2003 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: Packard, Benjamin H
Project Period: March 1, 2001 through February 28, 2005
Project Period Covered by this Report: March 1, 2002 through February 28, 2003
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Water , Ecosystems

Objective:

The overall objective of this research project is to develop a suite of molecular, biochemical, cellular, and tissue level indicators that provide rapid assessment and advanced warning of environmental stress in estuarine/coastal habitats. The particular emphasis is on assessment of reproductive parameters because: (1) rapid and accurate techniques are not readily available; (2) biomarkers associated with reproductive impairment can be early warning indicators of stress; and (3) reproductive impairment can be directly linked to effects on populations through modeling efforts. The research proposed here is integral to the overall goals of the Pacific Estuarine Ecosystem Indicator Research Consortium (PEEIR), which are to establish indicators that environmental managers can use for: (1) developing an approach for synthesizing indicators into technically defensible assessments of wetland health and integrity; (2) determining biotic integrity for fish and invertebrate populations within wetland communities; and (3) determining toxicant-induced stress and bioavailability for wetland biota. 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 PEEIR.

Progress Summary:

Development of a Rapid Indicator of PAH and/or Phthalate Contamination

Previously we established that the sea urchin embryo development bioassay could be used to identify possible polycyclic aromatic hydrocarbon (PAH) contamination under laboratory conditions using individual PAHs as well as complex mixtures (Pillai et al., 2002). We have continued the development of this indicator by showing that there are very few classes of compounds that induce the PAH-like response of exogastrulation. These include phthalates (dibutyl phthalate), some of the PAHs, and the positive control lithium chloride. Selected metals, organochlorine pesticides, pyrethroids and polychlorinated byphenyls (PCBs) do not induce this specific developmental abnormality (see Table 1).

Table 1. List of Contaminants Tested for Their Ability to Induce Exogastrulation in Sea Urchin Embryos.

List of Contaminants Tested for Their Ability to Induce Exogastrulation in Sea Urchin Embryos

The usefulness of the assay with field samples was demonstrated by testing elutriates of sediments collected at Carpenteria Salt Marsh (stations A-D), where exogastrulation was observed in 2002. In 2003, sediments from stations A-C again showed exogastrulation induction (Figure 1). Chemical analyses are being conducted, but initial screening in 2002 showed the presence of phthalates.

Percentage of Embryos Showing Exogastrulation When Exposed to Sediment
  Elutriates from Carpenteria Salt Marsh. Both 2002 and 2003 sediments from the
  same stations showed activity.

Figure 1. Percentage of Embryos Showing Exogastrulation When Exposed to Sediment Elutriates from Carpenteria Salt Marsh. Both 2002 and 2003 sediments from the same stations showed activity.

Sediments were obtained through the U.S. Environmental Protection Agency Narrangansett Laboratory from the Elizabeth River in Virginia, which is contaminated with creosote. Elutriates from this site also showed exogastrulation and served as a positive control for the Carpinteria sediments. Finally, embryos (in semi-permeable enclosures) were outplanted at a PAH-rich site in a local marina to demonstrate that the assay can be used in situ.

Indicators of Exposure to Organic Contaminants: P4501A Enzyme Expression

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. 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 detoxication 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. Liver samples from mudsuckers collected at all sites have been analyzed using SDS-PAGE followed by Western blotting with the antibody to P4501A. The assay has been refined since 2002 to be highly specific. Analyses with crab hepatopancreas is continuing using the same antibody.

Initial analyses indicate that, as a group, outplanted (12 weeks) Carpinteria Salt Marsh fish show higher P450 levels than those at the other sites. In particular, station C6 shows the highest levels both in collected fish and in outplants. A complete analysis by individual fish, including sex, sexual maturity, and histology, is underway to tease out this biomarker response. It should be noted that we would expect Stege Marsh fish, which are exposed to the highest level of contamination throughout their life histories, to have decreased P450 expression as part of a compensatory response as they adapt to a high organic contaminant load. This has been observed in other fish in several studies. Complete analysis of data is underway and involves a significant effort to mine these raw data.

Indicators of DNA Damage as Assessed Using the Comet Assay

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 usually will lead to cell death. We have been assessing DNA strand breaks in blood cells of fish and crabs from the different marshes using the Comet assay that determines the percentage of DNA migrating from nuclei under electrophoretic conditions. Animals from Stege Marsh, the most contaminated site, show significantly elevated DNA stand breaks in their hemocytes, and this is seen at station S. Fish at other stations did not show an increase in DNA damage above reference levels. Analyses are continuing for all fish. Large numbers of samples have been prepared and are being analyzed using a new software package.

