Research and Development
National Health and
Environmental Effects Research laboratory
Science Reuort
1, l&iillS J
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CONTENTS
Click on title to link directly to article.
RESEARCH
Page
Most Midwestern Amphibians Are at Low Risk for UV Effects 3
Fecal Bacterium Causes Coral White Pox Disease 4
Pfiesteria shumwavae Kills Fish by Predation, Not Poisoning 5
Drinking Water Disinfection Bv-Product Causes Cancer in Rats 6
Metabolism of Water Disinfection Bv-Product Studied in Rat and Human Liver 7
Drinking Water Disinfection Bv-Product Decreases Fertility in Male Rats 8
Work Continues on Endocrine Disruptor Screening Program 9
Ozone Sensitivity of Bean Plants Varies Throughout the Development Cycle. 11
Laboratory Sediment Toxicitv Tests Validated in the Field 11
TECHNICAL ASSISTANCE
Japanese Scientists Visit Three NHEERL Ecology Divisions 13
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RESEARCH
Most Midwestern Amphibians Are at Low Risk for UV Effects
Exposure of amphibians to ultraviolet radiation (UVR) has been suggested as a
cause of declines in populations and increases in the presence of hindlimb
malformations. Dr. Gerald Ankley, Dr. Stephen Diamond, Joseph Tietge, Gregory
Peterson, Gary Holcombe, Kathleen Jensen, David DeFoe, and Ryan Patterson of the
Mid-Continent Ecology Division of the National Health and Environmental Effects
Research Laboratory (NHEERL), with colleagues from the University of Minnesota's
Natural Resource Research Institute, compared levels of UVR exposure in 26
midwestern wetlands with levels that caused adverse effects in controlled outdoor
experiments on three midwestern amphibian species: northern leopard frogs (Rana
pipiens), green frogs (R. clamitans), and mink frogs (R. septentrionalis). Assuming that
the wetlands in the study accurately represent all northern midwestern wetlands, the
results suggest that most amphibians in the region currently are at low risk for UVR
effects.
This research is the first to use a risk assessment approach to address the
question of whether UVR harms amphibians in nature. The results contribute to
understanding the role of changing UVR levels in wetlands and provide perspective on
other hypotheses suggested to explain reductions in amphibian numbers, such as
pesticide exposure, disease, parasitism, and natural causes.
Three consecutive papers were published in the July 1, 2002, issue of
Environmental Science and Technology (ES&T) under the umbrella title of "Assessment
of the Risk of Solar Ultraviolet Radiation to Amphibians." In "Part I. Dose-Dependent
Induction of Hindlimb Malformations in the Northern Leopard Frog (Rana pipiens)," Dr.
Ankley and co-authors present UVR threshold levels derived from controlled outdoor
experiments. In "Part II. In Situ Characterization of Solar Ultraviolet Radiation in
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Amphibian Habitats," Gregory Peterson and colleagues survey factors that control the
UVR dose in 26 northern Minnesota and Wisconsin wetlands. In "Part III. Prediction of
Impacts in Selected Northern Midwestern Wetlands," Dr. Diamond's group compares
the experimental UVR effects levels with estimates of UVR exposure for the 26
wetlands.
The authors conclude that currently there is a slight risk of amphibian mortality,
and a somewhat higher risk of malformation, in 2 of the 26 wetlands studied. Risk of
malformation was also apparent in one additional wetland. However, factors such as
climate change, animal behavior, and variation among wetlands can alter exposure
levels, suggesting that UVR should not be ignored in future consideration of possible
effects on amphibians and other aquatic organisms. [ES&T, 2002, 36:2853-2858;
36:2859-2865; 36:2866-2874].
Fecal Bacterium Causes Coral White Pox Disease
Massive outbreaks of coral diseases over the last several decades may be
causing the substantial declines in the biodiversity and abundance of reef-building
corals. The greatest losses on Caribbean and South Florida reefs have been among
the branching elkhorn and staghorn corals, Acropora palmata and Acropora cervicornis.
