Download EXECUTIVE SUMMARY: PEEIR and the Resident Species

PEEIR
Pacific Estuarine Ecosystem Indicator Research Consortium
EXECUTIVE SUMMARY:
PEEIR and the Resident Species
Approach to Ecological Risk
Assessment
Issue
Managers have long hoped for an integrated and
practical approach to evaluate effects of toxic substances
on resident estuarine species. Assessing toxicant
responses in relation to the multiple stressors affecting
our coast is also a significant goal, but it remains out of
reach of current methodologies. For example, a “triad”
approach, comprised of analytical chemistry, toxicity
testing, and benthic surveys, is currently recommended
for management of contaminated sediments, but this
scheme is limited by poor linkage to the ecology of
resident species, poor detection of chronic effects, and
lack of consideration of multiple stressors. Nevertheless,
this has been the best available management technique
for more than a decade. A multitude of methods
now exist to evaluate sublethal and chronic effects in
estuarine species; yet, these are almost never utilized
by managers. What are the barriers to implementation
of updated techniques? Are the advantages worth the
effort required to validate a complementary approach
and develop management alternatives?
Major impediments include lack of consensus about:
•
what methods to use and how to integrate the results
and interpret their meaning;
•
where and when multiple lines of evidence are
required to complete a viable management
framework.
Major advantages include evaluation of:
•
effects in resident species, eliminating extrapolation
from nonresident to resident species;
•
chronic and sublethal effects, which are possibly
widespread but are not being evaluated;
•
•
•
•
long term effects of contaminant mixtures;
multiple stressors;
multiple time scales or relevant spatial scales;
linkage to effects on populations.
To realize these advantages, a new generation of
ecological indicators is required. Managers need
practical and effective techniques for a wide range of
applications, from the management of contaminated
sediments to more effective wetland restorations. A
novel integrated science approach has to be employed
that links the disciplines of ecology and toxicology in a
rigorous manner. Toward that end, the Pacific Estuarine
Ecosystem Indicator Research (PEEIR) consortium devised
the Resident Species Portfolio (RSP) approach.
Approach and Rationale
PEEIR has developed a nested portfolio of indicators for
resident salt marsh species, and our findings demonstrate
the feasibility of ultimately creating such portfolios for any
coastal or estuarine habitat type. Our RSPs are based on
the principle that effects of stressors occur in individual
organisms; yet, it is often populations and communities
that managers care the most about.
PEEIR developed indicators and integration tools that:
•
refine our ability to identify stressors as well as
characterize their effects on individual physiology, and
•
allow physiologically-based indicators to be related to
population and community phenomena.
Indicators were integrated at multiple levels of biological
organization (e.g. cellular, physiological, organismal,
population, community) in carefully selected resident
California salt marsh species (Figure 1).
A research partnership between University of California, Davis, Bodega Marine Laboratory
and University of California, Santa Barbara
Funded by U.S. EPA Science To Achieve Results (STAR) EAGLE Program Grant No. R82867601
PEEIR
Pacific Estuarine Ecosystem Indicator Research Consortium
We chose:
•
the mudsucker Gillichthys mirabilis and the shore
crab Pachygrapsus crassipes as sentinel species
because they are abundant, they inhabit mud
burrows and remain in the same sites (~30m range)
for juvenile and adult phases;
•
the marsh plants pickleweed, Salicornia, and
cordgrass, Spartina, because they comprise the
majority of the plant biomass in the marsh, they are
readily sampled, and they are an important conduit
for food chain transfer of heavy metals;
•
community and/or ecosystem-level
investigations in microbes using molecular
techniques, evaluation of nutrient enrichment
in the marsh food chain using stable isotopes,
and estimation of changes in benthic and bird
community diversity using parasites as a surrogate
indicator -- because each of these provided a
snapshot of marsh condition that could not be
obtained with standard survey techniques.
Figure 1. The PEEIR Conceptual Model is based on nested indicators in
resident salt march species.
Exposures to toxic substances and/or nutrient
enrichment were assessed in target tissues as well as
in the analyses of sediment or water. Evaluation of
exposure and effects in the same tissues was a critical
component of our approach.
