain
the mechanism responsible for the  altered
reproductive function.

Immunotoxicity  Tests.      Studies   are
underway by scientists in our Experimental
Toxicology   Division  to   evaluate   the
transgenerational   effects   of  endocrine
disruptors (specifically, dioxin) on immune
function. After exposing pregnant rodents
to dioxin, we  are assessing immunotoxic
responses in  the  pups, or  fetuses, by
 NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996
                                                                                    8

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measuring the functional integrity of cells
involved in immunity (e.g., natural  killer
cells and B and T lymphocytes). In studies
conducted, in FY96, we found that exposed
pups display alterations in the proportion of
T-cells in the thymus.   We also  found a
persistent  suppression  of T-cell-mediated
response. We are now trying to establish a
linkage  between these  two immunotoxic
events.

Ecological Tests. To better understand the
ecological impact of endocrine disrupters on
wildlife species, ORD has established a new
program  that addresses  the  ability  of
chemicals  to  disrupt hormonal control of
reproduction  and  development  in  fish,
amphibians, and  aquatic invertebrates. One
reproductive  outcome pf endocrine disrup-
tion  in   wildlife   is  the   induction  of
vitellogenin  in  oviparous  males  (those
belonging to species that hatch their eggs
outside the body, such as fish, reptiles, and
birds). Vitellogenin is an  estrogen-inducible
protein normally found only in  females; its
presence in males is taken as an indication
of  exposure  to environmental  estrogens.
Conventional  methods of detecting vitello-
genin are performed in  vivo, making them
relatively  expensive and  labor-intensive,
especially  for  use  as  screening  tests.
Consequently, NHEERL  is  attempting  to
develop in vitro  assays that could serve as
rapid, simple, and  inexpensive substitutes
for the in vivo tests.

For example,  our Atlantic Ecology Division
is developing a  novel  in  vitro screening
assay that uses laser cytometer technology
to  measure a chemical's affinity  for the
estrogen receptor in oviparous vertebrates.
Initially,  we  applied the laser cytometer
technique to a mammalian (rat) cell line in
which the number of receptors is known;
during  FY96, we  successfully  detected
receptor  binding. In FY97, the  method will
be  adapted to a fish cell line  of cultured
hepatocytes.  Receptor binding affinity will
be  compared  to vitellogenin production,
with the goal of using the method to detect
environmental  contaminants  that  may
interfere with egg production.

In FY97, investigators in our Gulf Ecology
Division will begin studies to determine the
suitability of an  in  vitro yeast  estrogen
system as  a screening tool  for estrogenic
activity. Estrogenic chemicals will be tested
in yeast cells engineered to contain genes
that code for the human estrogen receptor
and for a "reporter"  protein that  indicates
receptor binding. Results will be compared
to those obtained using the in  vivo  fish
vitellogenin assay.    Our   analysis   will
determine  the  feasibility of  using  this in
vitro assay in the  pre-registration process
for new chemicals.

Scientists  in our  Mid-Continent  Ecology
Division are developing in vivo toxicity tests
that examine endpoints other than vitello-
genin induction. Endpoints include those at
the subcellular  (hormone),  tissue  (histo-
pathology),  and whole  organism  (sexual
differentiation,  fecundity)   levels.    Test
organisms  include  small   fish  (fathead
minnow,  medaka,  and  zebrafish)   and
representative  freshwater   invertebrates
(e.g.,  molluscs, midges).    A variety of
estrogenic   chemicals  with   known  or
suspected mechanisms of action are being
tested to determine exposure windows and
endpoints  most  susceptible to  adverse
effects. This research is in its initial stages
of  development,  and  results  are   not
expected  until  FY97  or thereafter.   An
important emphasis  of this project is the
linkage  of  different  endpoints   to   one
another as well  as the correlation of results
from  the  organism  to  population-level
effects.

Reported declines in  regional and global
populations of amphibians  have raised
concerns  regarding  the possible role of
environmental contaminants on reproductive
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and developmental outcome. Scientists in
our  Mid-Continent  Ecology  Division  are
conducting studies to characterize "normal"
endocrine processes in amphibians and to
identify species that could serve as models
for assessing disruption of developmental/
reproductive processes.  Currently, we are
defining the "base-line" endocrine status for
two  amphibian species,  Rana pipiens and
Xenopus  laevis, emphasizing  endocrine
systems modulated  by thyroid hormones
and retinoic acids.  Chemicals with known
or suspected  modes of action are  being
administered  to perturb  the  endocrine
system.   This is  presently a laboratory-
oriented project, but in the future it could
involve field studies of  indigenous species.

Transgenerational  effects  of  endocrine
disruptors   in   wildlife  also  are   being
investigated.  These studies will provide
valuable information on the potential  of
endocrine  disruptors  to  produce   latent
reproductive or developmental effects. In a
project initiated in FY94, we fed marine fish
(mummichogs) a diet spiked with various
concentrations  of  dioxin.   Eggs   from
exposed females were hatched in clean sea
water.  Over the next two  years,  offspring
were raised to adulthood and assessed for
reproductive  capacity   (egg  production,
percent fertilization, larval survival, etc.).
Data are in the  process of-being analyzed
and  will  be  used  in  a multigenerational
model  of  dioxin  effects  on  population
dynamics.

In another transgenerational study, investi-
gators in our Atlantic Ecology Division are
evaluating  the  effect  of  EDCs  on  early
development  in  estuarine fish by studying
retinoid homeostatic regulation.  Retrnoic
acid is a powerful teratogen and regulator
of early development.   Its balance  in the
body can  be  affected  by changes  in the
activity  levels  of  the  cytochrome P450
enzymes,   which   metabolize   chemical
contaminants  such as EDCs.  During FY95-
96, we developed a novel, non-destructive
assay that permits measurements of P450
enzyme activity over time in embryos of fish
exposed to EDCs.  The embryos are allowed
to mature to larval stage, and observations
of effects (including hatch rate and success,
growth  and  survival, and lesion character-
ization)  are  then made.  This approach is
unique in that it permits us to evaluate the
relationship  between EDC exposure, P450
enzyme  induction,  and  transgenerational
health effects. In FY96, this technique was
applied  to  a field study  in which fish
collected  from a  Superfund site  highly
contaminated with  PCBs were evaluated.
Results  are  presently being  analyzed.  In
FY97, we will begin to characterize retinoid
status in fish embryos in  an  effort  to
correlate this endpoint  with changes  in
enzyme  activity levels and developmental
abnormalities.

During  FY94, a  transgenerational  study
using freshwater  fish  (trout  and  other
species) was initiated to investigate the
effects  of dioxin and  related chemicals  on
early life stage development and survival.
Different  exposure  scenarios,   including
translocation  of  dioxin from the adult
female  to oocytes, exposure of fertilized
eggs to  waterborne dioxin, and injection of
dioxin into fertilized eggs, were studied  by
researchers  in our  Mid-Continent Ecology
Division.  In FY95, we reported that early
life  stages  are  the most susceptible  to
dioxin  and   that  toxic potency  is not
influenced  by exposure route.   We also
demonstrated that  maternal transfer  of
dioxin was sufficient to cause dose-related
effects in offspsring.  Although we found
trout to  be the  most  sensitive species
tested,   a phylogenetic  basis to species
sensitivity   was  not  apparent  from  our
studies.

BIOMARKERS

In  FY96, scientists  in  our Reproductive
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996
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Toxicology Division reported the discovery
of a sperm membrane protein in the male
rat that appears  to  be a  biomarker for
fertility.   This protein,  also  common  to
humans, is compromised upon exposure to
chemicals that disrupt endocrine status and
is highly correlated with reduced fertility.
Our findings  have  received  widespread
commercial interest and are the subject of a
recent EPA patent application.  Currently,
we are attempting to develop an antibody
to this sperm biomarker that will enable us
to screen for  endocrine disruptive activity
and reduced fertility.

During  FY95-96,  scientists in  our  Gulf
Ecology Division conducted in vivo studies
in fish  using  vitellogenin induction as  a
biomarker for endocrine disruption.   Two
highly sophisticated analytical techniques-
the  Western  blot assay,  which screens
serum for vitellogenin, and the ELISA assay,
which   quantitates   the   amount   of
vitellogenin present-were used to determine
whether a fish was estrogenized. In FY96,
we reported that male carp  captured in an
area  known   to   be  contaminated   by
estrogenic chemicals exhibit vitellogenin
induction and reduced serum testosterone
concentration.

The above-mentioned  vitellogenin-induction
assay utilizes  blood  samples.   In  many
studies, however, the fish are too small to
provide sufficient blood for testing. For this
reason, we initiated  research in FY96 to
determine whether the liver, which is readily
obtained even from small fish, can be used
in the development of biomarkers. We are
studying two  endpoints: the liver estrogen
receptor and vitellogenin gene mRNA. If we
can determine that the  estrogen receptor
and/or the vitellogenin gene are activated
prior to  our  ability to  measure serum
indicators of estrogenic activity,  it may be
possible to use these endpoints as  early
indicators  of  response  to   estrogenic
chemicals.

Additional  biomarkers   of  reproductive
dysfunction in wildlife have been identified
for future study, including plasma steroid
hormone levels,  Jiver estrogen  receptor
levels, and retinal  necrosis  in  oviparous
animals.    In  FY97, researchers in  our
Atlantic Ecology Division will initiate studies
to  determine  which of these  endpoints
successfully reflects exposure to estrogenic
chemicals.    Initial  studies  will  involve
juvenile fish,  which produce  low levels of
endogenous  hormones,  making  results
easier to analyze.  This research will then be
expanded to include adult fish, and we will
determine  whether any  of the  indicators
presage  an   adverse  effect   (such  as
reproductive dysfunction).  In future years,
the diagnostic indicators will be applied in
field situations to obtain  a snapshot of the
reproductive    status   of    indigenous
populations.

In a field study conducted by Oak  Ridge
National Laboratory with funding from EPA,
fish from a river receiving  pulp and  paper
mill  effluent, which  contains dioxin,  were
investigated over a seven-year period.  The
studies preceded and accompanied exten-
sive modernization of mill facilities to reduce
contaminant  discharge.  The goal of the
project was to determine which biological
indicators  were predictive of population-
level effects.  The condition of fish prior to
mill  modernization  was  consistent  with
effects  attributed  to  EDC   exposure,
including a sex ratio skewed towards male
fish,  alterations  in reproductive  hormone
levels, and  an  absence  of young  fish
suggesting near total reproductive failure.
Following,  modernization of  the mill, we
observed that  these  endpoints  trended
towards  normality,  and  fish communities
have become more diverse coincident with
decreases in body burdens of dioxin.
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                    ENDOCRINE  DISRUPTORS
      PREDICTIVE  MODELS RESEARCH PROGRAM
NHEERL defines predictive models research
as  research  that  produces  data  on
mechanisms of  action,  pharmacokinetics,
and dose-response for use in developing
models that can estimate target dose and
effect.
ISSUE
How can we better estimate the human
health and environmental risks associated
with endocrine disruptors?
Risk assessments often rely on predictive
models that estimate dose and effect from
experimental  toxicity  information.   The
reliability of  the derived  risk estimates
depends on the soundness and accuracy of
the models as well as the strength and
scope of the toxicity data. To enhance the
precision of  risk  estimates for endocrine-
mediated effects,  advances in modeling are
required. These  advances, in turn, rely on
improved  understanding  of the  basic
processes involved in endocrine disruption.
Another issue relevant to risk assessment
involves exposure  to  mixtures of  EDCs.
One approach for  assessing  the risk of
mixtures is the Toxic Equivalency  Factor
(TEF)    method,    which   features   an
assumption of additivity for toxic effects.
However, it  is uncertain whether the TEF
method, which is applied  to mixtures of
chemicals  possessing a common mode of
action (e.g., dioxin congeners), is valid  for
all EDC mixtures.

PROGRAM IDESCRIPTIpH f

The primary objective  of  this research
program  is   to  produce data  that  will
facilitate the development  of biologically
plausible risk models.  To accomplish this
objective, our  Laboratory  has  initiated
research in three areas.  Research is being
conducted to understand the key events, or
MECHANISMS OF ACTION,  involved in
endocrine-mediated toxicity.  l?oth receptor-
and  non-receptor-based  mechanisms of
endocrine disruption are  under  study.
PHARMACOKINETIC STUDIES are being
conducted to describe the behavior of an
EDC as it is metabolized, distributed to
target  tissues, and eliminated from the
body. These data are vital to securing more
accurate predictions of tissue and cellular
dose, especially  at critical and  sensitive
early life stages.  Finally, we are producing
better ways  to assess the risks associated
with exposure to MIXTURES of endocrine
disruptors.   We  are developing  TEFs for
single chemicals, estimating the toxicity of
EDC mixtures based on  these TEFs, and
comparing  our predictions to  observed
effects.
MECHANISMS OF ACTION

Reproductive and Developmental Toxicity.
Significant new mechanistic data were
reported during FY95 by  scientists in our
Reproductive Toxicology  Division.  They
found that p,p'-DDE binds to the androgen
receptor,  thereby blocking the  action of
endogenous  androgens in the body  and
resulting in abnormal reproductive develop-
ment in male offspring.   This  important
discovery represents a new mode of action
for endocrine disruptors  and will greatly
facilitate  the risk assessment process for
this class of chemical.

We also are characterizing the mechanisms
involved in responses mediated  by the Ah
receptor, its binding partner (Ah receptor
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nuclear translocator, or ARNT), and the
glucocorticoid receptors. Palate cells from
embryonic tissues are being cultured in vitro
and treated with developmental toxicants,
such as dioxin.  Gene expression patterns in
the developing tissues  are then analyzed
across time, and dose-response profiles of
gene expression are produced.  Both mouse
and human cells are included in our studies,
permitting   interspecies  comparisons   of
response.  During FY95, we  characterized
gene expression patterns in the human cell
culture.   Initially,  we used immunohisto-
chemical  staining  techniques and  in situ
hybridization  to  identify  the  proteins
expressed in embryonic tissues.  Currently,
however, we are  attempting to quantify
gene expression through a more sophisti-
cated  technique called  PCR  (polymerase
chain  reaction).    PCR analysis of the
samples  was completed during FY96, and
statistical analysis of results is in progress.
Our plans are to expand this work in FY97
to examine the mechanisms involved in the
synergy between glucocorticoid hormones
and dioxin and their effects on developing
palates.  This new avenue of research will
help elucidate the interplay between gluco-
corticoids  and EDCs,  which is important
because  of the role hormones play as a
trophic factor in normal development.

To complement this in vitro study, scientists
in our Experimental Toxicology Division plan
to assess the role of the Ah receptor and  its
binding partner in  vivo.  Studies will  be
initiated in FY97 in which several EDCs will
be tested  in  a transgenic  mouse model
lacking the Ah receptor and  in mice  in
which the Ah receptor is present. We will
examine  a variety  of  endocrine-related
endpoints, such as developmental toxicity
(expression of  ARNT in palate shelves,
hydronephrosis), reproductive dysfunction
(sex differentiation)  and immune response
(functional  integrity  of  immune  cells).
Ultimately,  we plan  to use this transgenic
model to  study the pharmacokinetics  of
EDCs,  thereby  building  the  basis for
extrapolation of animal and in vitro test data
to humans.

We also are studying Ah receptor-mediated
mechanisms involved in the promotion of
endometriosis, a painful reproductive dis-
order in women and  a major  cause of
infertility. Several years ago, investigators
in our Reproductive  Toxicology  Division
developed  a  surgically-induced model of
endometriosis in the mouse.  We are now
using this model to investigate how  EDCs
promote  endometriosis and  impair  early
pregnancy.  Chemicals that mimic estrogen,
including dioxin and  dioxin-like compounds,
are being studied.  During FY96, we found
that chemicals  promote  the growth of
endometriotic lesions in a manner consistent
with  their relative  affinity  for  the Ah
receptor. Conversely, chemicals that do not
bind to the Ah receptor have no effect on
endometriotic lesions.   Plans  are  now
underway  to  develop  an in vitro human
endometrial cell line capable of  detecting
chemicals that disrupt uterine function.

In addition to receptor-based mechanisms of
toxicity, we also are studying non-receptor-
mediated  mechanisms   of  endocrine
disruption.   Using  female rodents, we are
investigating   the   effects   of  EDCs  on
ovulation. Estrogenic chemicals that affect
brain neurotransmitter  events are  being
evaluated for their impact on the hormonal
control of  ovulation.   Special attention is
being  paid to  the importance  of  nor-
adrenergic   transmitter  activity   in the
hypothalamus  and its involvement  in
luteinizing  hormone  secretion from the
pituitary,  which   represents  a  critical
endocrine event required for oocyte release.
During  FY95-96,   we  found that  when
ovulation is disrupted by an  EDC and the
egg is overripe  when  fertilized,  there  are
reductions in litter size and developmental
effects,  such as neural tube defects,  in
offspring.
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Neurotoxicity.   Mechanisms  involved  in
EDC-induced developmental  neurotoxicity
are  being  studied  by scientists in  our
Neurotoxicology Division.  The objective is
to examine the relationship between thyroid
hormone  disruption  and developmental
abnormalities of the central nervous system
(CNS). Chemicals selected for study include
dioxin, PCBs,  and PCB mixtures.  Experi-
ments conducted during FY94-95 showed
that  developmental  exposure  to  PCBs
caused persistent hearing  loss (ototoxicity)
in adult  rats; this effect was  associated
with  reduced  levels of circulating thyroid
hormones.  During FY96, we found that the
hearing deficits could be partly ameliorated
by replacing T4, a thyroid hormone, during
exposure. This suggests that PCB-induced
auditory deficits involve thyrotoxic actions.
We  plan  to conduct  additional tests  to
confirm whether decreases in T4 levels are
accompanied by ototoxicity. If so, this will
imply  that  there are  important non-Ah
receptor   mechanisms   underlying   the
neurotoxicity of some endocrine disruptors.

The effects of  endocrine  disruptors  on
regulation   of  body   temperature   and
metabolism, both of which are controlled by
the hypothalamus, also are being studied.
During FY95,  we found that exposing rats
to dioxin during early development reduced
their  core  temperature by-decreasing the
homeostatic set-point.   This  finding  is
important because it suggests that funda-
mental metabolic processes may be altered
by developmental exposure to endocrine
disruptors.    It  also  suggests  that the
hypothalamus, and possibly the thyroid/'are
potential target sites for endocrine-mediated
developmental neurotoxicity.  To  further
explore this possibility, we have designed a
series of tests to provide  basic information
regarding the role of endocrine disruptors,
including  thyroid  inhibitors,  in  thyroid
hormone production and brain development.
Effects  on   the  hypothalamic-pituitary-
adrenal axis of the brain,  on brain develop-
ment,  and on  learning and  memory  are
being evaluated in an effort to  elucidate  the
relationship    between   neuroendocrine
mechanisms and CNS function. Preliminary
results from  this  research  indicate that
interfering  with Jhe actions of  thyroid
hormone can  severely  compromise  brain
development, leading to smaller brain size,
cognitive deficits,  and  improper  develop-
ment of the auditory system.

Ecology. To help  predict risks to wildlife
from exposures to EDCs, NHEERL scientists
will initiate two large  projects  in FY97.
Investigators in our  Gulf Ecology  Division
will be conducting an integrated assessment
of endocrine disruptor effects on the  life
cycles of marine fish.   The goal of  this
research is to  develop well-characterized
model test  systems capable of analyzing a
variety of endocrine-mediated effects.   Our
research will focus upon tests that identify
specific reproductive, developmental,  and
physiological responses in the context of
complete life histories. In this  way, the  gap
between molecular definition, biochemical
action, and ecological consequence can be
bridged.  A suite of small-sized fishes  will
be observed following exposure to selected
EDCs, and effects on fertilization, growth,
spawning,  embryonic   development,   and
reproductive  success   will  be  recorded.
Behavioral as well as physiological functions
will be studied.

Also   in  FY97,  scientists   in  our  Mid-
Continent  Ecology  Division will  begin
developing in vitro test systems using cells
and tissues  from  aquatic  organisms to
assess critical steroid  receptor  systems.
Chemicals  shown  to  activate   receptor-
mediated   transcriptional  processes   in
mammalian tests will be evaluated for their
ability  to  activate  receptors  in aquatic
organisms.  The data will then be used to
determine  how closely mammalian tests
predict perturbations of steroid  receptor-
based processes in aquatic species. These
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tests   will   additionally   be   used  to
characterize various aspects of chemical
metablic  activation  and   bioavailability
related to reproductive and developmental
endpoints.

PHARMACOKINETIC STUDIES

Scientists in our Experimental Toxicology
Division  are  in  the  initial stages  of
developing a physiologically based pharma-
cokinetic  (PBPK)  model in  rodents that
focuses on critical periods of development
in the reproductive, immune, and central
nervous  systems.   During  FY95,  we
investigated    the    disposition    and
pharmacokinetics  of  dioxins in  the  adult
female rat, and in FY96 we published  our
PBPK model.  We are now  broadening  our
investigation to study pharmacokinetics in
pregnant and fetal rats. In FY97, we plan
to characterize  the tissue  distribution of
dioxin  from  the  maternal  to  the  fetal
compartment. When  developed, the  PBPK
model will  be extended to  humans  in an
effort to predict whether human fetal or
neonatal target  organs are  exposed to
potentially toxic levels of dioxins.

