earch and Developm
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ational Health a
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EPA600/R-02/036
May 2002
www.epa.gov/nheerl
An of
for
Fiscal Year 2001
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory
Research Triangle Park, NC 27711
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notice
The U.S. Environmental 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 processes and has been approved for publication as an EPA document.
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abstract
This Annual Report showcases some of the scientific activities of the National Health and
Environmental Effects Research Laboratory (NHEERL) in various health and environmental effects
research areas. Where appropriate, the contributions of other collaborating research organizations
inside and outside EPA are acknowledged. The report is an indicator of progress and accomplishments
that NHEERL has made in Fiscal Year 2001 in achieving the Agency's and ORD's strategic goals.
NHEERLs highlighted research is organized under these goals. Specific research areas included for this
year are: (1) Particulate Matter, (2) Air Toxics, (3) Drinking Water, (4) Aquatic Stressors,
(5) Pesticides, (6) Global Change, (7) Ecosystems Protection, (8) Human Health Protection,
and (9) Endocrine Disrupters.
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letter from the director
EPA's mission is to protect human health and to safeguard the environment. As EPAs scientific
arm, the Office of Research and Development (ORD) provides research, leadership, and advice on
scientific issues to EPA. As one of five laboratories and centers in ORD, the National Health and
Environmental Effects Research Laboratory (NHEERL) is charged with investigating the impacts of
environmental stressors on both human and ecosystem health, the degree of harm caused by the
stressors, and the factors that affect the degree of harm. The range of achievements highlighted in
this report reflects NHEERL's support to ORD and EPA.
EPAs research efforts are organized according to strategic goals outlined in its Strategic Plan: Clean
Air (Participate Matter and Air Toxics); Clean Water (Drinking Water and Aquatic Stressors); Safe
Communities (Pesticides); Climate Change (Global Change); and Sound Science (Ecosystems Protection,
Human Health Protection, and Endocrine Disrupters). ORD's research is planned and pursued to
support EPAs strategic goals in an integrated fashion. NHEERL supports that plan with
multidisciplinary teams of scientists dedicated to unraveling the many complex factors relating to a
specific problem. For example, this report showcases the work of epidemiologists, toxicologists,
analytical chemists, and others from across several divisions within NHEERL to determine the
effects of arsenic in drinking water.
Using a risk-based investigative approach, NHEERL provides scientific data from its human health
and environmental effects research to support ORD's mission to inform regulatory programs and
make sound policies to fulfill EPAs efforts to safeguard human and ecosystem health. We are
pleased to share some of our most important findings with you.
Lawrence W. Reiter, Ph.D.
Director, National Health and Environmental Effects Research Laboratory
Office of Research and Development
Research Triangle Park, North Carolina
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table of contents
introduction
particulate matter
air toxics
19
drinking water
aquatic stressors
31
pesticides
global change
43
ecosystems protection
53
human health protection
endocrine disrupters
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\
vancing Knowledge
For a Purpose:
Deciphering the Link Between
Invironmental Stressors and their Effects
on Human Health and Ecosystems
Highlights cm tcomplishments Made During Fiscal Year 2OO1
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 EPAs 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 national and
international research communities. Based in
Research Triangle Park, NC, NHEERL has nine
divisions in six states and a work force of over 700
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
research organizations around the world.
NHEERL research undergoes the highest levels of
independent scientific review and scrutiny, and
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Corvallis, OR
- If
Research Triangle Park, NC
**
Chape! Hill, NC
NHEERL
research centers
are located in several
geographically important
areas around the nation.
Gulf Breeze, FL
1
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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
scientific organizations.
In researching health and ecological risk,
NHEERL's organizational structure enables
scientists to develop innovative methods and
solutions to complex problems
in an integrated manner. 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 work force 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 particulate
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 particulate matter
• mechanisms of toxicity of air pollutants
• advances in drinking water safety research
• susceptibility of children 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
As the largest research center within EPA's Office of
Research and Development, NHEERL has nine divisions.
NHEERL headquarters and five health research divisions are
located in Research Triangle Park and Chapel Hill, North
Carolina. Four ecology research divisions are located in
ecologically significant areas around the country.
NHEERL Health Research Divisions
• Environmental Carcinogenesis (RTF, NC). Studies the associations
among environmental contaminants and cancer.
• Experimental Toxicology (RTF, NC). Examines the toxicity of
environmental contaminants to specific organ systems and bodily
functions.
• Human Studies (Chapel Hill, NC). Conducts epidemiologic and
clinical research on the human response to environmental
contaminants.
• Neurotoxicology (RTF, NC). Studies the effects of chemical and
physical agents on the nervous system.
• Reproductive Toxicology (RTF, NC). Develops methods used to study
the reproductive and developmental effects of environmental
contaminants.
NHEERL Ecology Research Divisions
The ecology research divisions assess the condition of regional
ecosystems—including terrestrial and aquatic environments—and study
the effects of pollution and other stressors on these ecosystems.
• Atlantic Ecology (Narragansett, RI). Atlantic seaboard ecosystems.
• Gulf Ecology (Gulf Breeze, EL). Gulf of Mexico ecosystems.
• Mid-Continent Ecology (Duluth, MN and Grosse He, MI). Inland and
freshwater ecosystems.
• Western Ecology (Corvallis and Newport, OR). Pacific coast
ecosystems.
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P articulate matter (PM) is one of the
six criteria air pollutants for which
EPA has established National
Ambient Air Quality Standards (NAAQS). The
term particulate matter refers to airborne solid
particles and liquid droplets. Based on particle
size, particulate matter is categorized as ultrafine
(0-0.1 micron in diameter), fine (0.1 - 2.5
micron), and coarse (2.5 - 10.0 micron). PM10 is
an older term that refers to particles less than 10
microns in diameter and includes coarse, fine, and
ultrafine particles. Generally, particles larger than
10 microns in diameter are trapped in the nasal
passages and do not make their way into the lungs.
EPA has established NAAQS for both fine and
coarse particles (in the form of PM,9). Coarse
particles can aggravate respiratory conditions such
as asthma. Generally, coarse particles enter the
air from dust generated by vehicles traveling on
unpaved roads, materials handling, and crushing
and grinding operations. The major source of fine
particles is fuel combustion from vehicles, power
plants, and industries.
Elevated levels of particulate matter have been
associated with increased mortality and
I
hospitalizations for heart
and lung conditions;
these associations are
stronger for fine particles
than coarse particles.
Because of its widespread
distribution and potential
impact on many people,
particulate matter continues to be one of the
highest research priorities at EPA.
Scientists at NHEERL
are world leaders in
particulate matter
research.
The NHEERL particulate matter research program
is multidisciplinary and includes three major
categories of studies: epidemiologic, human
clinical, and laboratory studies using animals and
tissue culture cells. The program is currently
investigating the
• health effects associated with particulate
matter,
• groups of people most likely to be affected,
physiological mechanisms underlying these
effects, and
toxic components of particulate matter.
NHEERL researchers conduct epidemiologic
studies in groups of people, toxicologic studies in
the laboratory, and clinical studies in human
volunteers, all of which contribute to the scientific
understanding of how particulate matter causes
health problems. Epidemiologic studies are often
the first to point toward an association between an
environmental exposure and a health problem.
When this occurs, scientists conduct laboratory and
clinical studies designed to clarify and characterize
the apparent association. The results from these
studies can then be used to identify additional
important endpoints to measure when conducting
future epidemiologic studies.
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2.9 47.2 107.8 206.7
Average CAPS Concentration (|i/m3
TOXIC COMPONENTS OF
PARTICULATE MATTER
A series of epidemiologic studies conducted by
university scientists in Utah Valley, Utah, found
an association between health problems and high
PM1Q levels, which were associated with operation
of a steel mill in the valley. The health problems
included increased hospital admissions for
respiratory conditions and increased deaths due to
respiratory and cardiovascular diseases.
Subsequently, to pinpoint the factors responsible
for this observed association, NHEERL scientists
obtained air quality monitoring filters from the
Utah Valley for the time period overlapping that
of the epidemiologic study. This included one
year during which the steel mill was closed due to
a labor dispute (year 2), the year preceding the
closure (year 1), and the year following mill
reopening (year 3). The researchers used water to
extract particulate matter components from the
filters. These extracts were used in a variety of
studies. The NHEERL Annual Report of
Accomplishments for Fiscal Year 2000 described
the results of several studies, the findings of which
were consistent with the epidemiologic studies.
Both clinical and laboratory studies found that
extracts of particulate matter collected when the
steel mill was operating caused more lung
inflammation than the particulate matter
collected when the steel mill was closed. (Lung
inflammation reversed in all study participants.)
Since the same amount of material from each of
the three years was used for the experiments, these
results demonstrate for the first time that the
chemical composition of particulate matter plays
an important role in its toxicity.
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Image STS006-114-063 courtesy of Earth Sciences & Image Analysis Laboratory, NASA Johnson Space Center
(http://eol.jsc.nasa.gov)
Research published in 2001 revealed a mechanism
whereby particulate matter causes lung
inflammation. A study conducted by scientists
from NHEERL and the University of North
Carolina at Chapel Hill using epithelial cells
lining the human airway identified a specific
cellular signaling pathway that increased the
production of inflammatory proteins when it was
activated by exposure to Utah Valley particulate
matter extracts.
Also in 2001, NHEERL scientists and collaborators
at CUT Centers for Health Research reported that
rats developed significant acute lung injury and
inflammation after extracts of years 1 and 3 were
put into the lungs. (The effects were largely
resolved within 96 hours.) Year 2 extract did not
cause appreciable lung damage. Because the degree
of lung damage was correlated with metal content
of the extracts, researchers hypothesized that
soluble metals may be responsible for some of the
adverse health effects observed.
This research approach
links associations first
identified in
epidemiologic studies to
animal toxicologic
studies and human
clinical studies, which-
taken together-provide
information on the
mechanisms by which
particulate matter
causes health problems.
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Other projects conducted by researchers at
NHEERL, the University of Rochester
School of Medicine and Dentistry, and the
University of North Carolina at Chapel
Hill investigated the extracts' metal
content, the specific effects of the extracts
on respiratory cells, and the mechanisms of
cellular injury. The researchers found that,
compared to year 2, years 1 and 3 extracts
contained more soluble iron, copper, and
zinc. A study examining the response of
human respiratory tract cells in the
extracts found that those cells exposed to
extracts from years 1 and 3 generated the
highest levels of oxidants (Figure 2) and
inflammatory proteins (Figure 3). These
findings add to the mounting evidence
that the type and amount of water-soluble
metals present in particulate matter may be
an important factor determining the toxic
effects of particulate matter in humans.
