Environmental <:
EPA600/R-0"0/101
November 2000 .-
National H^Fth and
EnvirotimentaI Effects
Resgarchll^boratbTy
,,
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EPA600/R-00/101
November 2000
National Health and Environmental
Effects Research Laboratory
An Annual Report of
Accomplishments for
Fiscal Year 2000
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory
Research Triangle Park, NC 27711
US EPA Office of Research and Development
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notice
The U. S Envnronmental Protection Agency through its Office of Research and Development
conducted and managed the research described in this report. It has been subjected to the Agency's
peer and administrative review and has been approved for publication as an EPA document
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abstract
This Annual Report showcases some of the research activities of the National Health and
Environmental Effects Research Laboratory (NHEERL) in various health and environmental effects
research areas. The report is an indicator of progress and accomplishments that NHEERL has made
in Fiscal Year 2000 to meet the requirements of the Government Performance and Results Act
(GPRA) NHEERL's highlighted research is organized by these goals. Specific research areas
included for this year are: (1) Particulate Matter, (2) Air Toxics, (3) Drinking Water^ 4) Aquatic
Stressors, (5) Pesticide Residues in Food; (6) Pesticides in the Environment; (7) Global Climate
Change, (8) Ecosystems Protection, (9) Human Health Protection, and (10) Endocrine Disruptors.
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letter from the director
™ f °RD'S Nati°nal Heakh ^ Environme^al Effects Research Laboratory
(NHEERL), are charged with determining the impacts of environmental stressors on human
and ecosystem health, the degree to which those stressors cause harm, and the many factors
both internal and external, that influence the degree of harm. Our research agenda is driven
by the need either to provide scientific information to inform specific regulatory decisions or
to resolve uncertainties in the risk assessment process and characterize the state of the
environment.
This Annual Report highlights some of NHEERL's research accomplishments which address
key human health and environmental effects issues. In addition, the report demonstrates the
progress that NHEERL has made in meeting the requirements of the Government Perfor-
mance and Results Act (GPRA). The highlighted accomplishments are presented by the
strategy goals contained in the Agency's strategic plan. These include: (1) Clean Air
(Particular Matter and Air Toxics); (2) Clean Water (Safe Drinking Water and Aquatic
Stressors); (3) Food Quality Protection - Safe Food; (4) Safe Communities; (5) Climate
Change; and (6) Sound Science. Through a program of integrated multi-disciplinary re-
search NHEERL scientists have addressed key scientific questions facing the Agency in
these high priority environmental areas. For example, why do elevated exposure levels affect
some populations more than others? How do pesticide residues on foods affect human
health? How will global climate changes affect ecosystems along coastal regions?
Using a risk-based approach, our research provides EPA the scientific data needed to inform
pohcies and regulatory programs that protect human health and the environment We
recognize the critical and complex nature of our research and its role in contributing to the
on-going effort to safeguard human health and the natural environment. We are proud to
share some of our most important findings with you.
Lawrence W Reiter, Ph.D.
Director, National Health and Environmental Effects Laboratory
Office of Research and Development
Research Triangle Park, North Carolina
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table of contents
i
introduction
5
particulate matter
II
air toxics
17
drinking water
23
aquatic stressors
29
pesticide residues in food
33
pesticides in the environment
37
global climate change
43
ecosystems protection
49
human health protection
55
endocrine disrupters
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National Health and Environmental Effects
Advancing
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The National Health and Environmental Effects
Research Laboratory (NHEERL) is an important arm of
the U.S. Environmental Protection Agency's (EPA)
Office of Research and Development (ORD). NHEERL
is EPA's focal point for scientific research on the adverse
effects of pollution and other stressors on human health
and ecosystem vitality. Our scientists provide
information essential to effective risk assessment, which
is the scientific basis for regulatory and policy decisions.
NHEERL provides vital leadership in both the national
and international research communities. Based in
Research Triangle Park, NC, NHEERL has nine
.So,. ... ••-.-.- • ........
divisions in six states and a work force of over 700
"Ssif -• "
federal employees. Five health divisions are centrally
located in Research Triangle Park and Chapel Hill, NC,
and four ecology divisions are based in ecologically
significant regions (Atlantic seaboard, Pacific coast,
Great Lakes, and the Gulf of Mexico) to address
national and regional ecological risk assessment issues.
"-.
Our scientists conduct in-house research as well as
participate in collaborative studies with academia, state
governments, other federal agencies, and other research
organizations around the world. NHEERL research
undergoes the highest levels of technical review and
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scrutiny, and our results are published in peer-reviewed
journals, reports, and other media as a means of
communicating our scientific progress and
accomplishments to the public and scientific community.
NHEERL scientists also regularly present research
findings at symposia, hold membership and leadership
positions on scientific committees and workgroups, and
participate in various nationally and internationally
recognized environmental organizations.
In researching health and ecological risk, NHEERL's
structure enables scientists to develop innovative
methods and solutions to complex problems in an
integrated manner. For example, our focus is not
necessarily pollutant-specific. Data extrapolated from
both animal and human studies are incorporated into
computer models that are used in real-world
applications. The combination of a scientifically diverse
workforce and highly specialized facilities enables
NHEERL to stay on the cutting edge of health and
environmental effects research. Currently, major
research activities are focused on the harmful effects of
paniculate matter and endocrine-disrupting chemicals,
and some of our most important projects are described
and showcased in this report. It is important to note
that this report is not a comprehensive summary of all
research completed at NHEERL during this year, but
rather, it highlights some of our recent accomplishments
in the following areas:
• health effects of airborne paniculate matter
• mechanisms of toxicity of air pollutants
H advances in drinking water safety research
• susceptibility of young individuals to certain
pesticides
• influence of pesticides on the developing immune
system
• effects of global climate change on ecosystems and
human populations
• assessment of the condition of aquatic and terrestrial
ecosystems
Hi influence of genetic factors and prior exposures on
sensitivity to pollutants
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The Clean Air Act requires EPA to review the public
health risks of paniculate matter and the other
criteria pollutants every five years. If warranted by this
review, the National Ambient Air Quality Standards
(NAAQS) are revised. Based on new epidemiologic
evidence of increased illness and death associated
with particulate matter, EPA revised the NAAQS for
paniculate matter (PM) in 1997. EPA established new
standards for fine particles, PM25, particles 2.5 um in
diameter or smaller, and revised existing standards for
PM10, particles smaller than 10 urn.1
Clean Air
Baltimore has air quality representative of urban
areas in the eastern United States. Previous
epidemiologic studies found a correlation between
increased particulate matter levels in the air and
hospital admissions due to heart conditions, but no
mechanism of action was suggested. Pollution levels,
including the fine (PM25) and coarse (PM252Q)
fractions and other gaseous pollutants, were
measured inside and outside the retirement facility.
Cardiac function was assessed by measuring beat-to-
beat heart rate variability, a factor associated with
autonomic nervous system control of the heart.
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particulate matter
Scientists at NHEERL are world leaders in particulate
matter research. NHEERL's PM research program is
multidisciplinary and includes three major categories
of studies: epidemiologic studies of humans, clinical
studies of humans, and laboratory studies using tissue
cultures and animal models. NHEERL's research is
designed to discover
• who is affected by particulate matter exposure.
• how people are affected by particulate matter.
• the physiologic and molecular mechanisms by
which particulate matter causes toxicity.
• the toxic components of particulate matter.
EPIDEMIOLOGIC STUDIES
In cooperation with scientists at the University of
North Carolina at Chapel Hill, NHEERL researchers
studied the effects of ambient air PM on cardiac
function in a retirement center in Baltimore, Maryland.