To enhance our  pharmacokinetic analyses,
we  are conducting  studies using  two
transgenic  mouse models,  each of which
lacks  a different  receptor system for EDCs.
One  model, discussed in  the  preceding
section, lacks the Ah receptor.  The  other
lacks the CYP1A2 receptor, which is  the
binding protein  for  dioxin  in  the  liver.
Without this protein, dioxin fails to bind to
liver tissue, which affects both pharmaco-
kinetics and resulting toxicity.  Development
of  the  CYP1A2  "knockout"  model  was
initiated during FY95-96, and in FY96 we
began   our  pharmacokinetic   studies.
Comparisons  between  transgenic   and
normal  mouse models  will enable  us to
better understand the pharmacokinetics of
endocrine disruptors, thereby facilitating the
development of more  reliable risk models.
Because aquatic organisms ingest contami-
nants via food, their  water milieu,  and
sediments,  it  is important to  know  the
relative  amount of chemical accumulation
from each exposure route in order to esti-
mate total dose and effect.  During FY94-
95, we began a series of studies evaluating
the uptake and elimination  kinetics of dioxin
in  medaka, a small fish species. Thusfar,
our studies have predicted a steady-state
bioconcentration factor from water to fish
tissue of over 500,000.  This value is much
higher   than  previously  reported  in  the
literature, suggesting a greater potential for
dioxin   accumulation   than   previously
estimated. We are now examining sediment
contaminant   bioavailability  and  dietary
contributions in an effort to more accurately
predict  bioaccumulation  and  toxicological
effect.

MIXTURES

To improve our ability to  assess the risk of
exposure to mixtures of EDCs, scientists in
our Experimental Toxicology Division have
conducted a study in which  human body
burdens of dioxins and dioxin-like congeners
(dibenzofurans and PCBs) were estimated.
During  FY95,  we determined the relative
potencies of  the  congeners using  sub-
chronic studies in experimental animals and
estimated the  total toxic  equivalency.  We
then compared the body burdens of dioxins
that produce effects in experimental animals
to body burdens associated with effects in
humans.  The TEF method  was used to
calculate body  budens of dioxins in humans.
We found that for effects that have been
clearly  associated with dioxin  exposures,
such  as chloracne  and  the  induction of
CYP1A1, humans and  animals  respond at
similar body burdens. These and additional
estimations  were central to the Agency's
Dioxin Reassessment, enabling  predictions
of  risk posed by the  levels of exposure
experienced by the general population.
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Investigators in our Mid-Continent Ecology
Division also are evaluating the feasibility of
using  TEFs  for  assessing the  risk  of
chemical mixtures. Chemicals that share a
common mode of action  (congeners  of
polychlorinated dibenzodioxins, furans, and
PCBs,  all of which are thought to act
through the Ah receptor) were selected for
study. W« are testing these chemicals for
toxicity  using  an ecologically  relevant
endpoint:  early   lifestage  mortality  in
rainbow trout.  From the results,  we have
established  tentative  TEFs  and  have
compared  these  values   to   TEFs  for
mammalian  species.   During  FY95,  we
found that the TEFs for dioxins and furans
in  fish are similar to those in mammalian
species.  However, the potency of some
PCBs is lower in fish relative to  mammals.
This suggests that inter-species differences
should  be  taken  into   account  when
assessing the risk of exposure to mixtures
of endocrine disrupters.
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National Health and Environmental Effects Research Laboratory
        DRINKING WATER
       HEALTH EFFECTS RESEARCH
             ANNUAL REPORT




             OCTOBER, 1996

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                          CONTENTS
Introduction	3

Summary of the Drinking Water Health Effects Research Program  	4

FY95-96 Highlights  	6

Waterborne Pathogens Research Program	7
          Epidemiology Studies
          Clinical Studies

Disinfection By-Products Research Program	9
          Human Studies
          Toxicology Studies
          DBP Mixtures

Other Priority Contaminants Research Program   	14
          Arsenic
          Aluminum

Conclusion  	15
NHEERL DRINKING WATER ANNUAL REPORT 7996

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                       INTRODUCTION
pp.pp.OSE;,
The purpose of this report is to communicate results from the Drinking Water Health
Effects Research Program of EPA's National Health and Environmental Effects
Research Laboratory (NHEERL).
The report contains

      • a summary of the NHEERL Drinking Water Research Program, including an
      explanation of its regulatory and programmatic context, its overall goal, the
      rationale for the program, and the research strategy

      • a  section that  highlights recent accomplishments (FY95-96 Program
      Highlights)

      • a description of the NHEERL Drinking Water Research Program, by program
      area, including a summary of research findings and anticipated progress for the
      near future.
COMMENTS WELCOME

The format of this report is still evolving, and we welcome feedback. Readers with
comments or requests for further information are encouraged to contact

      Fred Hauchman, Assistant Laboratory Director
      National Health and Environmental Effects Research Laboratory (MD-51A)
      U.S. EPA
      Research Triangle Park, N.C. 27711

      Phone: (919) 541-3893 or FAX: (919) 541-0642
      E-mail: HAUCHMAN.FRED@EPAMAIL.EPA.GOV
NHEERL DRINKING WATER ANNUAL REPORT 1996

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                        DRINKING WATER
          RESEARCH  PROGRAM  SUMMARY
REGULATORY AND PROGRAMMATIC
The  Safe Drinking  Water  Act  (SDWA)
requires  EPA  to identify  and  regulate
drinking water contaminants that may pose
a risk to human health.  EPA's rule-making
activities are supported by its Office of
Research and Development  (ORD),  which
maintains  a  multidisciplinary   research
program in drinking water.   Research on
health  effects is conducted by NHEERL, a
research arm of ORD.   NHEERL  provides
critical scientific data, methods, and models
that  address major uncertainties in the
Agency's  risk  assessment  process for
drinking water, leading to more scientifically
sound, cost-effective regulations.  Health
research on drinking water contaminants at
NHEERL is part of a multidisciplinary, multi-
Laboratory/Center research program in ORD
that   includes   the  National  Exposure
Research  Laboratory,  the  National  Risk
Management Research  Laboratory,  the
National   Center   for   Environmental
Assessment, and the National Center for
Environmental   Research   and  Quality
Assurance.
To ensure that sound scientific information
is available to characterize the nature and
magnitude  of the health  risks  posed by
microbial   pathogens,   disinfection  by-
products (DBPs), and other priority drinking
water contaminants.

lATIQNAfJE    *
For almost 100 years, public water supplies
have been treated with disinfectants, such
as chlorine, to reduce the risk of infectious
disease from waterborne pathogens.  Water
disinfection has been  highly effective in
reducing the incidence  of certain diseases,
such as cholera and typhoid.  However, the
continued occurrence of waterborne disease
outbreaks demonstrates that contamination
of drinking water with pathogenic bacteria,
viruses, and parasites still poses a health
risk when treatment is inadequate. The use
of disinfectants, while reducing microbial
risks, creates new potential  risks  as
chemical by-products are formed during the
treatment  process.    Some  of   these
disinfection by-products (DBPs) have been
shown to cause cancer and  other  toxic
effects in  experimental animals.  In humans,
however,  the scientific evidence of adverse
effects is  inconclusive. Research is required
to obtain  sufficient  understanding  of the
health  risks posed  by these  and   other
drinking water contaminants.
EPA has  conducted health  research on
drinking water since the 1970s.  Research
has addressed an array of contaminants,
including inorganics such as lead, industrial
solvents such as trichloroethylene, microbial
pathogens, and DBPs. The risk assessment
paradigm  of  the  National  Academy  of
Sciences  (NAS), which  consists of four
fundamental steps  (hazard  identification,
dose-response   assessment,    exposure
assessment,  and  risk  characterization),
provides the research context for NHEERL's
drinking water research program.  Emphasis
is placed on health research in two areas of
the risk assessment paradigm:

       •  Research in the area of hazard
identification  focuses on the development
of methods and data that can demonstrate
an  association  between  exposure  and
NHEERL DRINKING WATER ANNUAL REPORT 1996

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effects.
       •    Research   supporting  dose-
response assessment seeks to explain the
events  that  link  exposure and  effects.
These events  form  the  basis  for  the
predictive models used to quantify risk.

NHEERL's  research  program  in  drinking
water includes toxicological  studies  in
experimental animals,  clinical  studies  in
human volunteers, and epidemiology studies
in  selected   communities  to   resolve
important  drinking  water issues.    The
toxicological   studies  are  designed  to
characterize  the  various endpoints  of
potential concern (e.g., cancer, reproductive
toxicity, neurotoxicity), and to evalute the
biological  processes by which key drinking
water contaminants cause their  effects.
This information is then  used to support the
development of biologically based dose-
response (BBDR) and physiologically based
pharmacokinetic (PBPK) models of human
response for the highest priority contami-
nants.  The human studies, on the other
hand, are designed to improve the tools we
use in epidemiology studies and to improve
our understanding of waterborne disease
outbreaks and potential chemical-associated
health risks.  Drinking water contaminants
under   study   include:   WATERBORNE
PATHOGENS, such  as Norwalk virus and
Cryptosporidium; priority DISINFECTION BY-
PRODUCTS,  such   as  haloacids  and
trihalomethanes;  and  OTHER  PRIORITY
CONTAMINANTS, such as arsenic, which
occurs naturally in some source waters, and
aluminum,  which  is widely used  as  a
coagulant in water treatment.  During FY95-
96, a research plan for Microbial Pathogens
and  Disinfection  By-Products in  Drinking
Water was developed by ORD. This plan is
being used by NHEERL as a blueprint for its
microbial and DBP health effects research
program. An ORD research plan for arsenic
is presently under development.
NHEERL DRINKING WATER ANNUAL REPORT 1996

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     NHEERL  DRINKING WATER RESEARCH
          FY95-96 PROGRAM HIGHLIGHTS
The goal of this research is to characterize the human health risks associated with
exposure to waterborne pathogens found in drinking water.

      *• We made important advances in the development of serological tests that
      detect infection by Cryptosporidium. These tests will be used in drinking water
      epidemiology studies to help characterize population exposures to  this
      important pathogen.
The goal of this research is to develop data, methods and models to support
assessments of the health effects caused by individual DBFs and mixtures of DBFs.

      *• Our studies demonstrated for the first time that dichloroacetic acid is a
      hepatocarcinogen in the rat.

      *• We showed that dichloroacetic acid induces a unique ras oncogene mutation
      in hepatocellular carcinomas in rodents.  This information may help elucidate
      the mechanism involved in the activation of the cancer process for this
      chemical.

      *• We discovered a novel, glutathione-mediated metabolic pathway  for
      bromodichloromethane that leads to  the generation of genetically active
      metabolites.  This important finding may help characterize  the genotoxic
      mechanism involved in  the carcinogenicity of this DBF.

      *• We implemented a number of the research recommendations of a 1993
      expert panel workshop and a 1995 work-in-progress workshop on potential
      reproductive and developmental effects of DBFs.

OTHEH PBKWTY CONTAMINANTS f^SEAKCHJPS 141-
The goal of this research is to  assess the toxicity of arsenic, a contaminant found in
some  source waters, and aluminum, a widely  used coagulant for removing solids
during water treatment.

      >  Our studies showed that glutathione is critical to a metabolic pathway
      leading to the detoxification of arsenic.  This finding is an important step in
      understanding the factors that affect the variable sensitivity of humans to
      arsenic.
NHEERL DRINKING WATER ANNUAL REPORT 1996

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                WATERBORNE PATHOGENS
                     RESEARCH PROGRAM
ISSUE
What are the human health risks associated
with exposure to microbial pathogens found
in drinking water?
Although drinking water disinfection has
been highly effective in reducing the risk of
certain  waterborne  diseases,  microbial
pathogens   continue to cause occasional
disease  outbreaks  when  treatment  is
inadequate.  While the disease symptoms
caused by pathogens are generally known,
limited information is available on the doses
and conditions that produce disease.
NHEERL's research  program on  drinking
water pathogens is designed to improve our
understanding   of  waterborne  disease
outbreaks   in   the  U.S.   population.
Accordingly,    we    are    conducting
EPIDEMIOLOGY STUDIES to  evaluate the
causes   of  waterborne   illnesses,  the
magnitude of risk, and the impact of water
treatment alternatives and  source  water
quality on  disease  rates. - We also are
performing CLINICAL  STUDIES to determine
the virulence (or  infectious dose)  of key
pathogens and the impact of host factors,
such as immune status, on infection.
EPIDEMIOLOGY STUDIES

Health effects associated with differences in
source water quality and treatment process.
In  FY93, NHEERL  embarked on  a  multi-
phased study to examine the  impact of
source  water  characteristics  and  water
filtration  on the incidence of  waterborne
disease. The basic study design involves a
comparison  of  the  health  status  of
individuals before and after the addition of
filtration  units  in  the  home or  at  the
treatment plant.  In the first phase of this
research   effort   (FY93-94),    NHEERL
conducted a survey to identify communities
planning to upgrade their treatment plants
via  filtration.   Based on the survey, we
selected  eight  study  sites  for  possible
inclusion  in the project; an additional  10
were targeted for study in  later years.  In
the second phase of the research effort,
completed during FY95, data were collected
on  source water quality parameters  and
filter installation schedules. In FY96, we
gathered   information   on   community
demographics, completed the site selection
process,  and  initiated a pilot test of  an
epidemiological study design.

NHEERL and several outside organizations
are assisting  EPA's  National  Exposure
Research Laboratory (NERL) in an analysis
of the  impact of water quality, treatment
process,    and    distribution     system
contamination  on  endemic   waterborne
disease rates  in a community in  Quebec,
Canada.     A   35%   excess   risk   of
gastrointestinal illness was  reported in this
community in a previous investigation. The
objectives of the study are to confirm or
refute  the original  reports of  illness,  to
determine the source of the illness, and to
find suitable indicators of the health effects.
Participating households have been grouped
according  to   different  water  treatment
practices,  and in FY95, disease rates for
these household groups were examined. A
final report on the  project  is expected in
FY97.
NHEERL DRINKING WATER ANNUAL REPORT 1996

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Immunological   assays    for    use   in
epidemiology studies.  NHEERL is exploring
the possible use of serological tests in field
studies of  waterborne infectious disease.
At  issue is whether immunoiogica!  tests,
which can detect antibodies to pathogens in
the serum  of exposed individuals, can be
used   as  a  tool  to   characterize the
prevalence of waterborne disease.  In FY93,
a  collaborative effort involving  NHEERL,
NERL,  the Centers for Disease Control and
Prevention (CDC), and the Lovelace Medical
Foundation  was initiated.  We compared the
ability  of two analytical  tests (ELISA and
Western Blot) to characterize the  immune
response to infection by Cryptosporidium, a
protozoan parasite responsible for several
serious outbreaks in the  U.S.  in recent
years.  Sera collected during a 1992 disease
outbreak in  Oregon  were tested, and results
showed  that  the  Western  Blot  was the
preferred method for identifying  cases  of
cryptosporidiosis. Full-scale serosurveys are
now underway at both the community and
national  levels to examine the  risk  of
infection by Cryptosporidium as a function
of water treatment method, source  water
quality and demographics.

Waterborne   disease   surveillance   and
reporting.  Since 1971, EPA and the CDC
have compiled information on waterborne
disease  outbreaks  in  the  U.S.    This
surveillance program  provides  important
information  on   deficiencies  in   water
systems and on etiologic agents associated
with  outbreaks.    During  FY94-95, we
developed  and presented a training course
on  surveillance   and   investigation  of
waterborne disease outbreaks.  In FY96, a
report  characterizing the status of reported
waterborne disease in the U.S. for the years
1993-1994 was published, and a summary
of waterborne disease surveillance activities
in the U.S. was published.
CLINICAL STUDIES

Infectious dose of  Norwalk virus.   The
Norwalk  virus,  which  produces  gastro-
enteritis,  is believed to be responsible for
numerous waterborne disease outbreaks in
the U.S. In Phase I of this project, begun in
FY94, 45 adult volunteers were exposed to
various  doses  of Norwalk  virus  in  an
attempt to characterize the virulence of the
pathogen  and   evaluate  the impact of
immune status on infection  and  disease.
After dosing, each individual was evaluated
for clinical symptoms of gastroenteritis,
shedding of virus in the stool, and immune
response (sero-conversion).   Results from
Phase I are being used to characterize the
dose-response  of  the virus, which  will
enable us to expand our research efforts to
a larger study population and examine in
greater detail the range of outcomes at low
doses of the virus (Phase II).

Infectious dose of Cryptosporidium. EPA's
National  Exposure  Research  Laboratory,
with assistance from NHEERL, is conducting
a  clinical study of  Cryptosporidium to
determine the  infectious  dose   of  this
important waterborne pathogen.  In FY94,
scientists  at  the  University  of  Texas
exposed volunteers to Cryptosporidium, and
infectivity  and  immune  response  were
evaluated.   Doses as low as 30  parasite
oocysts were shown to cause infection in
humans,   and   symptom occurrence  and
disease severity were found to be unrelated
to dose.  In FY95--one year after the initial
exposure—individuals  were   re-challenged
with the parasite to examine the  ways in
which   additional  exposure  modulates
response.   Results   currently  are being
analyzed,  and  in FY97 an assessment will
be made  of the possible protection from
reinfection offered  by  an initial  parasite
challenge.
NHEERL DRINKING WATER ANNUAL REPORT 1996
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              DISINFECTION  BY-PRODUCTS
                      RESEARCH  PROGRAM
 What are the toxic effects associated with
 disinfection by-products, and what are the
 toxicologic bases for these adverse effects?
 Significant gaps exist in our knowledge of
 the adverse health effects caused by DBFs
 in drinking water.  Several epidemiology
 studies   have   suggested    possible
 associations    between   exposure    to
 disinfected water and  cancer or adverse
 reproductive  outcomes, but the findings
 have been inconsistent and causality has
 not been established.  Toxicological studies
 using experimental animals have shown that
 a   number   of  DBFs  cause   cancer,
 reproductive toxicity, and other effects, but
 the effects occur at concentrations higher
 than those typically found in drinking water.
 Moreover,  the toxicity of  many DBFs
 remains unknown or poorly characterized.
 Accurate assessments of the risks posed by
 DBFs and an understanding of the biological
 basis for observed effects are needed.

 PROGRAM  DESCRIPTION

 NHEERL  is   conducting  health  effects
 research on  DBFs in three distinct areas.
 HUMAN STUDIES are providing new data to
 characterize community risks, improve tools
 for  epidemiology studies,  and  advance
 methods for managing health and exposure
 data. TOXICOLOGY STUDIES in laboratory
 animals are  providing information on the
 toxicity (carcinogenic,   neurotoxic,   and
 reproductive/  developmental  effects)  of
 individual  DBFs and on the biological  and
 physiological processes involved in the toxic
 response.   These data are then used to
 support the development and  evaluation of
'predictive models of effect.  Finally, we are
evaluating ways in  which MIXTURES of
DBFs  can affect toxicity.   This  area of
research  addresses the adequacy  of the
additivity   assumption   used   in  risk
assessments    of    drinking    water
contaminants.

HUMAN STUDIES
Development  of improved  tools for field
research.  NHEERL is supporting work to
develop and validate models to improve
estimates  of  exposure  in  epidemiology
studies, thereby enhancing our ability to
relate exposure to effects. A mathematical
model  for predicting individual  household
exposure to trihalomethanes (THMs) based
on water  treatment process, distribution
system characteristics, and water transit
time  was evaluated in FY95.  The model
was used to estimate exposure at different
points  along the distribution system.   A
report is currently being drafted.

Improving methods for managing health and
exposure  data.   The utility  of  a data
management  system  (called Geographic
Information Systems, or CIS) for studying
the  potential  impact  of   DBFs   on
reproductive  health  is  currently  being
analyzed. A pilot study using GIS has been
conducted in  two  Colorado communities
that  use  either  chlorination  or  chlor-
amination  for water  disinfection.   The
relationship between adverse reproductive
outcomes  (specifically, low birth  weight)
and the concentration of residual chlorine or
other parameters at various points along the
distribution system is being analyzed.   In
FY95, statistics on health outcomes were
NHEERL DRINKING WATER ANNUAL REPORT 1996

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transformed   into   GIS   format,   and
preliminary epidemiologic analyses  were
conducted  in the chlorinated community.
During FY96, distribution system modeling
data and health information were integrated
into the  analysis.   A final report  on this
project is expected in FY97.

NHEERL  also is addressing  methodologic
issues involving the merger of databases.
National  databases on  health  (National
Maternal  and Child Health Survey, 1988)
and exposure (Federal Database Reporting
System)  are being  used in our study.  In
FY95, in  collaboration with EPA's National
Center  for  Environmental   Assessment,
scientists began to merge information on
adverse reproductive outcomes (e.g., fetal
death and low birth weight) with exposure
data  (MCL violations for 1987-1989)  to
isolate possible associations.  A final report
on this analysis is anticipated  in FY97.

Support for Ongoing Studies.  The State of
California,  with partial  EPA  support,  is
conducting a prospective study of drinking
water and  spontaneous abortions.  Three
types of information will be used to assess
potential risks:   1) quarterly  reports  of
drinking  water utilities; 2) self reports  of
subjects; and 3) analysis of DBPs based on
drinking  water samples from the  tap.  A
final report is expected early"in FY97.

The State of New Jersey also is conducting
a study  with partial  EPA support to re-
examine    the    findings   of    earlier
investigations that suggested an association
between neural  tube defects  and elevated
levels of trihalomethanes,  nitrates,  and
certain volatile solvents. This study is using
refined methods, such  as biomarkers,  to
relate exposure and effects.  A final report
will be submitted in FY97.