0.5 1
Oxidant Concentration
(measured as absorbance at 532 nm)
Extract
Metal chelator (deferoxamine)
Hydroxyl radical scavenger (dimethylthiourea)
Hydroxyl radical scavenger (dlmethylsulfoxlde)
1.5
FIGU
reactive 3~ „
(Household bleach and hydrogen peroxide are familiar
oxidants.) In the body, antioxidants neutralize or "scavenge"
oxidants, rendering them harmless. In this cell culture test,
adding a metal chelator or a hydroxyl radical scavenger to
the extract before exposing cells to particulate matter
extracts dramatically reduced oxidant production by cells
exposed to years 1 and 3 extracts. (The metal chelator
removed the metals from the extract, whereas the hydroxyl
radical scavengers neutralized hydroxyl radicals, one type
of oxidant, as they were produced by the cells.)
FIGURE 3. In cell cultures,
one measure of the
inflammatory response is the
amount of inflammatory protein
released following cell injury.
This graph shows production of
the inflammatory protein
interleukin-8 (IL-8) following
exposure to control (0 dose)
and three doses of Utah Valley
dust extracts. Note that IL-8
production increased as dose
increased. Production of
interleukin-6, another
inflammatory protein, followed
a similar pattern.
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The Clean Air Act and Amendments
dentified 188 hazardous air pollutants
[HAPs, also known as air toxics) that
are emitted from a variety of stationary, mobile,
and indoor sources. Significant doubts remain
regarding the health effects of hazardous air
pollutants. Of the 188 HAPs, EPA has not yet
developed cancer risk information for 143,
reference doses (RfD, for ingestion exposure) for
134, or reference concentrations (RfC, for
inhalation exposure) for 167. Appreciable
uncertainty accompanies the risk estimates for
many HAPs for which EPA has some health risk
information. Over the past decade, EPAs air
toxics regulatory program established technology-
based standards for specific industry categories.
Future regulatory actions and nonregulatory
guidelines will require an improved understanding
and quantification of the health risks that air
toxics from single and multiple sources pose at
multiple geographic scales
(e.g., local, regional).
To reduce uncertainties in
future risk assessments,
EPA needs more research
on adverse health effects
of air toxics. To this end,
EPA has an Air Toxics
Research Strategy
designed to meet four
long-term goals of EPAs
Air Program providing:
1. methods and information to support
assessment of the health effects and risks from
exposure to air toxics at national, regional,
and local scales;
2. measurements and models that will reduce
uncertainty of estimated mobile source
emissions and estimates of human exposure
and health effects associated with mobile
sources of air toxics;
3. methods to support residual risk assessments
and risk management strategies of stationary
sources, including major and area source
categories; and
4. health effects information and validated
emissions characterization and transport
models that estimate and provide guidance on
management of risks from indoor air toxics
based on building type and indoor activities.
AIR TOXICS IMPLEMENTATION PLAN
In support of EPAs Air Toxics Research Strategy,
NHEERL is developing an Air Toxics
Implementation Plan to guide its research in this
area. Most of NHEERL's air toxics research efforts
address the first goal mentioned above. NHEERL
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u
scientists conduct research to determine the
health and ecological risks and dose-response
relationships associated with exposure to air
toxics. Researchers at NHEERL are working to
fill current knowledge gaps regarding
mode of action, dose-response
relationships for acute and chronic
exposures, susceptibility of sensitive
subpopulations, and mixture interactions.
Historically, approaches to filling these
gaps have relied upon toxicity data
obtained from laboratory animals or
human studies. NHEERL is also taking a
newer approach through development of
physiologically based pharmacokinetic (PBPK)
models that facilitate dose-response assessments.
Current areas of air toxics research
activity at NHEERL include
irritant and pulmonary effects of aldehydes
and halides,
neurotoxic effects of volatile organic
compounds, and
carcinogenic effects of polycyclic organic
matter and hydrocarbons at low doses.
©
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NHEERL's Research
Planning Process
For each high-priority research area, NHEERL
develops an implementation plan. Once ORD
has identified the priority topics, these multi-year
plans provide a mechanism for prioritizing
research projects and link directly to EPA goals
and ORD strategic plans. To enhance research
integration with EPA programs and goals,
representatives from all ORD Laboratories and
Centers participate in developing implementation
plans. Also, representatives from EPA Program
Offices and Regions participate to ensure that
NHEERL is conducting research that supports
EPA's mission. At two-year intervals, each
implementation plan is reviewed and revised
based on new scientific findings and changing
research needs.
An implementation plan consists of a research
framework and research plans for specific
projects. The research framework is usually
established at workshops and meetings attended
by scientists and program managers. The
research framework
• describes the problem EPA faces,
• discusses how the NHEERL mission relates to
the problem,
• identifies principal scientific uncertainties,
• lists the uncertainties that NHEERL can
address or problems NHEERL can solve, and
• outlines major research approaches/steps
needed.
Research plans for specific projects are
developed by NHEERL divisions in response to
the implementation plan's research framework.
They are reviewed by a steering committee to
ensure their relevancy and responsiveness to
EPA needs.
ORD priorities for NHEERL
determine state of
science and new or
remaining agency
needs
develop
responsive
research
framework
conduct research
report results & review
progress
(All-Investigators'
meeting every 2 years)
revise research
framework & plans
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The PBPK model can be
used to improve risk
assessment for
unintentional exposures
to MTBE.
contribution of each route of exposure to observed
MTBE body burdens.
Scientists at NHEERL, the National Exposure
Research Laboratory (NERL), and the Centers for
Disease Control (CDC) collaborated to study the
pharmacokinetics of MTBE and its primary
metabolite, tertiary butyl alcohol (TEA). Studies
in rats formed the foundation for human PBPK
modeling. Based on the measurements made in
these rat studies, the researchers developed a
rat-to-human extrapolation model and
subsequently validated this PBPK model
with human volunteers. MTBE was most
rapidly absorbed following ingestion and _o
inhalation exposure. Dermal uptake was
slower and dependent on the MTBE
concentration. Compared to inhalation or
dermal exposure, a greater TBA:MTBE ratio
was observed following oral ingestion,
providing evidence of significant first-pass
metabolism for this route of exposure.
Regardless of the route of exposure, about
half of the MTBE was exhaled, with the rest
metabolized to TEA.
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Twenty-four hours after exposure, blood MTBE
concentration was very low, but blood TEA
concentration was still about 27% of peak,
indicating that TEA was metabolized and
eliminated more slowly than MTBE. Figure 1
shows the correlation between observed blood
MTBE concentrations and the levels predicted by
the PBPK model. The model accurately simulated
MTBE and TEA blood concentrations for all
three routes of exposure. Therefore, the PBPK
model can be used to improve risk assessment for
unintentional exposures to MTBE via inhalation,
dermal contact, and ingestion. With further
refinement, the model is expected to play an
important role in policy decisions that attempt to
balance the health benefits of using MTBE to
reduce ozone and carbon monoxide in the air
with the potential increased risk of cancer from
MTBE exposure.
MTBE Blood Concentrations
• Inhalation data
Inhalation model
• Oral Data
Oral Model
* Dermal data
Dermal Model
10
10 20
Time (h)
30
Figure 1. Correlation between MTBE blood
concentrations and modeled MTBE blood levels for
one participant.
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The 1996 Amendments to the Safe
Drinking Water Act (SDWA) require
iPA to conduct research to provide a
scientific foundation for regulatory standards that
limit contaminants in drinking water.
Contaminants that EPA is studying include
waterborne pathogens, man-made chemicals,
naturally occurring elements, and disinfection
by-products.
Arsenic is an element that occurs in several types
of rock formations, particularly in areas of past or
present volcanic or geothermal activity. In several
regions of the United States, arsenic leaches from
rock into groundwater that is used for drinking
water. Health problems associated with
arsenic ingestion include cardiovascular
disease, strokes, peripheral neuropathy (a
disorder of the nerves), diabetes,
abnormal fetal development, and several
types of cancer. While studies in foreign
countries have demonstrated that very
high levels of arsenic in drinking water
can be harmful, the minimum dose and
minimum duration of exposure required
to cause health problems have not been
established.
Studying the adverse health effects of
exposure to arsenic in drinking water is a major
research priority at EPA. At NHEERL, scientists
are conducting cutting-edge research investigating
the metabolic processes by which the body
transforms arsenic and the molecular mechanisms
by which arsenic causes cancer and other health
problems. Other scientists are characterizing
arsenic exposure in specific communities in the
United States.
ARSENIC MODE OF ACTION AND
METABOLIC FATE
A cooperative effort studying the mechanism of
action of arsenic (As) compounds and the role of
metabolism in the element's toxicity and
carcinogenicity involves scientists from NHEERL,
the University of North Carolina at Chapel Hill,
the University of British Columbia in Vancouver,
and the Polytechnical Institute of Mexico.
New analytic technique developed
This research team developed an analytic
technique to distinguish between methylated
arsenic compounds with arsenic in the +3
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(trivalent, As111) and +5 (pentavalent, As^O
oxidation states. This is important because the
oxidation state of an element in a compound may
Methylation, the addition of a methyl group to
a substance, is a common step in the body's
metabolism of foreign or toxic substances.
Methylated compounds are often less toxic to
the body than the original chemical. Scientists
have long thought that the methylation of
arsenic is a detoxifying reaction that produces
compounds less toxic than inorganic arsenic.
New research conducted by NHEERL
scientists and collaborators suggests that
some methylated arsenic compounds may be
more toxic than inorganic arsenic.
dramatically affect the compound's reactivity and
toxicity. In studies using this technology, the
research team identified and quantified methyl
As111 and dimethyl As111 in cultured cells, urine
specimens from people
exposed to inorganic
arsenic, and other
biological samples. The
oxidation state (As111 versus
As^O of arsenic metabolites
influences their toxicity.
Therefore, the ability to
distinguish between and
measure As111 and Asv
metabolites in tissue
cultures, tissue samples, and
urine is critically important
to studies of arsenic metabolism and toxicity. This
analytic ability is also important to epidemiologic
studies of people exposed to naturally occurring
arsenic in drinking water and/or food.