Previous studies have associated reduced heart rate
variability with sudden cardiac death, increased risk of
developing coronary artery disease, and increased risk
of death from all causes among survivors of heart
attacks. In the Baltimore study, increased ambient
PM25 levels were associated with decreased heart rate
variability. The association was more pronounced in
individuals with pre-existing cardiovascular
conditions. Although this study did not allow
researchers to conclude that day-to-day PM25
variations are definitely associated with day-to-day
risk of cardiovascular disease, this was the first study
to relate daily variations in particulate matter levels
with cardiac autonomic control. The results suggest
one potential mechanism for the association between
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increased paniculate matter levels and hospital
admissions for cardiovascular conditions. A similar
study is under way in Fresno, California, a community
with air quality representative of urban areas in the
western United States.
CLINICAL STUDIES
Although epidemiologic studies have consistently
demonstrated associations between elevated ambient
paniculate matter levels and increased morbidity and
mortality, the pathophysiological mechanisms
underlying these adverse health effects have not been
well characterized. Also, the specific effects of
paniculate matter on susceptible subpopulations have
not been well characterized. To address these issues,
NHEERL researchers study the health effects of air
pollution on human volunteers. Under constant
medical supervision, volunteers are exposed to known
amounts of concentrated ambient air particles
(CAPS) in a state-of-the-science exposure chamber.
Studying healthy young adults, NHEERL scientists
found that particulate matter levels equivalent to
those found in many major metropolitan areas could
cause mild lung inflammation and could increase
factors associated with blood clotting. Supporting the
S-induced Change
Blood Cells i
NHEERL scientist monitors a volunteer who is being exposed to
concentrated ambient air particles (CAPS).
In the lung, inflammatory white blood cells in
bronchoalveolar lavage fluid increase as
concentrated ambient air particle (CAPS)
concentration increases.
findings of the epidemiologic study in
Baltimore, healthy elderly volunteers
exposed to CAPS experienced decreased
heart rate variability. Ongoing CAPS
exposure studies are being conducted on
potentially susceptible populations,
including volunteers suffering from
asthma or chronic obstructive pulmonary
disease (COPD).
Other scientists at NHEERL are
studying the distribution of inhaled
particles in the lungs. Because inhaled
particles deposit variably in different
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regions of the lung, some areas may receive a dose
large enough to cause harm, even though particle
concentration in the air may be relatively low. Local
regions of the lung that receive greater doses are likely
to be affected first. Localized damage may lead to
subsequent health effects. NHEERL researchers have
studied the deposition patterns of ultrafine particles
(less than 0.10 (Jm in diameter) in healthy young
adults and older adults. Interestingly, they found that
ultrafine particles resemble coarse particles (3 to 5
(Jm) in their deposition pattern. Dose per unit surface
area of the lung is largest in the most proximal lung
regions and decreases with an increase in lung depth.
Also, peak surface dose in local areas is 3 to 9 times
greater than the calculated average lung dose. These
and other results suggest that the localized dose may
be a crucial factor in lung injury and subsequent
health effects, and that peak surface dose may be a
useful measure for estimating potential health hazards
of ambient particulate matter.
0.16-
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• ap = 0.04 pm
dp = 0.06 |jm
dp = 0.08 Mm
dp = 0.10(jm
Vt = 500 ml
Q = 250 ml/s
0.00
100
200 300 400
Volumetric Lung Region, ml
500
In the lung, local deposition of inhaled particles
increases with increasing lung volume up to a
point, after which deposition decreases with
increasing lung volume and depth. Also, the
deposition pattern of ultrafine particles {< 0.10 urn
in diameter) resembles that of coarse particles (3 to
5 |a.m in diameter].
Vt = tidal volume; Q = respiratory flow rate; d =
particle diameter. p
0.04
0.06 0.08 0.10 1.00
Particle Diameter, (jm
3.00 5.00
For each size of particle studied, peak local
deposition dose is many times greater than the
average surface dose.
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LABORATORY STUDIES
A labor strike that closed a steel mill in the Utah
Valley for a year provided opportunities to evaluate
the loxicity of ambient air particles. Previous
cpidemiologic studies found a reduction in hospital
Smog in the Utah Valley. Note the exhaust plume
in the center of the photo.
admissions for respiratory conditions when the
mill was closed compared to when the
mill was operating. NHEERL
scientists, in collaboration with
academic researchers, obtained
paniculate matter (PM10)
samples from a Utah Valley air
monitoring station for the year
before (year 1), the year during
(year 2), and the year after
(year 3) the steel mill closure.
Year 2 dust had the lowest
concentrations of soluble iron,
copper, and zinc, and generated
the lowest number of oxidants
(reactive compounds believed
to be a major cause of lung
tissue damage). Human volunteers were exposed to
particulate matter from each of the three years. Dust
from years 1 and 3 caused significant lung injury and
inflammation, whereas dust from year'2 caused
minimal injury. Like the Baltimore and CAPS studies,
this study is among the first to demonstrate a
correlation between epidemiologic and toxicologic
particulate matter research.
This study, and additional laboratory research, suggests
a potential pathophysiological mechanism for the
health effects reported in the Utah Valley. NHEERL
investigators demonstrated that cultured human lung
cells exposed to particulate matter from years 1 and 3
produced significantly higher levels of inflammatory
mediators than cells exposed to particulate matter
4 ,
•-,3 .
An NHEERL scientist monitors heart rate and blood pressure during
inhalation exposure of rats with genetic systemic hypertension to fine
ambient air particles from Ottawa, Canada.
8
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Two NHEERL scientists use morphometric analysis and single-cell calcium imaging to demonstrate how
particulate matter affects the sensory nervous system in initiating airway inflammation.
from year 2. Researchers are currently using this in
vitro system to investigate whether transition metals
in Utah Valley particulate matter cause the
inflammation.
In addition to clinical and laboratory studies in
humans, NHEERL scientists used several rodent
models of human diseases to study the mechanisms by
which particulate matter causes adverse health effects.
Rats with hereditary systemic hypertension and
cardiac disease serve as a model for humans with high
blood pressure and cardiovascular disorders. In one
study, these rats were exposed to residual oil fly ash, a
type of particulate matter that has a high metal
content and is emitted from power plants. Compared
to normal rats, rats with systemic hypertension
exhibited exacerbated pulmonary injury, a reduced
antioxidant defense response, and adverse
electrocardiograph changes. Other models used by
NHEERL scientists to study the health effects of
particulate matter include a mouse model of allergic
airway disease, a rat model of chronic bronchitis, and
a rat model of pulmonary hypertension. In an exciting
new area of research, NHEERL scientists are using
rodent models to study the neurologic mechanisms
involved in particulate matter-induced lung
inflammation.
1 Several issues related to particulate matter standards
are undergoing Supreme Court judicial review,
including the PM10 standard. This standard will likely
be revised to be PM25_10, particles smaller than 10 um
but larger than 2.5 um in diameter.
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The Clean Air Act Amendments (CAAA) of 1990
mandate regulations for air toxics. EPA's air toxics
research program provides information that quantifies
air toxics health risks and thereby directly supports
establishment of these regulations. The air toxics
research program investigates and assesses risks posed
by toxic air pollutants from major stationary sources
such as industries, urban area sources such as dry
cleaners, mobile sources including passenger
automobiles, and indoor sources associated with
houses and buildings. The objective of EPA's air toxics
program is to significantly reduce the risk of cancer
and other serious adverse health effects caused by air
toxics through continuous reductions in emissions.
Two major areas of research by NHEERL scientists are
(1) carcinogenic mechanisms of air toxics and
(2) concentration and time exposure dynamics in the
development of risk assessment models.
Qean.Air
air toxics
CARCINOGENICITY: POLYCYCLIC
AROMATIC HYDROCARBONS
Polycyclic aromatic hydrocarbons (PAHs) are
environmental pollutants found worldwide as
constituents of complex mixtures in air, water, waste
sites, and food. PAHs are specifically listed as
Hazardous Air Pollutants (HAP) in the Clean Air
Act Amendments of 1990, and are regulated
contaminants under the Safe Drinking Water Act and
the Clean Water Act. Sources of PAHs include
tobacco smoke and emissions from diesel vehicles and
industrial processes such as aluminum production, coal
gasification, coke production, and iron and steel
founding. Many, but not all, PAHs are known rodent
carcinogens and probable human carcinogens.