Workshops.   In FY93, NHEERL and  the
International  Life Sciences Institute (ILSI)
convened  an  expert  panel  to  review
published epidemiologic and experimental
data  on  the  reproductive/developmental
effects  of DBPs  and to  help  guide the
development of a research strategy.  The
expert  panel  concluded  that  currently
available data provide an inadequate basis
for identifying DBPs as a reproductive or
developmental hazard, but  that specific
types of research could be  conducted to
strengthen the scientific basis for such an
assessment. Since this workshop, NHEERL
has implemented a number of the research
recommendations of the panel in the areas
of epidemiology methods development and
toxicology studies on individual  DBPs. In
FY95, a drinking water reproductive effects
work-in-progress  workshop  was held to
review ongoing laboratory and field studies
being conducted  by NHEERL and outside
groups.   It was  noted that considerable
progress had been made in  improving the
state-of-the-science   since   the   FY93
workshop.

TOXICOLOGY STUDIES

Reproductive and Developmental Toxicity.
Reproductive screening studies of DBPs are
being conducted  both intramurally  and in
collaboration with the National Toxicology
Program (NTP) at the National Institute of
Environmental Health Sciences (NIEHS). A
wide range of chlorinated,  brominated, and
chlorobrominated  by-products  are  being
evaluated in male and female rodents.  One
DBP of particular interest  is  dihromnargtir
acid,  which has  been shown  to produce
effects  on both  the male  reproductive
system  and  the  developing   fetus  at
experimental exposure levels.    We are
performing  a thorough  evaluation  of the
reproductive and developmental toxicity of
this chemical in the male  rodent.  Studies
utilize a  broad  range of exposures and
multiple    reproductive    assessments.
Observations have included alterations in
NHEERL DRINKING WATER ANNUAL REPORT 1996
                                                                                 10

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sperm morphology and motility, as well as
in  mating   and   spermatogenesis,  at
experimental  doses.   Research  is now
underway  to determine  whether  these
effects  are  the  result of  compromised
endocrine balance.  Findings were published
in FY95 on reproductive competence and
sperm  quality.     We  are   presently
investigating  developmental  mechanisms
that may be involved in DBF-induced neural
tube and heart defects.  Specifically, we are
attempting to  determine  whether these
effects may be a consequence of changes
in protein  kinase activity during critical
stages of embryonic  development.  Also
during    FY95,    reproductive    and
developmental toxicity studies showed that
experimental   doses   of   hrnmate   and
diphlnrnhrnmnmfithanfi reduce sperm counts
in  rodents.   These  chemicals  are now
scheduled  for more  rigorous testing  for
effects on  fertility. Finally,  we evaluated
reproductive function in rodents exposed to
brnmndinhlnromftthane  and   found   a
significant impairment in sperm motility.

To   prioritize    chemicals    for   more
comprehensive testing in vivo  (and to
hypothesize  a   mechanistic   basis  for
developmental toxicity), we  conducted a
Quantitative Structure-Activity Relationship
(QSAR) study during  FY95 on a  series of
mono-, di-, and tri-haloacetic acids.  QSAR
analyzes the relationship between  key
structural properties of a chemical and its
outcome in toxicity tests. We are  focusing
Specifically on dinhlnrnanetir aniri (DCA) and
its chloro/bromo analogues, which  we have
shown to be embryotoxic in the in vitro
whole mouse  embryo  culture  system.
QSAR modeling  is being carried out in
tandern with ongoing experimental studies.
Our   results   in   FY95  showed   that
lipophilicity and electronic properties are
important determinants in  the induction of
neural tube  defects.   Based  on  these
findings,  we  are conducting   in   vivo
developmental screening tests in  which
comprehensive  fetal evaluations  (assess-
ments  of  soft   tissue  and  skeletal
abnormalities) are being performed.

Carcinogenicity.     Tests  conducted  by
NHEERL  during  FY94-95  showed that
hrnmpriirhlnrnmethane   induced  hepatO-
cellular (liver) cancer, but only at the lowest
dose tested. Studies are now underway to
try to explain this unexpected finding.   A
chronic bioassay using potassium  hrnmate
was   completed   in  FY95,  and  our
pathological analyses confirmed reports by
others that bromate induces cancer of the
kidney, thyroid,   mesentery,  and  large
intestine. Mechanistic studies are planned,
with an emphasis on the ability of  bromate
to induce  oxidative damage in  DMA, alter
thyroid function, and affect cell proliferation
and  death.   Finally, one of  our  most
important  findings  in this research area
involves diphlornarptir: arid.  During FY95,
we demonstrated for the first time ever that
DCA is a hepatocarcinogen in rats.

Several priority DBFs will be  evaluated  by
the NTP in their two year (chronic) cancer
bioassay program in rodents.  This research
will  be conducted  in  collaboration with
scientists  from  NHEERL   In  FY95, we
initiated a subchronic carcinogenicity study
of sodium  chlorate  in rodents to assist  in
the design  and implementation of a chronic
bioassay,   which  is scheduled   to  be
performed by NTP in FY97.

Neurotoxicity. During FY95, we completed
a series of experiments  in rodents on the
neurotoxic  effects  of riinhlnrnarptic arid
(DCA), an  important haloacid  commonly
found in disinfected drinking water.  These
studies involved  acute,  subchronic, and
chronic exposure  of male rats to DCA.
Following  acute  exposure,  effects  were
negligible;  however, following subchronic
and   chronic   exposures,   there  was
NHEERL DRINKING WATER ANNUAL REPORT 1996
                                   11

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preliminary evidence of neurotoxicity (limb
weakness  and  deficits  in  gait).    High
concentrations of DCA produced hindlimb
paralysis,  and although neuropathological
assessments indicated localized damage to
the spinal  cord, no damage to the nerve
that innervates the hindlimbs was observed.
A  more  extensive  assessment  of  the
neuropathological effects is in progress to
correlate  nervous system damage  with
behavioral  effects.  This  analysis  will be
completed in FY97.

Biologically  based  dose-reponse  (BBDR)
models. NHEERL is actively engaged in the
development of  BBDR  models to help
explain the biological/physiological events
involved in toxic responses  to  drinking
water contaminants. In one project, we are
developing a BBDR model for carcinogenesis
by  conducting  tumor  studies in rodents
while      simultaneously    measuring
biochemical,      immunohistological,
pathological,   genetic,  and   mechanistic
endpoints.   Special  attention has  been
focused on dinhlnrnap.etir.  arid (DCA) in an
effort  to explain  the progression of DCA-
induced liver lesions and  cancer.  During
FY95,  we helped define several  critical
parameters required for a  BBDR model for
DCA. In studies of ras oncogene activation
(which can initiate the cancer process)  in
the liver cells of  mice, we showed that a
unique mutation occurs in response to DCA.
This finding is significant because it may be
possible to use this "fingerprint" of genetic
damage to demonstrate exposure to DCA
and to explain a possible mechanism(s) for
carcinogenesis.  Our research on apoptosis
(programmed cell death) showed that DCA
suppresses cell  death in  liver cells, thus
increasing the  number of cells at risk  of
transformation and possibly enhancing the
chances for  tumorigenesis.   And finally,
research  in FY95 examining  the  role  of
endocrine disruption in DCA induction  of
liver  cancer  in   rodents has  indicated
alteration  of  steroid hormone levels and
changes in steroid metabolism and receptor
activity.   Our model development is  an
ongoing   effort,   and   as   additional
mechanistic results become available, we
will integrate these data with the tumor
data to develop  a  mathematical construct
that represents a BBDR model for humans.

Physiologically   based   pharmacokinetic
(PBPK)  models.   PBPK  models,  which
describe the behavior of a chemical as it is
metabolized,  distributed to target  organs
and tissues, and  eliminated from the body,
are being developed by NHEERL to facilitate
the extrapolation  of  toxicity data  from
animals to humans. In FY95,  considerable
progress  was   made  to   support  the
development   of  a   PBPK  model  for
hrnmnriirhlnrqmethanfj (BDCM). Parameters
being  studied   include  tissue  partition
coefficients,   primary   pathways   of
metabolism, metabolic rate constants, and
macromolecular  binding in target organs.
During FY95, we  found that glutathione
plays an important protective role in acute
BDCM toxicity in rodents, presumably due
to macromolecular binding.  In addition, one
of our  most  important findings was the
discovery of a novel, glutathione-mediated
metabolic pathway for BDCM  that leads to
the   generation    of   genetically   active
metabolites. Though believed to be a minor
pathway, this finding is significant because
it  suggests  there  may  be  a  genotoxic
mechanism   of   action    for   BDCM
carcinogenicity that is different from that of
chloroform,  another  important   trihalo-
methane.     Ongoing  studies   are  also
examining the involvement of cytochrome
P450 isozymes on BDCM metabolism and
toxicity.  An initial  PBPK model is expected
in FY97, and in subsequent years, we hope
to    characterize    human    brominated
trihalomethane  metabolism to  refine our
model.
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DBP MIXTURES

Drinking water invariably contains mixtures
of  DBFs.   In an  effort to  improve our
understanding of the interactions that may
occur  in  these  mixtures,  NHEERL   is
investigating binary and quaternary mixtures
of DBFs.  The first class of drinking water
contaminants  selected for testing  is the
trihalomethanes (THMs).  In a collaborative
effort with the EPA's National  Center for
Environmental   Assessment,    we   are
examining  hepatotoxicity  using various
combinations of THMs.  A matrix of dose
levels has been constructed so that effects
can be observed at different combinations
of dose.  Experiments on binary mixtures
(BDCM  and chloroform)  were initiated  in
FY95 in rodents,  and results are  in the
process of being analyzed. In subsequent
years, other binary combinations of THMs
will be assessed.  For those mixtures that
exhibit synergistic effects in  experimental
animals,     pharmacokinetic    and
pharmacodynamic studies will be conducted
to elucidate underlying mechanism(s). We
also have begun  to  study additivity for
mixtures   of   four   THMs   (BDCM,
chlorodibromomethane,  bromoform, and
chloroform).    We  are  predicting  the
response of the  mixture a priori based  on
additivity of the dose-response curves for
each individual chemical; the response of
the   mixture   is   then    determined
experimentally  and   compared  to  the
expected response.  We initiated the single-
chemical experiments in FY95, and, based
on the dose-response information obtained
from these studies, we began our mixtures
experiments in  FY96.  The purpose  is to
evaluate  the adequacy of the  additivity
assumption for dose and effect.

Additional  mixture  studies  have  been
conducted by NHEERL in collaboration with
EPA's National Risk Management Research
Laboratory (NRMRL)  using a simple assay
for   genetic  toxicity.     The   use  of
genotoxicity  testing  in  evaluations  of
alternative disinfectants received  increased
attention  recently when  Finnish  studies
demonstrated a positive correlation between
the mutagenicity of chlorinated drinking
water and certain human cancers.  In FY94,
drinking water samples were  collected by
NRMRL from treatment plants utilizing a
variety  of  water disinfection  practices
(ozonation, chloramination, chlorination, and
treatment with chlorine dioxide).  In FY95,
scientists  from  NRMRL  and   NHEERL
analyzed  the samples  for mutagenicity.
Ozonation did not enhance the mutagenic
potency of raw water, but treatment with
either  chlorine   or  chloramine  greatly
increased  mutagenic  potency.     Using
molecular techniques,  we then identified for
the first  time the spectrum of mutations
produced by these samples.  We found that
the mutation spectra  of the drinking water
samples resembled the spectrum  produced
by  MX,   a  compound  present  at  low
concentrations   in  drinking   water  but
extremely important  to its mutagenicity.
This indicates that the  mutation  spectrum
produced by a complex mixture reflects the
dominance of one (or a few) class(es) of
chemicals within the mixture.
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         OTHER  PRIORITY CONTAMINANTS
                     RESEARCH  PROGRAM
ISSUE
What health effects result from exposure to
arsenic and aluminum in drinking water?
Arsenic, a naturally occurring source water
contaminant,  is  important from  both  a
regulatory and public health  perspective.
Several studies have linked arsenic-tainted
drinking water to cancers  of the skin and
internal  organs,  such  as  the  bladder.
Aluminum compounds, which are used as
coagulants for removing solids during water
treatment, are generally considered safe at
levels found in drinking water.  However,
concerns have been raised regarding the
possible  involvement   of  aluminum  in
neurodegenerative   diseases,   such   as
Alzheimer's or Parkinson's  disease.
           , DESCRIPTION
The objective of this research program is to
provide  quantitative toxicity data on the
adverse  health effects caused by ARSENIC
and ALUMINUM.   We are  studying the
metabolism of arsenic and the biochemical
mechanisms involved in its toxicity to help
explain  how it induces cancer and other
effects  in humans.  For aluminum, our
research is  aimed  at  understanding the
modes of action involved in neurotoxicity.
ARSENIC
NHEERL is addressing several key scientific
issues that impact the risk assessment for
arsenic in drinking water. Studies are being
conducted to provide a better understanding
of  the   dose-response  relationship  for
arsenic,  the relationship between arsenic
metabolism and toxicity, and the factors
that may affect the variable sensitivity of
humans    to    arsenic.        Recent
accomplishments  in this  area include the
development   of   improved   analytical
methods for the study of arsenic disposition
and the development of an in vitro system
to characterize its  methylation.  During
FY94, we studied the enzymatic basis of
methylation to evaluate factors that may
influence inter-individual variation in arsenic
metabolism.    In  FY95,  we  published
important  information  on the  role  of
glutathione  in   the  methylation   and
detoxification of arsenic.  We currently are
drafting major portions of a research plan
for ORD to guide future work in this area.

ALUMINUM
A review  of the literature conducted in
FY95 by our laboratory concluded that the
association  between   aluminum   and
Alzheimer's disease  is   weak.  Although
aluminum is neurotoxic, no mechanism has
been offered to explain the wide variety of
effects observed  in experimental animals.
We are therefore conducting research to
identify biochemical mechanisms of action
and to develop data for use in BBDR models
to explain aluminum-induced neurotoxicity
in humans.   In  FY95,  we showed that
aluminum   alters   signal  transduction
mechanisms in brain tissue in vitro. These
results  may  help  us  understand  how
aluminum  causes deficits in learning and
memory.  We also showed during FY95 that
aluminum per se  does not  alter long-term
potentiation, indicating that aluminum may
have more general, non-specific effects on
brain   function.    Future  research  will
examine  behavioral  and  neurochemical
changes in vivo  following developmental
exposure to aluminum in drinking water.
NHEERL DRINKING WATER ANNUAL REPORT 1996
                                                                             14

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                         CONCLUSIONS
The accomplishments of NHEERL scientists on the health effects of drinking water
contaminants discussed in this report reflect the considerable progress that has been
made in improving the scientific basis for decision-making. These advances also lay
the foundation for  future progress in resolving key uncertainties in  health risk
assessment.  It is through the implementation of a multidisciplinary, mission-oriented
drinking water research program, which  emphasizes the study of high risk, high
uncertainty public health problems of relevance to EPA's Office of Water, that our
goals are being achieved.
NHEERL DRINKING WATER ANNUAL REPORT 1996
15

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National Health and Environmental Effects Research Laboratory
             AIR TOXICS
         HEALTH EFFECTS RESEARCH
              ANNUAL REPORT




             SEPTEMBER, 1996

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                           CONTENTS
Introduction	3

Summary of the Air Toxics Health Effects Research Program  	4

FY95 Highlights 	6

Air Toxics Hazard Identification Research Program	7
           Molecular/Biologic Methods
           Health Databases

Air Toxics Dose-Response Assessment Research Program  	9
           Exposure-Response Relationships
                 Respiratory Toxicity (Phosgene)
                 Neurotoxicity (Trichloroethylene)
                 Developmental Toxicity (Methanol)
                 Cancer (POM/PAHs)
           Risk Models
           Mixtures

Air Toxics Problem-Specific Studies Research Program	 13
           MTBE
NHEERL AIR TOXICS ANNUAL REPORT 1995

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                        INTRODUCTION
The purpose of this report is to communicate results from the Air Toxics Research
Program of EPA's National  Health and Environmental Effects Research Laboratory
(NHEERL).
The report contains

      •  a summary of the  NHEERL Air Toxics Research Program, including an
      explanation of its regulatory and programmatic context, its overall goal, the
      rationale for the program, and the research strategy

      • a section which highlights recent key findings (FY95 Program Highlights)

      • a description of the NHEERL Air Toxics Research Program, by program area,
      including a summary of research accomplishments and anticipated progress for
      the near future.
COMMENTS WELCOME                                     {j-n

The format of this report is still evolving, and we welcome feedback. Readers with
comments or requests for  further information are encouraged to contact:

      John Vandenberg, Assistant Laboratory Director
      National Health and  Environmental Effects Research Laboratory (MD-51A)
      U.S. EPA
      Research Triangle Park, N.C. 27711

      Phone: (919) 541-4527 or FAX: (919) 541-0642
      Internet: VANDENBERG@HERL45.HERL.EPA.GOV
NHEERL AIR TOXICS ANNUAL REPORT 1995

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                              AIR TOXICS
             RESEARCH PROGRAM  SUMMARY
REGULATORY AND PROGRAMMATIC
CONTEXT
The Clean Air Act (CAA) authorizes EPA to
regulate  a  wide  variety  of toxic  air
pollutants,   including   motor   vehicle
emissions and  hazardous  air pollutants
(HAPs) emitted from stationary sources.  It
requires  EPA  to  set  technology-based
standards for  pollutant  sources  and,  if
warranted,   health-based   standards  for
residual  risk  following   installation  of
Maximum Achievable Control Technology.
To  help  the Program  Office  meet these
requirements, EPA's Office of Research and
Development (ORD) maintains an Air Toxics
Research Program that develops methods
and  models to  improve  air  toxics  risk
assessment and provides chemical-specific
data on priority contaminants.
PROGRAM
To  develop improved  methodologies  for
assessment of health risks from exposure to
air toxics.

RATIONALE:                 ;?;' <    ;
Millions  of Americans, particularly those
living in urban environments, are exposed to
hazardous air pollutants.  The health risks
posed  by these exposures are potentially
high, according to the National Academy of
Sciences (NAS), but substantial  scientific
uncertainty remains regarding these risks.
For  example, although some air contami-
nants are known to be toxic in laboratory
studies,  little is known about the effects
that may  be  caused  in  humans  at  the
relatively  low  concentrations  normally
present in ambient air.  Research is needed
to  address the  poorly understood issues
surrounding the health risks of air toxics.
RESEARCH STRATEGY
To ensure that the Agency is equipped with
scientific and technical data relevant to the
formulation of sound environmental policy,
ORD operates a research program founded
on the principles of risk assessment.  In the
area of  health effects, the  program  is
patterned   after  the   risk   assessment
paradigm  of the  National Academy  of
Sciences (NAS). This paradigm consists of
4   steps-hazard   identification,  dose-
response assessment, exposure  assessment,
and  risk  characterization—that  provide
information for risk management decisions.
NHEERL's research programs in Air Toxics
adhere   to  this   risk-based   strategy,
emphasizing  three   types of   research
activities:
       • Research in the  area of hazard
identification focuses on the development
and utilization of methods to identify health
hazards.
       •    Research  supporting  dose-
response assessment seeks to explain the
events linking  exposure  to effects.  These
events  form the basis for the  predictive
models used to quantify risk.
       • There also may be instances when
scientific data on a particular contaminant is
required.    In such  cases,  short-term
problem-specific research is conducted  to
systematically   collect   and    analyze
information regarding   specific  gaps  in
knowledge.

The  objective  of  NHEERL's   research
program in Air Toxics is to provide toxicity
data and new methods for evaluating the
cancer and non-cancer risks for HAPs. Our
efforts are focused on a set  of chemicals
and source categories believed to present
the greatest potential threat to  public health
 and/or which serve as the best models for
 NHEERL AIR TOXICS ANNUAL REPORT 1995

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methods development.   In the area  of
HAZARD IDENTIFICATION, we are exploring
new areas in air toxics epidemiology. We
are developing molecular/biologic methods,
such   as   biomarkers,   to   strengthen
conventional epidemiology approaches, and
we are utilizing existing health databases to
help shape epidemiology research questions.
Research  in  DOSE-RESPONSE ASSESS-
MENT is designed to anticipate and address
the  most   problematic   air  toxics  risk
assessment issues faced by EPA, such  as
estimating exposure-response relationships
over a range of exposure  concentrations
and durations, predicting  human response
to these exposures, and  estimating risk
from  common  air  pollutant  mixtures.
Finally,  we  are  conducting  PROBLEM-
SPECIFIC STUDIES to provide human health
data for a fuel additive  (MTBE) recently
associated with health complaints in some
parts of the country.
NHEERL AIR TOXICS ANNUAL REPORT 1995

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              NHEERL AIR TOXICS  RESEARCH
                FY95 PROGRAM HIGHLIGHTS
HAZARD IDENTIFICATION RESEARCH  We demonstrated that  urine metabolite  analysis is a highly sensitive measure
      of recent exposure to polycyclic aromatic hydrocarbons (PAHs) and may serve as an
      effective biomarker for PAH exposures in epidemiology studies.
DOSE-RESPONSE ASSESSMENT RESEARCH, (pg 9}
The goal of this research is to better understand the factors that influence exposure-response
relationships for a set of  representative air toxics so that improved empirical methods and
quantitative, biologically based risk models can be developed.

      *• In studies of respiratory toxicity, we demonstrated that phosgene impairs pulmonary
      host defenses and enhances sensitivity to bacteria/ infection in the lungs of mice.

      •> We found that inhalation of trichloroethylene (TCE), a volatile organic solvent, causes
      a unique mid-frequency hearing loss in test animals.

      > We showed that the peak concentration of TCE in blood, as estimated by our PBPK
      model, is a good indicator of acute neurotoxic effects.

      *• Using rodents,  we found a direct link  between DNA adducts formed in lungs and
      induced mutations in lung tumor oncogenes for certain PAHs.   These findings were
      substantiated when we linked airborne combustion products with human carcinogenesis
      via DNA adducts in target tissues.
The goal of this research is to assess the human health effects of a fuel additive, methyl tertiary
butyl ether (MTBE).