Toxic activity investigated
In 2001, the collaborating scientists reported that
three trivalent arsenic compounds — inorganic
As111, methyl As111, and dimethyl As111—inhibited
the enzyme thioredoxin reductase in cultured rat
liver cells. As a key enzyme in the cellular
response to oxidative stress, thioredoxin reductase
plays a critical role in the response of cells to a
wide range of toxic agents. Exposing cells to
various doses of inorganic As111 and methyl As111
caused a concentration-dependent inhibition of
thioredoxin reductase. As the concentration of
inorganic As111 increased in the medium, the
concentrations of methyl arsenic and dimethyl
arsenic metabolites inside the cells also increased.
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As the intracellular concentration of arsenic
compounds increased, thioredoxin reductase
inhibition increased. Methyl As111 was more potent
than inorganic As111 and inhibited thioredoxin
reductase activity at much lower concentrations
(see Figure 1). The consequences of thioredoxin
reductase inhibition are unknown. Thioredoxin
reductase and thioredoxin (one of the molecules
with which thioredoxin reductase interacts) play
important roles in regulating cell growth and
genetically programmed cell death. These
processes are critically important to the growth
and survival of tumor cells. Further studies of the
influence of arsenic on the thioredoxin reductase-
thioredoxin system may shed light on the role of
arsenic in cancer development.
New genotoxic mode of action discovered
Also in 2001, the researchers reported that
trivalent methylated arsenic compounds caused
direct damage to free DNA and to DNA in
cultured cells. This is significant because
• no interactions of an arsenic compound with
DNA had been reported previously;
• the mode of action of arsenic in
carcinogenicity had been thought to be
through indirect mechanisms, rather than by
direct damage to DNA;
• an implicit assumption underlying arsenic risk
assessment had been that the methylation of
arsenic is a detoxification process. This
assumption is now in question; and
• these findings imply that people who
methylate arsenic efficiently may be at
increased risk of cancer when exposed to
arsenic in drinking water or food.
Incubation Time (hr)
Incubation Time (hr)
FIGURE 1. Relationship between intracellular concentration of arsenic compounds and thioredox
reductase activity when cultured cells were exposed to arsenic compounds for 24 hours, (a) 10 u,IV
inorganic arsenic As'", (b) 1 u,M methyl arsenic As'". Both inorganic and organic As'" inhibited
thioredoxin reductase. (At the concentrations used, none of the arsenic compounds killed cells.)
Symbols: = inorganic arsenic; = methyl arsenic; = dimethyl arsenic; = thioredoxin reducta
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The research team conducted two series of
laboratory studies involving the trivalent (As111)
and pentavalent (As^O forms of inorganic arsenic,
methyl arsenic, and dimethyl arsenic. In a DNA
nicking assay, only free DNA that had been
exposed to trivalent methylated arsenic (methyl
As111 and dimethyl As111) showed evidence of
breaks — "nicks" — in either one or both strands of
DNA. Further, DNA damage was dose-dependent;
the more extensive DNA damage occurred at
higher concentrations. In a single-cell gel (SCG)
assay using human lymph cells, the trivalent
methylated forms (methyl As111 and dimethyl As111)
caused appreciable damage to cellular DNA,
whereas both trivalent and pentavalent inorganic
arsenic (As111 and As^O caused only slight DNA
damage. Pentavalent methylated forms (methyl
Asv and dimethyl As^O were basically inactive.
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FIGURE 3. In the alkaline single cell gel (SCG) assay, as fragments of damaged DMA migrate in the electric field, they
produce the appearance of the tail of a comet. These figures are representative of those seen in (A) control, up to 1
mM methyl Asv or dimethyl Asv (no DMA damage); (B) 1 mM inorganic As'" or inorganic Asv, or 10 mM methyl As'"
(some DMA damage); and (C) exposures as low as 23 mM dimethyl As'" (appreciable DMA damage).
While the study results do not rule out the
possibility that inorganic arsenic compounds may
be genotoxic, the findings suggest that methylated
trivalent arsenic compounds, produced in the
body during the metabolism of inorganic arsenic,
may cause direct damage to cellular DNA. The
researchers plan further studies examining the
genotoxicity of arsenic compounds and the
possibility of a link between arsenic-induced DNA
damage and the development of cancer following
exposure to inorganic arsenic.
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13
ARSENIC EXPOSURE IN A COMMUNITY
After a previous study suggested that locally
harvested shellfish may contain large amounts of
arsenic, a Native American Tribe requested EPAs
assistance in determining the extent of Tribal
members' exposure to arsenic in their food and
drinking water. Scientists from NHEERL,
National Exposure Research Laboratory (NERL),
and the EPA Regional Office are working with the
Tribal council to conduct a study of families that
have resided in the community of interest for at
least 12 months. (The Tribe has requested
anonymity.)
All members of each participating household went
to the community medical center for enrollment
in the study. (Children in diapers are the only
family members not eligible to participate.)
Information about each individual's diet in the
previous four days, water consumption habits,
exposure to substances that affect arsenic
metabolism, and underlying health conditions was
obtained via questionnaire. Participants provided
blood and urine samples at enrollment and
samples of urine from the first morning void for
the two days following enrollment. Also,
participants were asked to record every food item
Gathering clams at low tide.
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Arsenic Concentrations in Groundwater
ities with arsenic concentrations
eding 10 jxg/L in 10 percent or more
mples.
ities with arsenic concentrations
sding 5 |ag/L in 10 percent or more
imples.
ities with arsenic concentrations
eding 3 ng/L in 10 percent or more
mples.
Counties with fewer than 10 percent of
samples exceeding 3 j^g/L, representing
areas of lowest concentration.
Counties with insufficient data in the
USGS data base to make estimates.
States: U.S. Geologic;
consumed for a 24-hour period that coincided
with the morning urine samples. Each household
also collected a sample of water from the kitchen
tap. One household member from each
participating family was asked to provide a sample
of all seafood, fish, potatoes, and rice consumed
during the study period. The data collection
phase of the project was completed in 2001.
Fish and shellfish samples will be analyzed for the
different forms of arsenic using techniques
recently developed by EPA researchers. Water,
blood, and urine samples will be analyzed for
arsenic and selenium, which is believed to affect
the way the body metabolizes arsenic. Urine from
the oldest member of each household will be
analyzed for a series of other metals in addition to
arsenic.
This project is the first
EPA study to examine
the association between
different forms of
arsenic in seafood and
water and the forms of
arsenic circulating in
blood and eliminated
in urine.
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i
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1
photo courtesy of University of Mi
Natural Resources Research
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The Clean Water Act requires states,
;erritories, and tribes to report the
condition of surface waters to EPA every
two years. EPA then reports to Congress on the
condition of the Nation's waters. States,
territories, or tribes set water quality standards and
designate the uses (e.g., drinking water supply,
swimming, fishing) of each water body. In the
reports to EPA, surface waters that do not meet
water quality standards are listed as impaired in
their ability to support the designated uses.
Maintaining ecological integrity in aquatic
ecosystems involves protecting these natural
resources from degradation of habitat, reduction
in diversity of plant and animal species, and
disruption of ecosystem functions. To this end,
environmental managers must be able to
assess the condition of an aquatic resource,
• determine the degree of impairment,
diagnose the cause (s) of impairment (the
stressors),
• forecast the effects of changing stressor levels,
and
design and implement restoration and
maintenance strategies.
AQUATIC STRESSORS
IMPLEMENTATION PLAN
Within NHEERL, a research implementation plan
is a mechanism for prioritizing research efforts to
ensure that projects are relevant and responsive to
EPAs needs. (See the Air Toxics chapter for a
discussion of implementation plans.) In 2001, the
NHEERL Aquatic Stressors Implementation Plan
was completed and readied for external peer
review. This Plan outlines research goals and a
Nationwide, 44% of
stream or river miles;
49% of lakes,
reservoirs, and ponds;
98% of Great Lakes
shoreline miles; and
42% of estuaries have
been designated as
impaired. (National
Coastal Condition
Report, 2001)
multi-year timetable for research projects on
stressor diagnostics and four types of stressors
(habitat alteration, nutrients, toxic chemicals, and
suspended/bedded sediments). NHEERL's aquatic
stressors research is focused on (I) diagnosing the
causes of aquatic ecosystem impairment and (2)
investigating stressor-response relationships.
Knowledge of stressor-response relationships will
provide the scientific foundation to guide
remediation and restoration activities and will
enable resource managers to forecast the benefits
and/or consequences of changing stressor levels.
Specific projects are being developed to
investigate the influence of the four types of
stressors on fish and shellfish populations. The
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A harmful algal
bloom (white and
tan areas in the
middle of the
water body) off the
central California
coast in the late
spring of 1998.
photo courtesy of Dr. Vera Trainer, NOAA/NWFSC Seattle
steering committee for the Plan included
representatives from each of ORD's laboratories
and centers, EPA Regional Offices, and EPA's
Office of Water.
STRESSOR DIAGNOSTICS
One research area identified in the Aquatic
Stressors Implementation Plan is development of
diagnostic tools to identify chemical and
The goal of EPA's Total Maximum Daily Load
(TMDL) Program is attainment of water quality
standards. The TMDL is a written, quantitative
assessment of water quality problems and
contributing pollutant sources. It is prepared by
the state or local water quality manager. The
TMDL
• identifies the need for point and nonpoint
source controls,
• provides a basis for action to restore a water
body,
• specifies the quantity by which a pollutant
needs to be reduced,and
• allocates pollutant load reductions within a
watershed.
nonchemical stressors over a range of geographic
scales. In addition to information about individual
water bodies, information about stressors at region
and watershed levels is vital to planning integrated
restoration and remediation programs. In 2001,
NHEERL drafted the Stressor Diagnostics portion
of the Aquatic Stressors Implementation Plan to
guide this research. Working with other ORD Labs
and Centers, the tools developed under this Plan
will enable resource managers to
• define the primary causes of impairment,
assign responsibility for the observed effects
among the various stressors, and
• assess potential interactions among stressors.
In addition to providing the foundation for
ecosystem maintenance and restoration programs,
the information gained by using these tools will
support EPA's Total Maximum Daily Load Program,
Superfund, and other regulatory activities.
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The Lake Michigan eutrophication modeling
study addressed questions that included:
• Which media (atmospheric deposition,
tributaries, or sediment resuspension) are the
major sources of phosphorus to Lake
Michigan?