Complex environmental mixtures containing PAHs
have been classified as respiratory carcinogens in
humans.
In discovering new
metabolic pathways
leading to the
of cancer,
ists use
ical
pfluding gas
ihy, mass
'and nuclear
-.jtesonance
. scientist
ple for oas
and mass
|ggpectrgme|ry analysis.
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Because of their structural diversity and wide range of
carcinogenic potencies, PAHs are an ideal class of
chemicals for studying the relationships between
structure and potency, metabolism, biotransformation,
biomarkers of dose, biomarkers of effect, and
biological activity. In previous studies, NHEERL
scientists discovered that individual environmental
PAHs varied in their potential to cause lung tumors
by a factor of over 200, a critical finding for PAH risk
assessment.
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In Fiscal Years 1999 and 2000, NHEERL researchers
studied the structural features of PAHs that confer
carcinogenicity by examining the mechanism by
which dibenzolfl.flpyrene (DB[a,QP) induces cancer.
Dtbenzo[a,/]pyrene, the most carcinogenic PAH yet
discovered, possesses carcinogenic activity that far
exceeds that of benzo[a]pyrene, the archetypal PAH.
(See graph.)
A comprehensive project evaluated the mutagenic
and lung carcinogenic effects of DB[a,I]P in bacteria,
mammalian cells, and mammals. These studies were
complemented with mode-of-action investigations
using computational research on DB[
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Trichloroethylene (TCE) is
a neurotoxic volatile
organic compound. In this
study, TCE neurotoxicity
was measured by
evaluating hearing loss,
visual function, and
behavior. Tissue dose of
TCE at the time of
neurological testing was
estimated using a
physiologically based
pharmacokinetic model,
which describes the
Signal detection behavior and auditory threshold, actual (• •) versus
predicted (—J by Haber's rule, C x t.
distribution of a substance to specific target tissues
over time. The scientists found that, if the simplest
form of Haber's rule was used to predict neurotoxic
outcomes across different exposure durations, the risk
would be overestimated when extrapolating from
short to long exposures and underestimated when
extrapolating from long to short exposures. For acute
effects of TCE on behavior and visual function, the
peak TCE concentration in the blood at the time of
neurologic testing was a good predictor of observed
performance on neurologic tests. However, cumulative
exposure, measured as the area under the blood TCE
concentration curve, did not correlate well with
outcomes. This study demonstrates that models
incorporating tissue dose estimates for varying
exposure scenarios will provide more accurate risk
assessments than models considering only the external
exposure parameters of concentration and time.
Similar results have been found in analogous studies
that examined changes in tissue sensitivity over time
in the lungs, female reproductive system, and
developing fetus.
Visual evoked potential amplitude
(VEPA) is a measure of brain activity
in response to visual stimuli. Peak
TCE concentration is a more
accurate predictor of VEPA than the
area under the cumulative TCE
exposure curve.
0 100 200 300
TCE Area Under the Curve (mg/l x hr)
0 100 200
Peak TCE Concentration (mg/l)
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"!,' "IL1 liHA.^ ==BtffiS-3« =—•r -
This equipment is used to test
rodent visual
neurophysiological responses
during inhalation exposure to
volatile organic compounds.
In experiments conducted
with this unique apparatus,
momentary blood and brain
concentrations of the test
substances correlated closely
with changes in brain
function.
This unique
experimental system
allows NHEERL
scientists to assess
rodent behavior during
inhalation exposure to
volatile organic
compounds.
Experiments conducted
in this laboratory
demonstrated that a
traditional approach to
risk assessment based
on Haber's rule may
misrepresent the risks
of behavioral deficits
associated with
exposures that vary in
concentration and
duration.
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The Safe Drinking Water Act Amendments of 1996
mandate that EPA provide a stronger scientific basis
to support future regulatory decisions and that EPA
conduct research on several specific drinking water
contaminants. Current drinking water research
priorities include disinfection by-products, waterborne
pathogens, and arsenic. There is also a growing
emphasis on unregulated chemicals and
microorganisms listed on the EPA's Contaminant
Candidate List. NHEERL research in two of these
areas, waterborne pathogens and naturally occurring
arsenic, is highlighted below.
Clean Water
Community water treatment facilities use a variety of
techniques to remove or destroy microbial pathogens.
These techniques may include filtration and
disinfection with either chlorine or an alternative
disinfectant such as ozone. EPA's Surface Water
Treatment Rule of 1989 requires all communities that
use surface water as a drinking water source to filter
their water unless special criteria are met. The
addition of filtration to municipal water treatment
facilities in response to the 1989 rule created unique
opportunities for research on waterborne disease
occurrence.
• ^—* ^M ^* * *^^
drinking water
WATERBORNE PATHOGENS
The nature and magnitude of endemic waterborne
diseases are not well characterized in the United
States. Because these illnesses tend to be self-limiting,
causing relatively minor symptoms in most
individuals, they are rarely seen by the medical
community. However, they may pose a serious health
threat to certain groups, particularly young children,
the elderly, and the immune compromised. The more
common waterborne pathogens are Shigella (and other
bacteria), Cryptosporutium, Qiardia, and enteric viruses.
Based on a survey of water utilities affected by the
Surface Water Treatment Rule, NHEERL scientists
selected a community for a pilot intervention study.
Information on gastrointestinal symptoms was
collected before and after installation of a filtration
system at the community water treatment plant.
Because gastrointestinal illness is more common in
children than adults, only families with one or more
children were included in the study. Preliminary
results showed a significant reduction in the rate of
gastrointestinal illness after filtration was instituted.
The risk of gastrointestinal illness was 1.8 times
greater before filtration than after filtration. One-third
(34%) of the gastrointestinal illness that occurred
before filtration was attributed to the lack of
filtration. Additional analyses are evaluating other
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changes in community health that followed addition
of filtration to the water treatment process. A larger,
similar study was launched in Fiscal Year 2000 in the
Seattle-Spokane area and an additional study is
planned for a community in Texas.
ARSENIC
Arsenic occurs naturally in water and soil in certain
regions of the United States, particularly in those
areas with volcanic or geothermal activity. Prior
research has linked the ingestion of arsenic with
cancer of the skin and internal organs. Also, evidence
suggests that arsenic exposure may be associated with
noncancer effects including cardiovascular disease and
diabetes. Based on research conducted by NHEERL
and other scientists, EPA recently proposed a new
Maximum Contaminant Level (MCL) of 5 pg/liter for
arsenic in drinking water. The MCL for arsenic has
been 50 pg/liter, a standard that was established in
1942.
A water filtration
plant under
construction in
Seattle, WA.
NHEERL scientists conducted an epidemiologic study
in Millard County, Utah. The study population was
assembled from historic records of the Church of
Latter Day Saints (Mormons). This population was
chosen due to the expectation that Mormons adhere
to lifestyle practices—including abstention from
drinking alcohol and smoking tobacco products—that
reduce the likelihood that these potentially
18
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confounding factors might have influenced the
observed results. All information was obtained from
existing records, including death certificates. Arsenic
exposure was not measured directly, but was estimated
based on historic measurements of arsenic in the
drinking water. (In the wells of this region, inorganic
arsenic levels vary from 2 jug/liter to more than 600
(Jg/liter.) Since mortality records were examined for
cause of death, scientists did not obtain information
on other sources of arsenic exposure. Therefore,
although the results of this study suggest an
association between arsenic exposure and adverse
health effects, they do not firmly establish a cause-
and-effect relationship. Obtaining data only from
existing records imposes limitations on study design
and interpretation of results, but it is a time-efficient
and cost-effective way to identify issues worthy of
further investigation.
Mortality in the study population was compared with
that expected in the Utah general population.