       > In a study investigating complaints  of illness associated with MTBE, our scientists
       demonstrated that ambient levels of MTBE have no significant effect on normal, healthy
       individuals. This study was awarded the EPA bronze medal.

       >  We sponsored a workshop on MTBE in which a panel of scientific experts from
       industry, academia, and government provided guidance to EPA on the feasibility and
       design of epidemiology studies of MTBE-exposed populations.
 NHEERL AIR TOXICS ANNUAL REPORT 1995

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                               AIR TOXICS
HAZARD IDENTIFICATION  RESEARCH  PROGRAM
NHEERL   defines  hazard  identification
research  as  research to characterize the
association    between    environmental
exposure and adverse effect.
Does exposure to ambient concentrations of
HAPs result in demonstrable health effects?
At  present,  there is  virtually no direct
observational evidence (i.e., epidemiologic
data) to link ambient HAP exposures with
health effects.  This is due, in part, to the
relatively small population sizes exposed to
HAPs (compared to criteria  pollutants,  for
instance), to limited exposure information,
and   to  the   expense   of   adequate
epidemiology studies.   Better tools  are
needed to identify and characterize these
risks.
PROGRAM DESCRIPTION
NHEERL is engaged in two research projects
to identify the  health hazards associated
with exposure to HAPs.  In the first project,
we are developing MOLECULAR/BIOLOGIC
METHODS to identify potentially hazardous
air pollutants and to facilitate field studies
of  urban  air  pollution.  Computational
chemistry   and    molecular   modeling
techniques are  being   used  to  explore
correlations   between   the    structural
properties of a  chemical and  its toxicity.
Biomarkers are  being developed to bridge
the association   between exposure  and
effects.  Biomarkers under study  include
DNA and protein adduct formation in target
tissues  and metabolite  analysis in human
body fluids. In the second research project,
HEALTH DATABASES on disease incidence
in urban areas are being evaluated to relate
exposure and effects.  We are using a data
management tool. Geographic Information
Systems (GIS), to interface existing health
effects databases (e.g.. State Birth Defect
Registries)  with  emissions  monitoring
information to identify geographic locations
that qualify for more detailed field studies.
           PROGRESS
MOLECULAR/BIOLOGIC     METHODS.
NHEERL  is  using  quantum  mechanical
studies of the structure  and reactivity of
PAHs (and their metabolites) to identify
biologically active chemicals and to describe
the interaction  that occurs between the
reactive chemical and its biological target.
In FY95, we  found that the electrostatic
properties of diol epoxides (metabolites) of
a   PAH   called   benzo(c)phenanathrene
influence its ability to interact with DNA
and  may explain observed  differences in
carcinogenic potency. These studies are
giving us insight into the  chemical features
associated with enhanced toxicity. We will
continue to apply this method to other air
toxics and their metabolites in an effort to
identify classes of  chemicals that pose a
potential  health hazard  and  to  better
understand the mode of action at the target
site.

We also  are developing tools (biomarkers)
for  use  in  environmental epidemiology
studies  to improve estimates of human
exposure and biologically effective dose. In
FY95,   we  made  several advances  in
biomarker development.    We  improved
methods for separating and identifying DNA
adducts using highly sophisticated analytical
techniques  (32P-postlabeling  and  HPLC
analysis). These techniques permitted us to
NHEERL AIR TOXICS ANNUAL REPORT 1995

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identify DNA adducts formed in mouse skin
and lung tissue following topical treatment
with  diesel  exhaust  extracts.     These
adducts  were  formed  by  nitroaromatrc
compounds present in the exhaust.

Another focus for biomarker research  by
NHEERL is  analysis  of body  fluids for
indicators of exposure  to  air toxics.  In
FY95, we published results from a study in
which  we  analyzed urine  collected from
individuals   belonging   to  a  population
associated with high lung cancer mortality.
We found a correlation  between exposure
to unvented smoke and PAH metabolites in
urine.   Further  evaluation of the data
showed that urine metabolite analysis is a
highly sensitive measure of recent exposure
to PAHs.

HEALTH DATABASES.  An emerging area of
research for NHEERL is the use of health
databases  to  help  shape  epidemiology
research   questions   and   hypotheses.
Reproductive/developmental and  pulmonary
health effects will  be  considered  first
because  existing databases appear to  be
more suitable for evaluating these effects.
Our  overall objective over the next couple
of  years  is  to   determine  whether  a
geographic area exhibiting a high incidence
of  health  effects  is  also  likely to  be
characterized   by    high     emissions/
concentrations of specified HAPs.
 NHEERL AIR TOXICS ANNUAL REPORT 1995
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                                AIR TOXICS
   DOSE-RESPONSE ASSESSMENT RESEARCH PROGRAM
NHEERL defines dose-response assessment
research  as  research  to  identify  and
describe the basis for the effects caused by
exposure  to  environmental  stressors or
chemical contaminants.

ISSUE

How  can risk  assessments of  toxic air
pollutants be strengthened?
EPA is mandated by the Clean Air Act to
assess  the  risks  posed  by  toxic  air
pollutants. Risk assessments frequently rely
on predictive models that estimate human
risk using experimental toxicity information.
The reliability of the derived risk estimates
depends on the  soundness and accuracy of
the models  as  well  as the strength and
scope of  the   toxicity  data.   Improved
methods and models are needed to enhance
the precision of risk  estimates,  and new
toxicity data are needed to facilitate model
development.
PHOGRAMjpESCRiPTlON

The primary objective  of this  research
program is to develop new approaches to
risk assessment for air toxics that anticipate
and address the problematic issues faced by
EPA's  Office of Air and Radiation  (OAR).
Specifically,  we are examining  various
combinations of exposure concentration and
duration to explain EXPOSURE-RESPONSE
RELATIONSHIPS. A range of health  effects
and chemical class combinations have been
selected for initial analysis: for respiratory
toxicity, we are using a  representative
irritant gas (phosgene); for neurotoxicity, a
solvent (TCE); for developmental toxicity, a
non-chlorinated  hydrocarbon   (methanol);
and  for cancer,  polycyclic organic  matter
(POM). Using these data, we are developing
empirical  and  biologically  based   RISK
MODELS to provide quantitative estimates
of response as a function of dose.  We also
are  conducting research to determine how
interactions   among   chemicals   in   air
MIXTURES affect risk.
EXPOSURE-RESPONSE   RELATIONSHIPS.
Short-term, high-concentration exposures to
toxic air  pollutants may induce toxicity
disproportionate to that caused by long-
term, low-level exposures. Recognizing this
distinction, ORD has anticipated difficulties
in assessing risks  from short-term expo-
sures. To  address this problem, NHEERL is
conducting  research   to   improve  the
scientific basis for  risk assessments from
acute exposures and is working with the
National Center for Environmental Assess-
ment  (NCEA)   to   apply   results  in
development of new acute risk assessment
methodologies. One approach to improved
assessment is to analyze toxicity at various
combinations of chemical concentration (C)
and  time (t).  According to Haber's "Law,"
the product of this relationship (Cxt) should
yield a constant effect  on health;  this
assumption    underlies   current    risk
assessment  efforts.    Our  research is
designed  to  determine  whether   this
assumption is valid across short-term and
intermittent exposures to air toxics.
       Respiratory  Toxic/ty: Irritant Gases
(Phosgene).   Phosgene is a  respiratory
irritant and a highly reactive air toxic. Using
various   chemical   concentrations   and
exposure times, NHEERL is examining the
effects  of phosgene on  pulmonary host
defenses and disease susceptibility, with an
emphasis on animal-to-human extrapolation.
In FY95,  scientists  in our  Experimental
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Toxicology Division  used  an  established
rodent model  for allergic sensitization to
show that phosgene impairs the immune
system.   Acute exposure  was shown to
decrease resistance to bacterial infection
and to enhance susceptibility to tumor cell
challenge; these effects appear to be due to
impaired alveolar macrophage activity and
natural killer cell activity, respectively. This
valuable  mechanistic  information  will  be
used  to  improve extrapolations of human
response.  Subchronic exposure  studies
showed that phosgene impairs clearance of
bacteria  from  the lungs  and  increases
inflammatory  response.   We  also found
during FY95  that  the  concentration of
phosgene is more important than  time of
exposure in determining response.
       Neurotoxicity:   Organic  Solvents
(Trichloroethylene, or TCE). Exposures to
volatile organic compounds (VOCs),  such as
solvents,  can  cause  neurotoxic  and
behavioral effects in humans. In FY94, we
initiated a project to better  define the
exposure-response relationship for solvents.
TCE was selected as the model compound.
Our objective is to describe the relationship
between C and t for a variety of neurotoxic
endpoints—including  indices of  complex
behavior, visual function,  and  ototoxicity
(hearing   loss)—as  a  function  of  acute
inhalation exposures in rodents.
       Behavior was  measured  using   a
vigilance test (response  time to a  signal
light  for  food  reward) developed by our
Neurotoxicology Division in FY94.  In FY95,
we applied this test to  airborne TCE and
evaluated  Cxt relationships.   Preliminary
findings suggest that TCE toxicity results
from  a combination of concentration and
time,  but  that concentration  is  more
important than time  in  determining the
strength of response.
       The effect of TCE  on visual function
was analyzed by measuring visual evoked
potentials  (VEPs).   Using an  inhalation
chamber, we exposed  rats in FY95 to TCE
vapors  while  simultaneously  recording
VEPs.    Collected  data  are  now  being
analyzed.
       Ototoxicity tests  of inhaled TCE
were   conducted   during   FY94-95  to
characterize hearing loss across a range of
hearing frequencies.  While most previous
studies of ototoxicity to organic solvents
have demonstrated hearing deficits at high
frequencies, we found during  FY95 that
TCE causes a unique hearing loss  in the
mid-frequency range, sparing  function at
lower and higher frequencies.  Research in
this area continues as we determine the Cxt
relationship  for  a   variety of  exposure
durations (acute to subchronic).
       Developmental    Toxicity:   Non-
chlorinated   Hydrocarbons   (Methanol).
Methanol represents an important chemical
under the Clean Air Act because it has been
proposed as an alternative fuel  for motor
vehicles and is a likely  fuel for fuel cell
technology cars of the future. Moreover, it
is listed among the 189 toxic air pollutants
requiring regulation by EPA.   In the early
1990s,  scientists  in  our Reproductive
Toxicology Division showed that methanol
adversely   affects   fetal   development:
inhalation of methanol by  pregnant mice—at
concentrations relevant to humans—resulted
in multiple birth defects.  Research on this
phenomenon continues,  and in FY95  we
completed   work  on   the   toxicity  of
methanol's principal metabolite, formic acid.
Using an in vitro embryo culture system, we
showed that methanol, not formic acid, is
the proximate developmental toxicant. We
also  completed  a   blood  profile of Cxt
effects for a variety of methanol exposure
scenarios, and we defined the critical period
for  induction  of  skeletal and  visceral
malformations.  In the upcoming year, we
will be narrowing the search for the early
events in  the pathogenesis of  the birth
defects caused by methanol, with specific
emphasis placed  on Cxt studies and gene
expression in exposed embryos.
        Cancer:     Polycyc/ic     Organic
Matter/Polycyclic Aromatic Hydrocarbons
 NHEERL AIR TOXICS ANNUAL REPORT 1995
                                                                                  10

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(POM/PAHs): PAHs are toxic components
of  many  environmental  contaminants,
including  combustion  emissions.   In  an
effort to uncover the mode of action for
PAH toxicity, we are studying a set of PAHs
and their metabolites for their ability to form
DNA  adducts  and   to   morphologically
transform cells.   Using  mouse embryo
fibroblasts, we studied an extremely potent
PAH, dibenzo(a,l)pyrene (DBA) in FY94 and
compared   its   toxicity   to   that   of
benzo(a)pyrene (BAP).  We concluded that
DBP  is  significantly  more active  as  a
morphological transforming agent than BAP.
In addition,  its  adducts are different from
those of BAP, which may provide a clue to
its higher carcinogenic potency.  In  FY95,
we  expanded   our  study   to  include
dibenzo(a,h)anthracene (DBA). Collectively,
the results from  these analyses will help
explain  the  mechanisms  underlying PAH
carcinogenesis.

In  an   important   project  in  humans
conducted by  scientists  in our  Human
Studies  Division   using  human  autopsy
samples,  we reported  in FY95 that POM
(combustion  products) can be  linked to
human carcinogenesis via the formation of
DNA adducts.

RISK  MODELS.   Using exposure-response
data  obtained from the research projects
described above, NHEERL is developing non-
cancer and cancer risk models to improve
the   scientific   basis   for  health   risk
assessment.
       Scientists   in   our  Experimental
Toxicology Division are in the initial stages
of developing a  biologically based dose-
response  (BBDR) model  for  pulmonary
toxicity using phosgene as the prototype for
irritant gases. We are collecting exposure-
response data for various Cxt combinations
in order to understand  why concentration,
rather than time, drives the toxic response.
NHEERL scientists are working with NCEA
to reflect these results through development
of an  acute  reference  exposure  (ARE)
methodology.  The use  of ARE method-
ologies will assist in  assessing the health
risks of acute exposures for phosgene as
well as for other compounds.
      To  improve  predictions  of  the
neurotoxic  effects  of  volatile  organics,
NHEERL  is  developing  a  physiologically
based  pharmacokinetic  (PBPK) model in
rodents to  estimate  human risk.   During
FY95, we used our model to estimate blood
and brain concentrations  of TCE and its
metabolites    in    order   to   predict
neurotoxicity.   The  accuracy of  these
predictions is  being determined based on
various measures of  neurotoxic outcome,
including  complex behavior, visual function,
and hearing loss. During FY95,  we showed
that  model  estimates  of  the   peak
concentration of TCE in blood were a good
indicator of neurotoxic risk across exposure
scenarios   and   neurotoxic    outcome
measures. In the future, parameters  of the
PBPK model will be scaled up  to produce
rational predictions of human risk based on
laboratory findings.
       As a result  of our discovery  in the
early  1990s that  inhalation of  methanol
caused   multiple  birth  defects  in  mice,
scientists in our Reproductive Toxicology
Division proposed a 5-year plan to develop
the  Agency's  first  BBDR  model   for
developmental toxicity  to be  used in
methanol risk assessment.  During  FY95,
NHEERL  moved closer   to  achieving its
scheduled development  of this model by
confirming the heightened sensitivity of the
mouse embryo culture system relative to
the rat in terms of developmental toxicity.
This evidence supports the relevancy of the
mouse  model for  predicting  effects in
humans  exposed  to methanol.    When
completed,  this model   will represent a
significant   step  forward  in  efforts to
evaluate   environmentally  induced  birth
defects.
       One  of the uncertainties  in cancer
risk assessment of PAHs is the validity of
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                                    11

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the assumption that all environmental PAHs
are equal in potency to benzolalpyrene
(BaP).   To help reduce  this uncertainty.
scientists     in     our    Environmental
Carcinogenesis  Division have studied the
lung   tumorigenic    potency   of   five
environmental PAHs in mice.  The relation-
ship between lung DNA  adducts, specific
mutations in lung  tumor oncogenes, and
lung tumorigenic potency induced by the
PAHs was evaluated.  We found that for
certain PAHs, there is a direct link between
their ability to form DNA adducts, mutate
oncogenes, and induce tumors.  A set of
DNA adduct(dose)-tumor(response) relation-
ships has been developed for these 5 PAHs
for use in risk assessment; this information
will be used to develop  cancer potency
estimates.

MIXTURES.  Urban air is a mixture of gases
and  pollutants;  rarely,  if  ever,  does
exposure involve  a solitary contaminant.
For this reason, NHEERL is studying  the
effects of chemical interactions in air using
mixtures   of    common    urban   air
contaminants.      Scientists   in    our
Neurotoxicology Division are examining the
interactive effects of mixtures of VOCs on
hearing. Several volatile solvents—including
TCE, xylene, and toluene—were selected for
testing.  A multifactorial  study in rodents
was   designed,   and   a   matrix   of
concentration  combinations was tested.
During  FY95, we found  that inhalation
exposure to mixed xylene, toluene and TCE
caused hearing deficits in the mid-frequency
range, sparing function at lower and higher
frequencies.   This information  is useful
because it suggests a common mechanism
of  toxicity for  environmentally  related
solvents.
 NHEERL AIR TOXICS ANNUAL REPORT 1995
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                              AIR TOXICS
                 PROBLEM-SPECIFIC STUDIES
NHEERL defines problem-specific studies as
research to characterize the effects induced
by a specific environmental perturbation or
chemical contaminant.

issue-
What are  the adverse effects  associated
with exposures to the fuel additive, MTBE?
Areas of the country unable to meet the
National  Ambient  Air  Quality Standard
(NAAQS)  for   carbon  monoxide  must,
according to the Clean Air Act of 1990,
consider  addition  of oxygenates to auto
fuels to reduce carbon monoxide emissions.
In 1992, the addition of an  oxygenate called
methyl-tertiary-butyl ether (MTBE) coincided
with  complaints   of  illness,   including
headache, nausea,  sore  throat, and  eye
irritation.  Concerns about the potential
health risks  of  MTBE  have   prompted
research in this area.

PROGRAM  DESCRIPTION^!     ?  7"
In response to emerging concerns regarding
the health risks posed by MTBE and other
fuel  additives, ORD  organized  a  joint
government-industry research  program for
health  risk assessment.   To  address the
uncertainties identified by the  assessment,
NHEERL  has  initiated  a  series of clinical
research  studies  to  evaluate  the health
effects   of  MTBE  in  normal,  healthy
individuals and has designed a study of
sensitive  subgroups of individuals (those
who complain of MTBE symptoms).  Study
participants are exposed for short duration
to environmentally relevant concentrations
of MTBE, and various measures of sensory
and physiological response are recorded and
analyzed.
PROGRAM PROGRESS. ?&;-!« ^ ,;:. -:

During FY94, a group of healthy volunteers
was recruited  by  our Human   Studies
Division to participate in a clinical study of
acute health effects induced by exposure to
MTBE.     Individuals  were  exposed  in
specially designed air chambers to levels of
pure   MTBE   approximating   a   typical
exposure during refueling. Cognitive testing
was conducted  during  exposure,  and
objective  measures  of  eye  and  nose
irritation were  obtained  pre-  and  post-
exposure.  Pharmacokinetic studies of blood
levels of MTBE and its principal metabolite,
tertiary butyl  alcohol  (TBA),  also  were
performed. Symptom questionnaires were
administered.  In FY95, we published the
findings of this important study,  which
earned the prestigious Bronze Medal from
EPA.   Our results showed that MTBE at
ambient levels has no significant effect on
normal, healthy subjects.  These findings
have  allayed some concerns  about the
potential risks posed by MTBE and have
facilitated its continued use in efforts to
control carbon monoxide and its attendant
health effects. In FY96, we plan to follow
up our pharmacokinetic studies to determine
whether TBA persists in the blood following
repeated exposure to MTBE.

We also conducted a workshop on MTBE in
FY95.   Panel members included  experts
from industry, academia, and government.
Results from studies of MTBE exposure and
related health effects were presented, and
a  report  was   prepared  by  the  panel
providing guidance to EPA on the feasibility
and design  of epidemiology studies on
populations exposed to  MTBE.
NHEERL AIR TOXICS ANNUAL REPORT 1995
                                   13

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National Health and Environmental Effects Research Laboratory
    INDOOR AIR RESEARCH
             ANNUAL REPORT




               JUNE, 1996

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                         CONTENTS
Introduction	3




Summary of the Indoor Air Research Program  	4




FY95 Highlights	5




Indoor Air Biocontaminants Research Program	6




Indoor Air Organic Vapors Research Program	9
NHEERL INDOOR AIR ANNUAL REPORT 1995

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                       INTRODUCTION
PURPOSE
The purpose of this report is to communicate results from the Indoor Air Research
Program of EPA's National Health and Environmental Effects Research Laboratory
(NHEERL).
CONTENT     -    ;';:***,.     ,  -   ,  -   '..!**  ,,;;!   \./

The report contains

•    a summary of the Health and Environmental Effects Research Program for
     Indoor Air, which describes the regulatory and programmatic context of the
     research program, the overall program goal, the rationale for the program, and
     the research approach

•    a section which highlights recent key findings (FY95 Program Highlights)

•    a more detailed description of the NHEERL Indoor Air Research Program, by
     program  area, including a  summary  of  research  accomplishments  and
     anticipated progress for the near future.
COMMENTS WELCOME

The format of this report is still evolving, and we welcome feedback. Readers with
comments or requests for  further information are encouraged to contact

     Sue McMaster, Assistant Laboratory Director
     National Health and  Environmental Effects Research Laboratory (MD-51A)
     U.S. EPA
     Research Triangle Park, N.C. 27711

     Phone: (919) 541-3844 or FAX: (919) 541-1440
NHEERL INDOOR AIR ANNUAL REPORT 1995

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                              INDOOR AIR
          RESEARCH PROGRAM SUMMARY
               AltfD
EPA's  Indoor  Air  Research Program  is
authorized by the Radon Gas and Indoor Air
Quality Research Act of  1986 (Title IV of
the    Superfund    Amendments    and
Reauthorization Act, or SARA). The Agency
has no regulatory jurisdiction over indoor
air; rather, its  goal is to  inform the public
debate. EPA's Office of  Air and Radiation
(OAR),   which  disseminates   advisory
information  on  indoor   air  quality  to
consumers, building  owners,  and public
health professionals, relies on the Office of
Research  and  Development   (ORD)  to
provide scientific data for this process. The
bulk of  ORD's resources for  indoor  air
research  are  appropriated  to areas  of
greatest   uncertainty:   health   effects,
exposure assessment, and  risk manage-
ment.  Health effects research, which is
designed to improve understanding of the
noncancer  health  risks  associated  with
indoor air  pollutants, is  conducted  by
NHEERL

           GOAL

To determine the health risks  posed by low-
level mixtures of organic vapors and indoor
allergens.