• Are specific tributaries major contributors?
What are the nearshore zone effects of these
tributaries?
• What is the history of phosphorus loads to
Lake Michigan?
• Do total phosphorus loads and ambient
concentrations of phosphorus and chlorophyll
a (measure of phytoplankton numbers) meet
the Canada-U.S. Water Quality Agreement?
• If increases or decreases in phosphorus loads
occur in the future, what are the forecasted
consequences or benefits?
The Lake Michigan Mass Balance Study is a
collaboration among EPA's Great Lakes National
Program Office and a number of EPA, federal,
state, academic, and private partners. As part of
this larger study, NHEERL and other ORD
scientists developed three computer models to
examine the relationship between phosphorus and
phytoplankton (microscopic plant-like aquatic
organisms, including many types of algae) in Lake
Michigan. In 2001, the eutrophication databases
were completed, the models were calibrated, and
simulation studies were conducted.
EUTROPHICATION MODELING
One type of aquatic stressor is eutrophication due
to human activities. In this context,
eutrophication refers to high
levels of nutrients in water
bodies from sewage discharge
or agricultural or urban runoff.
Eutrophication may produce a
number of effects, including
algal blooms, which may
shade out submerged
vegetation and ultimately
cause fish kills due to reduced
dissolved oxygen levels.
Nitrogen and phosphorus are
nutrients that may be found
in excess in surface waters as a
consequence of fertilizer-rich Lake Michigan
Monitored
agricultural and urban runoff. Tributaries &
Associated
Watersheds
The model simulations showed good agreement
with observed data. The simulation results
indicated that monitored tributaries emptying
Menominee River
Fox River
Sheboygan River
Milwaukee River
Grand Calumet River
St. Joseph River
Kalamazoo River
Grand River
Muskegon River
Pere Marquette River
Manistique River
Color shaded areas indicate watersheds
0 100 miles
|
100 kilometers
©
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into Lake Michigan were the largest contributors
of phosphorus to the lake. Also, substantial
amounts of phosphorus were present in Lake
sediments. Sediment resuspension contributed
appreciably to phosphorus levels in the water.
Both phosphorus loading data and model
simulations indicated that phosphorus loadings
have been stable for the last decade.
coastal zones were being degraded due to localized
phosphorus inputs even though lakewide target
levels were met. The Mass Balance Study
scientists concluded that target nutrient levels for
nearshore and coastal environments, especially
near tributary inputs, may need to be re-evaluated
to provide adequate ecosystem protection for
these areas.
Targets for phosphorus loading and ambient water
concentrations established by the Canada-U.S.
Water Quality Agreement were being met on a
lakewide basis. However, localized phosphorus
concentrations varied. Model applications using
the finest resolution indicated that nearshore and
Zebra mussels and Bythotrephes, a
zooplankton, are two non-native species
that are influencing Great Lakes nutrient
dynamics, the lower levels of the aquatic
food chain, and fish populations. Zebra
mussels filter phytoplankton (microscopic
plant-like algae) for food. Because of their
tremendous filtration capacities, they
remove phytoplankton, particles, and
nutrients from the water column and
deposit them in sediment. By filtering large
amounts of phytoplankton, the zebra
mussels compete with zooplankton, some
larval fish, and foraging fish for the same
food source. Similarly, Bythotrephes
consumes other zooplankton and
competes with zooplankton, larval fish,
and foraging fish for this food source. In
both cases, selectivity and competition for
the same food sources appear to be
adversely affecting both foraging and
predatory fish populations, especially
when these food sources are required at
critical life stages.
Environmental managers have expressed concern
that phosphorus loadings to Lake Michigan will
increase in the future due to human population
increases and land use changes. The
eutrophication model forecasted that a
phosphorus loading increase of 20 percent or more
would challenge targets for lakewide phosphorus
loading, ambient phosphorus concentration, and
steady-state phytoplankton population. This
m
* A
zebra mussel photo courtesy of University of Michigan's
Center for Great Lakes and Aquatic Sciences
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Logend
urban
agricultural
forest
water
wetlands
situation could produce symptoms commonly
observed in eutrophic water bodies: algal blooms,
taste and odor problems in drinking water,
submerged vegetation loss, food chain structure
alterations, and water clarity reductions. The
model predicted that reduced dissolved
oxygen, another frequent consequence of
eutrophication, was unlikely to be a problem
even if phosphorus loadings increased by
20 percent. (Dissolved oxygen in Lake
Michigan is considered to be saturated or
supersaturated, which is characteristic of
cold, deep lakes with low productivity.
Even if productivity increased
substantially, dissolved oxygen would not
decrease greatly.)
0 25 50 100 150
ZOO
: Kilometers
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Many of NHEERL's research projects
directly support EPA's decisions to
regulate pesticides and toxic
chemicals under the Federal Insecticide,
Fungicide, and Rodenticide Act and the Toxic
Substances Control Act. In order to develop
regulations that protect public and environmental
health, policymakers need scientific information
about a chemical's persistence in the environment
and its toxicity to humans and other animals.
Researchers at several NHEERL divisions are
investigating the means by which pesticides may
cause health problems, including immune system
suppression, cancer, nervous system dysfunction,
and endocrine disruption. These studies examine
the role of variables such as the dose and duration
of exposure to the pesticide and the
developmental stage — fetus, newborn, immature
young, mature adult — of the exposed individual.
Another area of research is development of
computer models that can predict the toxicity of
new chemicals based on their similarity to other
chemicals whose toxic activity has been
determined in laboratory tests.
Safe Food
While the health effects of short-term
exposure to high doses of pesticides have
been studied fairly well, the potential adverse
consequence of chronic exposure to lower
pesticide levels has not been thoroughly
investigated. Researchers at NHEERL have
been collaborating with scientists from North
Carolina State University to study long-term
exposure of rats to the organophosphorous
pesticide chlorpyrifos. The primary purpose
of the study is to determine what, if any,
adverse health effects may be caused by
chronic exposure and what pattern of
exposure is most harmful. The scientists also
hope to learn if chlorpyrifos must enter the
brain and spinal cord to cause problems or if
a body burden limited to the organs and
peripheral nerves can cause adverse health
effects. The scientists assessed a large
variety of endpoints including visual,
neurophysiological, nervous system, and liver
function; learning and memory; overt toxicity;
pathology of all major organs; and tissue
distribution of chlorpyrifos and its metabolites.
In 2001, the exposure phase of the study was
completed and end-of-exposure assessments
were conducted.
Based on previous studies, EPA recently
revised the risk assessment and risk
mitigation measures for chlorpyrifos. Sale of
products containing chlorpyrifos for use by
homeowners and in schools, parks, and other
settings where children may be exposed
ended December 31, 2001. Some uses of
chlorpyrifos are still allowed;
results of the current study may
have important implications
for professional pesticide
applicators and agricul-
tural workers.
HEPTACHLOR
Heptachlor is an organochlorine compound that
was used as an agricultural and domestic pesticide
from the mid-1960s to the early 1980s. In 1974,
EPA canceled its registration for all uses except
subterranean termite and fire ant control,
treatment of field corn, seeds and bulbs, citrus,
and pineapple. By 1983, EPA had phased out
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0)
many of the remaining registered uses, including
that for pineapples. The U.S. manufacturer
voluntarily discontinued heptachlor production in
1988. The chemical is still of interest because
heptachlor and its major metabolite, heptachlor
epoxide, are stored in fat and persist in the
environment. In fact, heptachlor epoxide is more
toxic and more biologically persistent than
heptachlor.
In a cooperative venture with the Hawaii
Heptachlor Research and Education Foundation
and the National Institute of Environmental
Health Sciences, NHEERL scientists studied the
effects of perinatal-plus-juvenile exposure to
heptachlor on the developing immune systems of
rats. Researchers exposed pregnant rats to
heptachlor from midgestation through post-natal
day 7. Pups were exposed to heptachlor from 8 to
42 days of age. In addition to litter size, pup
growth rate, pup survival, reproductive system
characteristics, reproductive capacity, and several
other indicators of health, several immune system
functional endpoints were evaluated in the pups.
The most significant immune system finding was a
dose-dependent suppression of the antibody
response at 8 weeks and 26 weeks of age (Figure 1).
These findings are consistent with other studies
that point toward a predisposition for the male
When most uses of heptachlor were canceled in 1974, the
pineapple producers in Hawaii were granted an extension until
December 1982 that enabled them to use up existing stocks of
the pesticide. During the same time period, green chop—the
chopped leaves of pineapple plants after the fruit has been
harvested—was promoted as an economical feed for dairy
cattle. In early 1982, high levels of heptachlor epoxide were
found in a routine milk test at a state Health Department
laboratory. This finding eventually resulted in a series of 11
recalls of milk, other dairy products, and meat from dairy cattle.
The University of Hawaii had founded a Pesticide Hazard
Assessment Project (PHAP) in the 1960s. Shortly after the
recalls were begun, the director of PHAP started testing breast
milk that was donated to a milk bank during 1982 through 1984.
In 350 samples, the average concentration of heptachlor
epoxide in human milk fat was 100 ppb. The maximum was
438 ppb. By comparison, the federal standard for heptachlor
epoxide in drinking water is 0.2 ppb.
Two class action lawsuits were filed by two local environmental
groups and approximately 100 mothers and children. In 1986,
the Hawaii Heptachlor Research and Education Foundation,
whose purpose is medical monitoring, scientific research, and
education regarding the potential health effects of heptachlor
exposure, was founded as part of the settlement of these
lawsuits.
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Corn oil 30^igH/kg/d 300 ^ig H/kg/d 3000^igH/kg/d
FIGURE 1. The immunosupressive effect of perinatal-plus-juvenile exposure to heptachlor (H).
Sheep red blood cells were used as an antigen to induce a specific, measurable, antibody
response. The production of antibodies after contact with an antigen requires the coordinated
function of several different immune cells. The lowest dose was selected to produce heptachlor
epoxide levels in the rats' milk comparable to levels that had been found previously in human
milk, (a) Primary IgM antibody response at 8 weeks of age. (b) Secondary IgG antibody
response at 26 weeks of age.
immune system to be suppressed by certain
organochlorine chemicals. However, these studies
are preliminary in nature, and it is too early to
draw any conclusions regarding potential
implications for humans exposed to
organochlorine pesticides. Future research will
determine if exposure to heptachlor influences
susceptibility to infectious disease in rats.
