Final 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. , 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
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 was 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 were 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.

Summary/Accomplishments (Outputs/Outcomes):

Reproductive Impairment of a Salt Marsh Fish as an Indicator of Pollutant Effects

Issue. Organisms living in salt marshes often are exposed to environmental stressors derived from urban, industrial, or agricultural activities, and these exposures may be continual, seasonal, or even just over a single tidal cycle. Resident marsh organisms have not been used for management of wetlands, and thus the health of endemic organisms in wetlands is largely unknown. Exposure of organisms to chemical stressors can be through sediment and water, as well as food exposure routes. Organisms living within the marsh channels and plains that integrate exposures and effects of contaminants are excellent indicators for determining the impacts of contaminants on resident species, without the extrapolations inherent in toxicity testing and other common methods currently in use.

Approach and Rationale. Our approach has been to establish a direct link between contaminants and reproductive impairment using a ubiquitous resident fish. To test the broad applicability of this approach, we tested this in five California marshes over 600 km of coastline. Integrating biological responses of exposure to contaminants at the level of reproduction provides a direct link to predicting population level effects of stressors.

Findings and Impact. The longjaw mudsucker, Gillichthys mirabilis, is a gobiid fish that is common to salt marshes in California. It is an extremely hardy species that occurs even at contaminated sites. Gillichthys spends its entire juvenile and adult life within the same marsh and typically lives within a 30-50 m home range. They establish mud burrows in the banks of channels within the marsh, and this also is where reproduction occurs. Reproductive impairment was a sensitive indicator of habitat condition in the salt marshes studied, and classic reproductive impairment, as well as endocrine disruption, were observed.

The findings include the following:

  • Males can be distinguished from females based on jaw length (Figure 1), enabling identification of sex without sacrificing fish, which is critical for studies of endocrine disruption.

Figure 1. Adult Gillichthys mirabilis (top). Males (left) can be distinguished from females (right) by their longer jaw (red arrow).

  • Males and immature fish from more contaminated sites showed the abnormal endocrine disruption response of choriogenins (or egg coat proteins) circulating in their plasma based on the use of a standardized antibody response. This response is considered normal only in sexually developing females.
  • Fish from contaminated sites showed an increased incidence of ovotestes (also known as intersex) where both ovarian and testicular tissues were present.
  • The ovaries from fish from contaminated sites showed both an increased incidence of tumors and an increased incidence of apoptosis or programmed cell death, a cellular response to toxic insult in which damaged cells are removed from the tissue in an attempt minimize the incidence of tumor formation.
  • Suites of indicators can be integrated using multivariate statistics to characterize the percentage of fish in a marsh with impaired condition.
  • We propose that this approach can be used in a standard portfolio of indicators to identify “at risk” wetlands and to assess their overall condition. This may be most valuable for marsh restoration and mitigation, as well as for selected total maximum daily load applications.

Applications. The applications of the research include the following:

  • Reproductive impairment in the mudsucker is a valuable tool for assessing the health of salt marshes, and their highly limited home range provides a means for establishing a map of fish health at specific stations within a marsh.
  • The assessment of endocrine disruption in mudsuckers is rapid, and sampling can occur in the field or the laboratory. In addition, individuals can be repeatedly sampled to assess temporal responses.
  • The contaminants responsible for endocrine disruption, as well as reproductive impairment, can be investigated once the biological responses within a marsh are established.

This general approach has been validated at several sites in California, but its applicability for issues relating to specific contaminant stressors or restoration actions may require refinement of the overall approach.

Endocrine Disruption in a Salt Marsh Fish as an Indicator of Wetland Condition

Issue. Contamination of many wetlands by industrial chemicals, many of which are endocrine disrupting compounds (EDCs), has resulted in fish and invertebrates exhibiting alterations in reproductive function. Hormones such as natural and synthetic estrogens and many other chemicals such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), plasticizers (phthalates), dioxins, pesticides, personal care products, and pharmaceuticals released from sewage treatment plants can disrupt endocrine function. Chemical analysis of sediments may not be an effective approach for identifying EDCs, because it is costly and will not reflect bioavailability of the chemicals; however, managers must prioritize and control the release of hundreds of “emerging contaminants.” A characterization of the physiological condition of resident species that includes both indicators of endocrine disruption and of overall reproductive health can provide a clear picture of the organismal impacts of EDCs and reproductive contaminants.