RATIONALE

Individuals in the United States spend 90%
of their time indoors; however, surprisingly
little is known about the health  risks of
indoor  air,   particularly  for   noncancer
endpoints.   Diverse  symptoms are asso-
ciated with contaminants from indoor air
sources, but neither the determinants  of
these  symptoms  nor   the   long-term
consequences  of exposure are  known.
These  factors  have  led  EPA's  Science
Advisory Board (SAB) to rank indoor air as
one of the top 5 health risks in comparative
risk studies.
To ensure that the Agency is equipped with
scientific and technical data relevant to the
formulation of sound environmental policy,
ORD operates a research program founded
on risk-based principles.  In the area of
health  effects,  the  program  explicitly
conforms to the risk assessment paradigm
described  by the  National Academy  of
Sciences (NAS) in  1983.   This paradigm
consists of 4 steps  (hazard  identification,
dose-response    assessment,    exposure
assessment, and risk assessment) that drive
risk  management decisions.   NHEERL's
research programs adhere to this risk-based
strategy.   In  our  Indoor  Air  Research
Program, we are studying the predominant
classes of biological and chemical indoor air
contaminants suspected of causing adverse
health  effects.   Our approach  is (1)  to
characterize the  determinants  of  allergic
response to BIOCONTAMINANTS,  and  (2)
to understand the  relationship between
exposure and effect for indoor ORGANIC
VAPORS.   The guiding philosophy of the
program is to progress from identifying the
health  effects of concern to  developing
methods that measure effects to producing
dose-response models that relate exposure
and response.
NHEERL INDOOR AIR ANNUAL REPORT 1995

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          NHEERL INDOOR AIR RESEARCH
            FY95 PROGRAM  HIGHLIGHTS
The goal of this research is to characterize the health risks posed by indoor allergens,
with special emphasis on the house dust mite.

      *•  Scientists in NHEERL provided the first direct evidence of a link between
         N02 and asthma.  Using a rodent model of dust mite-induced asthma, N02
         was found  to exacerbate various biochemical events associated  with
         immune response and to produce clinical measures of asthmatic symptoms
         (decreased lung function).
 ORGANIC VAPORS BESpARGH fejr'9!

 The goal of this research is to develop methods that measure the health effects of
 indoor organic vapors and to apply these  methods  to the study of potentially
 susceptible subpopulations.

      *• Our  published findings on  the health  effects  of carpet  emissions
        contradicted reports by a commercial testing firm showing severe signs of
        toxicity in mice. We replicated the tests conducted by the commercial firm
        and demonstrated that the carpet emissions caused no significant adverse
        effects in test animals.

      *• A NHEERL-sponsored workshop identified promising methods for assessing
        the neurotoxic health complaints associated with indoor air exposures.

      * We  identified a  subpopulation  of humans with alleged  increased
        susceptibility  to the effects of indoor air pollution (individuals  reporting
        Multiple Chemical Sensitivity, or MCS). A detailed evaluation of the health
        of these individuals in the unexposed state was completed.  This cohort will
        be compared to other groups in upcoming indoor air studies.
NHEERL INDOOR AIR ANNUAL REPORT 1995

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                           INDOOR AIR
BIOCONTAMINANTS RESEARCH PROGRAM
 NHEERL defines biocontaminants research
 as  research  that  leads  to  a   better
 understanding of the relationship between
 biocontaminants  and  associated   health
 effects, including the extent  to  which
 biocontaminants and other pollutants may
 interact to alter the health risk posed by
 either individually.
 ISSUE
 What are the health risks posed by indoor
 biological  contaminants,   or  allergens?
 What is the involvement of cofactors in
 allergic response?
 Asthma morbidity and mortality are on the
 rise  in the  United  States  and other
 industrialized   countries.      Mounting
 evidence,  led  by epidemiology studies,
 suggests that indoor allergens are involved
 in causing  or  exacerbating asthma.   Of
 particular interest is the house dust mite,
 which is one of the most prevalent indoor
 allergens.   These microscopic organisms
 are known to trigger allergic  reactions;
 they  also  have been  associated  with
 asthma.  Other pollutants, such as NO2 or
 ozone, may exacerbate  allergic response.
 For  example,  it has been shown that
 children  in homes with  gas stoves  or
 kerosene heaters, which emit N02,  have
 more respiratory  illnesses than children in
 homes without these features.
 The purpose of this research program is to
 provide a  clearer  understanding of the
 health effects  associated  with  indoor
 exposure  to  biocontaminants, including
 allergens (such as the dust mite), molds,
 and  other microbials.  The  role of  bio-
contaminants in causing and exacerbating
asthma is a primary research focus. The
research  combines  field,  clinical, and
laboratory   studies   to    1)   develop
quantitative tools to measure BIOLOGICAL
RESPONSE to  indoor  allergens, and  2)
determine the importance of COFACTORS
in the exposure-dose-response relationship.
The  near-term  goal is  to  resolve  these
issues for one of the most prevalent  indoor
allergens, the house dust mite. Dust mites
were selected  because  significant  indoor
human exposures occur,  the  exposures
have been associated with asthma, and the
antigen can be  measured quantitatively.
BIOLOGICAL  RESPONSES.  To  improve
understanding  of the biological  effects
produced by allergens, NHEERL is engaged
in  research   that  will  lead  to  better
measures  of  effect and  more accurate
models  for  predicting  dose-response
relationships.  Our approach includes the
development of animal models of allergic
lung disease and clinical and field studies
to evaluate human response to allergen
challenge.
      Animal models ~  Because many
important  research  questions are difficult
to answer in  humans, animal  models for
allergy  to  indoor  biocontaminants  are
essential.  Scientists  in our Experimental
Toxicology Division are  developing two
rodent models of allergen-induced asthma:
the  rat model,  which has been under
development for several years  and is near
completion, and the mouse model, which
began its development in FY95.
       Our rat model  successfully mimics
the pattern of allergy-induced asthma in
 NHEERL INDOOR AIR ANNUAL REPORT 1995

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 humans,  exhibiting   features  such   as
 eosinophilic    inflammatory    response,
 mucous secretion, and hyperreactivity to
 antigens leading  to  constriction of  the
 airways (similar to the wheezing response
 in  humans).  The production of immuno-
 logic disease in our model was  confirmed
 in  FY94 when we demonstrated that  the
 allergic state could be transferred to naive
 (nonallergic) rats.  During FY95 we began
 to  learn more about allergic response at the
 cellular  level  by using our rat model to
 characterize the  events  correlated with
 development    of    allergic    disease.
 Specifically, we began to study cytokines.
 Cytokines are soluble factors produced by
 cells   that    modulate   immune   and
 inflammatory response.  We are using PCR
 (polymerase chain reaction) techniques to
 detect  the  induction  of  a  variety  of
 cytokines,   which   helps   explain   the
 mechanism(s) involved in immune response
 and facilitates extrapolation of biochemical
 response  to the  disease  state.   It is
 anticipated that  the  rat  model  will  be
 completed in FY96, and it will be used in
 subsequent  years  to characterize  the
 effects and dose-responses for a variety of
 other  biocontaminants.
       We initiated a similar effort in FY95
 to  develop a model of  allergen-induced
 asthma  in the  mouse, which offers  the
 advantage of having a more clearly defined
 set  of   immunological  parameters   to
 measure.  The development of this model
 will continue  in FY96.
       Human studies — With humans, as
 with our animal models,  the objective of
 our research  is to better understand  the
 allergic/inflammatory response to an indoor
 allergen challenge. In FY93, scientists in
 our Human  Studies  Division launched a
 clinical  study of allergic asthmatics  to
 characterize their response to the house
 dust  mite  antigen.    The  antigen  is
 administered  via   nasal  challenge  (i.e.,
 applied directly into the nose rather than
breathed into the  lungs).  One  of  the
responses, reported in FY94, is an increase
in  eosinophils,  a blood cell that  signals
allergic response. We continued our nasal
challenge studies in FY95 by  evaluating
whether the  lower  airways  respond
similarly to the upper airways.  If a similar
response  to   dust  mite  allergen  is
demonstrated in the lower airways, it will
imply that the  dust mite can increase
allergen-induced morbidity in asthmatics.
      This clinical study was coupled  with
a field  study (also initiated in FY93) in
which we collaborated with investigators
at the University of North Carolina's Center
for  Environmental  Medicine  and  Lung
Biology  to  measure  house  dust  mite
antigen  in the homes of allergic asthmatic
children. We found that the degree of mite
contamination  in  the  homes  did   not
account for  differences  in   wheezing,
bronchial hyperrreactivity, or lung function.
During  FY94, we shifted  our focus from
children  to  adults,   where   controlled
inhalation  exposures  to  allergens   are
feasible.  Initial studies, using grass allergy
as a prototype,  verified that exposure to
grass  allergens  can  produce a  substantial
decline   in  pulmonary   function    in
asthmatics.  Future plans call for continued
clinical research on allergic, asthmatic,  and
normal  children  to  better  define   the
biological response to allergen challenge.

COFACTORS. Although cofactors such as
gender, age,  and pollution are  known or
presumed to affect dose-response relation-
ships, little is known of the involvement of
these cofactors in indoor allergen-induced
disease.  We are testing, in  laboratory
animals and  in   humans, the  hypothesis
that exposure  to  ubiquitous  pollutants,
such  as N02, Q, and volatile organics,
predisposes  an   individual    to   the
development  of indoor   biocontaminant
allergies and/or exacerbation of symptoms.
      Laboratory  animals  -  In  FY95,
NHEERL INDOOR AIR ANNUAL REPORT 1995

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 NHEERL,   in  collaboration   with   the
 University of North Carolina, provided the
 first direct link beween N02 and asthma.
 Although  exposure to NO2 alone does not
 trigger   an   asthmatic   attack,   we
 demonstrated that it exacerbates asthmatic
 responses when  administered to rodents
 sensitized with dust mite antigen. Various
 measures of immune response, including
 antibodies, allergen-activated lymphocytes,
 and inflammatory  cells in  the mucous,
 indicated  enhanced sensitivity.  Another
 critical observation was decreased lung
 function,  which  is a clinical measure of
 asthmatic symptoms.   We are currently
 exploring  the mechanism(s)  by which NO2
 exacerbates allergic disease.  Building upon
 what  we  have learned about cytokines in
 our rodent model, we have begun exploring
 cytokine responses in co-exposure studies.
 In this way, we  hope to better correlate
 cellular response  with the disease state.
       Similar  co-exposure  studies are
 being conducted with ozone to examine its
 interaction with  dust mite antigen.  This
 research was initiated in FY94, and results
 thusfar indicate that ozone is not as potent
 as NO2 in modulating immune response.
       Human studies — In  FY93, we
 began an important study  in humans to
 evaluate the interactions between indoor
 allergens  and chemical air  pollutants.
Using nasal challenge, we exposed dust
mite-sensitive asthmatics to ozone, a lung
irritant.  In FY95, we published several key
findings. A comparison of the pulmonary
responses  of   asthmatics  and  non-
asthmatics showed that asthmatics appear
to be  more  susceptible to ozone than
normal,  healthy individuals. Ozone seems
to sensitize asthmatics  to the effects of
biological contaminants, predisposing them
to  a   more   severe   allergic  reaction.
Moreoever,  we found  that asthmatics
experience a different kind of inflammation
(eosinophil-driven) than normal individuals
(neutrophil-driven).

While the issue of asthma morbidity and
mortality is far from resolved, our studies
in both  animals and humans indicate that
interactions  between   common   indoor
pollutants  may  produce  unexpectedly
severe adverse effects.
NHEERL INDOOR AIR ANNUAL REPORT 1995
                                                                                   8

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                              INDOOR AIR
    ORGANIC VAPORS  RESEARCH  PROGRAM
 NHEERL defines organic vapors research as
 research   that   leads  to   a   better
 understanding of the relationship between
 organic  vapors  and  associated   health
 effects,  including the extent to  which
 organic vapors and other pollutants may
 interact to alter the health  risk posed by
 either.
   SUE
 Do indoor organic vapors pose a human
 health risk?  If so, what is the magnitude
 of this risk and what factors influence risk?
 Exposures to organic vapors indoors are
 common, and many signs/symptoms have
 been  attributed  to  such   exposures.
 Predominant among these symptoms is
 sensory irritation. However, despite many
 apparent associations,  the  relationship
 between sensory irritation and exposure to
 organic  vapors  is unclear.   Part of the
 difficulty  in  establishing  relationships
 between exposure and effect results from
 the    lack   of   adequate   means   to
 quantitatively  measure  effects.    An
 additional   difficulty  is  that  not  all
 individuals exposed to indoor pollutants are
 equally susceptible; allergies, asthmatics,
 and  individuals  who   report  multiple
 chemical sensitivity  (MCS)  may be  at
 greater risk of adverse  effects than the
 general public.  To protect the health of
 individuals who may be most vulnerable to
 the  effects  of  indoor  pollutants,  it is
 necessary to identify and study susceptible
 subpopulations  and to  understand  the
 determinants of  susceptibility.
 The primary emphasis of this research is to
develop    objective,    QUANTITATIVE
METHODS  with  which to measure the
symptom-based effects of organic vapors.
Our  approach  is to use laboratory and
clinical studies to assess sensory irritation,
neurotoxicity,    immunotoxicity,    and
respiratory health effects associated with
indoor  exposures to organic vapors.  As
adequate assessment  methods  become
available, they  are  enlisted  to  address
questions regarding the toxicity of VAPOR
MIXTURES  and  to evaluate responses in
SUSCEPTIBLE SUBPOPULATIONS who may
be at greater risk of adverse response than
the general  population.

PROGRAM PBQGRESS?   ;  :

QUANTITATIVE METHODS. Using experi-
mental  animals and human volunteers,  we
are  developing  quantitative  tools  to
measure  the effects of organic  vapors.
Our  laboratory  is interested  in  sensory
irritation-which manifests  itself in the
eyes,  nose,  and   throat—as   well  as
pulmonary,  or lung, irritation.  A collateral
goal  of our  research is to understand and
describe  the  neurological  and  immune
components of  these  responses,  which
often  defy  detection  by conventional
assessment means.
       Experimental  animals  —  During
FY94-FY95, NHEERL successfully tackled
a politically charged indoor air problem. A
commercial  animal-testing  firm  claimed
that  it  had  scientific evidence to validate
anecdotal accounts of adverse  health
effects resulting from exposure to carpet
emissions. The testing facility stated that
it had found an association between carpet
emissions and severe  signs  of toxicity
(heart and nervous system conditions) in
NHEERL INDOOR AIR ANNUAL REPORT 1995

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 mice.   In FY94,  NHEERL  initiated   an
 effort to replicate the findings described in
 the report.  In a study marked by extensive
 scrutiny   from   both   the    scientific
 community and the public, NHEERL  and
 other  ORD scientists  characterized  the
 chemistry  and microbial  emissions from
 three  "problem" carpets.   Following  a
 carefully controlled experimental paradigm
 designed to replicate the procedures used
 by the private  firm,  we were able to
 demonstrate that exposure of mice to
 these  emissions did  not result  in  any
 significant adverse effects.  In FY95, we
 published our findings, and the controversy
 abated.
       NHEERL is also  interested in identi-
 fying promising experimental  approaches
 to assess the complaints  of neurotoxicity
 associated with indoor exposure to  organic
 vapors.   To this end, we sponsored a
 workshop  in  FY95  to provide scientific
 input to EPA's research planning process
 with regard to the development of animal
 models of nervous system vulnerability to
 indoor  air  pollutants.   Workshop  partici-
 pants   discussed  experimental  design
 schemes  and  susceptibility and  offered
 recommendations  for future  research
 efforts.  Their  recommendations included
 the development and use of animal models
 of  susceptibility  to  indoor  air,  the
 identification    of   objective   outcome
 measures for human health consequences,
 and evaluation of mechanisms involved in
 susceptibility.
       Human   volunteers   —  Through
 clinical   studies,   our   laboratory    is
 attempting to derive quantitative methods
 for  assessing  sensory irritation  and to
 understand the  relationships  among  the
 assessment  measures.    In  FY94,   we
 invented a device (the video corneascope)
 to assess eye irritation  that measures,
 noninvasively, tear film break-up time.  We
 also developed a quantitative, computer-
 based method  for estimating eye redness.
These objective measures of eye irritation
are now being applied to other research
studies in which irritancy is an outcome.
      In addition to being uncomfortable,
irritation  may  interfere with performance
and productivity/  To address this issue,
we initiated studies  in FY94 to evaluate
the impact of sensory irritation on  task
performance  in  humans.  Two  types  of
studies are being performed. In one, we
are  producing  sensory  irritation   with
electrical stimulation, and in the  other we
are using noise as a surrogate for  irritation.
The effect of  both stressors on perform-
ance  is   being  assessed.    Preliminary
experiments were completed in FY95, and
data are undergoing analysis.

VAPOR   MIXTURES.   The  goal of  this
research  is  to  develop  methods  that
predict the critical   features of mixtures
that contribute  to  the  adverse  health
effects from indoor air.  Our intent  is  to
better  understand  the  mechanisms  of
mixtures toxicity.  Previous work by our
Laboratory (prior to 1993) showed that a
mixture of organic vapors could produce
subjective responses of eye,  nose  and
throat irritation  in normal healthy males;
we also  showed, for the first time,  that
exposure to a vapor mixture could produce
objective signs  of  inflammation in the
nose.  In FY93, our data  showed  that
perception of odor and sensory irritation
were independent of one another, and that
eye, nose,  and throat irritation  were
independent  from one another.  These
differences   suggested  that   dissimilar
mechanisms were at work, and that the
onset of one type of sensory irritation did
not  presuppose another.   In FY94, we
collaborated  with investigators at  Yale
University to study the factors involved in
these responses by  examining whether
mixture components behave additively or
synergistically  with  regard  to sensory
irritation. The results, published in FY95,
NHEERL INDOOR AIR ANNUAL REPORT 1995
                                                                                 10

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 suggest  that  the  degree of  additivity
 among components of  a vapor mixture
 varies from sub- to supra-additivity, and
 the factors affecting response may include
 carbon-chain  length, lipid solubility, and
 the number of compounds comprising the
 mixture.

 SUSCEPTIBLE SUBPOPULATIONS.  Among
 those who may be especially sensitive to
 the effects of indoor  air pollutants are
 individuals who report multiple chemical
 sensitivity (MCS).   MCS is a  condition
 described by individuals  who allege that
 their health has been impaired by chemical
 exposure  and that low-level exposures can
 trigger the adverse reactions.  The goal of
 this research project is to identify a subset
 of individuals who claim to have MCS, to
 conduct careful clinical characterizations of
 the  condition,   and   to  utilize   this
 experimental   population  in  studies of
 response to  indoor pollutants.   During
 FY94,  we  collected  data  on  persons
 reporting MCS symptomatology based on
exposure history and symptoms, medical
history   and  examination,  psychiatric
evaluation and a profile of medication, and
psychological  and  physiological  tests.
During FY95, individuals who met the
criteria for  MCS were identified, and we
completed  a detailed evaluation  of the
health  of   these  individuals  in  the
unexposed  state.   In FY96  we plan to
collect additional information that will help
characterize the differences between this
group  and  healthy individuals.    It  is
anticipated that the MCS cohort  will be
used  as a subgroup  in a  number of
upcoming  indoor  air studies  in  which
researchers  will  investigate  responses,
such as nasal irritation, of MCS and control
populations  to organic vapors and  other
indoor pollutants. These studies will help
further define the factors contributing to
MCS symptomatology.
NHEERL INDOOR AIR ANNUAL REPORT 1995
                                   11

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 National Health and Environmental Effects Research Laboratory
     GLOBAL CLIMATE CHANGE
HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH
             ANNUAL REPORT




                MAY, 1996

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                         CONTENTS
Introduction	3

Summary of the Global Climate Change
     Health and Environmental Effects Research Program	4

FY95 Highlights  	6

Global Climate Change Problem Formulation Research Program  	8
           Ecological Effects
           Indicators of Climate Change

Global Climate Change Determinants of Effect Research Program	11
           Mechanistic Research
           Predictive Models

Global Climate Change Problem-Specific Studies Research Program	14
           Stratospheric Ozone
                Human Health Effects
                Environmental Effects
NHEERL GLOBAL CHANGE ANNUAL REPORT 1995

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                       INTRODUCTION
PURPOSE
The purpose of this report is to communicate results from the Global Climate Change
Research Program of the EPA's National Health and Environmental Effects Research
Laboratory (NHEERL).
CONTENT.;;   .  ;--....;, ,-  .,.-;. •  ••.•'     ., •         ...     ;. v:>-:.-;;    ,

The report contains

      • a summary of the Health and Environmental Effects Research Program for Global
      Climate Change, which describes the regulatory and programmatic context of
      NHEERL's research program, the  overall program goal, the rationale for the
      program, and the research approach

      • a section which highlights recent key findings (FY95 Program Highlights)

      • a more detailed description of the NHEERL Global Climate Change Research
      Program, by program area, including a summary of research accomplishments and
      anticipated progress for the near future
COMMENTS WELCOME

The format of this report is still evolving, and we welcome feedback.  Readers with
comments, suggestions, questions, or requests for further information are encouraged to
contact

      John Vandenberg, Assistant Laboratory Director
      National Health and Environmental Effects Research Laboratory (MD-51A)
      U.S. EPA
      Research Triangle Park, N.C.  27711

      Phone: (919) 541-4527 or FAX: (919) 541-0642
NHEERL GLOBAL CHANGE ANNUAL REPORT 1995

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                 GLOBAL CLIMATE  CHANGE
            RESEARCH PROGRAM SUMMARY
               AND PROGRAMMATIC
As a result of the Global Change Research
Act of 1 990,  a comprehensive government-
wide program  (the  U.S.  Global  Change
Research  Program,  or  USGCRP)  was
instituted to improve our ability to understand
and respond to global change. EPA supports
this  effort   through  its  Global  Change
Research Program.  Several  EPA  offices,
including  the  Office  of   Research  and
Development (ORD)  in cooperation with the
Office of Policy,  Planning  and  Evaluation
(OPPE) and  the Office of Air and Radiation
(OAR), are assessing the ecological impacts
of climate change to inform the policy-making
process.*  NHEERL is  contributing to this
effort by conducting research to characterize
the effects  of global  climate change  on
ecological resources.

