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2004 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
Current Institution: University of California - Davis , University of California - Santa Barbara
EPA Project Officer: Levinson, Barbara
Project Period: March 1, 2001 through February 28, 2005
Project Period Covered by this Report: March 1, 2003 through February 28, 2004
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000)
Research Category: Ecological Indicators/Assessment/Restoration

Description:

Objective:

The overall aim of this component of the 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. This component’s particular emphasis is assessment of reproductive parameters because rapid and accurate techniques are not readily available for endemic marsh organisms, 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 proposed research is integral to the overall goals of Pacific Estuarine Ecosystem Indicator Research (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. The objective in this proposal 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 PEEIR.

This Biological Response to Contaminants (BRC) report primarily focuses on our fish indicator species, Gillichthys mirabilis, whereas the Ecosystems Indicator Component (EIC; subproject R828676C001) annual report focuses on the crab and clam indicator species. It should be noted, however, that research on all three species, as well as the plant indicators, are a continuous collaborative effort between all of the PEEIR components.

Progress Summary:

Reproductive Impairment in an Endemic Fish From California Wetlands

We are taking a multifaceted approach for the study of reproductive impairment in an indicator fish species, the long jaw mudsucker (Gillichthys mirabilis). Our integrated approach will eventually enable identification and prioritization of reproductive contaminants in urban- and agriculture-impacted estuarine environments. We have been employing an in vitro reporter gene bioassay for estrogenicity (and aryl hydrocarbon receptor activity) with in vivo expression of reproductive gene products (choriogenins or egg shell proteins), organismal-level reproductive health (ovarian/liver apoptosis, ovarian tumors, reproductive status), and population-level impacts in the sentinel fish, Gillichthys. The research project to date has taken a broad approach to studying adverse reproductive physiological effects so that the responses measured are to the broad range of environmental chemicals present in the wetland sites, rather than focusing on a short list of known reproductive toxicants. Through collaborations with the Biogeochemistry and Bioavailability Component (BBC; subproject R828676C003) and the EIC (subproject R828676C001), we are now trying to link site chemicals with exposure, reproductive health, and population-level parameters through modeling approaches.

The BRC component of the PEEIR program has developed Gillichthys as a sentinel fish indicator for wetland condition. Our approach has involved quantifying molecular, cellular, and biochemical responses in individual organisms collected from stressed and less-stressed wetlands and along possible gradients within a marsh and between marshes. In addition to fish collected from field sites, we conducted experiments in the field that involved outplanting fish at specific stations for up to 3 months. This was followed by a suite of analysis measurements that included molecular, biochemical, and physiological parameters that all will be directly linked to growth/condition indices at the level of individual organisms. We also have measured known contaminants at the outplant/collection stations. Indicators have included: (1) DNA strand breaks in red blood cells using the Comet assay; (2) acetylcholinesterase enzyme activity to assess organophosphate and carbamate pesticide exposure; (3) apoptosis or programmed cell death in multiple tissues; (4) cytochrome P450 enzymes to quantify exposure to many types of organic contaminants; (5) levels of metal and organic contaminants in subsets of matched fish; and (6) choriogenin proteins in male/non-reproductive fish to evaluate xenoestrogen exposure. Individual fish and population-level health parameters have also been measured and will be related to biomarker results. Multivariate analyses relating biomarker responses to growth impairment and fish condition are a key part of the integration indicator effort. These analyses are used to ultimately derive indicators based on relationships among multiple response parameters and stressor profiles.

Development and Application of Indicators for Endocrine Disruption in an Endemic Fish

Endocrine disrupting compounds (EDCs) can mimic endogenous hormones or inhibit normal hormonal activity of endocrine and neuroendocrine systems. Numerous chemicals, including polychlorinated biphenyls (PCBs), dioxin, DDT, insecticides, fungicides, alkylphenols, synthetic estrogens, and others, can interfere with vertebrate endocrine systems (Soto et al., 1994). Some of these chemicals can act as estrogens (estrogenic EDCs) and can cause feminization, thus limiting the reproductive capacities of exposed species. Organisms impacted by estrogenic EDCs include humans, birds, fish, amphibians, and reptiles. In aquatic environments, the source of steroidal estrogens and estrogenic EDCs is thought to be primarily human sewage and industrial activities. For studies of endocrine disruption, we have been 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.