Additional studies will define the critical time
periods for exposure and the cellular and
molecular mechanisms by which heptachlor
influences immune system development.
TRIAZINE HERBICIDES
Atrazine is a widely used triazine herbicide. An
estimated 68 to 72 million pounds of atrazine were
used in the United States in 1995, primarily to
control annual grasses and broadleaf weeds in the
cultivation of food crops and conifers. In the
heavily farmed Midwest, many drinking water
sources, including groundwater, contain triazine
herbicides. Because atrazine has induced
mammary tumors in female laboratory rats and is
so widely used, concerns about potential adverse
health effects in humans have arisen.
Many compounds that cause cancer damage the
DNA of cells; that is, they are genotoxic.
Previous in vitro and in vivo studies of atrazine's
potential for genotoxic activity produced
conflicting results. In an attempt to determine if
three triazine herbicides— atrazine, cyanazine,
and simazine — are genotoxic, NHEERL scientists
conducted a series of three experiments. Only
one study found small to negligible amounts of
DNA damage in the white blood cells of mice
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TOXICITY MODELS
An important component of EPA's mandate to
protect public health and the environment is to
assess the potential risks that new or existing
pollutants may pose to humans and ecosystems. A
number of groups within EPA use structure-
activity relationship (SAR) concepts to establish
toxicity testing requirements and to support
regulatory actions. A central assumption of SAR
methods is that structurally similar chemicals
likely act through a common mechanism of
action.
To date, a large amount of SAR research has
focused on developing predictive SAR models for
rodent carcinogenicity. This is due to the
regulatory importance of carcinogenicity in
assessing the risk of environmental chemicals and
the tremendous investment of time and money
required for two-year rodent carcinogenicity
studies. Collaborating with an international SAR
expert from the Institute Superiore in Rome, Italy,
an NHEERL scientist recently coauthored a
review and critique of available SAR and artificial
intelligence models for predicting rodent
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(A
Within a group of
chemicals, if differences
in chemical properties or
structural features can be
related to changes in
biological activity, this
knowledge may be used
to predict the activities of
new chemicals with
similar characteristics.
carcinogenicity. The more complex models
supplemented chemical structure information
with biological information derived from cell and
tissue culture studies and other sources. The
review described the various types of models in
use, their limitations, and their relative success in
recent prediction
"contests." The review also
indicated potential
directions for model
improvement.
In more direct SAR
applications, NHEERL
researchers have modeled
key metabolic steps and
identified molecular
mechanisms for well-
defined chemical classes and biological endpoints.
For example, scientists from NHEERL, CUT
Centers for Health Research, Meijo University in
Japan, and the University of Missouri at St. Louis
collaborated to study a series of organophosphate
pesticides and related compounds capable of
interacting with the male hormone (androgen)
receptor in cells. The project yielded a theoretical
model that linked specific structural features of
the chemicals with differences in androgen
receptor activity. This knowledge can be a useful
component of an SAR strategy to screen similar
types of chemicals for androgenic activity. As
SAR technology develops, it is likely to become a
very important tool when screening chemicals for
endocrine-disrupting activity. (See the chapter,
Endocrine Disrupting Chemicals, for information
on NHEERLs work to develop laboratory-based
screening tests.)
In an SAR project that has potentially much
broader scope and impact, NHEERL investigators
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are developing and promoting a database standard
for public toxicity databases that includes
chemical structures. This standard format will
enable scientists and others to search across and
within these databases by defining specific
structural characteristics in the search parameters.
The goal of this effort is to enlist the toxicology
and modeling research communities in creating a
EPA and ORD are continually working to
improve the efficiency of toxicological
assessment. In this context, NHEERL's SAR
researchers have been:
• evaluating and providing guidance to the
scientific community in the use of SAR
methodologies for toxicity screening;
• using computational and SAR approaches
to study mechanisms of toxicity for
specific classes of environmental
chemicals; and
• spearheading development of community
database standards that improve public
access to and use of existing toxicity
data in SAR application and model
development.
decentralized network of Web-accessible, standard
toxicity databases that can be downloaded and
used in an unrestricted manner by persons in
government, academia, public interest
organizations, and industry. These databases will
span multiple toxicity endpoints (e.g., cancer,
nervous system dysfunction, and immune
suppression) and will be located at widely
distributed sources such as EPA, other federal
agencies, and selected academic sites. This project
has the potential to greatly improve the ability to
explore and model public toxicity information
from a chemical structure perspective.
r
o
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As a participant in the U.S. Global
Change Research Program, EPA's role
is to assess the impact of global
climate change on ecological and human health
and to assess strategies for adapting to climate
changes. During these assessments, climate
change is viewed as one of many stressors that
may interact to cause adverse effects. Several
specific questions are being addressed in EPA's
Global Change Research Strategy.
What are the potential consequences of
climate change and climate variability on
human health, ecosystems, and social well-
being in the United States?
What are the indicators of climate change at
population, community, and ecosystem levels
of organization?
How can one identify future ecological
vulnerabilities on a range of spatial scales
resulting from the joint effects of changes in
climate, sea level, and other stressors such as
pollutants and land use?
How do climate-induced changes like
temperature, moisture, and
atmospheric composition affect the
biology of ecosystems?
How are human and ecosystem
exposures to UVB radiation
changing and what are the effects of
these exposures?
This coral shows evidence of the bleaching
that occurs when symbiotic algae are lost,
which is one consequence of environmental
stress associated with global climate change.
fish, mammals, and amphibians. Recently
completed projects examined the impact of global
climate changes on terrestrial ecosystems.
Specifically, scientists investigated the qualitative
and quantitative effects of elevated carbon
dioxide, temperature, and ozone on tree growth
and on biogeochemical processes in forests. Field
NHEERL scientists have been studying
the effects of global climate change in a
variety of ecosystems across the nation,
including coral reefs, wetlands, and
forests. They have investigated the
impact of climate change on a variety of
plant and animal species including birds,
Carbon dioxide and tropospheric
ozone are two major pollute ~'~
associated with industrialize
and urbanization. Carbon die
is necessary for plant gro
However, at elevated levels, it is
a major greenhouse gas th~*
contributes to global warm.
Elevated levels of ozone in the
troposphere (the atmospheric
layer nearest the ground) have
been associated with damac~ '~
forests and health problerr
humans. NHEERL scientists
studied the separate
combined effects of these
stressors on forest ecosystems in
the Pacific Northwest.
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13
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O
studies were conducted across a transect in the
Pacific Northwest that included coastal and
Cascade Range Douglas fir-hemlock forests and
western juniper forests. Multi-year experimental
studies were conducted in controlled-environment
chambers where climatic factors and chemical and
physical characteristics of the soil were monitored.
Two research efforts combined experimental and
computer modeling methods to determine the
effects of climate change on plant and soil
processes. One study examined the effects of
elevated carbon dioxide and temperature on a
Douglas fir seedling ecosystem. A second study
investigated the effects of
increased carbon dioxide and
ozone on a Ponderosa pine
seedling ecosystem. The studies
measured carbon and nitrogen
inputs, reservoirs, fluxes, and
losses. Data collected in these
experiments were used in the
computer models, which provide a
consistent analytic framework and a
conceptual basis for (1) integrating
diverse measures into an internally
consistent framework, (2) relating
stressors to probable effects, and
(3) making meaningful
extrapolations across scales of time,
space, and biological organization.
H In Douglas fir seedlings, increased
temperature caused several
physiological changes that affected growth.
However, there was no net influence on seedling
biomass because the increases in growth that
occurred in response to some physiologic changes
were offset by the growth-stunting effects of other
physiologic changes. Elevated carbon dioxide
increased water use efficiency but did not alter
plant growth or carbon uptake and distribution
through the test ecosystems. Subsequent modeling
studies confirmed that low soil nitrogen limited
the response of Douglas fir seedlings to elevated
carbon dioxide levels.
Nutrient Cycling and Global Change
Ecosystem stability depends on the regular cycling of nutrients, water,
and energy through different components and levels of the system. As
key indicators of ecosystem function, the cycling of carbon, nitrogen, and
water through living systems can be used to study the impact of
environmental stressors. Previous studies found that elevated levels of
carbon dioxide in the atmosphere stimulated cycling of carbon and
nitrogen but inhibited cycling of water; elevated ozone inhibited cycling
of all three substances.
©
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Stem diameters for
ponderosa pine
seedlings exposed
to elevated levels of
carbon dioxide
and/or ozone.
ACLO = ambient carbon
dioxide, low ozone.
ACEO = ambient carbon
dioxide, elevated ozone.
ECEO = elevated
carbon dioxide,
elevated ozone.
ECLO = elevated carbon
dioxide, low ozone.
o
Apr 1,98 Jul1,98 Oct1,98 Jan 1,99 Apr 1,99 Jul 1,99 Oct1,99 Jan 1", 2000
In Ponderosa pine seedlings, elevated carbon
dioxide caused an increase in growth even though
soil nitrogen was low, suggesting that different
plant species vary in their response to changing
climatic conditions. Elevated ozone levels
decreased both shoot and root growth.
These NHEERL projects support EPA's global
climate change research program by
providing (1) biological data on basic ecosystem
processes, the biogeochemistry of terrestrial
systems, and the effects of climate change factors
on these processes and systems; (2) a
parameterized model (TREGRO) for plant growth
simulations; and (3) a parameterized model
(GEM) for biogeochemical simulations. These
models will be available for public and private
groups to use in future assessments of the effects of
global climate change.
An NHEERL scientist
climbs to great
heights to
monitor the
health of
a forest.
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As part of its mandate to protect the
environment, EPA conducts and
sponsors ecosystems research. The
goal of this research is to provide scientific
leadership and the knowledge necessary to assess,
improve, and restore-at multiple geographic
scales-the integrity and sustainability of various
types of ecosystems. This research program has
four fundamental areas: monitoring, processes and
modeling, risk assessment, and risk management
and restoration.
CONDITION OF THE NATION'S
ESTUARIES
The National Coastal Condition Report, finalized
in November 2001, results from a cooperative
venture among many offices and agencies. The
Office of Wetlands, Oceans, and Watersheds
(EPA Office of Water) and the ecology divisions
of NHEERL were the lead organizations in
producing this report. The report is based largely
on data collected during ongoing federal and state
coastal monitoring programs. Information was
obtained from numerous sources, including EPAs
Environmental Monitoring and Assessment
Program (EMAP), the National Oceanic and
Atmospheric Administration (NOAA), the U.S.