*EPA is also a member of the Intergovernmental
Panel on Climate Change (IPCC). In October of
1995, this United Nations-sponsored  group
released a report acknowledging for the first time
a link between human activities and climate
change and noting the vulnerability of ecosystems
and human health to such changes.
To provide information for policy makers that
will  reduce  the  scientific  uncertainties
surrounding the nature, rate, and magnitude
of ecological  response  to global  climate
change.

RATIONALE
Levels  of  greenhouse   gases   in  the
atmosphere are  irrefutably  on  the  rise.
Atmospheric CO2, for example, has increased
13% in less than 40 years. There is prevailing
scientific  agreement that the  continued
accretion of greenhouse gases will lead  to
significant   changes   in   climate-driven
conditions, such as rises in temperature and
sea level, shifts  in  the  composition and
geographic distribution of ecosystems, and
adverse human health effects.  However,
uncertainty remains regarding the rate and
magnitude of the projected changes, as well
as the processes that regulate or modulate
change.
RESEARCH
To ensure that the Agency is equipped with
scientific and technical data relevant to the
formulation of sound environmental policy,
ORD operates a research program founded
on risk-based principles. In the area of health
effects, the program explicitly conforms to the
risk assessment paradigm described by the
National Academy of Sciences (MAS) in 1 983.
This paradigm consists of 4 steps (hazard
identification,  dose-response  assessment,
exposure    assessment,    and     risk
characterization) that drive risk management
decisions. For ecological effects, the research
program follows the framework for ecological
risk assessment developed by EPA in 1 992,
consisting of problem formulation, analysis
(characterization of exposure and effects),
and   risk  characterization.     NHEERL's
research programs adhere to these  risk-
based strategies.  Three types of research
activities are emphasized:
       • Research in  the area of hazard
identification,  or  problem  formulation,
focuses on the development of methods that
can  provide  evidence of  an association
between exposure and effects.
       • Research characterizing  dose-
response seeks to identify and describe the
events linking exposure to effects. These
events, referred to here as determinants of
effect, form  the  basis for  the  predictive
models used to quantify risk.
       • There also may be instances when
scientific data on a particular contaminant or
 NHEERL GLOBAL CHANGE ANNUAL REPORT 1995

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stressor are required. In such cases, short-
term problem-specific research is conducted
to  systematically   collect  and   analyze
information that  will fill specific gaps  in
knowledge.

To characterize response to global  climate
change,  NHEERL  has  established three
research areas  that follow  the  precepts
discussed above. In the area of PROBLEM
FORMULATION, we  are assessing  the
ecological effects of climate change and are
identifying early warning signs to alert us to
ecological decline. Our DETERMINANTS OF
EFFECT  research is designed to evaluate
the events responsible for the interactions that
occur between ecosystems, atmospheric CO2,
and climate change, and we are developing
and utilizing predictive models to describe the
dynamics   of  global  change.    Finally,
PROBLEM-SPECIFIC STUDIES have been
conducted  to   provide   qualitative  and
quantitative data on the adverse health and
environmental effects caused by the depletion
of stratospheric ozone.
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      GLOBAL CLIMATE CHANGE RESEARCH
              FY95  PROGRAM  HIGHLIGHTS
The goal of this research is to improve our ability to identify and foresee the ecological
consequences of global climate change.

      > Our studies of a  fish species (Rivulus marmoratusj common to mangrove
      ecosystems indicated that its distribution may serve as a biological indicator of
      global climate change.

      * A technique for determining thermal requirements for freshwater fishes was
      developed and utilized to demonstrate that redistribution of fish species may
      represent a major impact of climate change.

      * Scientists from our Western Ecology Division were invited to lead two international
      scientific teams responsible for preparing key chapters in the report released this
      year by the IPCC.
DETERMINANTS OF EFFECT RESEARCH (pg 11)
The goal of this research is to better understand the factors that influence climate change,
atmospheric CO2 concentrations, and ecosystem vitality and productivity.

      *• As one of the major accomplishments of the entire U.S. Global Change Research
      Program, our Atlantic Ecology Division, in collaboration with extramural scientists,
      showed that the net loss of carbon from the terrestrial biosphere during the late
      1980s may be close to zero, indicating that carbon losses due to deforestation must
      be offset by carbon accretion elsewhere in the terrestrial biosphere. Following the
      Mt. Pinatubo eruption in 1991 and a transient period of global cooling, the biosphere
      became a net sink for CO2.

      *•  Along with extramural collaborators, we  demonstrated that atmospheric
      concentrations of CO2 are modulated by changes in the atmospheric supply of iron
      to  remote ocean areas; limitations in aeolian flux of iron constrain the growth of
      phytoplankton, thereby reducing marine CO2 uptake.

      > We used our predictive models to show that over the next 100 years, increasing
      amounts of CO2could be added to the atmosphere due to accelerated changes in
      the distribution of global vegetation
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6

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 he goal of this research is to investigate the effects of stratospheric ozone depletion.

      *• We showed that pigmentation in humans, which safeguards against sunburn and
      skin cancer caused by UV exposure, does not protect against immune suppression;
      however, some types of sunscreen are effective in mitigating suppression of the
      immune system.

      * Whereas studies using greenhouses and growth chambers had shown that UV-B
      had significant effects on rice production, our studies of rice grown under realistic
      field conditions (with a higher UV-A to UV-B ratio believed to stimulate cell repair)
      demonstrated that UV-B does not pose a major risk to crop yield.
NHEEFIL GLOBAL CHANGE ANNUAL REPORT 1995
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                  GLOBAL CLIMATE CHANGE
 PROBLEM FORMULATION RESEARCH PROGRAM
NHEERL  defines  problem  formulation
research  as  research to demonstrate a
causal  connection between exposure and
effect.
What   are   the   enduring   ecological
consequences of global climate change? Are
there early warning signs in the ecosystem
that presage change?
Changes in the global climate have begun to
have a significant impact on ecosystems. For
example, rising water temperatures resulting
from  global warming are responsible for coral
dieback and bleaching in  the  tropics.
Although our knowledge regarding the impact
of  climate  change  is  improving,  our
understanding of effects is limited. Changes
that  naturally or seasonally occur within an
ecosystem  can   confound  our ability  to
discriminate between effects that are short-
term and those that are more enduring, and
research  is  needed  to   clarify   these
differences.   In   addition,  indicators are
needed to alert us to impending ecological
decline. Bioindicators quantify the health of a
population or ecosystem and can  help
diagnose the most probable cause(s) of the
observed effects.

PROGRAM DESCRIPTION

To  characterize   ecosystem  response  to
climate change, NHEERL has established two
complementary areas of research.  We are
conducting   ECOLOGICAL   EFFECTS
research to evaluate the protracted response
of terrestrial  and  aquatic  ecosystems  to
climate variability and change, and  we are
developing methods that hasten detection of
the effects of global climate change, known as
INDICATORS research.  Indicators research
is an emerging field of interest  and one that
represents an area of growth for NHEERL. At
present,  we  are  evaluating  biological
indicators that possess a  high degree of
sensitivity  and specificity  for  ecosystem
damage. The goal is to identify and validate
relevant measurements that can serve as
prognosticators of change.
PROGRAM PROGRESS?
ECOLOGICAL EFFECTS. One of the ways in
which we  are characterizing  the  abiding
effects of climate change on ecosystems is
through experimental assessments of forest
productivity. Forest productivity is a gauge of
terrestrial ecosystem health and is measured
in terms of ecophysiological responses, such
as  the  rate of tree  growth,  death,  and
replacement. The life stage most vulnerable
to climate change is seedling establishment;
therefore, we have selected Douglas fir tree
seedlings for our studies of productivity. We
are exposing the seedlings to elevated levels
of CO2 (at varying temperatures) in controlled
environmental chambers maintained by our
Western Ecology Division for  studying the
effects of environmental stresses on plant and
soil ecological processes. These chambers,
collectively  referred to as  the  Terrestrial
Ecophysiological  Research  Area (TERA),
measure plant shoot, plant root, and litter/soil
processes  in  real  time.  Our research is
unique in that the effects of changes in both
CO2  and  temperature (as  well as other
variables, such as moisture) can be  studied
simultaneously.  Studies  were initiated  in
1993, at which time  the  seedlings were
planted  and  measurements  of survival,
growth,  and CO2 uptake  were begun.  In
FY95, we reported that as temperatures rise
(along with  enhanced CO2),  there is  an
increase  in   soil  respiration  with   an
accompanying release  of  CO2 into  the
atmosphere. Because this release is greater
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than the uptake of CO2 by the plant during
growth, there is a net increase in atmospheric
CO2.  This result suggests that increases in
temperatures will exacerbate climate change
by increasing atmospheric CO2. Increases in
temperature also  were  shown to  cause
asynchronous tree growth;  trees "break bud"
at an  irregular rate  when  temperatures
increase,  and  the  result  is  a  distorted,
deformed tree.  These chamber studies are
due to be completed in FY97, at which time
the trees will be harvested.

In a  related  vegetation  study,  NHEERL
scientists,  working  in  cooperation  with
scientists from the Desert Research Institute
in  Reno,  NV,  conducted  a  year-long
investigation beginning in FY93 on the effects
of elevated CO2 (in combination with nitrogen
fertilization) on the growth of the ponderosa
pine.  Scientists used a miniaturized camera
system   to  study  root  proliferation  and
asssociated fungal structures.   In FY95  we
published our findings.  We reported that,
contrary to some assumptions, higher levels
of atmospheric CO2  enhanced   seedling
growth, rooting depth, intensity of rooting and
fungal  colonization.     Apparently,  CO2
stimulates processes in the rhizosphere (the
root/soil complex) that enable the plant to gain
access  to additional nutrients.  Our data
support accumulating evidence suggesting
that processes in the rhizosphere are key to
the response  of vegetation "to  stress (see
"Indicators of Climate Change" below).

We are characterizing the effects of climate
change on fresh water ecosystems by studying
shifts in the composition and/or geographic
distribution of  aquatic species.   We are
attempting to  predict  such  shifts  using
estimates  of   the   tolerance  of  aquatic
organisms-specifically, freshwater fishes~to
changing habitat conditions, such  as  rising
water temperatures. Researchers in our Mid-
Continent  Ecology  Division  developed  a
technique  for  determining  the   thermal
requirements for freshwater fishes based on
available  field  measurements,  and then
utilized this technique to evaluate the potential
effects of rising water temperatures on the
distribution of 57 species of freshwater fish.
Our results, published this year, suggest that
redistribution of fish species may emerge as
a major impact of climate change. We found
that  temperature  shifts predicted  by  a
doubling of atmospheric CO2 would result in a
50% reduction in the existing habitat for cold-
and  cool-water fishes.   EPA's  Office  for
Policy, Planning and Evaluation used these
findings to estimate losses in revenue spent
on sport  fisheries in U.S. streams of up to
$320 million annually.

Also, we are  examining coastal ecosystem
effects of climate change.   Scientists in our
Atlantic   Ecology  Division   are  addressing
future ecological vulnerabilities along the U.S.
Atlantic  coast in  a project called "coastal
futures."  The goals of  our coastal  futures
research  are to anticipate the environmental
implications  of human  demographic and
climate-related changes along the Atlantic
coast and  to reduce  key  uncertainties
regarding the ecological risks associated with
these changes. Currently, research is being
conducted to address the combined effects to
coastal ecosystems of projected increases in
nutrient loading resulting from coastal zone
development  and increases in temperature
resulting from global warming. This research
will contribute to advances in regional and
state level vulnerability assessments.

INDICATORS   OF CLIMATE   CHANGE.
NHEERL is conducting  research to identify
biological  indicators   that  may   herald
ecosystem vulnerability to climate change. In
cooperation with researchers at the University
of Miami, scientists in our Gulf Ecology
Division are studying a coastal zone habitat-
the mangrove ecosystem~to identify aquatic
indicators of climate  change.   Mangrove
habitats  buffer   insular  and  continental
coastlines,  and their  response to climate-
induced  changes  could  influence  future
coastal zone  environments. The mangrove
ecosystem, which  is endemic to the central
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Florida coast, was  identified in  the  IPCC
report as an  ecosystem particularly at risk.
We are studying this model coastal wetland at
different levels of scale (species, habitat, and
landscape) to enhance  our ability to identify
ecosystem  processes  that  may presage
climate change. We  have begun by studying
a fish species (Rivulus marmoratus) common
to  mangrove  ecosystems  to   determine
whether it can be used as a surrogate for
mangrove  ecosystem  sensitivity.  During
FY95, we reported that the distribution of this
fish may serve  as  a biological indicator of
global climate change. These findings, along
with  those generated at the habitat  and
landscape levels, will be used to develop
models that address extrapolations of scale,
i.e., how effects compare as one moves from
species to habitats to landscapes.
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                   GLOBAL CLIMATE CHANGE
  DETERMINANTS OF EFFECT RESEARCH  PROGRAM
NHEERL defines determinants of effect
research as research to identify and describe
the bases for the health and/or environmental
effects caused by exposure to environmental
stressors or chemical contaminants.
What factors control, temper, or regulate the
ecological effects caused by climate change?
Levels  of  atmospheric  CO2  and  other
greenhouse gases are on the  increase,
driving a global-scale climate change that is
stressing ecosystems. Ecosystems respond
to climate-induced stress in a variety of ways,
some  of   which  further  impact   CO2
concentrations.  For  example, increases in
CO2  are known to stimulate plant growth,
increasing the sequestration of carbon by the
biosphere.  However, other factors, such as
tree  mortality, offset  these processes and
drive CO2 back into the atmosphere. Until the
fundamental bases for the ecological effects
of climate  change are  understood and the
processes  affecting the  flux of CO2 between
the  biosphere  and  the  atmosphere  are
explained,   substantial   uncertainties   will
remain  with regards  to  climate change
projections.

PROGRAM DESCRIPTION

The objective of this research program is to
achieve  a better understanding of  the
interactive processes linking climate change,
atmospheric CO2 (and other climate-induced
stressors),  and ecosystem response. We are
especially interested in ascertaining the ways
in which ecosystems modulate atmospheric
CO2  and thereby accelerate climate change.
To achieve our objective, we are conducting
MECHANISTIC RESEARCH, in which we are
analyzing  the  events  that  regulate  these
interactive processes, and we are developing
PREDICTIVE   MODELS  that   integrate
information  on the changing features of
ecosystems, atmosphere, and  climate to
explain the dynamics of change. Much of our
research focuses on the factors that control
the flux of carbon, a surrogate for tracking
greenhouse  gases,  through the  global
system.  Our research is helping to identify
major sources and sinks for greenhouse
gases and is closing critical scientific gaps in
our understanding of the  global carbon
budget,   which   will  increase   scientific
confidence in climate change predictions.
PROGRAM PROGRESS

MECHANISTIC  RESEARCH.     Marine
ecosystems   represent   an   important
component of the global carbon cycle.  To
explain their role in modulating atmospheric
CO2, we are studying factors (and associated
mechanisms)  that influence the oceanic
uptake of CO2. One factor is phytoplankton,
which utilize  CO2 for growth and thereby
remove it from the atmosphere.  It is believed
that growth of phytoplankton is constrained in
several open ocean areas by low levels of iron
flux from the atmosphere.  Our Atlantic
Ecology  Division  along  with  extramural
collaborators  have tested this hypothesis to
determine whether changes in iron flux in the
ocean in these  remote areas  modulate
marine  production  and,  consequently,
atmospheric CO2 concentrations.  This year,
we  reported  that carbon  fixation in  major
regions of the open sea is indeed limited by
low aeolian flux of iron; therefore, past and
future changes  in iron flux to the  ocean
surface  may  affect  atmospheric concen-
trations of CO2.

NHEERL also  is  conducting  mechanistic
research on terrestrial  ecosystems  by
studying the movement and sequestration of
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carbon in soils and in  the  rhizosphere of
plants (the root/soil complex).  Specifically,
scientists in our Western Ecology Division are
examining processes  that occur within the
rhizosphere   to   affect   plant   growth,
metabolism, and carbon utilization.  During
FY93 and FY94, we used a unique culturing
system to quantify the uptake and  allocation
of  carbon  by conifer  (ponderosa   pine)
seedlings. We were among the first to directly
measure carbon flux through all major pools
of the rhizosphere.  In FY94, we reported that
the symbiotic associations that exist between
the root system and its associated fungi have
a profound effect on the cycling  rate and size
of forest carbon pools.  This infers that the
role of forests in the global carbon cycle would
be  altered world-wide if these rhizospheric
associations were affected by global climate
change.    At the  regional  scale,  these
associations  could be  affected  by  other
anthropogenic stressors, such as tropospheric
ozone.  We therefore initiated a  project in
FY94 to obtain a mechanistic understanding
of rhizosphere response to stressors  using
tropospheric ozone as the test agent. In FY95
we  demonstrated  that ozone alters the
movement of  carbohydrates in ponderosa
pine seedlings, often reducing the amount of
carbohydrate  allocated  to  roots  and their
associated fungi.  As a result, the  seedlings
are more susceptible to nutrient and moisture
stress.  These findings  collectively suggest
that perhaps the most significant mechanism
for carbon release in plants exists within the
rhizosphere.    Moreover,   anthropogenic
stressors such as ozone  may magnify the
effects  of increases  in  temperatures and
regional  drought predicted  to result from
climate change.

Chemistry methods developed by extramural
collaborators of our Atlantic Ecology Division
are improving our  understanding of the
processes involved in CO2 cycling and are
enabling us to more precisely estimate trends
in biologically mediated carbon fluxes.  In the
past eight years, carbon measurements have
been  made  at  a  growing  network  of
monitoring stations, with joint support from
EPA and the National Science Foundation.
The results suggest that in the late 1980s, the
net  loss  of carbon  from  the terrestrial
biosphere may have been close to zero, and
that following the eruption of Mt. Pinatubo in
1991, the biosphere became a net sink  for
CO2. It has been postulated that following the
Mt. Pinatubo eruption, increased iron fluxes to
the  Southern  Ocean stimulated  marine
production,  and the subsequent transient
global  cooling  during the  next  few years
reduced  respiratory CO2 losses  from the
terrestrial biosphere.  Post eruption, the rate
of  atmospheric   CO2  increase   slowed
dramatically as a  result of these biospheric
responses.  This finding was recognized as
one  of the major accomplishments  for the
entire U.S. Global Change Research Program
in FY95.

PREDICTIVE MODELS.  NHEERL is  actively
engaged in the development of global climate
models that improve our ability to extrapolate
experimental findings  to real life situations.
Whereas current  climate models treat the
biosphere  as  a  static  system, we are
pioneering   the  development  of   more
comprehensive and dynamic  models that
integrate information on changing features of
the biosphere, the atmosphere, and climate to
more accurately describe the associations
between ecosystem structure, carbon content,
vegetation,   hydrology,  and  atmospheric
composition.

Since 1992, scientists in our Western  Ecology
Division   have   been  involved   in   the
development of models  that  simulate  the
transient response of terrestrial ecosystems to
climate change.   A  key  feature of  these
models is their ability to integrate the time lag
between climate change and response. We
simulated the  lags associated  with  forest
dieback, migration, and regrowth to predict
the timing and nature of forest response to
climate change.  In FY95 we published  our
findings, which revealed that over the next
100 years, increasing amounts of CO2 could
 NHEERL GLOBAL CHANGE ANNUAL REPORT 1995
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be  added  to  the  atmosphere  due  to
accelerated changes in  the  distribution  of
global   vegetation.      Redistribution   of
vegetation  will likely be driven  by forest
dieback and wildfires.  We also developed
and applied another model that simulates the
transient effects  of  delayed  migration  of
species and ecosystems under rapid climate
change. During FY95, results from our model
projected decreases in the diversity of tree
species as changes in climate and land use
accelerate. This suggests that most of the
species that dominate (and hence control)
ecosystem integrity are vulnerable to dramatic
reductions in number over the next 50 to 100
years.