Program design and sampling activities were
thoroughly integrated, with over 30 research
laboratories quantifying indicator responses in each
of these species synoptically. Fish indicators were
measured by removing all necessary tissues from an
individual fish, conducting the appropriate laboratory
analyses, then finally integrating all data for each
individual fish. Essentially, we would take the fish apart
and put it back together again!
We sampled seven sites, over 400 km of coast in
northern and southern California (Figure 2). Sampling
was conducted at fixed stations within sites at three
tidal elevations. Field and laboratory experiments were
also undertaken to elucidate key relationships among
indicators and to further characterize specific exposures.
An important feature of the RSP approach is that
variables needed to quantify the population-level
significance of indicator responses via modeling
investigations were carefully considered in the overall
sampling design.
The integrative methodology employed in PEEIR could
be used to make significant improvements in ecological
risk assessment. Imagine a clinical study that neither
controlled for confounding variables such as age or
lifestyle and sex, nor measured all experimental variables
in the same subject! Yet, this is exactly the type of
design that is frequently applied to coastal assessments.
They are most often a patchwork of indicators applied
in various species. This makes it impossible to ascribe
relative effects of multiple stressors statistically or to link
biomarker responses to populations. To reiterate, key
integrative concepts included the use of:
•
selected resident species for a specific habitat type to
simplify the species selection process;
A research partnership between University of California, Davis, Bodega Marine Laboratory
and University of California, Santa Barbara
Funded by U.S. EPA Science To Achieve Results (STAR) EAGLE Program Grant No. R82867601
PEEIR
Pacific Estuarine Ecosystem Indicator Research Consortium
•
integrative sampling with most effects measured
simultaneously in individual organisms to make feasible
integrative statistics related to chronic effects and
multiple stressors, as well as to make more humane use
of fish tissue samples;
•
integrative statistics to discern the relative
significance of responses to multiple stressors, to relate
chronic effects to contaminant exposures, and to ascribe
population level effect;
•
ecological research and modeling to elucidate and
further validate the population-level significance of key
responses;
•
interplay between laboratory and field efforts
to refine relationship of chronic effects to specific
contaminant responses.
Hence, this type of integrative approach could be a
valuable new paradigm in ecological risk assessment that
puts more “eco” into ecotoxicology.
Findings and Impact
Fish
The mudsucker (Figure 3) proved to be an excellent
sentinel species for evaluating effects of contaminants
in California salt marshes (Link to Fish Reproductive
Impairment). We developed an integrative statistical
approach to expressing fish condition using a synthesis
of biomarkers and various morphometric measurements
(Link to Fish Integrated Indicators). In addition,
indicators with a more specific application were
developed for use in charting the effects of endocrine
disrupting compounds as well as contaminants that
cause genetic damage and tumors. Responses along
these two potential pathways of effect were developed
as practical indicators (Figure 4).
Figure 3. The longjawed mudsucker, Gillichthys mirabilis, was a
valuable sentinel fish species for studying salt marsh condition.
Figure 2. Aerial photo showing locations of PEEIR research sites
along the California coast.
•
The presence of female egg shell proteins
(choriogenins) in male and immature fish is an abnormal
response; and by measuring its frequency in a costeffective microplate assay, an early warning of endocrine
disruption was obtained (Link to Fish Endocrine
Disruption).
•
More severe effects were noted in the presence of
ovotestes, or fish with both ovary and testis in the same
animal. These data were obtained by simply dissecting
the fish and observing the anatomical changes.
•
With respect to genetic and cellular damage that can
lead to tumor formation, we measured the frequency of
cell suicide, or apoptosis, as an early warning indicator
using a commercially available kit (Link to Fish Apoptosis).
•
Tumor prevalence, a more severe response, is assessed
with standard histopathology that is widely available in
contract laboratories.
Both the integrative statistics and modeling investigations
were used to evaluate population-level significance of
biomarker and physiological responses (Link to Fish
Integrated Indicators, Link to Modeling Populations).
The development of all of these responses as indicators,
and the testing of modeling assumptions, involved a
combination of integrated laboratory and field studies to
understand the physiological and ecological mechanisms
responsible for changes in indicator values.