Geological Survey (USGS), the U.S. Fish and
Wildlife Service (FWS), and state and tribal
agencies. Most of the data upon which this report
is based were collected in estuaries, the highly
productive regions where freshwater rivers and
streams meet the ocean. Adequate information
was available to fully assess only the estuaries of
the Northeast, Southeast, and Gulf of Mexico.
Conclusions drawn about West Coast estuaries and
the Great Lakes represent partial assessments based
on available data. The estuaries of Alaska, Hawaii,
and island territories were not evaluated due to lack
of data. The National Coastal Condition report
may be viewed on EPAs Web site at http://
www.epa.gov/owow/oceans/cwap/downloads.html.
Seven primary indicators were used to evaluate
the condition of estuarine waters: water clarity,
dissolved oxygen, eutrophic condition, fish tissue
contaminants, benthic condition, sediment
contamination, and coastal wetlands (Figure 1).
Although additional ecological indicators were
used by some monitoring programs, these seven
were the ones used most widely and consistently.
Based on the monitoring data, each indicator was
assigned a value of good (=5), fair (=3), or poor
(=1) for each coastal region — northeastern,
What is an Estuary?
An estuary is a coastal region where a river or
stream empties into the ocean, mixing fresh
water with salt water. Estuaries are influenced
by tides but are protected from the full force of
ocean currents and storms by reefs, barrier
islands, and/or projections of land, mud, or
sand. Because the rivers and streams carry
nutrients from the land to the sea, and ocean
tides keep the nutrients from settling out,
estuaries are among the most productive
ecosystems on earth. An estuary generates
more organic matter each year than a
comparable area of forest, grassland, or
farmland. Estuaries and the associated
wetlands support a great diversity of living
organisms and are the nurseries of numerous
marine animals, including most of the
commercially important fish and shellfish
species. The protected coastal waters
characteristic of estuaries also serve as ports
and harbors for shipping, commercial fishing
operations, and recreational use.
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southeastern, Gulf of Mexico, West Coast, and
Great Lakes. The seven indicator values were
then averaged to assign an overall rating for a
region. In calculating the national scores, each
region's score was weighted by its area.
Based on available data, the overall condition of
the Nation's coastal waters was fair to poor.
Although about 56 percent of the U.S. estuaries
evaluated were in good condition to support human
use and aquatic life, about 33 percent were
impaired for human use, about 34 percent were
impaired for aquatic life, and about 23 percent were
impaired in their ability to support both human use
and aquatic life.
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Poor Condition
Water clarity is considered poor if less
than 10% of surface light reaches a depth
of 1 meter.
Ranking
Good: Less than 10% of the coastal waters have
poor light penetration.
Fair: 10% to 25% of the coastal waters have poor
light penetration.
Poor: More than 25% of the coastal waters have
poor light penetration.
a
Dissolved oxygen levels are considered
poor when concentrations are less than
2 ppm.
Dissolved
Oxygen
Gooey/ Less than 5% of the coastal waters have
poor dissolved oxygen.
Fair: 5% to 1 5% of the coastal waters have poor
dissolved oxygen.
Poor More than 15% of the coastal waters have
poor dissolved oxygen.
Areas with a greater than 40% decline in
wetland acreage from 1780 to 1980
and/or a greater than 10% decline from
the mid-1970s to the mid-1980s are
considered to be in poor condition.
Good: Less than 25% decline in wetlands' acreage
from 1780 to 1980 and/or less than 5% decline from
the mid-1970s to the mid-1980s.
Fair: Between 25% and 40% decline from 1780 to
1980 and/or between 5% and 10% decline from the
mid-1970s to the mid-1980s.
Poor: Greater than 40% decline from 1780 to 1980
and/or greater than 10% decline from the mid-1970s
to the mid-1980s.
Eutrophic condition is a measure
developed by NOAA that examines six
different eutrophication symptoms and
assigns a value of low, moderate, or high.
High eutrophic condition is equivalent to
poor condition for this indicator.
Good: Less than 10% of the coastal waters have
high eutrophic condition.
Fair: 10% to 20% of the coastal waters have high
eutrophic condition.
Poor: More than 20% of the coastal waters have
high eutrophic condition.
Sediment
Contamination
Sediment contamination is evaluated using
ERM and ERL criteria. ERM is the
concentration of contaminant that will result
in ecological effects 50% of the time. ERL
is the concentration of contaminant that will
result in ecological effects 10% of the time.
An estuary is in poor condition if it exceeds
one ERM criterion or five ERL criteria.
Good: Less than 5% of the coastal waters exceed
one ERM criterion or five ERL criteria.
Fair: 5% to 15% of the coastal waters exceed one
ERM criterion or five ERL criteria.
Poor: More than 15% of the coastal waters exceed
one ERM criterion or five ERL criteria.
A poor benthic index score indicates that
benthic communities are less diverse than
expected, populated by greater than
expected pollution-tolerant species, and
contain fewer than expected pollution-
sensitive species.
Good: Less than 10% of the coastal waters have a
low benthic index score.
Fair: 10% to 20% of the coastal waters have a low
benthic index score.
Poor: More than 20% of the coastal waters have a
low benthic index score.
Fish Tissue
Contaminants
An estuary is in poor condition for fish
tissue contaminants if more than 10% of
fish sampled have tissue residues greater
than FDA and international criteria, or more
than 20% of fish sampled have tissue
residues greater than EPA Guidance
Values.
Good: Less than 2% of the coastal waters have
poor fish tissue condition.
Fair: 2% to 10% of the coastal waters have poor
fish tissue condition.
Poor: More than 10% of the coastal waters have
poor fish tissue condition.
FIGURE 1. Indicators used to assess coastal condition.
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Overall National
Coastal Condition
Overall
Great Lakes
No Indicator
Data Available
Overall
Northeast
Good Fair Poor
Ecological Health
Water Clarity
Overall
West \7
O Dissolved Oxygen
Coastal Wetlands
Overall
Southeast
Eutrophic Condition
Sediment
Benthos
Fish Tissue
FIGURE 2. Overall national coastal condition and coastal condition by region.
Of the seven ecological indicators, only water
clarity and dissolved oxygen levels were rated
good overall. Poor light penetration was a
problem primarily in the western Gulf of Mexico
and western tributaries of the Chesapeake Bay. In
the Southeast, naturally high productivity and
strong sediment transport and resuspension
processes contributed to poor water clarity. The
water clarity indicator does not distinguish
between human-induced and naturally occurring
causes of poor clarity (Figure 2).
Dissolved oxygen is essential to support aquatic
life. Low dissolved oxygen levels are often
associated with large algal blooms. As large
amounts of algae die and sink to the bottom,
oxygen is consumed during the decay process.
Shellfish Growing Waters
Between 1990 and 1995, an increasing number of states
classified estuarine and nonestuarine waters according
to their suitability for growing edible shellfish. In 1995,60%
of the shellfish growing waters were classified as approved.
The most common pollution sources that limited shellfish
harvests were urban runoff, upstream sources, wildlife, and
wastewater treatment systems. At the time of the report,
19 of 21 shellfish growing states were involved in at least
one restoration project that either improved water quality,
restored habitat, or enhanced shellfish stocks.
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.5. ERA.
Fortunately, low dissolved oxygen levels were a
problem only in a few specific areas. Low
dissolved oxygen levels are one potential
consequence of eutrophication, an increase in
nutrient levels. The overall rating for
eutrophication was poor and scientists expect
eutrophic conditions to become worse in
70 percent of U.S. estuaries by 2020.
Based on data from the East Coast and Gulf of
Mexico, the overall rating for fish tissue
contamination was fair. Of the fish sampled,
26 percent had elevated levels of contaminants in
edible tissues. However, 22 percent were
contaminated with organic arsenic compounds
not considered to be toxic to humans. Therefore,
only 4 percent of sampled fish contained
nonarsenical compounds of concern to humans.
Fish sampled in the EMAP program were
examined for signs of disease and external
abnormalities. Bottom-feeding fish had the
highest frequency of disease. The number of fish
with multiple abnormalities was highest in areas
where sediments contained high levels of multiple
contaminants.
Benthic condition (as measured by quantity and
diversity of bottom-dwelling organisms) and
sediment contamination were poor overall. These
two indicators were related: 62 percent of the
estuaries that scored poor on benthic condition
also had contaminated sediments. Benthic
North- South- Gulf of West Great Alaska Hawaii Center- All U.S
east east Mexico Lakes minous U.S.
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communities in poor condition were
characterized by less diversity or
abundance of organisms than expected,
greater than expected pollution-tolerant
species, and/or fewer than expected
pollution-sensitive species. The most
common sediment contaminants were
pesticides, PCBs, and metals. The most
heavily contaminated sediments were in
the Northeast.
The coastal wetlands indicator earned a poor
rating overall. Scientists estimate that nearly
50 percent of the coastal wetlands in the lower
48 states have been lost in the 200-year period
between 1780 and 1980. During the mid- to
late-1990s, coastal wetland losses in the Southeast
and Gulf of Mexico continued at a high rate of
more than 1 percent per year (Figure 3).
State water quality assessments (required under
the Clean Water Act) and state advisories were
examined in preparing the National Coastal
Condition report. Although states used different
FIGURE 4. Percent of
beaches responding to
the survey that closed at
least once in 1998 (EPA).
monitoring techniques and methodologies, these
data provided important information about
coastal condition. In 1998, state water quality
reports suggested that 44 percent of assessed
estuaries in the continental U.S. were impaired by
some type of pollution or habitat degradation.
The most frequent impairments were for aquatic
life support, swimming, and fish consumption.
The major factors causing impairment were
pathogens, oxygen-depleting substances, metals,
and nutrients. The primary sources of impairing
pollutants were municipal point sources, urban
runoff or storm sewers, atmospheric deposition,
industrial discharges, and agriculture (Figure 5).