We also are developing models that simulate
spatial responses of terrestrial ecosystems to
climate change, e.g., geographical shifts  in
vegetation. From these models we are able
to  predict changes  in  terrestrial carbon
storage and displacement.  This year, we
developed spatial data on the distribution  of
young  to late  secondary forests in tropical
forested regions; these data will be used  to
describe how carbon requirements change as
vegetation is redistributed. We also applied
spatial  analysis techniques to describe factors
influencing ecosystem vitality and productivity.
We combined regional vegetation models with
estimates of predicted  human use of forest
resources and identified  regions and timber •
products vulnerable to climate change. Much
of this  research was conducted  in support of
the  IPCC.    Finally,  we  developed  and
published in FY95 a method for measuring
and mapping uncertainty in spatial simulation
model  results; this  method  improves our
ability to extrapolate experimental findings to
ecological assessments.
An important issue largely ignored by current
climate  models  is the  linkage that exists
between terrestrial and aquatic ecosystems.
We are  conducting hydrological modeling to
relate terrestrial  ecosystem structure  and
function with  aquatic community character-
istics.   The goals of this research are to
predict the response of the hydrological cycle
to climate-induced changes  in precipitation
and to understand the interactions between
hydrological   systems   and    regional
vulnerabilities.    We  are   collecting  and
mapping information on soil erosion, sediment
runoff, and nutrient transport to evaluate the
impact  of hydrological change  on  the
terrestrial  landscape.  In FY95, these data
were  entered into   models that  simulate
regional  moisture flow through  terrestrial
ecosystems and into streams  and rivers.  Our
results will be  used to  project habitat changes
and to help explain the dynamics that exist at
the interface between terrestrial and aquatic
ecosystems.

Delineating geographic  areas of ecological
similarity  based on  multiple  landscape
characteristics has become a  powerful tool for
evaluating  patterns  of  regional ecological
response and for extrapolating observations
from individual sites to regional scales. This
approach  was developed  by  a  NHEERL
scientist and  is  referred to as  "Omernik
ecoregions." In FY95, we continued to refine
this approach and applied our techniques to
develop a classification for the western corn
belt. These ecoregion classifications advance
the scientific basis for  evaluations of response
to global climate  change.
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                   GLOBAL CLIMATE CHANGE
PROBLEM-SPECIFIC STUDIES RESEARCH PROGRAM
NHEERL defines problem-specific studies
research as research to describe the nature
and magnitude of the effects induced by a
specific   environmental   perturbation  or
chemical contaminant.
What are the adverse effects associated with
the depletion of stratospheric ozone?
Stratospheric ozone shields the earth from.
ultraviolet (UV) radiation, thereby providing
protection  against  possible  health  and
ecological effects. As levels of stratospheric
ozone decrease, the integrity of this protective
atmospheric layer is threatened, increasing
the potential for UV exposure. Some of the
adverse  effects  resulting  from   UV-B
exposures are well known,  such  as  skin
cancer.  However, other effects, such as the
suppression of  immune function,  are not
clearly  understood.    Moreover,   many
uncertainties remain with regard to ecological
responses to increased levels of UV.
PROGRAM DESCRIPTION
NHEERL's  stratospheric ozone  research
program  seeks to  resolve key questions
surrounding  the HUMAN  HEALTH and
ENVIRONMENTAL EFFECTSfrom exposure
to excess UV radiation.  We have studied
suppression  of human immune function,
ocular effects, effects  on crop yields,  the
effect on the oceanic food chain, and possible
synergistic effects between UV radiation and
chemical toxicity. This research supports the
periodic effects assessments required by the
Montreal Protocol and  provides data to the
Program Office to explain the implications of
ozone depletion.
PROGRAM
HUMAN HEALTH EFFECTS. The goals of
these research efforts were to assess the
impact of UV radiation on the human immune
system  and  to  characterize the  health
implications of immune suppression. Using a
variety  of experimental  techniques,  we
evaluated whether the effects to the immune
system from UV-B exposures are local or
systemic, and we evaluated the capability of
sunscreens and Vitamin A to protect against
suppression of  immune function.    Our
research to date has shown that UV-B does
indeed suppress human immune function and
that the suppression is systemic in nature.
Additional studies  in humans conducted this
year demonstrated that pigmentation, which is
a safeguard against sunburn and skin cancer,
does not protect against immune suppression;
however, some types  of  sunscreen are
effective  in  mitigating suppression  of  the
immune system.

To  enhance  our understanding  of  the
immunologica! effects  of UV radiation,  we
developed animal models to study immune
function. We identified a strain of laboratory
mice with mechanisms of immune supression
similar to those found in humans and used
this  animal model to study the effect  of
immune suppression on disease suscepti-
bility.  In FY94 we found that several types of
infection were  altered by exposure to UV-B,
and   in  FY95  we demonstrated   that a
laboratory diet supplemented with Vitamin A
provided some protective effect against UV-
induced immune suppression.

We  also have conducted field  studies in
southern Chile, an area that offers a unique
opportunity to  study the health effects of
increased UV exposure due to its location
near  the seasonal Antarctic stratospheric
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ozone "hole". During FY95, we reported that
it is possible to perform standardized ocular,
dermatologic, and immunologic testing in the
field.   Despite anecdotal reports  of  blind
sheep, our studies of ocular effects in animals
found no short-term effects produced by UV
exposure.

ENVIRONMENTAL EFFECTS.  During FY95,
NHEERL   scientists  resolved  a  critical
question regarding the effects  of enhanced
UV radiation on the  production of  rice, the
world's   most   important  crop   species.
Previous studies by others using greenhouses
and growth chambers had shown that UV-B
had  significant effects on rice production.
However,  research conducted this year by
NHEERL found no such effects when rice was
grown  under realistic field conditions.  This
suggests   that  field-grown   rice  is   less
susceptible to the effects of UV-B (possibly
due  to  higher UV-A to  UV-B  ratios, which
stimulate   cell   repair).     Consequently,
increased UV-B  does not appear to pose a
major risk to rice yield.

Marine phytoplankton are at the base of the
oceanic food chain.   Reductions in their
productivity could have major impacts on the
structure   and/or   function   of   marine
ecosystems.   Phytoplankton are especially
vulnerable to increases in UV-B because they
inhabit  the  surface  zones that  are highly
exposed to  UV  radiation.  'During  FY95,
NHEERL scientists found that ambient levels
of UV-B  may  inhibit  the light  harvesting
efficiency and production of phytoplankton in
clear waters typical of the open sea.  Ship-
board  experiments  were  conducted  on
Antarctic phytoplankton during the period of
the ozone "hole" to assess maximum UV-B
impacts under field conditions.  Our results,
which are currently in press, indicate that
increased    UV-B   alters   the  pigment
composition of phytoplankton.  These findings
support laboratory-based data.  However,
because the effect is mitigated by cloud cover
and mixing  of phytoplankton in the  water
column, direct  application  of  laboratory-
derived dose-responses to the Antarctic may
overestimate effect. To assess the sensitivity
of  different  phytoplankton   physiological
processes to UV-B, we tested the effects of
UV-B  on nitrogen uptake. We reported this
year  that  ecological  assessments  based
solely on reductions in carbon fixation without
consideration of nitrogen may underestimate
the extent of damage to phytoplankton.

Finally, in studies of the effect of sunlight on
the toxicity  of  chemical  compounds,  we
reported during FY95 that the toxicity of some
aromatic hydrocarbons can be increased by
several orders of magnitude in the presence
of sunlight. UV light activates some of these
compounds  such  that toxicity  increases
directly  with  increases in light intensity and
energy.      Quantitative   Structure-Activity
Relationship (QSAR) studies are permitting us
to  predict the  relationship  between  the
structure of  a chemical and the  extent to
which sunlight increases its toxicity.
 NHEERL GLOBAL CHANGE ANNUAL REPORT 1995
                                     15

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     I
  *l*—^
 r*»tl
National Health and Environmental Effects Research Laboratory
  CRITERIA AIR POLLUTANTS: PM10




  HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH
              ANNUAL REPORT




              NOVEMBER, 1995

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                                    NHEERL PM,0 ANNUAL REPORT 1335
                          CONTENTS
Introduction	3




Summary of the PM10 Health and Environmental Effects Research Program ....  4




FY95 Program Highlights	5




PM10 Problem Characterization Research Program  	6




PM10 Dosimetry Research Program  	8




PM10 Causal Mechanisms Research Program	 10

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                                        NHEERL PMW ANNUAL REPORT 1995
                            INTRODUCTION
PURPQSE: The purpose of this report is to communicate results within EPA from the
Office of Research and Development (ORD) Particulate Matter (PM10*) Health  and
Environmental Effects Research Program.
CONTENT:  The report contains (1)  a summary of the Health and Environmental
Effects Research Program for PM10, which describes the ORD context of NHEERL's
research program, the overall program goal, the rationale for the program, and the
research approach; (2) a section that highlights some recent key findings (FY95
Program Highlights);  and (3)  a more detailed description of the NHEERL PM10
Research  Program,  by  program   area,   including   a  summary   of  research
accomplishments and anticipated progress for the near future.
COMMENTS WELCOME: The format of this report is still evolving, and we welcome
feedback.  Readers with comments, suggestions, questions, or requests for further
information are encouraged to communicate them to lla Cote, Assistant Director,
National Health and Environmental Effects Research Laboratory (MD-51A), U.S. EPA,
Research Triangle Park, N.C. 27711.  [Phone: (919) 541-3644 or FAX: (919) 541-
0642]
 PM10 refers to the most respirable air particles-those 10 micrometers and smaller.

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                                       NHEERL PM10 ANNUAL REPORT 1995
          PM10 HEALTH AND ENVIRONMENTAL EFFECTS
                  RESEARCH PROGRAM SUMMARY
EPA/ORD CONTEXT OF NHEERL'S PM10 RESEARCH PROGRAM: Sections 108-109
of the Clean Air Act (CAA) require EPA to periodically review and revise National
Ambient  Air  Quality Standards (NAAQS) for ambient air  pollutants, including
paniculate matter (PM). EPA's Office of Research and Development (ORD) provides
scientific support for this process.  ORD's PM10 research program, which is based
upon a peer-reviewed PM10 strategy, is designed to reduce uncertainties in risk
assessment and to provide sound scientific data to guide decision-makers in possible
revisions of the standard. Four areas where substantial uncertainties exist have been
identified by ORD and are the focus of its research efforts: health  effects research,
exposure research, source characterization, and  risk assessment.  This  document
summarizes the health effects research.
PROGRAM GOAL: To provide credible  PM  health effects data that reduce the
uncertainties in risk assessment and thereby guide revisions of the PM standard.
RATIONALE: Recent epidemiological studies of urban populations have suggested that
exposures to paniculate matter at levels below the current NAAQS may lead to
increased  morbidity  from pulmonary  disorders and  increased  mortality  from
cardiopulmonary diseases and cancer. Age and pre-existing cardiopulmonary disease
appear to be important factors in PM susceptibility. PM research is needed to provide
a more  cogent  explanation of the public health burden  incurred by exposure to
paniculate matter.
APPROACH:   Research   is   being   conducted  to  (1)  improve   PROBLEM
CHARACTERIZATION by providing more detailed analysis of existing epidemiologic
data and by initiating new epidemiologic studies utilizing more thorough assessments
of particle composition and exposures; (2) evaluate DOSIMETRY (exposure-dose
relationships) by measuring and modeling particle deposition in the lungs; and (3)
investigate CAUSAL MECHANISMS by determining the role of PM composition, size,
and physical properties on health effects.

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                                         NHEERL PM10 ANNUAL REPORT 1995
                       NHEERL PM10 RESEARCH
                     FY95 PROGRAM HIGHLIGHTS
Problem Characterization Research (pg 6)

The goal of this research  is to improve our understanding of the epidemiological
observations suggesting a relationship between increased mortality/morbidity and PM
exposure.

      >We assembled a peer-review advisory panel of widely respected extramural
      scientists to provide epidemiological guidance to EPA investigators.

Dosimetry Research (pg 8}

The goal of this research is to develop animal and human dosimetric models to better
understand the role of particle size and pre-existing conditions  on the health effects
of PM and to facilitate animal-to-human extrapolation.

      *• We  demonstrated that breathing pattern and airway resistance, not age or
      gender, are the most significant factors affecting particle depostion in the lung.

      >We formulated an artificial lung lining fluid that  simulates human lung lining
      fluid;  it is being  used to improve dosimetry estimates  and to elucidate the
      mechanisms of ox/dative stress in lung disease.

Causal Mechanisms Research (pg 10)

The goal of this research is to explain the biological mechanisms that evoke the health
effects associated with exposures to PM and its components.

      »• We found that the "fingerprint" of mutations induced by PM in human lung
      tumors differs according to the source of PM exposure.

      *• We  developed two rodent models of enhanced susceptibility to permit the
      evaluation of pre-existing inflammation as a risk factor in PM responsiveness.

      *• We  found that the amount of transition  metal present on air particles is
      directly related to the severity of oxidative lung damage.

      *•  We  showed that urban  air  particles  are  much more  cytotoxic and
      inflammatory than natural particles, such as asbestos or silica.

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                                        NHEERL PM10 ANNUAL REPORT 1995
   PM10 PROBLEM CHARACTERIZATION RESEARCH PROGRAM

NHEERL  defines problem characterization research as research to  identify and
describe  the  health and environmental risks posed  by exposure to environmental
contaminants.

Issue

Do exposures to PM at levels below the current national standard produce increased
mortality and morbidity?  If so, what health effects are caused by PM and  its
components?

Recent assessments of epidemiological data have shown significant associations
between  various measures of ambient paniculate matter and excess mortality and
morbidity, raising serious concerns that exposure to PM at levels below the currently
accepted standard  may  impose a heavy burden on  human health.  However,
uncertainties in the data, such as limited characterizations of exposure and possible
artifacts  in the statistical methodology, have clouded our understanding of these
observations.

Program  Description

This research is designed to help  EPA understand  the relationship  between PM
exposures and  health effects  (mortality  and morbidity) using  more advanced
biostatistics,  more  coherent mortality and  morbidity  measures, and  improved
characterization  of exposures.  Epidemiologists  in NHEERL are collaborating with
investigators in EPA's National Exposures Research Laboratory (NERL), the National
Center for  Evaluation and  Assessment  (NCEA), and  other public  and  private
organizations to re-analyze existing epidemiological databases using better exposure
data  and  additional  statistical  applications  and  to  construct  additional
morbidity/mortality data sets using improved exposure monitoring and biomedical
data.

Program  Progress

Re-analyze existing epideminlngical databases. We are re-evaluating particle data
collected by EPA from 1991-1992 using improved characterizations of exposure, and
we are integrating these data with mortality figures for the same period. This new
analysis  will significantly  enhance evaluations of the relationship between mortality
and  PM exposures.

Construct additional data sots. The objective of this research is to collect additional
data for  use in epidemiology studies  of the health effects of PM. To support this

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                                         NHEERL PM10 ANNUAL REPORT 1995
effort, we undertook a 10-year survey of size-specific ambient PM levels in 36 U.S.
locations; the survey was completed this year. The PM data collected in this survey
will be coupled with data on mortality and morbidity (lung function endpoints)  to
evaluate health effects relative to PM size.

New epidemiology studies, coordinated by NHEERL and jointly funded by Federal,
State, and academic contributions, are underway. We assembled an advisory panel
of expert extramural scientists to provide peer review and technical guidance for
these studies, and the panel is due to submit a final report on NHEERL's epidemiology
research plan in FY96.

Our multi-year cooperative venture on health research in China continues and is near
completion.  We  have collected data on lung cancer mortality in Xuan Wei, China
(where a high incidence of lung cancer has been observed) and are relating these
health statistics to various measures of PM exposure.  This analysis will improve our
understanding of the relationship between lung cancer and PM.  In  another study
underway in China, the relationship between PM and morbidity is being assessed
using health endpoints such as pulmonary function and respiratory illness.  During
1995, we were engaged in the sampling of PM in 4 cities of China with disparate
levels  of   ambient  PM  exposures.    Biomedical-demographic-socioeconomic
questionnaires were administered to households taking part in the study, and lung
function  data were collected  by performing spirometry tests in the children  of
participating households.  This  project, scheduled for completion in 1999, is being
performed in collaboration with  NCEA, which will assume responsibility  for the
remainder of its execution.

In  cooperation  with   researchers  in  the  Czech  Republic,  we  instituted  a
multidisciplinary health  research program in 1992 to document the relationship
between human health effects and PM exposures in a heavily industrialized region of
Eastern Europe.  We are  studying respiratory, neurobehavioral, reproductive, and
genetic effects in the Teplice  District, an  area of exceptionally high levels of PM.
Results generated this past year  indicate an increase in the prevalence of respiratory
symptoms in school children (compared to children living in less polluted districts);
neurobehavioral performance in this same population of children was not found to be
related to  air  pollution exposure.   Studies  are  underway to characterize the
relationship between PM  exposures, exposure to carcinogenic constituents in the air,
and biomarkers of dose  and genetic damage.

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                                         NHEERL PM10 ANNUAL REPORT 1995
               PM10 DOSIMETRY RESEARCH PROGRAM

NHEERL  defines dosimetry research as research that  elucidates the relationship
between  exposure and dose at the site of toxicity.

Issue

What is the relationship between PM exposure, dose, and the effects observed in
sensitive subpopulations?  How do the effects observed in laboratory animals relate
to human response?

Substantial uncertainties exist regarding the relationship between PM exposure, dose,
and  observed effects.  It is believed that air  flow in the lungs of  healthy and
susceptible individuals differs, thereby influencing the pattern of particle deposition
in the lung.  Particle deposition, in turn, determines dose and  contributes to the
effects of PM. Understanding deposition patterns and the clearance of particles from
the lungs of healthy and susceptible individuals is critical for assessing the potential
risks of PM.  Of additional importance  is the attendant development of mathematical
or computational models to explain dose distribution in the airways and to link data
collected in animals to humans.  Of particular interest are dosimetric models for
susceptible  subpopulations;  such models do not presently exist, in part because
toxicology data on PM has been  generated only in healthy animals.

Program  Description

To improve our understanding of PM exposure-dose  relationships, we are examining
particle deposition  and clearance in  the lungs  of humans and  laboratory animals
exhibiting normal and abnormal lung function.  Both monodispersed aerosols (aerosols
composed of particles of uniform size)  and polydispersed aerosols (particles of varying
sizes and more representative of real-world exposures)  are included  in our studies.
An  important component of our research is the effect of factors such  as  airway
obstruction,  age, and pulmonary disease on particle retention and deposition. Our
findings are then used to develop dosimetric models to estimate dose distribution in
the human lung.  These models are helping us predict exposure-dose relationships in
individuals with abnormal lung function and are assisting our extrapolation of animal
data to humans.

Program  Progress
       deposition and ntearanre. The objective of this research is to provide human
exposure-dose data for use in risk assessment and to help relate this data to the
effects observed at the site of toxicity. We are using particles differing both in size
(ultrafine, fine, and coarse)  and in size distribution (mono- versus polydispersed
aerosols) to measure deposition dose in models of the human airway, in experimental

                                      8

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                                          NHEERL PM10 ANNUAL REPORT 1995
animals, and in human volunteers.  Collectively, these research efforts will help us
understand the distribution and clearance kinetics of PM in the human respiratory
tract.

The airway models allow us to perform mathematical analyses of dose distribution
and to study how particle distribution is influenced by changing conditions, such as
airway obstructions, air  flow rates,  branching patterns, etc.  In addition, we have
developed an in vitro model in  which we formulated an artificial lung lining fluid to
simulate conditions in the  human lung; results from this study are  improving  our
dosimetry estimates.

Our animal experiments are  helping to explain particle clearance kinetics.  This year,
we completed a pilot study in dogs in which we evaluated the retention of particles
in the lung following intrabronchial deposition. Our research demonstrated complete
clearance  of particles from large airways  within 24 hours, which  confirms  the
conventional understanding of particle  clearance kinetics.

Our human studies are elucidating differences in particle deposition between normal
and  abnormal lungs.   We  have been  able to refine our research  by developing a
technique that can measure minor airways obstructions, which are ordinarily difficult
to detect using conventional lung function tests. This year, we showed that the most
significant factors affecting fine-particle deposition in individuals with normal lung
function are breathing pattern and airway resistance.  We are also studying the  role
of alveolar macrophages and cellular response in  particle clearance, and these data
are being analyzed.  In studies  of particle clearance kinetics in humans, we demon-
strated this year that "inert" particles cause a significant inflammatory reaction in the
human lung, lending credence to the theory that urban air particles may be potent
inducers of lung  damage and inflammation.  We also have examined variability in
particle deposition with respect to age (children vs.  young adult vs. elderly)  and found
that age appears to have only minor effects on deposition.  Over the next two years,
we plan to study particle deposition in three additional  susceptible population groups:
heavy smokers, asthmatics, and persons with chronic obstructive pulmonary disease.

Doslmetrin models.   The objective  of this research is  to  develop  more  realistic
estimates of dose distribution in  the human lung. Using the Cray supercomputer, we
are incorporating the data obtained from the studies described above to modify  and
expand existing dosimetry models. The  improved models are assisting predictions of
exposure-dose relationships under a variety of conditions, such as obstructed airways
and uneven ventilation. Our findings during the past year indicate that the total lung
deposition of particles increases with airways obstruction, suggesting an enhance-
ment of particle deposition in individuals with abnormal lung function.  These models
also will aid in the extrapolation of animal data to better define exposures of concern.
As human clinical and animal studies progress and more data become available, the
theoretical models will be validated and improved.

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                                         NHEERL PM10 ANNUAL REPORT 1995
        PM10 CAUSAL MECHANISMS  RESEARCH PROGRAM

NHEERL defines causal mechanisms research as research to identify and characterize
the physical, chemical and/or biological mechanisms whereby an environmental agent
may induce health or environmental effects.

Issue

What are the causal mechanisms that can provide a biologic explanation for the
epidemiologic observations of excess mortality and morbidity?

Despite human evidence linking low-level PM exposures with adverse health effects,
no plausible biological mechanisms for the observed toxicity have been offered.
Understanding causal mechanisms would help explain the recent findings relating PM
toxicity to particle size and composition and  would help account for variability in
susceptibility to  PM.   The issue  of susceptibility  is important because  sensitive
subpopulations, including individuals with  physiological abnormalities, pre-existing
disease, or weakened physical condition, may be predisposed  to unusual response,
injury, or death when exposed to particles.