Among men in the study population, prostate cancer,
hypertensive heart disease, and one type of
kidney disease occurred at higher rates than
expected. Among women in the study
population, hypertensive heart disease and
other types of heart disease occurred more
frequently than expected. No other health
outcomes in the study occurred at an increased
rate.
scientis
collects a water sample
frpmjhe kitchen sink of a ~ *
participatin
iri an epiclemiologic study.
urinary arsenic level as a potential biomarker of
exposure in a chronically exposed population. Arsenic
concentrations in the drinking water varied from 8 to
620 micrograms per liter. Arsenic intake was
estimated using two data sources: daily food diaries
kept by participants and inorganic arsenic levels in
the drinking water. In the body, inorganic arsenic is
metabolized into several forms. Researchers found that
all age and gender groups in the study excreted the
various forms of arsenic in the same proportions. (See
pie charts.)
Arsenic Profiles for Different Groups
Arsenic Speciation in Adult Females
Arsenic Speciation in Adult Males
70.3%
3.9%
12.0%
Arsenic Speciation in Male Children
70.8%
Other NHEERL scientists are exploring the
potential health effects of arsenic in drinking
water in more detail. One study examined
14.2%
Arsenic Speciation in Female Children
^72.0%
4.2%
11.0%
14.1%
3.8%
10.9%
13.3%
Regardless of age or gender, the same arsenic compounds
(species) were produced in similar proportions.
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This proportion also remained constant over all levels
of arsenic exposure. (See bar graph.)
The study also showed that the concentration of
arsenic in drinking water is a better predictor of
urinary arsenic excretion than estimates of arsenic
consumption calculated from daily food diaries. (See
line graph.) Therefore, urinary arsenic concentration
can be used to accurately estimate exposure in future
studies of the potential health effects of arsenic in
drinking water.
Arsenic Species Within Exposure Groups
DMA
MMA
As(lll)
As(V)
>130 (
51-130|jg/l
31-50^/1
0-30 (jg/l
In all exposure levels,
the same arsenic
compounds (species)
were produced in
similar proportions.
c concentration in arinKina w«"»i
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Various types of chemical, biological, and physical
stressors affect the health and sustainability of fragile
aquatic ecosystems. To improve ecosystem risk
assessment, EPA conducts research to identify and
assess the impact of aquatic stressors. Three critical
types of stressors are being studied by NHEERL
scientists: chemical stressors, such as persistent
bioaccumulative toxicants; nonchemical stressors such
as habitat alteration; and eutrophication, which
includes the effects of nutrient overload, hypoxia, and
harmful algal blooms.
providing a better understanding of risks to ecosystem
resources and processes, this research will promote
better and more ecologically sustainable choices by a
variety of decision makers.
NHEERL researchers
obtaining samples of
aquatic animals from a
stream.
Clean Water .
aquatic stressors
NHEERL's Wildlife Research Strategy addresses four
key areas of research where advances in science will
dramatically improve wildlife risk assessment
techniques and criteria methodology:
WILDLIFE RESEARCH STRATEGY
Over the past year, EPA's NHEERL finalized the
Wildlife Research Strategy, which describes the
strategic approach for NHEERL scientists to follow
when researching the effects of environmental
stressors on wildlife. The goal of the Wildlife
Research Strategy is to develop scientifically valid
approaches for assessing risks to wildlife populations
from multiple stressors.
Research conducted
under this program will
produce models,
methods, and findings for
EPA Program and
Regional Offices to use
when conducting wildlife
population risk
assessments. By
Extrapolation research will improve the basis
for predicting toxicological responses among
wildlife species and exposure scenarios of
concern.
Coordinated wildlife population biology and
wildlife toxicology research will improve
predictions of population dynamics in
spatially explicit habitats.
Methods research will advance techniques for
assessing the relative risk of chemical and
nonchemical stressors on wildlife populations.
The program uses a tiered approach to
developing tools, recognizing that different
tools are needed for screening-level
assessments and for scenario-specific risk
assessments.
Additional research will define geographical
regions and spatial scales appropriate for
wildlife risk assessments.
An NHEERL scientist is developing statistical and
ecological models using the estuarine benthic
amphipod Ampelisca abdita, a bottom-dwelling
crustacean, to evaluate sediment toxicity. These
models describe the relationship between classical
toxicological endpoints and population-level effects
measured in field monitoring programs.
23
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During Fiscal Year 2000, the Wildlife Research
Strategy underwent external review and was finalized.
In addition, the NHEERUs Wildlife Workgroup
Green Heron (Butorides striatus)
Bald Eagle
(Halraeetus
leucocephalus)
identified three case studies that will be used to
demonstrate the utility of models and methods
developed using this strategy. These three risk
assessment scenarios represent a broad diversity of
environmental issues. The first scenario will examine
the impact of acutely toxic agricultural insecticides on
representative bird species. This project will focus on
population-level effects in agricultural
landscapes that vary by cropping
practices and intensity of
insecticide use. The second
scenario will
examine the
role of mercury
, , ^HHiSF Frog (Ranidae)
and other
coexisting stressors on population
dynamics of common loons and other fish-eating
birds. The third scenario will examine the suite of
stressors that affect amphibian population dynamics.
The Wildlife Research Strategy is an excellent
example of how EPA scientists develop the methods
and procedures by which research is to
be conducted. Because the problems
addressed by this program are
multidimensional—requiring the
integration of researchers from
such diverse fields as toxicology,
population ecology, and spatial
modeling—this program is also
an outstanding example of the
integrated approach that EPA
uses to address complex
environmental issues.
24
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The figure below outlines the NHEERL conceptual approach for wildlife risk assessments, fo.cusing on the effects
component of the assessment process. Step I involves spatial and temporal characterization of stressors that may
adversely affect the population of concern, especially contaminant exposure, habitat suitability, and introduced
f
species. Results from Step 1 provide the input for Step 2, quantification of the exposure-response and habitat-
response relationships at the individual level The specific response variables estimated in Step 2 are spatially
explicit demographic rates of individuals within populations, such as fecundity and life stage-specific probability of
survival. These demographic rates in turn drive population models in Step 3, generating outputs describing
population growth rate or other appropriate population-level endpoints (for example, extinction probabilities). The
formula in Step 3 is a very simplistic way of saying that the population size at some time period (n) is related to the
population size at the previous time period (n(1) times M, the population growth rate, which integrates survival,
fecundity, immigration, and emigrauon. Finally, these population dynamics are integrated with the landscape
characteristics in Step 4 to determine habitat-specific population sources and sinks using spatially explicit modeling
platforms. Analysis of the cumulative population dynamics across the landscape provides the estimates of wildlife
risks from chemical exposure, habitat changes, introduced species, and other forms of disturbance in the landscape.
Step 1
Conceptual Approach
PCB Concentration
Step 2
nt = MnM
Step 3
Habitat
UnitB
Step 4
-------
DISSOLVED OXYGEN
Just as oxygen in the air is critical to the life of land
animals, oxygen dissolved in water is vital to the
survival of aquatic animals. Oxygen enters water from
the atmosphere by diffusion and by the action of wind
on the water surface. Floating and rooted aquatic
plants add dissolved oxygen to shallow waters that
receive enough sunlight to support photosynthesis.
Under normal conditions, temperature and salinity are
the two most important factors that influence the
amount of oxygen that can be dissolved in a body of
water.
Low levels of dissolved oxygen stress aquatic animals
and can cause death if the levels are low enough.
Eutrophication — increased nutrients in the water —
is the most common cause of low dissolved oxygen
levels. Wastewater discharges and agricultural and
urban practices that result in nutrient-rich runoff are
the activities most responsible for eutrophication of
streams, rivers, lakes, and estuaries. Increased nutrient
levels promote algal blooms. The subsequent
decomposition of dead algae consumes oxygen,
lowering the dissolved oxygen content of the water.
Massive fish kills in estuaries often make the news
headlines during the summer months, when the
effects of eutrophication are most severely felt.