A paper recently published in the Proceedings of the National Academy of Science
(PNAS) details the destruction of elkhorn coral populations, with losses in the Florida
Keys typically in excess of 70%. Serratia marcescens, a common intestinal bacterium,
was identified as the causal agent of white pox. Kathryn Patterson, a University of
Georgia, Athens, doctoral student in marine sciences, was the principal investigator.
She conducted the research at the Gulf Ecology Division under the mentorship of
co-author Dr. Deborah Santavy.
Serratia marcescens is a ubiquitous bacterium found in human feces, but it is
more commonly associated with hospital infections in immune-compromised patients.
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This species is also found as intestinal microbiota of other animals and as a free-living
microbe in water and soil. Serratia species are known to cause disease in both marine
and freshwater fishes and to pose a serious threat as an opportunistic pathogen to other
marine organisms. Sewage may be the source of S. marcescens contamination. The
S. marcescens strain (PDL100) isolated from white pox-affected elkhorn coral may also
be associated with pollution of fecal origin. At present, however, the origin, pathogenic
mechanisms, and host range of the PDL100 strain are unknown but under investigation.
EPA, the State of Florida, and the National Oceanic and Atmospheric
Administration have developed a Water Quality Protection Program (WQPP) for the
Florida Keys National Marine Sanctuary. Findings from this research will help
implement the WQPP and provide a rationale for improved treatment of wastewater and
stormwater in the Florida Keys. The study builds on the knowledge that corals are
highly sensitive to all forms of pollution. A new no-discharge zone designation now in
effect for all state waters within the Sanctuary will help curtail the dumping of harmful
waste by boats and ships, a key priority of the Sanctuary's Water Quality Protection
Program. [PNAS, 2002, 99:8725-8730].
Pfiesteria shumwayae Kills Fish by Predation, Not Poisoning
Pfiesteria are dinoflagellates, a type of microscopic algae implicated in certain
fish kills. Scientists at the Virginia Institute of Marine Sciences of the College of William
and Mary showed that Pfiesteria shumwayae kills fish by feeding directly on their skin,
not by releasing a potent toxin into the water. EPA is a contributor to the multi-agency
program that supported this research. Dr. Calvin Walker of the Gulf Ecology Division is
a co-author who worked on research design issues related to toxins. Earlier
researchers attributed several massive fish kills in mid-Atlantic estuaries and some
human health effects to toxins released in the water by Pfiesteria piscicida, a very
similar dinoflagellate, but the specific toxins were not identified.
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Three different approaches showed how Pfiesteria shumwayae kills fish.
(1) Using a P. shumwayae culture known to kill fish, Pfiesteria dinoflagellates were
removed from the culture by filtration and centrifugation, presumably leaving behind any
secreted toxins in the aqueous layer. When larval fish were added to the aqueous
layer, none was killed. (2) Fish kills were observed only when the larval fish were put
into direct physical contact with Pfiesteria dinoflagellates in water. (3) Using
videomicrography, high-resolution microscopy, and electron microscopy, large numbers
of Pfiesteria dinoflagellates were seen swarming toward the larval fish, attaching to
them, and feeding directly on their skin-in effect, skinning the fish alive. Thus,
Pfiesteria shumwayae is shown to kill fish by direct predation, not by release of toxins.
Much more research is needed before the effects of Pfiesteria on human health can be
explained in similar detail. [Nature, 2002, Aug. 5 issue on-line; 418:967-970].
Drinking Water Disinfection By-Product Causes Cancer in Rats
A lifetime exposure study in rodents, described in a recent paper by Michael
George, Donald Doerfler, Tanya Moore, Stephen Kilburn, and Dr. Anthony DeAngelo of
the Environmental Carcinogenisis Division (ECD) and colleagues at Pathology
Associates, Inc., found that bromodicholoromethane (BDCM), a major by-product of
chlorine disinfection of drinking water, is carcinogenic in the male F344/N rat.
A previous study by the National Toxicology Program found that BDCM
administered orally in corn oil increased kidney and colon cancer in male F344/N rats
and kidney cancer in male B6C3F., mice. These NTP findings are significant because
epidemiology studies point to an increased risk of colorectal cancer in humans drinking
chlorinated surface waters, and kidney cancer is rare in male mice.
