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
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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
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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
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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.
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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
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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
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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
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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
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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
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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.
NHEERL TROPOSPHERIC OZONE ANNUAL REPORT 1996
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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
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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
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National Health and Environmental Effects Research Laboratory
ENDOCRINE DISRUPTORS
HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH
ANNUAL REPORT
NOVEMBER, 1996
-------
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
-------
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 decadesas 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 effectsundescended 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
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996
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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
10
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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.
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996
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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
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996
12
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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
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996
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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.
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996
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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.
NHEERL ENDOCRINE DISRUPTORS ANNUAL REPORT 1996
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National Health and Environmental Effects Research Laboratory
DRINKING WATER
HEALTH EFFECTS RESEARCH
ANNUAL REPORT
OCTOBER, 1996
-------
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
exposureindividuals 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
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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
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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.
NHEERL DRINKING WATER ANNUAL REPORT 1996
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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.
NHEERL DRINKING WATER ANNUAL REPORT 1996
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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
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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
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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 characterizationthat 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
NHEERL AIR TOXICS ANNUAL REPORT 1995
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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
endpointsincluding 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 miceat
concentrations relevant to humansresulted
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
NHEERL AIR TOXICS ANNUAL REPORT 1995
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 solventsincluding
TCE, xylene, and toluenewere 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
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National Health and Environmental Effects Research Laboratory
INDOOR AIR RESEARCH
ANNUAL REPORT
JUNE, 1996
-------
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 throatas 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
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National Health and Environmental Effects Research Laboratory
GLOBAL CLIMATE CHANGE
HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH
ANNUAL REPORT
MAY, 1996
-------
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
-------
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.
NHEERL GLOBAL CHANGE ANNUAL REPORT 1995
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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
NHEERL GLOBAL CHANGE ANNUAL REPORT 1995
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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
NHEERL GLOBAL CHANGE ANNUAL REPORT 1995
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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
NHEERL GLOBAL CHANGE ANNUAL REPORT 1995
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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.
NHEERL GLOBAL CHANGE ANNUAL REPORT 1995
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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
NHEERL GLOBAL CHANGE ANNUAL REPORT 1995
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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
12
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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.
NHEERL GLOBAL CHANGE ANNUAL REPORT 1995
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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
NHEERL GLOBAL CHANGE ANNUAL REPORT 1995
14
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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
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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
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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.
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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.
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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.
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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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