Congress has mandated the establishment of national
minimal levels for dissolved oxygen. In FY 2000,
NHEERL researchers completed a dissolved oxygen
criteria document for saltwater, which describes
sensitivity to low dissolved oxygen levels in saltwater
animal species of the mid-Atlantic region. Most of
these species also occur along the southern coasts of
the United States. Because higher water temperatures
are expected to increase respiratory demand for
dissolved oxygen, and less oxygen is dissolved in warm
versus cold waters, scientists hypothesized that
southern populations may differ in their sensitivity to
low dissolved oxygen levels compared to northern
populations of the same species.
Grass shrimp (Palaemonetes Vuigans
'
To test this hypothesis, NHEERL researchers
compared sensitivity to dissolved oxygen in northern
and southern populations of two crustaceans (Say mud
crab, Dyspanopeus sayi and grass shrimp, Palaemonetes
vulgaris) and one fish (inland silverside, Menidia
beryllina). The scientists selected crustacean
populations from Rhode Island and Georgia and fish
populations from Rhode Island and Florida. Rhode
Island populations were tested at 18 to 20°C, whereas
southern populations were tested at 28°C.
Researchers conducted
acute exposure tests on
juvenile fish and on
larvae of all three
Heat-exchange system
in seawater trough that
controls test
temperature in
experiments with
marine and estuarine
organisms.
26
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species. All tests were conducted in
a flow-through system capable of
maintaining dissolved oxygen
content at any level between zero
and saturation.
In FY 2000, tests on all three
species were completed and showed
that northeastern and southern
populations of these three species
displayed the same sensitivity to
low dissolved oxygen levels.
(See graph.) This research
demonstrates that much of the data
used in establishing the mid-
Atlantic dissolved oxygen criteria can be used in
developing dissolved oxygen criteria for the coastal
waters of the southeastern U.S. and Gulf of Mexico.
Ongoing research will answer remaining questions
about applying the methods and data of the Mid-
Atlantic dissolved oxygen studies to other coastal
regions. This is one example of NHEERL research
that directly supports the development of regulatory
standards.
120T
Dyspanopeus sayi (Say mud crab)
Say mud crabs from
Georgia exhibited the
same sensitivity to
dissolved oxygen levels
as say mud crabs from
Rhode Island.
Rhode Island (18-20 °C)
Georgia (28 °C)
r~
3456
Dissolved Oxygen (mg/L)
—r~
5
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The Food Quality Protection Act of 1996 (FQPA)
mandates development of a single, health-based
standard for all pesticides in all foods and provides
special protection for sensitive subpopulations,
particularly infants and children. The FQPA also
directs EPA to consider all nonoccupational sources of
exposure, including drinking water, when setting
maximum allowable levels for contaminants. In
support of these mandates, NHEERL scientists are
actively studying age-related differences in response to
pesticides, the effects of aggregate and cumulative
exposures, qualitative and quantitative differences, and
improved methods for extrapolation. This research is
being conducted as a collaborative effort among
divisions within NHEERL.
response to a single oral dose five- to seven-fold lower
than the dose that caused toxic responses in adult
rats. The toxic response measured was either a
behavioral or biochemical abnormality consistent with
anticholinesterase exposure. This response correlated
with the age-related development of the enzyme
systems responsible for chlorpyrifos detoxification.
occuirjor some
- -"•
pesticides but not for
Food Quality Protection
pesticide residues in food
Researchers at NHEERL have developed a rodent
model to study how age influences the response of an
individual to a given pesticide dose. Chlorpyrifos
[Dursband} LorsbanfS; O,O-diethyl O-(3,5,6-trichloro-
2-pyridyl) phosphorothioate] is an anticholinesterase
chemical that has been one of the most widely used
organophosphate insecticides in the United States. In
studies of chlorpyrifos, young rats exhibited a toxic
However, rats exposed to methamidophos (Monitor^
O,S-dimethyl phosphoamidothioate), which is detoxified
by different mechanisms, showed no age-related difference
in toxic response. This comparison demonstrated clearly
that age-related differences in toxicity occur for some
pesticides but not for others. Further research is aimed at
understanding the underlying basis for these differences, so
that conducting studies on a chemical-by-chemical basis
may not be necessary.
-------
NHEERL scientists are using a variety of approaches
to study the effects of mixtures of pesticides that share
a common mechanism of toxicity. The researchers are
• developing statistical models to assess additive
and nonadditive interactions.
• conducting interaction experiments of two to five
pesticides.
• determining age-related differences in response to
pesticide mixtures.
• evaluating effects on behavior, neuromuscular
function, central nervous system
electrophysiology, thermoregulation, and
cardiovascular function.
In one pesticide mixture study, the toxicity of a blend
of chlorpyrifos and diazinon showed no deviation
from additivity.
30
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31
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People may be most vulnerable to certain classes of
environmental contaminants during in ute.ro
development, infancy, childhood, and/or adolescence.
Children may be more vulnerable
to environmental contaminants
than adults because of differences
in absorption, metabolism,
storage, and excretion, the sum of
which produces higher
biologically effective doses in
target tissues. Furthermore,
children and adolescents can
have greater exposure than adults
because of proportionately higher
food intake, dietary differences,
and activities that result in
greater contact with
environmental contaminants.
Many environmental health
threats to children may not be recognized because
scientific understanding of when and why children's
exposures and responses differ from those of adults is
PESTICIDES AND THE DEVELOPING
IMMUNE SYSTEM
EPA's Office of Prevention, Pesticides, and Toxic
Substances recently identified the need to evaluate
immune function in developing animals. In FY 2000,
NHEERL scientists validated a rodent model to detect
increased immune dysfunction in adults following
Safe Communities
pesticides in the environment
incomplete. The Food Quality Protection Act of 1996
and the Safe Drinking Water Act Amendments of
1996 require that EPA give special consideration to
children and other susceptible subpopulations when
establishing health-based standards, especially those
related to pesticides in food and water.
chemical exposure during immune system
development. This standardized procedure for
evaluating developmental immunotoxicity can be used
by other researchers and by the chemical industry to
investigate the developmental immunotoxic potential
of individual agents or mixtures. In this way, relatively
-------
small research projects completed in the laboratory
provide the means to collect millions of dollars worth
of data generated by chemical manufacturers.
"...following chemical
exposure during immune
system development, the
fetus, neonate, young
child, adolescent, and/or
adult may develop
chemical-induced immune
dysfunction that may
manifest as a susceptibility
to infectious diseases or
the development of cancer."
In validating this model, NHEERL researchers
exposed rats to the organochlorine chemical TCDD
(2,3,7,8-tetrachlorodibenzo-p-dioxin) and to the
organochlorine pesticides heptachlor and
methoxychlor. Pregnant females exposed to TCDD on
gestation day 14, resulting in late gestational and early
lactational exposure, gave birth to pups that exhibited
persistent suppression of T cell-mediated immune
responses. Male offspring tended to be more sensitive
to immune suppression than female offspring. In
separate studies of perinatal/juvenile exposure to
heptachlor and methoxychlor, NHEERL scientists
discovered that the amount of major metabolites of
these pesticides found in dam's milk and in lymphoid
and other tissues was directly proportional to immune
system suppression. That is, the greatest amount of
immune system dysfunction occurred in the rats with
the highest levels of pesticide metabolites. In contrast,
adult rats exposed to comparable doses of these three
chemicals did not exhibit immune suppression.
T cell-mediated immune functions are important in
the defense against bacterial, viral, and parasitic
infections, and against certain cancers. These studies
suggest that, following chemical exposure during
immune system development, the fetus, neonate,
young child, adolescent, and/or adult may develop
chemical-induced immune dysfunction that may ,
manifest as a susceptibility to infectious diseases or
the development of cancer. These studies also
underscore the need for further experimental and
epidemiological work in developmental
immunotoxicology to support EPA efforts in children's
risk assessment.