Approach and Rationale. Our approach was to focus on two types of reproductive impacts of EDCs in Gillichthys mirabilis (long-jawed mudsucker) collected from or outplanted at selected California marshes: (1) the presence of choriogenins (egg coat proteins) in male or immature fish and (2) the co-occurrence of ovarian and testicular tissue (ovotestes). These abnormalities were analyzed in conjunction with a cell reporter assay for endocrine disruption potential, as well as with direct chemical characterization of sediments from these marshes. The rationale for this approach was as follows:

  • Choriogenins (egg coat proteins) normally are produced in the liver in response to estrogen and are transported in the blood to the ovary of reproductive female fish. They normally are not found in male or immature fish, thus the presence of choriogenins in plasma of male/immature fish reflects exposure to EDCs. Choriogenins are detected easily in plasma with Western blot analysis or enzyme-linked immunosorbent assay and a choriogenin antibody. Fish can be marked and sampled repeatedly, allowing for numerous temporal assessments in order to monitor dynamic habitat condition, success of remediation, etc.
  • Ovotestes are gonadal tissues with characteristics of both testicular and ovarian structures, and reflect exposure to EDCs that are estrogenic or anti-androgenic. Dissection of fish and gross examination of gonadal tissue for ovotestes is routine. Histological examination of tissue sections for confirmation of ovotestes, as well as for the presence of tumors and apoptosis is useful. Tissue samples for histology can be archived and analyzed at a later date.

Findings and Impact. Fish from contaminated marshes exhibited several types of impaired reproductive function:

  • Choriogenins were detected in the plasma of a higher percentage of male/immature fish from marshes with higher contaminant levels (Figure 2a). Mudsuckers outplanted at selected stations reflected a similar trend (Figure 2b). The types of contaminants detected in sediments at sites where endocrine disruption occurs include PCBs, PAHs, DDT pesticides, metals, and phthalates.
  • The sediment estrogenic potential, as determined by a recombinant cell reporter assay, showed a similar response as choriogenins.
  • Ovotestes were found only in fish from the most contaminated marshes, Stege and Carpinteria (Figure 3a). Typically one of the paired gonads exhibited both types of tissue (Figure 3b).

Figure 2a. Wild Caught Mudsuckers from Stege Marsh (SM) and Carpinteria Salt Marsh (CS) Exhibited a Higher Incidence of Chorio­genins in Male or Immature Fish than Mudsuckers Collected from the Less Obviously Contaminated Marshes China Camp (CC), and Walker Creek (WC), Although a Few Fish from Tom’s Point (TP) also Had Choriogenins

Figure 2b. Naive Fish Outplanted for 3 Months at the Sites Indicated Showed a Similar Trend

Figure 3a. Ovotestes Were Found Only in Mudsuckers from Stege Marsh and Carpinteria Salt Marsh

Figure 3b. Mudsuckers from Contaminated Sites Showed Ovotes­tes Where the Gonads Exhibited Both Testicular and Ovarian Tissue (left). Normal females show the typical paired ovaries of equal size (right).

Applications. The applications of the research include the following:

  • Presence of choriogenins in mudsuckers is a useful indicator for managers interested in diagnosing or monitoring for the presence of endocrine disrupting compounds in salt marshes. The techniques involved are relatively noninvasive (involving blood collection) and are routine and relatively inexpensive, involving analysis of plasma for choriogenins using commercially available antibodies and common detection methods. Analyses of fish from other marshes in the SF estuary is underway.
  • The presence of ovotestes is a functional indicator of impaired reproduction because of EDC exposure in mudsuckers. Although sacrifice of fish is necessary, gross examination of gonads is inexpensive and simple to perform and can be followed up by histological examination of tissue sections where appropriate.
  • The EDC indicators developed here provide tools for managers to map the extent of endocrine disruption within or between marshes.