A research partnership between University of California, Davis, Bodega Marine Laboratory
and University of California, Santa Barbara
Funded by U.S. EPA Science To Achieve Results (STAR) EAGLE Program Grant No. R82867601
PEEIR
Pacific Estuarine Ecosystem Indicator Research Consortium
• In-depth research on crab movement and
demographics confirmed that movement in
juvenile and adult phases was quite limited;
and crabs were an effective indicator of
anthropogenic inputs of nutrients and other
contaminants into marshes at small spatial
scales (Link to Crab Sentinel Species, Nitrogen
Isotopes).
•
Demography (population size, size structure,
sex ratio) was a poor indicator of toxic effects
on crabs, but possible detrimental effects of
the invasive green crab, Carcinus maenus, were
detected (Link to Crab Sentinel Species).
Figure 4. Indicators of reproductive impairment in fish included markers
of endocrine disruption (choriogenins, ovotestis) as well as indicators of
tumorigenesis and apoptosis (cell death).
Crabs
Investigations using the shore crab, Pachygrapsus
crassipes (Figure 5), resulted in the development of a
simple and useful indicator (Link to Crab Reproductive
Performance). By quantifying crab embryo abnormalities
along with contaminant levels in the embryos, the
potential detrimental effects of contaminants at various
wetland sites can be evaluated.
In contrast to the findings on fish, biomarker
responses in crabs were not predictive of
contaminant exposure; and hence, integrative
statistics focused on the relationship between
detrimental reproductive effects and
contaminant exposure (Link to Crab Sentinel
Species). Shore crabs appear to be an excellent
sentinel species for monitoring the condition of
California coastal habitats, and we envision that
this approach could likely be extended to east
and gulf coast marshes using fiddler crabs (Uca
pugilator, Uca pugnax, Uca minax) and grapsid
crabs (Sesarma cinereum, Sesarma reticulatum).
•
Embryo abnormalities can be easily assessed with a
hand lens; and in our studies, the abnormalities were
indicative of other reproductive effects in the same
crabs.
•
The relationship between heavy metals in embryos
and the frequency of embryo abnormalities was stronger
when only the embryos in the outer portion of the clutch
were analyzed. Of course, this is because only the outer
portion is directly exposed to sediment. This observation
highlighted the need to evaluate exposure and effects in
the same tissue whenever possible.
Figure 5. Assessing crab embryo abnormalities in the field
is a practical indicator of salt marsh condition.
A research partnership between University of California, Davis, Bodega Marine Laboratory
and University of California, Santa Barbara
Funded by U.S. EPA Science To Achieve Results (STAR) EAGLE Program Grant No. R82867601
PEEIR
Pacific Estuarine Ecosystem Indicator Research Consortium
Plants
Ecosystems
PEEIR research on salt marsh plants was significant
because it resulted in indicators that can be applied at
multiple spatial scales (Figure 6). Although plants make
up the majority of the biomass of a marsh, indicators are
rarely developed for this component of the ecosystem.
A variety of integrative measurements for assessing
aspects of ecosystem health were considered. One
line of investigation utilized the diversity of trematode
parasites of snails to indicate diversity of other organisms
in salt marsh ecosystems, including benthic invertebrates
and birds (Link to Parasites). This method was validated
in PEEIR field studies in Southern California marshes
as well as in a marsh restoration. We determined that
larval trematodes in snails may be used to monitor
changes in biodiversity over time and among sites and
that the technique is more cost-effective than standard
field surveys of bird and invertebrate populations.
The Southern California Wetland Recovery Project is
recommending this technique for ongoing monitoring.
Remote sensing was used as a rapid and cost effective
tool to characterize species distribution patterns
among marshes (Link to Plants Remote Sensing). In
addition, remote sensing and analytical chemistry on
salt exudates from marsh plants were paired to develop
a novel indicator of metals mobilization across a marsh
landscape (Link to Plants Salt Exudates). Analysis of
metals in salt exudates is a rapid and cost-effective
technique.
Further methodologies for plants were developed
in the laboratory, but continued investigations will
be needed for field validation. For example, a new
biochemical method was developed that quantifies
the potential sequestration of toxic metals into food
webs (Link to Plants Metabolites). The method involved
electrophoretic analysis of phytochelatins, which are
peptides that tightly sequester toxic metals in plants.
Significant efforts were also made to link physiologic
responses of marsh plants to spectral changes that could
be observed by remote sensing (Rosso et al., 2005).