Of beaches responding
to the survey, the
percent closed in each
state at least once
in 1998:
| | 0-10
PI 11-50
51-100
No Data
Available
Beach Closure
in 1998
o
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Leading Pollutants/Stressors of Estuary Impairment
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Pathogens (Bacteria)
Oxygen-Depleting Substances
Metals
Nutrients
Thermal Modifications
PCBs
Priority Toxic Organic Chemicals
41%
47%
10 20 30 40 50 60
Percent of Impaired Estuarine Square Miles
Leading Sources of Estuary Impairment
Municipal Point Sources
Urban Runoff Storm Sewers
Atmospheric Deposition
Industrial Discharges
Agriculture
Land Disposal of Wastes
Combined Sewer Overflow
30 40 50 60
Percent of Impaired Estuarine Square Miles
FIGURE 5. Leading factors responsible for estuary impairment in 1998 and
sources of those factors.
EPA conducted a voluntary survey of beaches in
1998 (Figure 4). Of the 1,062 coastal beaches
that responded, 33 percent had an advisory or
closing at least once during 1998. Approximately
16 percent experienced at least one closing. The
major causes of beach closure included stormwater
runoff, pipeline breaks, and combined sewer
overflows (due to storm water and sewage being
transported in the same system).
CONDITION OF THE GREAT LAKES
NHEERL ecologists and other scientists are
collaborators in a study to assess the condition of
the Great Lakes. The lead organization is the
Natural Resources Research Institute
(NRRI) of the University of
Minnesota-Duluth, which received a
$6 million grant through EPA's Science
to Achieve Results (STAR) program in
January 2001. Other collaborators
include scientists at the University of
Minnesota-Twin Cities, Minnesota Sea
Grant, University of Wisconsin-Green
Bay, University of Wisconsin-Madison,
Cornell University (New York),
University of Windsor (Ontario,
Canada), John Carroll University
(Ohio), and University of Michigan.
EPA's Great Lakes National Program
Office has a representative on the
steering committee (Figure 6).
Environmental
indicators are
measurable biological,
physical, or chemical
characteristics that
reflect the overall
health of the
ecosystem or the area
being studied.
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The purpose of the study is to determine what
environmental indicators will most efficiently,
economically, and effectively measure the
condition, integrity, and sustainability of the
Great Lakes basin. The Great Lakes basin covers
200,000 square miles and contains approximately
18 percent of the world's surface fresh water.
Because the lakes are interconnected,
environmental changes in one area of the basin
have a ripple effect on other areas. The
environmental health of the basin affects, and is
influenced by the activities of, 36 million
residents. In addition to providing in-depth
information on the condition of the Great Lakes,
Kilometers
100 o 100
FIGURE 6. Landcover map
showing locations of institutions
participating in the Great Lakes
cooperative study as black dots
Pink: agricultural land;
green: forest.
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this study will serve as a model for studying and
monitoring other critical watersheds globally.
Close monitoring enables rapid recognition of
changing conditions, in turn enabling quick
action to correct adverse situations.
Based on previous research, EPA has identified
more than 80 environmental indicators to be
evaluated in the study. New indicators may also
be identified and assessed. The overall project is
organized into five major focus areas. Teams of
scientists from NHEERL and the participating
universities will investigate indicators of
environmental stress and ecosystem responses to
stress in each focus area:
• water quality and diatoms (one type of
microscopic algae),
• fish and macroinvertebrates (aquatic insects,
crustaceans, and worms),
• wetland vegetation,
• birds and amphibians, and
• chemical contaminants.
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Environmental Indicators
Two types of indicators will be evaluated,
those indicative of stressors and those
indicative of the environment's response to
the stressors. Examples of stressors include
land use change, climate change, point and
nonpoint discharges, nonnative species,
atmospheric deposition (e.g., acid rain), and
hydrological modifications. Indicators of
response to stress include changes in land
cover, water quality (contaminants and
nutrients), and biological populations and
communities (amphibians, birds, diatoms,
fish, macroinvertebrates, and aquatic plants).
The project spans four years and includes three
phases: examination of existing data, a pilot study,
and a comprehensive field study. To date, the
cross-organization teams have been sharing GIS-
landscape coverages, existing datasets, and
methods. In the fall of 2001, the research teams
met in Duluth to analyze the summer's pilot study
and establish the sampling frames for the
comprehensive field study. These sampling frames
will be the basis for evaluating the ability of the
different classes of indicators, either alone or in
combination, to detect changes in environmental
condition. While assisting the university
researchers in establishing the most efficient
study design, the initial pilot effort and
future studies will expand and extend
NHEERL's on-going investigations.
These projects include studies
of the coastal wetlands in
Lakes Superior and
Michigan, research on
persistent bioaccumulative
toxicants in the Great Lakes, and development of
fish and macroinvertebrate indicators for
nearshore and coastal zones. At the end of the
project, the investigators will recommend a
portfolio of indicators that is cost-effective and
that accurately reflects the condition of the Great
Lakes. These indicators will be used to further
study and monitor the Great Lakes basin.
In 2001, preliminary assessments were conducted
of Lake Superior, Lake Michigan, and Lake
Ontario. These initial assessments estimated
variability of the proposed indicators, verified
landscape classifications previously determined by
remote sensing technology, and determined
logistic constraints of access to sampling sites.
The data obtained will be used to develop the
sampling design for the more extensive studies to
be conducted in subsequent
years.
photo courtesy of James L. Lasswell,
Dept. of Entomology, Texas Ag. Exp.
Station, Stephenville, Texas
Aquatic insects
such as dragonflies
and other aquatic
invertebrates such as crayfish,
mussels and snails may be
valuable environmental indicators.
Loss of species diversity among
aquatic invertebrates has been linked
to Great Lakes coastal wetland
habitat degradation.
\
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In addition to protecting our environment,
EPA is charged with protecting human
health. This task is fulfilled by conducting
research on priority environmental chemicals,
assessing risks to humans and the environment
associated with environmental chemicals, and
establishing regulations and management actions
based on those risk assessments. A major emphasis
at NHEERL is to introduce new technologies that
expand the set of tools available to evaluate human
health risk following exposure to environmental
chemicals. New technologies developed at
NHEERL have important applications in this area.
Assays using a sperm protein patented by NHEERL
have the potential to identify when environmental
chemicals impair male fertility. Also, a new
Genomics Program will coordinate NHEERL
research on how environmental chemicals affect
the human genome—the body's genetic blueprint.
FIRST EPA BIOTECHNOLOGY PATENT
In 2001, an NHEERL scientist became the first
EPA researcher to obtain a biotechnology patent
on behalf of EPA. The patent is on the sperm
protein SP22, the nucleotide (DNA) and amino
acid sequence of SP22, and all recombinant
fragments thereof. A second patent is pending. It
covers the use of SP22 in fertility diagnostics of
humans and animals and other reproductive
technologies including contraception, artificial
insemination, and in vitro fertilization. NHEERL
will receive any royalties derived from commercial
licensing of the patents. The scientist-inventor is
entitled to one-third of the royalties, up to a
maximum of $ 150,000 per year.
SP22 was discovered during in vivo rodent studies
designed to identify molecular factors associated
with infertility. Male rats were exposed to four
chemicals known to reduce fertility. To improve
the likelihood that the study would detect
molecular influences on fertility, the chemical
doses used were low enough that sperm shape and
motility were either unaffected or only slightly
affected. (Abnormalities of sperm shape and
In this photomicrograph, sperm are attached to the zona pellucida
surrounding the egg. Only one sperm will penetrate the zona
pellucida and fertilize the egg.
motility are also often associated with reduced
fertility.) Mature sperm were collected and
surgically inseminated into female rats. Sperm
from the same collections were examined for
changes in shape, motility, and the type and
amount of specific proteins present on the sperm
membrane. Male fertility was measured as the
number of embryos implanted in the uterus
divided by the number of eggs ovulated
(determined by counting corpora Jutea of
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pregnancy on the ovaries) expressed as a
percentage. All four test chemicals used in the
study caused a decrease in fertility. Importantly,
levels of one sperm protein were also diminished
and were highly correlated with fertility
(Figure 1). Because this sperm protein was
22 kilodaltons in size, it was named SP22. Further
analysis of the data showed that the amount of
SP22 in sperm could be used to predict male
fertility.
In other NHEERL studies, antibodies to SP22
have identified the location of SP22 on sperm
from the rat, hamster, rabbit, bull, and human.
In addition, these antibodies have inhibited
fertilization of hamster eggs in vitro (Figure 2).
Similar results have been obtained with human
eggs and with rats (in vitro and in vivo studies).
Collectively, the data indicate that SP22 plays a
critical role in the initial interaction of the sperm
100
80
60
t
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40
20
-20
5,000
10,000 15,000
SP22 (I.O.D.)
20,000
FIGURE 1. The relationship between fertility and SP22 levels. SP22 is measured in integrated
optical density units (I.O.D.).
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In 2001, NHEERL established a Genomics
Steering Committee, which will initially propose
an in-house program for coordinating genomics
and proteomics research. An example of
interdivisional cooperation, the committee
consists of one representative from each health
division and two ad hoc
members. NHEERL
researchers have already
conducted a number of
individual projects applying
genomics and proteomics
knowledge to environmental
health risk assessment. The
new Genomics Program will
coordinate NHEERL research
efforts in these areas and will
formalize NHEERLs role in
this important field. In
addition to research on gene
expression patterns and the
resulting protein products,
the NHEERL Genomics
Program will also include
studies of how environmental
chemicals interact with genes
and influence gene
expression. This is consistent
with NHEERLs Strategic
Plan and EPAs role of
assessing the risk that
environmental pollutants
pose to human health.
Also in 2001, NHEERL joined the North
Carolina Biotechnology Center Consortium for
Genomics and Bioinformatics. This affiliation
will facilitate collaborative research between
NHEERL scientists and researchers at other
Consortium institutions.
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Example Genomics Projects at NHEERL
Use gene array data in rats to identify and examine suspected mechanisms of toxicity
for inhaled environmental pollutants, with the goal of distinguishing carcinogenic from
noncarcinogenic air toxicants.
Identify molecular alterations in cells lining the urinary bladder of rats after exposure
to disinfection by-products in drinking water. The urinary bladder is one of the principal
sites of cancer in humans exposed to drinking water disinfection by-products.
Describe the effect of two dietary antimutagens (substances that prevent genetic
mutation), vanillin and cinnamaldehyde, on gene expression in Salmonella, E. coll, and
cultured human liver cells.
Examine the effects of dietary folate deficiency on arsenic-induced genotoxicity in
mice. This work includes analyses of altered gene expression resulting from folate
deficiency, from arsenic exposure, and from the combination of folate deficiency and
arsenic exposure.