Ovarian Apoptosis and Tumors As Indicators of Reproductive Impairment in a Marsh Fish

Issue. Organisms living in salt marshes often are exposed to environmental contaminants derived from human activities, and reproduction is one of the most sensitive responses observed. Reproductive organ health and successful reproduction can be linked directly to population effects in many species. The responses of apoptosis, or programmed cell death (Figure 4), and tumor formation are typically highly conserved between species and are direct indicators of reproductive toxicant and/or carcinogen exposure.

Figure 4. Apoptosis of Programmed Cell Death. Damage to organelles such as mitochondria results in a highly specific cellular response that involves increases in calcium (Ca2+) and activation of a key enzyme, caspase. This ultimately results in highly regulated cleavage of DNA leading to cell death.

Applications. The applications of the research include the following:

  • Apoptosis in salt marsh fish is proposed as an early warning indicator of more severe impacts (tumors), and the technology established in the biomedical research field can be translated directly to fish ovarian tissue.
  • Apoptosis can be included in an integrated and multibiomarker approach for investigations of reproductive health of resident species.
  • Preservation of samples can be conducted during routine monitoring for subsequent histological apoptosis and tumor analyses.
  • The terminal dUTP nick-end labeling (TUNEL) assay is available in a kit form and could be used in both university and contract laboratories.

Approach and Rationale. Our approach has been to establish a direct link between contaminant exposure and reproductive impacts in a ubiquitous resident fish to assess habitat condition and diagnose stressors in five California salt marshes. Apoptosis was evaluated in histological sections of ovaries from fish collected in the different marshes. The TUNEL (terminal transferase (Tdt) + fluorescein-dUTP) assay is a cytochemical approach to identifying cells undergoing apoptosis within a tissue. We adapted the TUNEL assay for use with fish tissues.

Findings and Impact. The mudsucker, Gillichthys mirabilis (Figure 1, top), a gobiid fish that is common to salt marshes in California, is an extremely hardy species that occurs even at highly contaminated sites. Reproductive impairment based on apoptosis and tumor formation was a sensitive indicator of habitat condition in the salt marshes studied. The findings include:

  • Fish from the most contaminated marsh (Stege Marsh) showed a greater incidence of apoptosis in ovarian cells based on nuclear TUNEL labeling (Figure 5).

Figure 5. Apoptosis in Ovaries. Ovarian tissue showed an increased incidence of apoptosis in females from Stege Marsh (SM) compared to other locations. Granulosa (G) and thecal (T) cells both showed increased TUNEL positive nuclei.

  • Fish from two of the more contaminated sites (Stege Marsh, Carpinteria) also showed increased incidence of ovarian tumors that included teratomas as well as granulosa cell tumors (Figure 6).
  • The high incidence of apoptosis and tumors at the same site (Stege Marsh) may relate to the inability of fish to clear damaged cells rapidly enough to prevent tumorigenesis.

Figure 6. (a) Fish from Stege Marsh (SM) and Carpinteria Salt Marsh (CS) Showed Increased Tumors in Ovaries as Compared to Toms Point (TP) and Walker Creek (WC). (b) Histological section through an ovarian tumor.

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 Anderson SL, Cherr GN, Morgan SG, Vines CA, Higashi RM, Bennett WA, Rose WL, Brooks AJ, Nisbet RM. Integrating contaminant responses in indicator saltmarsh species. Marine Environmental Research 2006;62(Suppl 1):S317-S321. R828676 (Final)
R828676C002 (Final)
  • Abstract from PubMed
  • Full-text: Science Direct Full Text
  • Other: Science Direct PDF
  • Supplemental Keywords:

    watersheds, estuaries, ecological effects, bioavailability, ecosystem indicators, aquatic, integrated assessment, ecological effects, ecosystem indicators, , estuarine research, aquatic ecology, environmental indicators, ecosystem assessment, biological markers, biomarker, biomarkers, ecological assessment, ecological exposure, ecosystem condition, ecosystem health, ecosystem indicators, ecosystem integrity, environmental consequences, environmental indicators, environmental stress, environmental stressor, environmental stressors, estuaries, estuarine ecosystems, fish, plant indicator, statistical evaluation,, 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

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    Progress and Final Reports:

    Original Abstract
  • 2001
  • 2002 Progress Report
  • 2003 Progress 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