When ECD administered BDCM in drinking water to male F344/N rats and male
B6C3F., mice, there was no increase in the incidence of large cell adenocarcinoma in
the colon of the rat or kidney cancer in the mouse, contrary to the NTP results using
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corn oil. However, because of the increased prevalence and multiplicity of liver
neoplasms, BDCM administered in drinking water was judged to be carcinogenic in the
male F344/N rat. Because the dose-response curve was complicated by a bimodal
shape that can be only partially explained by an inhibition of BDCM metabolism in the
liver, additional work is needed to understand the underlying cause more fully.
These results can be used in the further development of a risk assessment for
BDCM, as required by the Safe Drinking Water Act. (See also the following article on
BDCM metabolism.) [International Journal of Toxicology, 2002, 21:219-230].
Metabolism of Water Disinfection By-Product Studied in Rat and Human Liver
Bromodichloromethane (BDCM), an important by-product of the drinking water
disinfection process, has toxic and carcinogenic effects in rodents at concentrations
much higher than those found in drinking water supplies. The Experimental Toxicology
Division (ETD) has developed a physiologically-based pharmacokinetic (PBPK) model
for BDCM in rats, which potentially can be extended to a biologically-based dose-
response model to extrapolate effects from rats to humans. A PBPK model predicts the
absorption of a chemical and its distribution to organs in a whole animal. To provide
essential information for developing the extrapolation model, Dr. John Allis, recently
retired from ETD, and Dr. Guangyo Zhao, an ETD post-doctoral fellow in the Curriculum
in Toxicology at the University of North Carolina at Chapel Hill, recently published two
papers that identified and quantitatively measured the important pathways for
metabolism of BDCM in the liver of both rats and humans.
Previous work in ETD revealed that several liver cytochrome P450(CYP)
enzymes, especially CYP2E1, are responsible for the vast majority of BDCM
metabolism in the rat. The scientists measured the kinetics of BDCM metabolism for
each CYP enzyme involved with metabolism of drugs and other foreign compounds in
rat and human liver. Metabolic activities of the important CYP enzymes were measured
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one at a time by two in vitro methods. The first used preparations made by recombinant
DMA techniques that produced a single CYP enzyme so that its activity could be
measured without interference from others. Both rat and human CYP enzymes were
tested. Because, unlike in rats, there is no information on BDCM metabolism in
humans, a second approach was used for the human CYP enzymes. In this case,
microsomes isolated from human livers were tested, with the activity of one CYP
enzyme measured while blocking the activity of the others with inhibitory antibodies.
Microsomes give a closer approximation to the whole animal than do the recombinant
enzymes.
Recombinant enzyme measurements confirmed that the three CYP enzymes
identified in the rat were indeed the most important ones. In both rats and humans,
CYP2E1 was dominant at low BDCM concentrations, but two other enzymes were
important at moderate BDCM concentrations. The measurements in microsomes
confirmed these conclusions. Although CYP2E1 is the most important BDCM-
metabolizing enzyme in both rats and humans, there are important differences between
the species for the other enzymes. These results are essential to the development of a
model to extrapolate effects from rats to humans. (See also the previous article on
BDCM carcinogenesis.) [Chemico-Biological Interactions, 2002, 140:137-153;
140:155-168].
Drinking Water Disinfection By-product Decreases Fertility in Male Rats
Bromochloroacetic acid (BCA) is one of the more prevalent disinfection
by-products (DBPs) formed when disinfectants are used to treat drinking water. In a
recent publication from the Reproductive Toxicology Division, Dr. Gary Klinefelter, Lillian
Strader, Juan Suarez, and Naomi Roberts described the effects of BCA on reproduction
in male rats. BCA adversely affected both the formation of normal sperm and the
fertility of treated male rats, as well as altered the level of SP22, a sperm membrane
protein identified earlier in Klinefelter's laboratory.
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Adult male rats were treated with BCA orally for 14 days, using a range of doses
from 8 to 240 mg/kg/day. Various sperm parameters and the level of fertility of the
treated rats were then measured. Fertility of the treated animals was also compared
with the level of SP22, a protein found on the surface of sperm. Both the fertility of
sperm and their SP22 level were decreased in all dose groups, setting the Lowest
Observed Adverse Effect Level at 8 mg/kg. Importantly, the decrease in SP22 was
correlated with the decrease in fertility.