Microbial ecologists in PEEIR assessed whether changes
in microbial community assemblages can be used
as an indicator of contaminant exposure and effects
(Link to Microbial Assemblages). Two techniques
were contrasted: terminal restriction fragment length
polymorphisms (TRFLP) and phospholipid fatty acid
profiling (PLFA). The techniques revealed similar, but not
identical, patterns in microbial assemblages between
marshes. Multivariate statistical analyses revealed that
microbial patterns were related to heavy metal rather
than organic contaminants. These also may be best
applied as indicators of long term adaptation to low
level contamination, since they appear to show greater
responses than common acute toxicity assessments or
sediment chemistry (Cao et al., 2006).
BAY
© Michael Rigsby
Figure 6. Metal-laden salt exudates from Spartina, (left) and remote
sensing of species distribution (Stege Marsh, above) were both used to
create novel indicators using marsh plants.
A research partnership between University of California, Davis, Bodega Marine Laboratory
and University of California, Santa Barbara
Funded by U.S. EPA Science To Achieve Results (STAR) EAGLE Program Grant No. R82867601
PEEIR
Pacific Estuarine Ecosystem Indicator Research Consortium
An additional contribution of the microbiology team
was a demonstration of how indicators of nitrogen
cycling in wetlands can be used to guide management
of nutrient inputs into wetlands (Link to Nitrogen
Cycling) and may contribute to wetland remediation
efforts.
Stressor characterization
At all phases of the PEEIR program, new methods
of stressor characterization in marsh systems were
identified. Some of these utilized the indicator
species directly, and others were simply linked by field
observations and laboratory and field experiments
to the indicators. Nutrient inputs into marshes were
evaluated using stable isotopes, and this provided
an integrated indicator of nutrient enrichment
(Link to Nitrogen Isotopes). For many applications,
the stable isotope technique is superior to the
traditional approach of measuring dissolved nitrogen
concentrations. Moreover, the PEEIR modeling team
has used the field data on proportions of stable
isotopes of nitrogen to better characterize trophic
interactions and flow of nitrogen in marshes (Link to
Modeling Value Added). Analytical chemistry of heavy
metal and organic contaminants on sediment and
tissues was extensive (Hwang et al., a, b, in press) and
involved innovative methods to characterize some
chemical stressors as well as to rank the relative level of
contamination among sites (Link to Sediment Quality
Objectives) or provide linkage to specific effects (Link
to Crab Reproductive Performance).
In the course of the fish research, we field-tested a
commercially available microplate assay for estrogenic
potential to verify the presence of endocrine disrupting
compounds in sediments. Linking the plate assay to
fish effects was significant because this scheme can
be used in the future to conduct bioassay-directed
fractionation studies known as Toxicity Identification
Evaluations (TIE) to ascribe the relative potency of
different chemical fractions. This would help to
pinpoint which chemicals should be the target of any
remediation activities (Link in Progress).
Pathogen contamination in coastal sites is a serious
problem that has resulted in beach closures that are
costly to the economy and pose risks to human health.
Figure 7. Principal components (PC) analysis of TRFLPs clearly
discriminated between waste sources generated from two sewage
samples (S) and feces from cattle (C), dog (D), human (H) and
seagull (G).
Furthermore, wetland restoration activities can remobilize
pathogens buried in sediment. As a portion of the microbe
investigations, PEEIR researchers worked with the Southern
California Coastal Water Research Project (Link to SCCWRP)
to compare several molecular techniques that might be
used to characterize microbes in wastewater and, after
further development, sediment samples as well. The TRFLP
technique was among the most powerful in discriminating
various sources of fecal contamination such as human,
cow, dog, and gull (Figure 7) (Link to Bacterial Pollution).
The next step is for researchers to improve the methods for
applications to coastal sediment and water samples.
Additional contributions on stressor characterization
include:
•
remote sensing methodology to assess changes in
marsh plane elevations attributable to climate change or
direct habitat alteration (Link to Plants Topographic);
•
A larval fish bioassay that improves upon standard
techniques by using growth rate as an endpoint (Rose et
al., 2005);
•
A bioassay that uses a specific developmental
abnormality in sea urchin embryos, called exogastrulation,
to characterize exposures to PAH (Pillai et al., 2003).
