2002 Progress Report: Pacific Estuarine Ecosystem Indicator Research (PEEIR) Consortium: Biological Responses to Contaminants Component: Biomarkers of Exposure, Effect, and Reproductive Impairment

EPA Grant Number: R828676C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R828676
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Pacific Estuarine Ecosystem Indicator Research (PEEIR) Consortium
Center Director: Anderson, Susan L.
Title: Pacific Estuarine Ecosystem Indicator Research (PEEIR) Consortium: Biological Responses to Contaminants Component: Biomarkers of Exposure, Effect, and Reproductive Impairment
Investigators: Cherr, Gary N. , Anderson, Susan L. , Denison, Michael , Griffin, Frederick J. , Nisbet, Roger M. , Snyder, Mark J. , Wilson, Barry W.
Current Investigators: Cherr, Gary N. , Anderson, Susan L. , Baston, David , Bennett, Bill , Brooks, Andrew , Denison, Michael , Green, Peter , Hwang, Hyun-Min , Jackson, Susan , Lewis, Levi S. , Morgan, Steven , Nisbet, Roger M. , Rashbrook, Vanessa , Rose, Wendy , Teh, Swee J. , Vines, Carol , Wilson, Barry W.
Institution: University of California - Davis , University of California - Santa Barbara
EPA Project Officer: Hiscock, Michael
Project Period: March 1, 2001 through February 28, 2005
Project Period Covered by this Report: March 1, 2001 through February 28, 2002
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Water , Ecosystems


The objective of this research project is to determine the efficacy of a suite of molecular-, biochemical-, cellular-, and tissue-level indicators to collectively predict ecosystem responses to contaminant stress. Biomarkers of reproductive impairment are important early-warning indicators of ecosystem impacts, but they need complete characterization and validation in an ecosystem context as proposed in Pacific Estuarine Ecosystem Indicator Research (PEEIR). This section's particular emphasis is on the assessment of reproductive parameters. Because rapid and accurate techniques are not readily available, biomarkers associated with reproductive impairment can be early-warning indicators of stress and reproductive impairment can be directly linked to effects on populations through modeling efforts. The research proposed here is integral to the overall objectives of PEEIR, which are to establish indicators that environmental managers can use to: (1) develop an approach for synthesizing indicators into technically defensible assessments of wetland health and integrity; (2) determine biotic integrity for fish and invertebrate populations within wetland communities; and (3) determine toxicant-induced stress and bioavailability for wetland biota.

Progress Summary:

Research conducted in this component of the project is comprised of both laboratory experiments and field sampling. Each of these activities is coordinated with all components of the project. Integration is achieved through use of common sampling sites, model organisms, and modeling and statistical procedures common to the PEEIR Consortium. We completed our first intensive field sampling in the summer and fall of 2002. When analysis is completed, synthesis of multiple responses will ensue. Below are presented initial data for several project components.

Site Characterization and Developmental Toxicity. One of the first activities was to characterize wetland sites with respect to toxicity, because there was little or no background information available. Sea urchin embryo development, an accepted bioassay that is very sensitive, was used to evaluate sediment elutriates from stations at selected sites. Toxicity gradients were detected and were independent of sulfide and ammonia levels (see Figure 1). These data have been used in combination with chemistry values and remote sensing outputs to inform site selection for the project.

Figure 1. Example of Toxicity of Sediment Elutriates From Carpenteria Salt Marsh (CSM). A decrease in normal development of sea urchin embryos indicates toxicity. Three samples at different elevations per station were assessed. Station C showed the most toxicity. Note the presence of abnormal development in the form of exogastrulae at "B-Low."

In addition to providing basic toxicological data for subsequent PEEIR efforts, the data from these initial studies provided us with clues as to what contaminants may be responsible for the observed toxicity. It was found that polycyclic aromatic hydrocarbons (PAHs) induce the specific developmental abnormality known as exogastrulation (Pillai et al., 2003). Recently, we found that phthalates also induce exogastrulation and they occur at high levels at Station B at CSM. We plan to utilize the exogastrulation assay for in situ experiments and the molecular marker associated with this developmental abnormality, ß-catenin, in other species because it is highly conserved. ß-catenin may prove to be a universal marker applicable in many species.

Biochemical and Molecular Responses. An important area of research has been the development of the incidence of apoptosis, or programmed cell death, as an indicator of stress as well as decreased reproductive output in fish. Cell death in liver and gonad can directly impair reproductive output. We have utilized a well-developed technique that measures DNA cleavage (the transferase-mediated dUTP nick end labeling [TUNEL] assay) on field and laboratory samples, as well as cysteine aspartate protease (caspase) activity as indicators of apoptosis. This latter indicator has broader potential as an early-warning technique, because caspase 3 activity is a crucial bottleneck in the apoptotic cascade. Using a fluorogenic peptide substrate (Asp-Glu-Val-Asp-AMC) specific for caspase 3 activity, we examined caspase activity in topsmelt hepatocytes exposed to cadmium (0, 10, and 100 ppb concentrations) for 12 hours. We detected significantly higher caspase 3 activity levels with increasing cadmium dose in topsmelt liver cell extracts, indicating increased apoptosis in these cells. The caspase assay also was employed with livers of fish from several sites, and it was found that sites with contamination showed increased caspase activity. Although preliminary at present, further characterization of filed samples is underway. In addition, the TUNEL assay is being used to corroborate the caspase data; for fish cells in vitro, the results are consistent with the caspase data.