Further work is underway to establish a No Observed Adverse Effect Level for
the effects of BCA on sperm. Current efforts are also focused on the effects of mixtures
of several of the known water DBFs such as BCA, dibromoacetic acid, and
dibromochloroacetic acid. Finally, an epidemiology study, in collaboration with the
University of North Carolina at Chapel Hill, is assessing the effects of water containing
various levels of DBFs on the quality of sperm from men drinking the water. SP22
levels and other parameters will also be evaluated. [Toxicological Sciences, 2002,
68:164-173].
Work Continues on Endocrine Disrupter Screening Program
In response to a mandate by the Food Quality Protection Act and to growing
concerns that environmental chemicals may adversely affect human health by altering
the endocrine system, EPA launched an endocrine disrupter screening program
(EDSP). Three NHEERL Divisions-Gulf Ecology (GED), Mid-Continent Ecology (MED),
and Reproductive Toxicology (RTD)-continue work on the Tier 1 and Tier 2 assays that
are likely to be included in the final testing battery. Tier 1 involves screening tests to
select endocrine-active chemicals for further study; Tier 2 involves detailed tests to
determine the mode of action of the chemicals selected in Tier I.
Dr. William Benson, Director of GED, is Acting Chair of the Endocrine Disrupter
Methods Validation Subcommittee (EDMVS), which provides guidance to the Agency on
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validation of EDSP screening methods and includes experts from government, industry,
and stakeholder organizations. This subcommittee also advises the Agency
on reducing animal use, modifying procedures to make them less stressful to test
animals, and replacing animals where scientifically appropriate. GED is the Agency
lead for development of a proposed Tier 2 assay. Dr. Chuck McKenney is leading a
team developing a standard multi-generation test using mysids, which are small, marine
invertebrates. In addition, Dr. Michael Hemmer and Larry Goodman of GED are
working with colleagues from MED on development of a fish life-cycle test under
consideration for Tier 2.
Dr. Gerald Ankley of MED is a member of the ecotoxicology Validation
Managment Group of the Organization for Economic Cooperation and Development,
which is charged with developing and documenting internationally harmonized EDSP
screening and testing methods for invertebrates, fish, amphibians, and birds. MED is
also the Agency lead for developing two of the proposed Tier 1 assays: a short-term
reproduction test with the fathead minnow (Pimephales promelas), and metamorphosis
assay with the African clawed frog (Xenopus laevis). This work is coordinated by Dr.
Ankley, Dr. Sig Degitz, and Joe Tietge. In addition, MED is involved in developing full
and partial life-cycle tests for Tier 2 assays using fish and frogs. Led by Dr. Patricia
Schmieder, MED has also created models for prioritizing chemicals to be screened and
tested.
Dr. Robert Kavlock, Director of RTD, is a member of EDMVS. Acting as
consultants on various key EDSP activities, several other RTD scientists have
presented their research to the EDMVS on a number of protocols, including the male
and female puberty assays by Dr. Ralph Cooper; the Hershberger assay and the in
utero and lactational assay by Dr. Earl Gray; estrogen receptor binding assays and
aromatase activity by Dr. Susan Laws; restricted feeding and puberty studies by Dr.
Tammy Stoker; steroid formation by Dr. Jerome Goldman; and androgen receptor
binding by Dr. Vicky Wilson.
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Currently, EPA anticipates that issuance of final guidelines for priority setting and
validation of the Tier 1 screen will be completed by 2003. Placement of orders to begin
screening of chemicals having high production volume is expected by 2003, and
completion of Tier II validation by 2005.
Ozone Sensitivity of Bean Plants Varies Throughout the Development Cycle
Five Western Ecology Division scientists (Drs. David Tingey, E.H. Lee, Wiliam
Hogsett, Jilian Gregg, and intern Justin Rodecap) recently published an article showing
how ozone sensitivity of the bush bean plant changes as the plant develops. Growing a
popular variety of bush beans (Phaseolus vulgaris) in open-topped field chambers, the
researchers used four different treatments on the plants. They found that ozone
exposures during the leaf production stage caused little injury to foliage and had only a
small impact on growth and bean yield. Ozone exposures during pod development,
however, significantly increased foliar injury and decreased both growth and yield.