PESTICIDES IN THE COMMUNITY
NHEERL researchers conducted a pilot study of
children in southern California's Imperial Valley to
evaluate organophosphate pesticide exposure and
potential health effects. Organophosphate pesticides
are used residentially and agriculturally, and are the
most commonly used class of insecticides in the
United States today. Because agricultural application
of pesticides is a primary potential route of human
exposure, the Imperial Valley, with its year-round
agricultural activities, was an ideal site for this study.
34
-------
Children two to four years of age
with flu-like symptoms who were
being seen by a physician at a health
clinic were eligible for the study.
With the informed consent and
assistance of the parent or guardian,
scientists obtained a urine sample
from each child for organophosphate
pesticide metabolite analysis.
Researchers also requested a finger-
prick blood sample to analyze for the
enzyme acetylcholinesterase, but the
blood sample was not required for
participation in the study. The two basic goals of the
study were to (1) estimate the occurrence of
unrecognized pesticide-related illness in young
children and (2) improve methods to evaluate the
prevalence of pesticide exposure among young
Aerial spraying of pesticides is a common practice in many
agricultural regions.
children. By the end of Fiscal Year 2000, data had
been collected and were being analyzed. This study
will provide important information about pesticide
exposure in children that will directly support EPA's
regulatory efforts.
-------
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In 1990, Congress established the U.S. Global
Change Research Program (USGCRP) to coordinate
federally supported research on global climate change.
As part of the USGCRP, EPA conducts assessments
research on the consequences of global climate
change to human health, ecosystems, air quality, and
water quality. Stressors of concern include (1)
increasing temperature, (2) decreasing stratospheric
ozone levels, which increase harmful ultraviolet
radiation exposure, and (3) changing land cover and
land use, which sometimes have unintended and
adverse consequences.
would have a major impact on coastal areas because of
the high population density of such regions,
substantial costs of defending shorelines and property,
detrimental effects on infrastructure (drinking water
supplies, waste management systems), and significant
loss of coastal wetlands, beaches, and recreational
facilities.
Climate Change
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COASTAL AREAS
Continued increases in concentrations of atmosphe
greenhouse gases can raise global surface
temperature—accelerating the'hydrological cycle,
changing the patterns of climate variation, raising the
sea level, and possibly altering the frequencies and
intensities of climate extremes. Risks to human health
and ecosystems may occur either as the direct result of
such climate changes (for example, heat-related
mortality or sea-level rise) or from the combined
effects of climate variation and other stressors
affecting air and water quality. A rising sea level
At NHEERL, global change research is supporting
EPA's ability to perform effects-based assessments that
integrate across ecological resources and geographic
scales and that address major classes of environmental
problems. NHEERL scientists are developing methods
and collecting data to characterize current ecosystem
conditions in mid-Atlantic and Northeast estuaries.
Their objective is to relate ecological conditions in
these estuaries to past and current characteristics
topography, vegetation type and coverage, and human
land use—of the associated watersheds.
-------
Projected climate extremes in the 21st century, such
as floods and droughts, may be of greater magnitude
than those experienced in the 20th century. Recent
results from NHEERL studies suggest that human
population trends and resulting modification of
watersheds in the mid-Atlantic region over the past
fifty years have amplified some risks associated with
climate extremes. Increased demands for water can
magnify effects of drought. Increased nutrient loading
to watersheds has substantially raised the nitrogen
flux-pcr-unit flow over the past century. Preliminary
results suggest that, in recent decades, hypoxic and
anoxic conditions in the Chesapeake Bay have
become more sensitive to climate variation, possibly
due to increased nitrogen loading to associated
watersheds. Similar effects have been observed in the
Gulf of Mexico. Since the late 1950s, annual delivery
of nitrate from the Mississippi River to the Gulf has
nearly tripled, resulting in an expanding hypoxic zone
on the Louisiana-Texas shelf. The findings of research
conducted by NHEERL and other USGCRP scientists
suggest that risk managers should consider adaptive
actions to minimize health and ecological risks
associated with both direct and indirect effects of
climate variability and change.
FORESTS
NHEERL scientists have analyzed the effect of
interactions of climate change, elevated carbon
dioxide, and ground-level ozone on biomass
accumulation. Modeling studies indicated that the
amount of carbon that can be stored as biomass under
increasing carbon dioxide levels is reduced with
increasing ground-level ozone exposure. A study of
Douglas fir seedlings showed that elevated
Distribution of summertime dissolved oxygen within one meter of
bottom sediments across estuarine waters in the Mid-Atlantic
Region Data were derived from daylight observations and do not
necessarily reflect nighttime depressions that may occur in some
areas (Condition of the Mid-Atlantic Estuaries EPA 600-R-98-147).
atmospheric carbon dioxide concentrations reduced
the levels of monoterpenes released by the seedlings.
Similarly, elevated temperatures also reduced
monoterpene release significantly. These findings
have major implications for Douglas fir forests because
monoterpene emissions are an important ecological
defense against leaf predation by insects.
ULTRAVIOLET RADIATION
Whereas high concentrations of ground-level ozone
pose risks to human and ecosystem health, high
concentrations of stratospheric ozone protect human
38
-------
and ecosystem health by absorbing harmful ultraviolet
(UV) radiation. Stratospheric ozone is being depleted
by chemical emissions, especially chlorofluorocarbon
compounds that have been widely used as refrigerants
and aerosol propellants. Exposure to ultraviolet
radiation has been associated with immune system
suppression, cataract development, and increased risk
of some skin cancers.
The EPA program UV-Net monitors ultraviolet
radiation at the Earth's surface. Data are collected
from monitoring sites
in 7 urban areas and 14
national parks, which
represent major
An ultraviolet (UVJ
radiation monitor.
ecosystem types. Park service personnel operate the
monitoring sites in the national parks, which also
collect air quality data. The UV radiation data from
this program is coordinated with data collected by
other agencies and made available to researchers
through the National UV Monitoring Center
(NUVMC). The NUVMC is a cooperative endeavor
of the EPA and the University of Georgia.
Other NHEERL researchers have studied the
influence of ultraviolet radiation on the toxicity of
polycyclic aromatic hydrocarbons (PAHs). PAHs are
ubiquitous pollutants that are found in tobacco smoke
and emissions from diesel vehicles, coal gasification
plants, and many industries. Scientists exposed the
segmented worm Lumbriculus variegatus to phototoxic
PAHs (anthracene, fluoranthene, and pyrene) singly
and in binary mixtures. Afterward, the worms were
exposed to ultraviolet light. The resulting toxicity
Locations of UV monitoring stations
-------
was described by a concentration addition model.
That is, exposure to PAHs and ultraviolet light was
more toxic than exposure to PAHs alone. This
research is just one example of how NHEERL
scientists are thinking about the potential interaction
of environmental stressors that are traditionally
studied by researchers in widely separated disciplines.
NHEERL ccologists are studying the decline of corals
in recent decades, especially in the Caribbean Sea.
Coral reefs have a very high biodiversity and
abundance of life. Coral losses have been attributed to
disease and coral bleaching — the loss of obligate
symbiotic algae from the coral tissue. Several causes of
bleaching and disease have been proposed, including
increased exposure to ultraviolet radiation, increased
water temperatures, higher nutrient concentrations,
and combinations of these stressors. Coral ecosystems
are sufficiently important that an Executive Order was
issued to improve understanding of the factors causing
coral loss, with the goal of curtailing further loss.
A Florida Keys field survey completed in FY 2000
determined frequency and distribution of coral
diseases and bleaching. The survey included
permanent sites that are revisited periodically to
monitor long-term trends. Data from additional survey
sites will allow comparison of different regions and
ef types. Data from all survey sites will be examined
re
for potential relationships with UV exposure,
temperature, and water quality.
NHEERL scientist examining
pecimens exposed to UV
radl3H6n-4n,,iLSplar simulator.