A research partnership between University of California, Davis, Bodega Marine Laboratory
and University of California, Santa Barbara
Funded by U.S. EPA Science To Achieve Results (STAR) EAGLE Program Grant No. R82867601
PEEIR
Pacific Estuarine Ecosystem Indicator Research Consortium
Applications
Implementation of the Resident Species Portfolio (RSP)
approach has three phases (Figure 8). The first phase is
selection of resident species appropriate for the habitat
type and major stressors in question. We found that two
animals with different ecological niches, but some shared
characterstics, together with one or two plant species
provided an informative grouping for salt marsh habitats.
However, selection of the number of species in a portfolio
would depend on the nature of the problems under
investigation. Species selection is informed by simple
ecological models and practical considerations such as
availability, ease of sampling, and reproductive strategy.
Once species are selected, then possible indicators or
measurements should be listed for further consideration.
The second phase of RSP implementation involves the
iterative development of a portfolio. This process is
somewhat analogous to the development of an investment
portfolio. Investment is also a complex process which can
be simplified by applying selected principles to a step-wise
decision making framework. The steps include:
STEP 1: Clarify the goal and timeframe of the investigation.
Just as an investment portfolio will vary dramatically
for different goals (college? retirement?) any study of
estuarine sites involves many choices. Is there a specific
regulatory deadline or question? Is this a long term
monitoring program with specific goals? Is this an
assessment of resident species in decline?
STEP 2: Invoke concepts of risk and uncertainty.
Like financial investment, the goal of estuarine studies is
to minimize uncertainty and maximize return. We want
the most information possible for the dollar invested,
with the least risk to the resources. Hence, managers
must clearly characterize the potential resource risks
and their magnitude (serious decline of a species? loss
of major habitat or small section of marsh?) as well
as the level of uncertainty regarding stresses on the
resource. Once this is accomplished, the size and type
of portfolio can be characterized.
STEP 3: Determine the appropriate level of diversification.
Typically, a financial portfolio will include a balance
of diverse investments. In choosing indicators for
RSPs, diversification is also an asset. In general, we
distinguish between three types of indicators:
•
Condition indicators which provide a general
indication of individual health (e.g. histopathology,
reproductive output, growth);
•
Diagnostic indicators which give clues as to
what stressors may be causing impaired condition
(acetylcholinesterase enzymes, endocrine disruption);
•
Stressor indicators which are used to quantify
stressor levels (contaminants in sediment or tissues,
nutrient levels, pathogens).
A poorly characterized problem involving a large
resource risk should result in the development of a
large portfolio with an emphasis on condition and
stressor indicators. Tissues for diagnostic indicators
should be collected, a subsample analyzed and the
remainder archived. In contrast, if the problem is an
assessment of a well-focused issue, then diagnostic
and stressor indicators would be used in a careful and
definitive analysis of what contaminants are causing
problems in the condition of a species.
STEP 4: Apply iterative process to final program design.
Figure 8 (Implementing the PEEIR Portfolio) illustrates
the simple iterative process that is used in undertaking
an investigation. This process is easy to implement
given two conditions. The first condition is that core
participants in three fields must be identified and
must be willing to work in an integrative manner. The
three core groups include: ecologists, environmental
chemists, and ecotoxicologists who work at multiple
levels of biological organization (cellular, physiological,
organismal, population, community). Secondly, a plan
for archiving of tissues and sediments is required,
usually necessitating long-term access to an ultracold
freezer.
A research partnership between University of California, Davis, Bodega Marine Laboratory
and University of California, Santa Barbara
Funded by U.S. EPA Science To Achieve Results (STAR) EAGLE Program Grant No. R82867601
PEEIR
Pacific Estuarine Ecosystem Indicator Research Consortium
Figure 8. The three phases of resident species portfolio development and implementation.
The third phase of RSP implementation is indicator
integration and interpretation, and it must blend
seamlessly with progress in the second phase. This
phase involves indicator interpretation and populationlevel interpretations. While it is tempting to prescribe
one approach to statistical integration for each species in
the portfolio, we have found that this is not productive
primarily because the different species and indicators
respond very differently to specific stressors. However,
for fish indicators, sequential multivariate analyses
represent an exciting possible approach for describing
relationships among indicators and between indicators
and stressors (Link to Fish Integrated Indicators).