Program Description

Our research on causal mechanisms is focused in two areas. First, we are assessing
the effects of particle size and composition on PM toxicity. In vitro methodology is
being used to test  specific mechanistic hypotheses of injury and response at the
cellular level; experimental animals are being used to investigate the role of particle
characteristics in lung inflammation and cancer; and human volunteers are being used
to evaluate specific biochemical and physiologic events resulting from PM exposures.
Two  particle-associated agents purported to play a  role in toxicity --  metals and
organic matter — are receiving special attention. Secondly, we are evaluating the role
of susceptibility factors, such as pre-existing lung disease, on  response to PM. We
are developing animal models of respiratory disorder  and  disease to represent
sensitive  human  subpopulations  and are  using  these models to  study enhanced
susceptibility to PM. Data also are  being collected  in humans with respiratory disease
(e.g., asthmatics) to explain predisposition to the effects of PM.

Program Progress

Partinie size/composition.  In vitro techniques are being used to  describe injury at the
cellular level and to devise mechanistic explanations of toxicity. We are exposing
lung cells and tissues (both animal and human) to a wide range of PM sources and
sizes to test specific hypotheses regarding  the  relationship  between toxicity and
particle size/composition.  Toxicity endpoints include oxidant formation, inflammation.
                                     10

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                                         NHEERL PM10 ANNUAL REPORT 1995
and  DNA damage  (discussed  below under  human studies).  Results this  year
demonstrated that urban air particles are much more cytotoxic and inflammatory than
natural particles, such as asbestos or silica.  In addition to providing a database on
the relative toxicities of particles from many sources, results from the in vitro studies
will strengthen animal-to-human extrapolations.

We are conducting animal studies to investigate the role of surface-related metals in
oxidant lung injury and inflammation and to evaluate the effect of particle-associated
organics on the induction of cancer. In  our studies of surface-related metals, we are
exposing animals to paniculate matter high in metal content (residual oil fly ash) and
are measuring various physiological and  biochemical indicators of damage, such as
oxidant formation.  We also have developed a method to adhere metals to  inert
particles to mimic the fly ash and have used these metal-coated particles in our animal
studies.  Our findings indicate that the amount of  metal present on air  particles is
directly  related  to the  severity of injury and that  lung damage appears  to occur
through  an oxidant mechanism. With regard to cancer, our studies have shown that
PM contains organic matter that is carcinogenic in animals.  We also have produced
evidence that particle size may play a  role in cancer induction.  These experimental
studies will serve as the basis for subsequent human studies.

Results  from our human studies suggest that exposure to increasing levels of PM
results in  elevated levels  of genetic damage (DNA adduct formation).   To better
characterize this relationship, we are developing biomarkers of PM exposure (urine
metabolites and  DNA adducts) and biomarkers of molecular effect (DNA adducts and
mutations at the k-ras gene  in human lung tumor samples),  with  complementary
studies conducted in animals.  By relating biomarkers of exposure to biomarkers of
effect and further linking these data to PM10 exposure profiles, a better understanding
of the sequence of events leading to health effects is being attained.  Results  have
been promising thusfar. Our group published findings in 1994 suggesting that small
particles may induce cancer via cell proliferation or other pathways not involving the
formation of DNA adducts. Within the  past year, we have shown that the spectrum
of mutations at the k-ras gene from lung tumors differs depending on the source of
PM exposure, indicating the possible involvement of different mechanistic pathways.

Susceptibility Factors
Due to the apparent strong association between pre-existing cardiopulmonary disease
and excess  mortality, we are developing  animal models of emphysema, pulmonary
and systemic hypertension, asthma, and infectious disease to represent  sensitive
human subpopulations. This year, two  rodent models of enhanced susceptibility
(pulmonary  hypertension  and pre-existent inflammation)  were developed to  help
explain the risk factors involved in PM responsiveness.  Studies of sensitive human
subpopulations,  such as  asthmatics  and persons with chronic obstructive  lung
disease, are just getting underway. Individuals will be exposed to ambient particles,
and we  will  measure indices of lung function and cardiovascular function.


                                     11

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                                         NHEERL PM10 ANNUAL REPORT 1995
Development of measurement techniques.  The objective Of this  research is  to
develop a continuous monitoring method to measure deposition of polydispersed
aerosols in the lung using laser optics adapted for inhalation studies. This technique
will permit a more accurate assessment  of particle inhalation,  respiratory tract
deposition, and dose distribution  of ambient PM.
                                     12

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National Health and Environmental Effects Research Laboratory
        PESTICIDES IN THE
DIETS OF INFANTS AND CHILDREN

  Peer-Reviewed Research on Priority Issues
            September, 1995

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                     PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
                            CONTENTS
Introduction	  3



Research Program Summary	  4



NHEERL Research Project Selection Process	  5



Summary of NRC Recommendations	  6



Research Progress



     Age-Related Differences in Sensitivity Research Program  	  7



     Toxicity Testing Guidelines Research Program	  9



NHEERL Research Projects



     The Effects of Pesticides on Reproductive Toxicity	 10



     The Effects of Pesticides on Learning and Memory 	 11



     The Biochemical Effects of Pesticides on the Central Nervous System  	 12



     The Effects of Pesticides on the Immune System and Allergic Response  ... 13



     Neurochemical Changes and Behavioral Effects Induced by Pesticides	 14

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                      PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
INTRODUCTION
      EPA scientists have long been concerned that infants and children might be
especially vulnerable to the toxic effects of pollutants. In 1993, these concerns were
reinforced by the National Research Council (NRC) in its report entitled Pesticides
in the Diets of Infants and Children. The NRC found that children may be at greater
risk  than  adults to the toxic effects of pesticide residues  on  foods.   It also
acknowledged that before sound estimates of the scope and magnitude of this risk-
could be made, important gaps in the scientific database on pesticides had to be
filled. The NRC identified areas where information was needed and proposed a
broad-based research strategy to characterize the unique susceptibility of infants
and children to the toxic effects of dietary pesticides.

      Research on the differential risks associated with exposures to pesticides is
an integral part of the intramural program at the National Health and Environmental
Effects Research Laboratory (NHEERL).  A multidisciplinary program  addressing
age-related differences  in pesticide toxicity is fundamental to NHEERL's on-going
research efforts.  In addition, NHEERL recently initiated a targeted, peer-reviewed
program to expand the scope of existing pesticide research to determine how young
and mature animals differ in their responses to exposure to pesticides.  Scientists
throughout NHEERL submitted research proposals in  a competition  for funding
under this program. The best research ideas are now being implemented.  Findings
will supplement the database on age-dependent toxicity to pesticides and explain
some of the principles guiding developmental toxicity. Results also will be used to
revise current testing guidelines and to reduce uncertainty in risk assessment.

      This document summarizes

            • the overall NHEERL research program in this area
            • the project selection process implemented by NHEERL
            • the NRC  recommendations
            •  research progress made by NHEERL in addressing the NRC
                            recommendations
            • the individual projects contributing to the  research progress
                 reported

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                     PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
RESEARCH PROGRAM SUMMARY
      An integrated and multidisciplinary research program addressing age-related
quantitative and qualitative differences in pesticide toxicity is part of the on-going
research program at NHEERL. This program consists of several research projects
within  the  Neurotoxicology, Developmental  Toxicology,  and  Environmental
Toxicology Divisions which compare the toxicity of representative pesticides in adult
and immature animals. Studies are currently underway to:

      •provide more complete toxicity data in animals exposed to pesticides during
      development

      • characterize qualitative and quantitative differences in response between
      young and adult animals

      • provide data to improve current testing guidelines

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                     PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
NHEERL RESEARCH PROJECT SELECTION PROCESS
      During FY95, NHEERL expanded the scope of its existing pesticide program
by initiating several new projects that address the research needs outlined in the
NRC report, Pesticides in the Diets of Infants and Children. These projects were
selected  based on the following  process  and will provide the foundation for
NHEERL's future research in this area.

      • A Steering Group was formed to develop a comprehensive research
      strategy responsive to the recommendations made by the NRC. The strategy
      pivots on 3 key issues identified by the NRC (described in greater detail on
      page 6):
           age-related differences in sensitivity to pesticides
           toxicity testing guidelines
           reducing uncertainties

      •A Request for Proposals (RFP) was developed and distributed internally to
      NHEERL scientists.

      • An extramural panel was convened to review the proposals for scientific
      merit.

      • A panel consisting of Assistant Laboratory Directors  and  Program Office
      Representatives reviewed the proposals for programmatic relevance.

      • Proposals were prioritized according to scientific merit and programmatic
      relevancy and funded as a function of available funds.

      • Five projects were selected and were initiated late in FY95.
A description of the proposals for which resources were allocated begins on page
10.

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                      PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
SUMMARY OF NRC RECOMMENDATIONS
Among the recommendations set forth by the NRC1 to safeguard the health of infants and
children were the following:

Assess Age-related Differences in Sensitivity to Pesticides (pp. 42-44; 105-110; 152-156)
      According to the NRC report, toxicity data on mature animals may not sufficiently
characterize the toxic effects of pesticides in young animals. The report recommended
that studies be  conducted  to  resolve the  issue of potential  differences in
susceptibility between young and adult animals, and that this research lead to the
development of appropriate models for evaluating pesticide toxicity in infants and
children. The NRC also found a paucity of data related to pesticide toxicity in developing
organisms, particularly with regard to neurotoxic, immunotoxic, and endocrine responses.
It was recommended that research be conducted to fill these data gaps and to
explain the  underlying principles  guiding  developmental  toxicity (such  as
pharmacodynamics, metabolism, and mechanisms of action).

Develop Toxicity Testing Procedures (pp. 105-110; 152-156)
      The NRC  observed that current testing guidelines do  not include standard tests on
immature animals as  part of the  basic evaluation  of pesticides for  toxicity.   It
recommended that a standard developmental assessment protocol be established
to facilitate the  systematic interpretation of pesticide toxicity studies in immature
animals. A redesign  and  expansion in scope of current test methods would be
required, especially in the areas of neurodevelopmehtal effects, immunotoxicity, and
reproductive/developmental toxicity. In cases where no such guidelines exist, as in
the visual system, procedures would need to  be developed and validated.  It was
recommended  that several representative classes of pesticides be included in
validation studies to compare responses in adult and immature animals.

Reduce Uncertainty (pp. 359-363)
      The evaluation of potential risks to infants and children due to dietary pesticide
residues requires consideration of several factors that can impact the risk assessment
process. To reduce the degree of uncertainty in estimates of risk, the NRC recommended
that physiological parameters and biochemical measurements in young and adult
animals be compared to assess age-dependent differences, that evaluations of age-
related differences  in absorption, metabolism, detoxification, and excretion of
pesticides (called PBPK modeling) be made, that the use of the benchmark dose for
risk assessment applications be explored, and  that toxicological data be generated
to help evaluate the adequacy of the uncertainty factor  used in calculations of risk
assessment.

1 Pesticides in the Diets of Infants and Children, National Academy Press, Washington, DC, 1993.

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                      PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
AGE-RELATED DIFFERENCES IN SENSITIVITY TO PESTICIDES
RESEARCH PROGRAM
Program Description
      A multidisciplinary evaluation of quantitative and qualitative age-related differences
      in pesticide toxicity is an integral part of the  existing research program within
      NHEERL.   Organ systems, behavior,  and biochemical  responses  are being
      examined to enhance our understanding of the principles of developmental toxicity.
      Special emphasis is being  placed on neurotoxic, immunotoxic, and endocrine
      responses; at issue are reproductive competency and function, neurobehavioral
      changes, neurochemistry, neural growth and differentiation, allergic response, and
      immune function.  New animal models are being developed that may more closely
      approximate responses in humans. Results from these studies also will address a
      number of issues of concern to risk assessors, including the extrapolation of animal
      data to human populations, the adequacy of the current uncertainty factor used in
      calculations of risk assessment, the identification of the most appropriate adverse
      effect for calculations of the reference or benchmark dose, and the determination
      of safe levels (tolerances) of pesticide residues.  In addition, studies of mechanisms
      of action will enhance biologically-based risk assessment models.
                           RESEARCH PROGRESS

Recent NHEERL Findings

      • Younger animals can be up to 21  times more sensitive than adults to the
      neurotoxic effects of a pyrethroid pesticide.

      •Younger animals are more sensitive to the lethal effects of some cholinesterase-
      inhibiting pesticides; however, the difference is usually less than a factor of 10.

      • The developing visual  system  is sensitive to the  effects of cholinesterase-
      inhibiting pesticides.

      •Some pesticides affect steroid hormones during development in rats, which can
      alter the onset of puberty.

Current Activity

      • Studies are underway to examine toxicokinetic and toxicodynamic factors
                      PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN

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AGE-RELATED DIFFERENCES, CONT'D
      that might explain age-related  differences in sensitivity to the cholinesterase-
      inhibiting pesticides.

      • On-going studies are testing the hypothesis that exposure to pesticides during
      development results in myopia and that the effect is age-dependent.

      • Cholinesterase-inhibiting pesticides have been shown to affect learning and
      memory in adult rats; studies to examine these effects in young animals are
      underway.

Near Future

      • Age-related effects wijl be evaluated using
           Physiological  parameters  (semen evaluations,   reproductive  organ
           changes,  neural  growth  and differentiation,  brain  weight, and  lung
           inflammation)
           Behavioral changes (effects on learning and memory)
           Biochemical measurements (endocrine markers,  immune  response
           endpoints,  DMA and  protein  synthesis, enzyme activity, and  hormonal
           measurements)
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                       PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
TOXIC1TY TESTING GUIDELINES
RESEARCH PROGRAM
Program Description
      Studies are underway that could lead to the revision of current testing guidelines for
      developmental toxicological endpoints, including teratogenic,  reproductive, and
      neurotoxicological effects. Established rodent models are  being used to  study
      exposure strategies, biochemical changes, and behavioral endpoints in an effort to
      improve existing test procedures.   In addition,  new test  methods are  being
      developed and validated to augment current guidelines. Representative classes of
      pesticides are being used to facilitate our understanding of the differences in
      response between young and adult animals.

                           RESEARCH PROGRESS

Recent NHEERL Findings

      •NHEERL research has helped OPPTS develop testing guidelines for assessing
      the  effects of pre- and perinatal exposures to  pesticides to predict outcome in
      infants and in children.

      • NHEERL  scientists helped draft the  developmental  neurotoxicity testing
      guidelines.

Current Activity

      • Studies to further refine and validate the procedures described in the testing
      guidelines continue.

      •Research on the visual toxicity of cholinesterase-inhibiting pesticides is underway.

Near Future

      • New test procedures for assessing differential sensitivity  to pesticides will be
      developed, including
            a rabbit model for reproductive toxicity testing
            new tests for learning and memory
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                       PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
NHEERL RESEARCH PROJECTS
The Effects of Pesticides on Reproductive Toxicity


General Approach
      This research will use male rabbits as a model to determine how pesticides (o,p-
      DDT and p,p'-DDE)  administered at a young age affect the development and
      function of the mature reproductive system. The rabbit, with its prolonged period
      of reproductive development, closely approximates the infancy-adolescence phase
      in humans. However, the rabbit model has not been used routinely in studies of this
      type; therefore, a subordinate objective of this research is the establishment of the
      rabbit as a relevant  animal model for study of male reproductive competency.
      Analyses will  include endocrine  markers, semen evaluations (sperm number,
      motility, and morphology), fertility, and reproductive organ changes.


Research Questions

      Is the reproductive system of the young more susceptible to pesticides than that of
      the mature animal?

      What exposure levels pose a risk?

      Is the rabbit a good model for reproductive toxicity?

      What are the effects of pesticides on endocrine markers, on semen (sperm motility,
      morphology), on fertility, and on reproductive organ development?

NRG Recommendations Addressed
      This study  should provide qualitative information on the  susceptibility of the
      developing reproductive system to pesticides, it should provide quantitative (dose-
      response) data that will enhance biologically-based risk assessment models, and
      it should provide mechanistic explanations useful for human  risk assessment.
      Moreover, results from this  study will be used to determine the appropriateness of
      the  rabbit model  for reproductive toxicity testing; these methods could then be
      applied to OPPTS testing guidelines.


Progress
      Project will be initiated late in FY96.

For more information contact: Gary R. Klinefelter, Ph.D.
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                       PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
NHEERL RESEARCH PROJECTS
The Effects of Pesticides on Learning and Memory
General Approach
      Research will  be  conducted  to  assess  age-related  differences  in  the
      neurobehavioral effects of pesticides.  Cholinesterase-inhibitiftg pesticides (carbaryl
      and chlorpyrifos) shown previously to affect learning will be administered to male
      and female rodents perinatally. The animals will then be raised to adulthood and
      evaluated for  learning deficits  and memory  impairments.   Complementary
      evaluations will be carried out in rodents exposed to pesticides as adults. Studies
      will be conducted to determine whether changes in learning and memory can be
      correlated with changes in neurochemical responses.
Research Questions

      Does early exposure to pesticides produce long-lasting effects on learning and
      memory?

      Can better tests be developed to detect the effects of pesticides on learning and
      memory?
NRG Recommendations Addressed
      By addressing age-related differences in sensitivity to the developing nervous
      system, this proposal responds to an area of concern specifically identified by the
      NRC report. Different heirarchical levels of learning and memory will be studied, the
      results of which may be used to document differences in risk based on age of
      exposure. Tests for cognitive function,  if found useful for evaluating the neurotoxic
      potential of pesticides, may lead to a revision of testing guidelines for new and
      existing pesticides.
Progress
      Project initiated late FY95.
For more information contact: Robert C. MacPhail, Ph.D.
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                      PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
NHEERL RESEARCH PROJECTS
The Biochemical Effects of Pesticides on the Central Nervous System
General Approach
      Research will determine whether exposure to cholinesterase-inhibiting pesticides
      (carbaryl and chlorpyrifos) results in toxic effects to the central nervous system
      (CMS) that are expressed differently in developing and adult animals. The specific
      aims of the study are the development of an exposure strategy, the measurement
      in young  and adult animals of diverse endpoints associated with growth  and
      differentiation of the CNS, and the determination of mechanisms of action.
Research Questions

      Are the biochemical effects of exposure to pesticides different in the young and
      adult CNS?

      Can mechanisms of action be identified?

      What is the effect of early exposure to pesticides on growth and differentiation of
      the CNS?
NRG Recommendations Addressed
      Due to the broad range of biochemical assessments that will be made, this research
      will contribute to the database on pesticide toxicity, which addresses the NRC's
      concern regarding  paucity of data.   Qualitative  (and possibly quantitative)
      differences in response between the mature and developing CNS will likely be
      observed. The in-depth assessment of biochemical markers of effect will be useful
      for risk assessment and will complement similar  projects with  predominantly
      behavioral components.
Progress
      Project initiated late FY95.
For more information contact: Stanley Barone, Ph.D.
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                       PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
NHEERL RESEARCH PROJECTS
The Effects of Pesticides on the Immune System and Allergic Response
General Approach
      The impact of pesticide exposure on the development of allergy to house dust mites
      will be studied in adult and young animals using the pesticides dieldren and
      carbaryl. An established rodent model for allergic sensitization will be used to test
      two hypotheses: 1) that exposure to pesticides promote the development of allergic
      sensitization to house dust mites, and 2) that this effect is greater in young animals
      than in mature animals.  Investigators will focus on a tightly drawn and clinically
      relevant set of immunological parameters to evaluate immune function, pulmonary
      hyperactivity, and lung inflammation.
Research Questions

      Does early exposure to pesticides impair the immune system?

      Can pesticides help trigger asthma?

      Are there biochemical changes (biomarkers) that represent an early indication of
      exposure to pesticides?
NRG Recommendations Addressed
      Data from these studies should indicate whether the young represent a sensitive
      subpopulation for immune response and whether pesticide exposures play a role
      in the development of allergic lung disease. Dose-response relationships will be
      used to extrapolate information from animal toxicology studies to pesticide-exposed
      human populations.  Finally, mechanistic information on immune responses will
      prove useful to risk assessment.
Progress
      Project initiated late FY95.
For more information contact: MaryJane Belgrade, Ph.D.
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                       PESTICIDES IN THE DIETS OF INFANTS AND CHILDREN
NHEERL RESEARCH PROJECTS
Neurochemical Changes and Behavioral Effects Induced by Pesticides
General Approach
      Research to test the behavioral and neurochemical effects of exposure to pesticides
      (chlorpyrifos and methoxychlor) will be performed in young and adult rats to explore
      age-related differences in neurotoxicity.  The effects of both acute and chronic
      (repeated) exposure to pesticides will be studied, and long-term and immediate
      effects will be observed. Changes in behavioral endpoints (cognitive development
      and learning) will be related to neurochemistry in the brain and thyroid.
Research Questions

      What are the long-term and immediate effects of exposures to pesticides and do
      they differ in mature and immature animals?

      How do behavioral effects (changes in learning and memory) relate to effects on
      neurochemical processes?

      What are the effects of early exposure to pesticides on the endocrine system?
NRG Recommendations Addressed
      This project will evaluate pesticide effects on the neural and endocrine systems,
      both of which were identified as areas of concern by the NRC.  Empirical evidence
      regarding qualitative and quantitative differences in the effects of pesticides across
      age will be generated, thereby helping to fill gaps in data.  By linking behavioral
      effects with effects on underlying  neurochemical processes, this research will
      contribute to the development of biologically-based approaches to risk assessment.
      This research  will also help determine the adequacy of current animal testing
      guidelines by addressing the  importance of age of exposure and by  possibly
      developing more sensitive biological or  behavioral  methods for use in these
      evaluations.
Progress
      Project initiated late FY95.

For more information contact: Mark E. Stanton, Ph.D.
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