40
-------
NHEERL scientists also participate in
the Coastal Intensive Site Network
(CISNet). As part of CISNet,
researchers are conducting field
transplant and laboratory studies of
corals and symbiotic algae. In these
studies, coral measurement endpoints
include bleaching, symbiotic algae
counts, tissue protein content, thymine
dimer production (related to UV
exposure), pigment formation (potential
protection from UV exposure), and
severity of diseases.
41
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In recognition that knowledge is vital to policy-making
decisions, EPA conducts ecosystems research to provide
the scientific understanding needed to maintain or
restore the integrity of ecosystems now and in the
future. This research provides information at multiple
geographic levels: local, watershed, state, regional,
tribal, national, and international.
NHEERL ecologists working in a coastal mud flat.
Hovercraft used to gain access to the research site
is shown in the background.
Scientists at NHEERL are leaders in ecosystem
research. The Agency's ecosystem research program
consists of four fundamental areas: monitoring,
ENVIRONMENTAL MONITORING AND
ASSESSMENT PROGRAM
The Environmental Monitoring and Assessment
Program (EMAP) was created to establish a scientific
basis for statistically valid, cost'effective assessments
of the condition of various ecosystems and to detect
trends at local, state, regional, and national levels.
Since its inception in 1988, scientists in the EMAP
have
• developed ecosystem-specific indicators and
monitoring designs.
• determined baselines for the health of some
aquatic ecosystems.
• developed technologies and procedures that
reduce monitoring costs.
• identified areas for restoration and remediation.
• transferred technology to some states for
collecting monitoring data, which could be
aggregated for national assessments.
"Proof-of-concept" studies, which validated the
EMAP's approach to ecosystem monitoring, were
conducted in the mid-Atlantic region and were
completed in 1999. Two new programs, the Coastal
2000 Initiative and the Western Pilot, were designed
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processes and modeling, risk assessment, and risk
management and restoration. Current NHEERL
research projects are establishing the baseline health of
ecosystems throughout the United States and are
developing new, efficient procedures for ecosystem
monitoring and assessment. '
Measuring light-harvesting efficiency of salt-marsh plants.
43
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Columbian
Acadian
Virginian
Carolinian
West Indian
The Coastal 2000 Initiative will supply
the EPA with important baseline data
about the health of our nation's estuaries.
For the Coastal 2000 Initiative, coastal areas of the United
States have been assigned to named provinces.
10 assess the health of aquatic ecosystems on a large
scale. They were launched in 1999 and continued
through 2000.
The Coastal 2000 Initiative will provide a
comprehensive, statistically valid estimate of the health
of the nation's estuaries at the state, regional, and
national levels. It involves a strategic partnership
among EPA, other federal agencies, 24 coastal states of
the United States, and Puerto Rico. In 1999,
NHEERL and state personnel assessed the condition of
small estuaries—those less than 250 square kilometers
in size—in California, Oregon, and Washington. In
2000, these assessments were extended to the Atlantic
and Gulf coasts with over 1,500 sites being sampled.
Initial findings are expected in 2001.
The Coastal 2OOO Initiative
provides the first national
environmental report card
on coastal ecosystem
health.
The Western Pilot is the largest, most
comprehensive study sponsored by the
EPA on the ecological condition of the
western part of the United States. It is a
collaborative effort of 12 western states,
native American tribes, universities, three
EPA Regions, and other federal agencies. The
objectives of this project are to establish the
baseline health of the widely varied aquatic
ecosystems throughout the west and to identify
environmental stressors associated with degraded
conditions in these systems. In Fiscal Year 2000, the
large estuaries of the west coast—Puget Sound, WA;
Columbia River Estuary, OR; and San Francisco Bay,
CA—were studied.
NHEERL scientist collecting samples in a stream.
44
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Index of biotic integrity (IBI) scores measure fish community health.
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The primary responsibility of the Environmental
Protection Agency (EPA) is to protect human health
and the environment. In doing so, EPA uses risk
assessments to identify and characterize
environmentally related human health problems.
Many uncertainties exist in the risk-assessment
process due to the complex relationship between
exposure to a chemical in the environment,
distribution of the chemical in the body, and the
toxic or adverse effects of the chemical in individual
humans. Current risk-assessment procedures rely
heavily on default assumptions that were made in the
absence of relevant scientific data. For example, EPA
often makes risk-assessment decisions based on
information derived from animal models.
on such default assumptions by providing a
mechanistically based understanding of toxicity and
the factors associated with susceptibility. NHEERL is
also developing models to account for exposure
scenarios that differ
with respect to media
(air, water, or soil),
route of exposure
(inhalation,
ingestion, or skin
absorption), temporal
dimensions, and
other factors.
Sound Science 1
human health protection
In the laboratory, animals have historically been
exposed to conditions that do not precisely mimic the
exposure conditions to which humans are subjected in
the environment. Three assumptions operating in this
scenario are (1) variation in exposure conditions does
not affect the validity of the results, (2) sensitivity to
the chemical is similar for humans and the animal
model, and (3) detoxification of the chemical is
similar for humans and the animal model. These and
other assumptions introduce uncertainty into the
process by which risk assessors attempt to determine if
a chemical poses a threat to human health. One
important objective of NHEERL is to reduce reliance
One of the greatest uncertainties in the risk-
assessment process is the fact that individuals differ in
their response to chemical exposure. Further, certain
identifiable subpopulations within the general
population may be differentially susceptible to a given
exposure level. For example, children may be more
sensitive than adults to the toxic effects of some
chemicals.
Research on susceptible subpopulations at NHEERL is
based on the hypothesis that variability in response to
environmental toxins is due in part to biological
variability.
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"One of the greatest
uncertainties in the risk-
assessment process is the
fact that individuals differ
in their response to
chemical exposure."
This variability depends on internal factors (gender,
race, age, and specific genetic factors) and on external
factors (prior exposures, pre-existing disease, activity
level, nutrition, stress, licit or illicit drug use, cigarette
smoking, alcohol use, or socioeconomic status). A
major emphasis of the program on human health
protection is to understand more clearly how
variability in human susceptibility alters response to
chemical exposures.
AIR POLLUTION AND ASTHMA
In the United States, the prevalence of allergic
asthma has been increasing steadily since 1980,
especially among adolescents and children 14 years of
age or younger. NHEERL scientists are using human
volunteers, epidemiologic studies, and animal models
to study how air pollution contributes to the
development and expression of allergic asthma.
NHEERL scientists have developed mouse and rat
models that exhibit many of the characteristics of
human allergic asthma. These animal models are
being used to
• test the hypothesis that air pollutants and other
environmental agents enhance the development
of allergic diseases.
• identify specific agents that trigger allergic
reactions.
• identify biological parameters that can be used to
rank the relative potency of triggering agents.
• study the mechanisms by which air pollutants
exacerbate the symptoms of allergic asthma.
Using a rat model of allergic asthma, NHEERL
researchers discovered that exposure to residual oil fly
ash (ROFA), a product of fuel oil combustion,
increased the allergic sensitivity to dust-mite allergen.
Allergic sensitivity is determined by measuring dust-
mite allergen-specific IgE antibody levels and
immediate bronchoconstriction response.
(See graph on facing page.)
«0-4yr
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• Total
The prevalence of asthma in the United States has been increasing since
1980, especially among children and youth 14 years of age and younger.
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Exposure to residual oil fly ash {ROFAJ stimulates the release of the pro-inflammatory cytokine TNF-alpha.
Exposure to either ROFA or TNF-alpha enhances [aj IgE antibody and fbj immediate airway response to dust-
mite allergen. These findings provide evidence that ROFA sensitization to dust-mite allergen is associated with
an increase of inflammatory cytokines in the lungs.
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"Research on susceptible subpopulations at
NHEERL is based on the hypothesis that
variability in response to environmental toxins
is due to biological variability."
A follow-up study determined that the metal
components of ROFA cause the allergic sensitization.
These asthma studies are an excellent example of how
NHEERL researchers are discovering the physiological
mechanisms underlying the relationship between
environmental contaminants and human health.