The approach also provides a pathway for relating
indicator responses to populations. Population modeling
creates value added in describing various mechanisms
of population response (Link to Modeling Value Added)
and potential relationship between incremental changes
in indicators and population endpoints such as size
structure, reproductive success and population growth
rate (Link to Modeling Populations).
The Portfolio Approach can be used in a variety
of resource investigations including: cleanup and
remediation of hazardous waste sites, wetland
restorations, sediment quality management, evaluation of
species declines, long-term monitoring of resources, Total
Maximum Daily Load (TMDL) investigations, assessment of
the relative success of regulatory activities, and evaluation
of the relative effects of emerging compounds.
A research partnership between University of California, Davis, Bodega Marine Laboratory
and University of California, Santa Barbara
Funded by U.S. EPA Science To Achieve Results (STAR) EAGLE Program Grant No. R82867601
PEEIR
Pacific Estuarine Ecosystem Indicator Research Consortium
Comparison of the RSP approach to a “Sediment Quality
Triad” framework indicates that the multiple lines
of evidence provided by the Triad and RSP method
together can strengthen ecological risk assessment and
reduce uncertainties significantly (Link to Sediment
Quality Objectives). A comparison to the Index of Biotic
Integrity (IBI) is also underway (Link to IBI Critique) and
has been coupled with a powerful new example, of
several indicators combined, to create a scorecard that
will improve management of San Francisco Bay (Link to
SF Bay Scorecard).
Summary and Conclusions
The RSP approach represents a practical framework
that can be implemented in a stepwise process, but
we caution that maintaining an integrated approach
is essential for success. Our most well-developed and
cost-effective indicators would be no more expensive to
utilize in management than a typical chronic toxicity test.
These include:
•
Fish reproductive impairment including endocrine
disruption, apoptosis, and histopathology
(contaminant effects)
•
Shore crab reproductive abnormalities (contaminant
effects)
•
Salt exudates from marsh plants (metal bioavailability
and food chain transfer)
•
•
Fish condition (multiple stressors)
Electrophoresis of phytochelatins to quantify metal
bioavailability (contaminants)
Our website will be updated as new publications on
these indicators and their applications emerge. Using the
three-phased process outlined above, the RSP method
can be implemented in a wide variety of management
applications. PEEIR has demonstrated the potential of this
concept for salt marsh ecosystems, but it can be applied to
other habitat types as well.
A promising new paradigm in ecological risk assessment
has been created with this novel blend of ecology and
toxicology (Figure 9). Integrated science is a powerful tool
needed to develop the next generation of indicators that
address the problems of scale, the simultaneous effects of
multiple stressors, and the true complexities of nature.
Contact Names
University of California, Davis, Bodega Marine Laboratory,
Susan L. Anderson, [email protected]
Gary N. Cherr, [email protected]
University of California, Santa Barbara,
Roger M. Nisbet, [email protected]
Trematode parasites of snails for diversity of birds and
benthic species (multiple stressors)
Additional indicators that are ready for use but require
University/Agency partnership or special techniques that
are not yet widely available include:
•
Stable isotopes of nitrogen in marsh fauna (nutrient
enrichment)
•
Remote sensing to map marsh plant species and
topography (multiple stressors)
•
Microbial diversity for long-term exposures
(contaminants/multiple stressors)
Promising new methodologies that would require further
field validation include:
•
•
TRFLP to assess the presence of microbial pathogens
in estuaries (pathogens)
Figure 9. The synergy between ecologists and toxicologists was
one of the keys to the success of the PEEIR program.
References Cited
Rosso, et al. 2005. Environmental Polution 137: 241-252.
Cao, et al. In press. Microbial Ecology.
Hwang, et al., a. In press. Chemosphere.
Hwang, et al., b. In press. Chemosphere.
Rose, et al. 2005. Environmental Toxicology & Chemistry 24:192-200.
Pillai, et al. 2003. Toxicology 186: 93-108.
Funding
See website for acknowledgment of matching funds from
NASA, CalFed, and UCTSRTP.
A research partnership between University of California, Davis, Bodega Marine Laboratory
and University of California, Santa Barbara
Funded by U.S. EPA Science To Achieve Results (STAR) EAGLE Program Grant No. R82867601