Identify genetic or molecular changes (biomarkers) that occur in genes in response to
environmental chemicals. The purpose of this research is to eventually assess potential
sensitivity of children to adverse outcomes following exposure to environmental
chemicals.
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EPA defines an endocrine-disrupting
chemical (EDC) as an exogenous
chemical substance or mixture that
alters the structure or function (s) of the endocrine
system and causes adverse effects at the level of
the organism, its progeny, populations, or
subpopulations. The Food Quality Protection Act
and Amendments to the Safe Drinking Water Act
of 1996 reflected growing concern about the
presence of potential endocrine-disrupting
chemicals in food, water, and the environment.
Passage of these laws required EPA to develop a
screening program to determine whether
individual substances may cause endocrine
disruption in humans.
EPA established the Endocrine Disrupter
Screening and Testing Advisory Committee
(EDSTAC) and charged the committee to provide
recommendations for a screening and testing
program. Based on these recommendations, EPA
created the Endocrine Disrupter Screening
Program, which focuses on providing methods and
procedures to detect and characterize
endocrine-disrupting activity
in pesticides, commercial
chemicals, and
environmental
contaminants. The use
of these standardized
protocols will help EPA
and industry efficiently
gather information
regarding endocrine-disrupting activity of the
estimated 87,000 chemicals in commercial use
and the many thousands of chemicals under
development. Based on this information, EPA
will be able to take appropriate action. NHEERL
scientists are playing a major role in the
development of these testing protocols.
PUBERTAL RAT PROTOCOLS
Puberty, the onset of sexual maturity, is a
developmental stage characterized by extensive
interactions among many hormones, organs, and
tissues. This time period is also distinguished by
increased sensitivity to environmental chemicals.
NHEERL scientists, in collaboration with program
representatives from EPAs Office of Prevention,
Pesticides and Toxic Substances (OPPTS), are
developing testing protocols to detect potential
effects of EDCs on puberty in the male and female
rat. These protocols are designed to detect when
a chemical exposure causes structural
malformation of sexual organs,
abnormal reproductive
function such as
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0 6.25 12.5 25 50 100 150 200
Atrazine (mg/kg)
0 12.5 25 50 100 200
Atrazine (mg/kg)
Figure 1. Effect of atrazine on puberty onset in (a) male and (b) female rats. Age is measured in
days. Puberty onset is determined by the age of preputial separation in male rats and the age of
vaginal opening in female rats. * indicates a statistically significant difference compared to controls
delayed onset of puberty, or abnormal function of
other endocrine organs such as the thyroid and
hypothalamus. (The hypothalamus is an area of
the brain that influences all
hormonal activity in the body.)
k NHEERL scientists published
background papers
describing the attributes
and weaknesses of the
male and female pubertal
rat protocols in 2000.
These papers raised
several issues that are
important to the
successful implementation
of these two protocols for
EDC screening.
Research activities at NHEERL in 2001 addressed
these issues. As part of the standardization and
validation process, NHEERL scientists assisted
OPPTS in coordinating the evaluation of these
protocols by an independent contractor. This step
is important because contractor laboratories will
likely be the major sites conducting the tests after
the protocols are finalized and implemented. One
key element in this process is the use of test
chemicals whose identity and endocrine-
disrupting activity are unknown to the contractor.
To date, compounds representing various classes of
endocrine-disrupting chemicals (e.g., estrogens,
anti-androgens, thyroid toxicants) have been
readily detected by the contractor using the
pubertal rat protocols. Future studies will use
expanded dose ranges to investigate the sensitivity
of these protocols by determining the lowest dose
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that causes an effect. The results will indicate
whether or not the pubertal rat protocols can
detect weak EDCs as well as potent ones.
NHEERL scientists used the male and female
pubertal rat protocols to determine if they could
identify chemicals with a central nervous system
mode of action. The protocols, conducted using
the chlorotriazine herbicide atrazine, showed that
puberty was delayed in both sexes (Figure 1). This
work showed, for the first time, that atrazine alters
male reproductive function during
development. It also showed that these
protocols are able to detect adverse effects
of atrazine in the female following a much
shorter duration of exposure than
previously reported. (See the Pesticides
chapter for more information on atrazine.)
laboratories. NHEERL scientists provided
information on optimum protocol procedures to
the laboratories, analyzed the data, and wrote a
report for OECD.
An in utero-lactational assay is being developed to
identify chemicals that affect the developing fetus
and the nursing newborn. NHEERL is developing
the protocols, selecting chemicals, and analyzing
results of tests that are conducted by a contractor.
Atrazine Risk Assessment
IN VIVO ASSAYS
The Hershberger assay is a 10-day test to
identify substances that act like male
hormones (androgens) and substances that
interfere with male hormones
(antiandrogens). This assay is being
developed as a cooperative venture
between EPA and the Organisation for
Economic Co-operation and Development
(OECD), a European organization, with
NHEERL as the lead laboratory. In 2001,
the first phase of an interlaboratory
standardization and validation study was
conducted with 17 participating
The human health risk assessment for a substance is a
determination of the danger it poses to humans. It is based
on health problems in humans and/or laboratory animals that
have been associated with exposure to the substance. Three
types of health conditions that carry great weight in the risk
assessment process are cancer, reproductive problems, and
birth defects. During risk assessment, different guidelines
are followed for agents that cause different types of health
problems. For agents that cause cancer, the mechanism by
which the agent causes cancer is emphasized during the risk
assessment process, particularly when extrapolating findings
in laboratory animals to humans.
Over the years, laboratory studies conducted in different
strains of rats produced conflicting results regarding atrazine's
ability to cause cancer. Atrazine caused cancer in only one
strain of laboratory rats, but not in others. Recent studies at
NHEERL confirmed that atrazine does not cause cancer by
damaging the DNA of cells, which is one mechanism by which
some chemicals cause cancer in humans. These results
suggest a hormonal mechanism of cancer production unique
to one strain of rats. Therefore, the risk assessment guidelines
for substances that cause cancer do not apply to atrazine.
However, two NHEERL research efforts indicate that risk
assessment guidelines for chemicals that cause reproductive
problems and/or birth defects may be more appropriate for
atrazine: (1) pubertal rat studies demonstrated atrazine's
endocrine-disrupting effects on sexual maturation and (2)
recent studies revealed adverse effects on prostate gland
development following neonatal exposure.
Risk assessment is a dynamic, not static, process. As the
studies on atrazine demonstrate, the risk assessment for a
chemical is subject to change as new information emerges.
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IN VITRO ASSAYS
In addition to assays in live rodents, NHEERL is
developing a variety of screening tests using
hormone receptors in cell and tissue cultures.
Substances that are similar in size and shape to a
natural hormone may bind to hormone receptors.
If this happens, the chemical may mimic the
hormone by stimulating the same response in the
cell as the hormone or it may inhibit hormone
activity by preventing the natural hormone from
binding to the receptor. Thus, identifying
chemicals that bind to hormone receptors is an
efficient way to distinguish between chemicals
that should be tested further (those that interact
with hormone receptors) and chemicals that are
unlikely to have endocrine-disrupting activity.
The in vitro assays under development at
NHEERL involve
• androgen receptors,
• estrogen receptors,
• genes regulated by androgens, and
• synthesis of steroid hormones.
Two androgen-receptor binding assays,
one that is cell-free and the other that
uses whole cells, have been developed
and are in use. Another whole-cell
screening assay uses receptors tagged
with fluorescent antibodies. Because
androgen receptors move to the DNA in the
cell nucleus after they have been activated, the
distribution of fluorescence in the cells indicates
whether or not the test substance bound to the
androgen receptor (see Figure 3).
How Steroid Hormones Work
Steroid hormones travel in the blood from the
tissue in which they are synthesized to specific
organs and tissues where they exert their
influence. Once inside a cell in the target organ
or tissue, a steroid hormone binds to a specific
receptor molecule. The receptor is activated
and relocates to the nucleus near the DNA. The
activated receptor then binds to a specific
promoter sequence in the DNA of the target
gene, which is either activated or inactivated.
The result of gene activation is the production
of specific proteins, which may be enzymes,
structural proteins, growth factors, or other
active substances, including other hormones.
An endocrine-disrupting chemical that binds to
a steroid hormone receptor may mimic the
natural hormone or it may prevent the natural
hormone from binding. In either situation,
normal hormone-cell interaction is disrupted.
Figure 2.
Simplified
schematic diagram
showing the DMA-
containing nucleus in a
cell. Compare to Figure 3.
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Using a novel approach, one NHEERL researcher
developed an assay that uses a virus to insert
androgen receptors into the cultured cells. This
process is efficient and the receptors remain very
sensitive to androgens. However, inserting
receptors into cells every time an assay is prepared
creates quality control issues due to variability
from one test to the next. In contrast to these
assays, another NHEERL scientist developed a
cell line that retains the receptors of interest
through cell replication cycles. These androgen-
responsive cell lines have been distributed to
other laboratories for validation studies. NHEERL
researchers are in the early stages of developing
analogous estrogen-responsive cell lines. A
different type of assay is being developed to detect
inhibition of steroid hormone synthesis in tissue
cultures. <»
Figure 3. In (a) and (c), androgen receptors exposed to a positive control and a positive test
substance migrated into the nucleus and attached to DMA, forming dense areas of fluorescence. In (b)
and (d), the receptors remained scattered about outside the nucleus, forming a more diffuse pattern of
fluorescence after being exposed to media alone and a negative control. These patterns help to
distinguish between EDCs and non-EDCs.
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Coming Soon...
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A report on the contamination levels of mercury in selected
finfish and shellfish in the estuaries of the United States.
A summary and comparison of multiple toxicities following
developmental exposure to pesticides: neurotoxicity,
immunotoxicity, and reproductive toxicity.
A report on waterborne disease outbreaks in the United
States in 1999-2000; this report will provide information
on causative agents, health effects, water quality, and
treatment issues.
A searchable database platform to enable researchers and
risk assessors to explore structure-activity associations
across toxicity endpoints of regulatory interest.
A report describing the significance for cancer risk
assessment of a newly discovered mode of action of
polycyclic aromatic hydrocarbons.
The development and application of in vivo immuno-
histochemical methods to verify that mechanisms of toxicity
observed during in vitro studies can be extrapolated to the
whole organism, enhancing the use of in vitro data in risk
assessment.
altered nutrient ratios on the occurrence of hazardous algal
blooms.
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www.epa.gov/nheerl
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