These results suggest that the greater sensitivity of the bean plants to ozone during
their pod-filling and maturation stages is the consequence of reduced energy levels
available for the normal processes of repair. Therefore ozone standards set to protect
vegetation must consider that ozone sensitivity changes with the stages of plant
development. [Water, Air, and Soil Pollution, 2002, 39:325-341].
Laboratory Sediment Toxicity Tests Validated in the Field
Amphipods are small, shrimp-like, sediment-dwelling, aquatic animals that are
commonly used to test sediment toxicity because of their high sensitivity to many
chemicals. Laboratory sediment-amphipod toxicity tests are used extensively to
determine the toxicity and bioavailability of chemical contaminants, the extent and
severity of pollution impacts, and for setting sediment-quality guidelines. Some critics,
however, question their ecological relevance, claiming that responses in a simple,
controlled laboratory test cannot be extrapolated to responses in the field. Dr. Steven
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Ferraro and Faith Cole of the Western Ecology Division conducted a field validation
study of two sediment-amphipod toxicity tests at a Superfund site in Seattle's Elliott Bay,
which is contaminated with polycyclic aromatic hydrocarbons (PAHs). They showed
that the predictive ability of their toxicity tests was linked to ecologically important field
effects, and that, under the proper conditions, the tests can be used for laboratory-to-
field extrapolation.
The field validations were conducted by testing relationships between the survival
of amphipods in each of two toxicity tests; each of seven observed field effects,
including changes in the number of species, abundance, and diversity of sediment-
dwelling animals; and total PAHs in the sediments at 30 field stations. Potential
confounding environmental variables were identified and accounted for. The findings
suggest that when their predictive ability is sufficient, sediment toxicity tests are likely to
be more cost-efficient than observational field studies for determining the magnitude
and spatial extent of chemical contaminant effects in the field. [Environmental
Toxicology and Chemistry, 21 (7): 1423-1437 (2002)].
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TECHNICAL ASSISTANCE
Japanese Scientists Visit Three NHEERL Ecology Divisions
Japan is considering extending its chemical management program for the
protection of human health to include environmental protection as well. Three
Japanese scientists representing the Ministry of the Environment visited several
Ecology Divisions between July 29 and August 9, 2002, to meet with NHEERL scientists
experienced in the development of toxicity bioassays and applications to ecological risk
assessment. These meetings were an important opportunity for international
technology transfer of methods and models developed at NHEERL to protect terrestrial,
freshwater, and marine ecosystems.
At three Ecology Divisions-Atlantic (AED), Mid-Continent (MED), and Western
(WED)-the visitors and NHEERL scientists covered a wide range of topics, including
development and application of bioassays, species currently used in bioassays,
institutions that conduct bioassays, test organism suppliers, results of chemicals tested,
bioassays in ecological risk assessment, sediment toxicity identification and evaluation
(TIE) techniques, future challenges for toxicity testing, and applications to ecological risk
assessment.
At AED, a series of informal presentations were given on the development of
water quality criteria, equilibrium sediment guidelines, and population models to
extrapolate from effects on individuals to effects on populations. The Japanese
scientists toured the facility, heard a presentation on AED's dissolved oxygen testing
system, and viewed the completion of a sediment TIE test.
At MED, additional topics included the development of quantitative structure-
activity relationship (QSAR) models to predict the toxic potential of chemicals and to use
in ecological risk assessments. The Japanese scientists also participated in a
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colloquium on the use of medaka data in risk assessments. Medaka, a fish native to
Japan, is one of three fish species used in Japan and internationally to assess the
endocrine-disrupting potential of chemical contaminants in the environment. Avian test
methods and the use of avian toxicity information in risk assessments were also
discussed.
At WED, the interchange focused on standard plant bioassays, various methods
for soil invertebrates, and approaches to soil microbial assays used in hazard and risk
assessments. Additionally, there were discussions on marine sediment bioassays and
tours of the greenhouse, plant growth chambers, and outdoor plots used in plant testing.
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