NHEERL's studies in human volunteers that were
completed in Fiscal Year 2000 confirmed that
asthmatics and nonasthmatics exhibit different
inflammatory responses in the lung when exposed to
air pollutants. Compared to nonasthmatics, asthmatics
responded to lower concentrations of air pollutants
and released larger quantities of a different set of
inflammatory proteins. Information from these and
other studies provides risk assessors with a very
important mechanistic basis for considering sensitive
subpopulations in the risk-assessment process. Other
diseases being studied for their potential to increase
susceptibility to toxin exposure include diabetes,
cardiovascular disease, and other pulmonary
conditions.
Preparing DNA samples for agarose gel electrophoresis to look for genetic polymorphism.
52
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NHEERL scientist analyzing DNA for the presence of base substitution genetic polymorphism.
GENETIC POLYMORPHISM AND
SENSITIVITY TO TOXINS
Genetic polymorphism, the existence of multiple
forms of a gene, is thought to be associated with
increased risk of adverse health effects following
exposure to some environmental contaminants.
NHEERL research on arsenic metabolism supports the
general hypothesis that variations in response to
chemicals are associated with polymorphism in the
genes responsible for chemical metabolism.
During animal studies, NHEERL scientists discovered
variation in the rate at which individuals methylate
arsenic metabolites. (Arsenic is detoxified in the body
when its metabolites are methylated). More
importantly, the researchers found that the rate of
metabolite methylation was associated with the rate at
which arsenic toxicity developed. Animals that
methylated arsenic metabolites slowly developed toxic
signs earlier than animals that methylated arsenic
metabolites rapidly. Based on this research,
epidemiologic studies will be conducted at NHEERL
to determine the role of genetic polymorphism in the
toxicity of arsenic to humans. Ultimately, this
research could lead to the identification of
biomarkers-measurable physiological parameters—that
would enable scientists to predict the likelihood of
increased arsenic sensitivity among certain
subpopulations.
53
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Endocrine disrupting chemicals (EDCs) are exogenous
chemical substances or mixtures that interfere with
the production, release, transport, metabolism,
binding, action, or elimination of the natural
hormones of the body. EDCs may adversely affect
individual organisms, progeny, populations, or
subpopulations. A broad range of substances,
including widely used pesticides and numerous
industrial chemicals, have been identified as potential
endocrine disrupters. Because of the potential scope of
the problem, the possibility of serious effects on the
health of populations, and the persistence of some
EDCs in the environment, endocrine disruption is
one of the six high-priority research areas identified in
EPA's Office of Research and Development Strategic
Plan.
Evaluating tissue
samples for evidence of
endocrine disrupter
activj|^ on reproductive
development inTine
fern? ""
Sound Science
endocrine disruptors
Regulatory control of endocrine disrupting chemicals
falls under four legislative acts. The Toxic Substances
Control Act (TSCA) and the Federal Insecticide,
Fungicide, and Rodenticide Act (FIFRA) provide
EPA with the authority to evaluate the toxicity of
industrial chemicals and pesticides. In addition, the
Food Quality Protection Act (FQPA) and the Safe
Drinking Water Act Amendments (SDWAA) of 1996
require the testing of pesticides and chemicals found
in food and water to determine their "estrogenic or
other endocrine effects in humans."
In the evaluation
of endocrine
disrupter activity
on pubertal
development in
the male rat, an
NHEERL scientist
uses a robotic
pipettor to
prepare serum
samples for
radioimmunoassay.
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As a consequence of these legislative directives, the
Endocrine Disrupter Screening and Testing Program
(EDSTP) was established to develop standardized
protocols for in vivo and in vitro assays to detect
chemicals that affect estrogen, androgen, and thyroid
function. NHEERL researchers have taken a lead role
in developing many of these assays. In Fiscal Year
2000, NHEERL scientists have been involved in a
number of projects related to this effort, including
developing or examining various in vitro tests for
identifying potential endocrine disruptor activity
(for example, competitive binding assays and
androgen/estrogen gene regulation tests).
finalizing protocols for screening assays in male
and female pubertal rats. These assays evaluate
endocrine disruption from chemical exposure
occurring during puberty, a developmentally
sensitive life stage.
working with the Organization of Economic and
Community Development (OECD) and serving as
the lead laboratory for the standardization and
validation of an in vivo test for androgenic
activity (the Hershberger Assay).
In vitro techniques
being used to assess
environmental
contaminants for
androgenic and/or
antiandrogenic
activity.
Other NHEERL researchers have developed a short-
term assay using the fathead minnow (Pimephales
promelas) to evaluate the effects of chemicals on
endocrine and reproductive function. The test starts
with reproductively mature minnows and examines
the effects of chemical exposure on hormone levels,
gonadal status, reproductive behavior, secondary
sexual characteristics, fecundity, egg fertility, and early
embryonic development. This test is cost-effective and
can be completed in a fraction of the time required
for a traditional full life-cycle study.
In addition to developing screening assays, NHEERL
scientists are studying the specific reproductive effects
caused by endocrine disrupting chemicals and the
mechanisms behind these reproductive effects. One
active area of research at NHEERL is the link
between chemical exposure and inflammation of the
prostate (prostatitis) in male rats, which is mediated
by altered prolactin levels. Prolactin is a pituitary
hormone that regulates many functions, including
brain development. In rats, prolactin provided in the
dam's milk influences development of the brain area
that ultimately regulates prolactin levels in the adult.
A decrease in milk prolactin levels during critical
brain development results in altered prolactin levels
in the offspring. The long-term consequence is
prostatitis when the young males mature into adults.
Atrazine is one of the chloro-S-triazine herbicides, the
largest group of herbicides sold in the United States.
NHEERL scientists discovered that atrazine acts on
the pituitary gland of nursing rats, suppressing
prolactin secretion in the milk. Male rats whose dams
56
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were exposed to atrazine during the first nine days of
lactation exhibited an increased incidence and
severity of prostatitis when mature.
Neonatal exposure to other substances, including
estrogen, can also alter prolactin secretion. Based on
this observation, NHEERL researchers hypothesized
that perinatal exposure to estrogenic compounds may
increase the risk of prostatitis in male offspring by
decreasing prolactin levels in the dam's milk. They
exposed pregnant rats late in gestation and their pups
during the first five days after birth to either the
hormone 17beta-estradiol, the insecticide
methoxychlor, or the pharmaceutical agent tamoxifen.
As adults, the male rats exposed to these chemicals
perinatally experienced a significantly higher
incidence and severity of lateral prostate
inflammation compared to controls. These and other
studies demonstrate a previously unrecognized critical
period of susceptibility to chemicals that affect the
pituitary gland.
NHEERL researchers
hypothesized that perinatal
exposure to estrogenic
compounds may increase
the risk of prostatitis."
NHEERL scientists are also studying the mechanisms
by which phthalates cause reproductive abnormalities
in animals. One of the most widely used phthalates is
di(2-ethylhexyl)phthalate (DEHP), a plasticizer used
in toys, food wraps, cosmetics, vinyl floors, and
medical products, including blood transfusion and
dialysis equipment. In previous studies, in ute.ro
exposure to DEHP, dibutyl phthalate (DBF),
butylbenzyl phthalate (BBP), and di-isononyl
phthalate (DINP) caused malformed sexual organs
and disrupted androgen-dependent processes in male
rat offspring. NHEERL studies of the antiandrogenic
effects of DEHP and its major metabolite found that
neither compound binds to the human androgen
receptor. (Binding to the androgen receptor is a
common antiandrogenic mechanism.) Other studies
indicate that inhibition of testicular testosterone
production is the most likely mechanism by which
DEHP disrupts sexual differentiation. These and other
studies identifying the specific mechanisms of toxicity
provide a scientific basis for the risk assessment of
human exposure to endocrine disrupting chemicals.
SJ
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United States
Environmental Protection Agency
Center for
Environmental Research Information
Cincinnati, OH 45268
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