For studies on endocrine disruption, we are applying immunologic assays to detect induction (estrogenic activity) of choriogenins (eggshell protein precursors) that are made by the liver in response to estrogenic compounds including environmental estrogens and estrogen mimics. For this research, we are applying both commercial antibodies and antibodies that we have developed as routine tools for detecting endocrine disruption in male and immature fish (see Figure 2). This approach is more broadly applicable than the more commonly used vitellogenin assay in fish because the choriogenins are more highly conserved and the antibodies can be used on a very broad range of fish species. By utilizing data on the presence of estrogenic and/or androgenic activities from sites determined by Dr. Denison's reporter bioassay (see below), subsequent chemical analyses and demographic data collected by the Ecosystem Indicators Component, we should be able to determine cause and effect relationships for reproductive impairment. These can be linked to populations in the modeling research underway in the Nisbet laboratory. We know of no other efforts that attempt to link endocrine disruptor screening tests to population-level effects in such a comprehensive manner.

Figure 2. Assessment of Choriogenins in Plasma From Fish Collected From Several Sites Using a Fluorescent Enzyme Linked Immunosorbent Assay Microplate Assay. The increased optical density indicates increased choriogenins. All samples were normalized to concentration of protein in the plasma. Initial studies suggest that fish from Stege Marsh (SM), the most contaminated site we are studying, show increased circulating choriogenins. This suggests that estrogenic compounds may be present.

A rapid screening tool for aryl hydrocarbon receptor (Ah receptor) activity (dioxinlike compounds), as well as for estrogenic and androgenic chemicals, is the Chemically Activated Luciferase Expression (CALUX) bioassay, which has been developed in Dr. Denison's laboratory. This assay uses cells transfected with constructs of the dioxin-response, estrogen-response, or androgen-response elements and fluorescent reporter genes. Studies have demonstrated an excellent relationship between toxic equivalents of dioxinlike compounds and CALUX bioassay values. These and other data demonstrate the utility of this bioassay system for the detection and relative quantitation of dioxinlike compounds and related toxic/bioactive halogenated aromatic hydrocarbons (HAHs) in multiple media and tissue samples. The CALUX bioassay system provides a relatively rapid and more cost-effective approach for the detection and relative quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxinlike HAHs and endocrine disruptors in sample extracts; this bioassay can be directly related to subsequent chemical analyses as well as biomarker responses (P450s and choriogenins) and ultimately population-level effects (reproductive impairment). Site sediments and pore waters now are being prepared for the CALUX system and the final development of this assay for estuarine systems will be an important contribution to the PEEIR program. We envision that this assay could be used by other EaGLE centers that wish to screen for endocrine disrupting compounds at selected sites and we have initiated discussions with the Great Lakes Ecosystem Indicator project on the use of this method as well as the choriogenin antibody approach. Discussion with the other EaGLE groups has taken place.

An indicator of contaminant stress can be DNA strand breaks. These strand breaks can be repaired but may lead to mutations or overall diminished energy budgets; unrepaired strand breaks will usually lead to cell death. We have assessed DNA strand breaks in blood cells from fish and crabs from the different marshes using the Comet assay that determines the percentage of DNA migrating from nuclei under electrophoretic conditions. This initial phase is for us to develop a baseline for DNA damage using an approach by which individuals may repeatedly be sampled. Figure 3 shows DNA damage in blood cells (hemocytes) from the shore crab (Pachygrapsus). Animals from SM show significantly elevated DNA strand breaks in their hemocytes. Similar results have been obtained in mudsucker blood cells from the same marsh. We now are applying this assay to gametes and embryos from these species.

Figure 3. DNA Strand Breaks in Hemocytes From the Shore Crab. Increased DNA in the "tail" of the comet indicates increased DNA strand breaks. SM shows the greatest degree of DNA damage as compared to Tom's Point (TP) and Walker Creek.

Figure 4. Fluorescence Images Show Control Fish Blood Cells With Little or No DNA Strand Breaks. Cells from a fish exposed to metals showing the characteristic comet tail indicate high levels of strand breaks.

Biochemical biomarkers of exposure have been suggested as early-warning indicators of marsh degradation once they are placed in the proper context and other data sets are available regarding the overall condition of the organisms (Cherr, 2002). Our studies have included the analyses of P450 enzymes in tissues as these tend to be responsive to exposure to many organic contaminants and have long been used as a biomarker in both laboratory and field studies. We have focused on CYP1A, which is involved in the detoxification of many hydrocarbons and related chemicals. We have raised an antibody to a highly conserved peptide domain of CYP1A and have found that it cross-reacts with both vertebrate and invertebrate tissues. The PEEIR program provides a novel opportunity to place these data in a population context and further discern their utility as indicators.