Indicators of Endocrine Disruption

For studies of endocrine disruption, we are applying immunologic assays to detect induction (estrogenic activity) of choriogenins (egg shell 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 we have developed as routine tools for detecting endocrine disruption in male and immature fish (Figure 2). This approach is applicable more broadly 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 also using data on the presence of estrogenic and/or androgenic activities from sites determined by Dr. Denison’s reporter bioassay, subsequent chemical analyses and demographic data collected by the Ecosystems Indicator Component, we should be able to determine cause and effect relationships for reproductive impairment. Dr. Denison has completed extractions of all sediments from 2003 collections and is conducting reporter bioassays at present. These data will be correlated with the choriogenin response as well as chemical characterization being conducted.

Presence or Absence of Choriogenins in Plasma of Male or Immature
  (No Gonad) Mudsuckers. As expected, Toms Point (TP) and China Camp (CC) show no response. 
  Specific stations at Stege and Carpenteria show an endocrine disrupting chemical
  response

Figure 2. Presence or Absence of Choriogenins in Plasma of Male or Immature (No Gonad) Mudsuckers. As expected, Toms Point (TP) and China Camp (CC) show no response. Specific stations at Stege and Carpenteria show an endocrine disrupting chemical response.

Indicator of Exposure to Contaminants: Programmed Cell Death (Apoptosis)

Another 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, as well as the health of individuals, based on histological condition and biomarker responses such as P450 and Comet. We have used a well-developed technique that measures DNA cleavage (the TUNEL assay) on field and laboratory samples, as well as caspase (cysteine aspartate protease) activity as indicators of apoptosis. We now have established a correlation between caspase activity and the TUNEL assay for fish exposed to cadmium; this is the first time both of these assays have been used in an aquatic organism. The TUNEL assay has been applied to livers of fish collected from all sites for 2002 and 2003. All data for each individual fish will be correlated with histological condition and biomarker responses to tease out individual variability in all responses and to determine station and site responses. These analyses will be completed later this year.

Developing a Larval Fish Model for Stressor Effects on Growth, Respiration, and Biomarker Responses

A series of experiments using larval topsmelt exposed to cadmium were conducted to develop indicators of contaminant-impaired growth and effects on physiology. These data are being used to develop a dynamic energy budget model for larval estuarine fish. Examples of the types of data collected include growth (based on weight and otoliths), respiration, food consumption, and apoptosis in different tissues.

Future Activities:

Analyses of samples and data will continue from 2002 and 2003 samples and results. New outplant experiments will be conducted this summer at select stations at Carpinteria Salt Marsh and Stege Marsh to define endocrine disrupting chemical responses further. In addition to choriogenin responses, we will analyze plasma using the cell reporter system to identify endocrine disrupting chemicals in mudsucker circulation. The dynamic energy budget modeling will proceed rapidly now that all of the organismal and physiological responses have been measured. Throughout the winter we will be working with the integration team to assess the most effective suites of stressor indicators using our animal models.


Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other subproject views: All 21 publications 3 publications in selected types All 3 journal articles
Other center views: All 139 publications 42 publications in selected types All 40 journal articles
Type Citation Sub Project Document Sources
Journal Article Pillai MC, Vines CA, Wikramanayake AH, Cherr GN. Polycyclic aromatic hydrocarbons disrupt axial development in sea urchin embryos through a β-catenin dependent pathway. Toxicology 2003;186(1-2):93-108. R828676C002 (2003)
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  • Supplemental Keywords:

    aquatic, indicators, biomarkers, wetlands, reproduction, cellular, molecular, biochemical, bioavailability, ecosystem, ecological impacts, estuary, watersheds, ecological effects, ecosystem indicators, 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 stresses, ecological risk assessment, wetlands, aquatic ecosystem, bioindicator, anthropogenic stress, biomarkers, ecosystem assessment, estuaries, wetland ecosystem, bioavailability, nutrients, trophic effects, ecosystem indicators, environmental indicators, coastal ecosystems, ecosystem restoration, aquatic ecology

    Relevant Websites:

    http://www.bml.ucdavis.edu/peeir/ Exit

    Progress and Final Reports:

    Original Abstract
  • 2001
  • 2002 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