Figure 5a-c. CYP1A Levels in Fish, Crabs, and Mudsuckers. (a) CYP1A levels in fish livers from SM, CSM, and TP. The stations at each marsh are designated by letter or number. Note the clear induction of CYP1A in fish from SM, particularly at stations A, B, and C. Stations D and F also show elevated P450 as compared to TP and CSM. (b) Crab hepatopancrease CYP1A levels are higher at SM than at TP. (c) Similarly, mudsucker liver CYP1A levels are higher at SM as compared to CSM or TP, which were not different from each other.

Relating Biomarker Responses to Populations. Biomarker responses such as apoptosis and endocrine disruption can be directly linked to reproductive outcome and population viability. Laboratory studies are ongoing (Project Investigators Anderson, Cherr, Nisbet, and graduate student Wendy Rose) to relate growth alterations to reproductive outcome and population viability in a variety of models. These eventually will include biomarker responses as well. For example, Dynamic Energy Budget models describe growth, development, and reproduction in single organisms in response to a specified food environment. Toxicant effects are represented through impacts on assimilation rates and efficiencies, and on respiration rates. We have been conducting sublethal exposures of young fish to cadmium, a contaminant present at several sites (see Figure 6). By assessing growth, food intake, respiration, and metal loads in tissues, we will be able to develop a model that predicts individual and population effects of chronic exposure to contaminant stress.

Figure 6. Effects of Chronic Cd Exposure (14 day) on Topsmelt Larval Growth. Fifty ppb and 100 ppb induce significant inhibition of growth based on length and weight.


Cherr GN. Can we develop and utilize indicators of ecological integrity to successfully manage ecosystems? In: Qualset CO, Rapport DJ, Ralston D, Lasley B, eds. Proceedings of Managing for Ecosystem Health, Third International Congress on Ecosystem Health. CRC Press; 2002. pp. 227-229.

Pillai MC, Vines CA, Wikramanayake AH, Cherr GN. Polycyclic aromatic hydrocarbons disrupt axial development in sea urchin embryos through a ß-Catenin dependent pathway. Toxicology 2003;186:93-108.

Future Activities:

Analyses of samples will continue for both 2002 and 2003. We are initiating a large number of outplants of mudsuckers and crabs at four sites to directly assess biomarkers, growth, and tissue contaminants in a controlled experimental design. There are critical experiments for the validation of annual sampling efforts. For reproductive studies, we will be assessing successful development in crabs, as well as the induction of spawning of sexually mature mudsuckers from different sites. This is the first time such approaches will be attempted.

After results become available from the sampling and experiments conducted in 2003, synthesis efforts will include: (1) integration of data with models; (2) statistical analysis relating biomarker responses to growth and reproduction of fish and invertebrates; and (3) the development of methodology recommendations useful to the U.S. Environmental Protection Agency (EPA) in the development of sublethal indicators of contaminant stress in wetlands. Discussions already are underway with EPA Region 9 regarding potential technology transfer for our novel and widely applicable methods related to endocrine disruption. The Denison group also has accepted samples from the west coast Environmental Monitoring and Assessment Program to perform the CALUX bioassay on sediment samples. We will determine whether this rapid screening method is ready for broader application.

Journal Articles:

No journal articles submitted with this report: View all 21 publications for this subproject

Supplemental Keywords:

aquatic, ecosystem indicators, biomarkers, wetlands, reproduction, cellular, molecular, biochemical, bioavailability, ecosystem, ecological impacts, estuary, watersheds, ecological effects, ecosystem integrated assessment., RFA, ENVIRONMENTAL MANAGEMENT, Water, ECOSYSTEMS, Ecosystem Protection/Environmental Exposure & Risk, estuarine research, exploratory research environmental biology, Ecosystem/Assessment/Indicators, Ecosystem Protection, Aquatic Ecosystems, Terrestrial Ecosystems, Ecological Monitoring, Ecological Indicators, Risk Assessment, anthropogenic stress, anthropogenic stresses, wetlands, aquatic ecosystem, bioindicator, ecological risk assessment, estuaries, ecosystem assessment, wetland ecosystem, biomarkers, nutrients, bioavailability, trophic effects, ecosystem indicators, coastal ecosystems, environmental indicators, ecosystem restoration, aquatic ecology

Progress and Final Reports:

Original Abstract
  • 2001
  • 2003 Progress Report
  • Final Report

  • Main Center Abstract and Reports:

    R828676    Pacific Estuarine Ecosystem Indicator Research (PEEIR) Consortium

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R828676C000 Pacific Estuarine Ecosystem Indicator Research (PEEIR) Consortium: Administration and Integration Component
    R828676C001 Pacific Estuarine Ecosystem Indicator Research (PEEIR) Consortium: Ecosystem Indicators Component
    R828676C002 Pacific Estuarine Ecosystem Indicator Research (PEEIR) Consortium: Biological Responses to Contaminants Component: Biomarkers of Exposure, Effect, and Reproductive Impairment
    R828676C003 Pacific Estuarine Ecosystem Indicator Research (PEEIR) Consortium: Biogeochemistry and Bioavailability Component