The development of more useful tools for assessing physiological responses to EDCs has been an area of focus of the PEEIR program. Organismal-level responses have been used for some time to assess endocrine disruption in the aquatic environment and have been compared to in vitro assay responses. These responses have included the abnormal expression of vitellogenin (yolk protein) in males and immature/juvenile fish. The transcript for the protein typically is quantitated in the estrogen-responsive liver and/or the protein itself is detected in the circulatory system as it is being transported to the ovary. Although vitellogenin mRNA expression is a useful measure in liver cells of estrogenicity, its assessment cannot be made without sacrificing fish. Vitellogenin protein can be measured in plasma collected from fish, however, vitellogenin antibodies (the probes used to detect the protein) are species-specific and generally need to be developed for each species of interest. Another specific biomarker of estrogenic activity are the egg envelope (chorion) proteins or choriogenins. The transcript and the protein of choriogenin (also known as zona radiata protein) have been used as biomarkers of xenoestrogen exposure as well. Because the zona pellucida domain of choriogenins is highly conserved in egg envelopes from invertebrates to mammals, antibodies constructed to this domain tend to cross-react across species. Therefore, the use of either commercial or lab-developed choriogenin antibodies can detect exposure to xenoestrogens when assessed in male or juvenile fish of different species.

Figure 1: Sexual Dimorphism: The Maxilla of Male Gilllichthys Are Longer and More Squared Off (gray arrow) than Those of the Female (A). Relationship between maxilla length and sex of fish (B).

For this research, we have been applying both commercial antibodies and antibodies that we have developed for use as routine tools for detecting endocrine disruption in male and immature Gillichthys. Our focus has been on male and immature fish, as it is abnormal for these to express choriogenins. We have coupled the choriogenin analyses with a reporter cell bioassay of sediment estrogenicity using the estrogen-responsive luciferase reporter gene bioassay ( Rogers and Denison, 2000). This assay uses a reporter plasmid vector containing the firefly luciferase gene under estrogen-inducible control of several estrogen-responsive DNA enhancer elements. Stable transfection of this vector into human ovarian carcinoma (BG-1) cells resulted in the isolation and characterization of a recombinant cell line (BG1Luc4E2) that responds to 17β-estradiol with the induction of luciferase activity in a dose-, time-, and chemical-specific matter. The utility of these recombinant cells as a bioassay screening system for environmental estrogens has been demonstrated by their response to known xenoestrogens ( Rogers and Denison, 2000) and EDCs.

Gillichthys is a sentinel species for reproductive health in wetland habitats for the flowing reasons: (1) It is an extremely hardy and ubiquitous fish in salt marshes along the west coast and has been used in numerous environmental physiology studies, including outplant experiments where contaminant stressors are present (Forrester et al., 2003). (2) It lives and reproduces in a single estuary (Yoklavich et al., 1991) in mud burrows and thus is exposed (directly and through food and is sediment they ingest) to contaminants associated with sediments. (3) Once larvae settle in a marsh, the juveniles and adults have a very limited range (~30 m) around their burrows, enabling exposure to be directly linked within a known spatial range (Brooks, 1999). (4) It is sexually dimorphic so that males and females can be identified in the field and sampled on site without sacrificing the fish (Figure 1). (5) It expresses detectable choriogenins and fish can be repeatedly sampled using small volume (20-50 µl) plasma collections (up to 2/month) without any adverse effects.

To experimentally address physiological and organismal responses of Gillichthys at Stege Marsh, we conducted outplant experiments in 2004. This involved collecting juvenile fish from a relatively unimpacted region of Carpinteria Salt Marsh, acclimating the fish under flowing water conditions of appropriate salinity and temperature for Stege Marsh, and outplanting in individual cages (12 replicates per station) for 3 months. Three fish from each station were used for chemical analyses and a number of biomarker endpoints were assessed, including choriogenins, P450 enzymes, blood cell Comet assays, histopathology, and so forth. Growth and apoptosis analyses still are being completed.

Our studies collecting or outplanting Gillichthys at four different marshes in California included the highly impacted (industrial organic chemicals, and metals) site, the Stege Marsh (Richmond, CA) in the San Francisco estuary, a moderately impacted (pesticides) site in southern California (Carpinteria Salt Marsh), and several reference marshes, including the San Francisco estuary (China Camp, Novato, CA) and Tomales Bay (Walker Creek and Toms Point). These studies have shown that Gillichthys collected from different stations at Stege Marsh showed distinct markers of endocrine disruption, including ovotestes in sexually mature fish (Figure 2) and choriogenins in the plasma of male and immature fish (Figure 3). The reference sites at China Camp, Walker Creek, and Toms Point showed little or no choriogenin response and a lack of ovotestes. Carpenteria Salt Marsh showed a marginal response. These data show that we can detect EDC effects in both wild collected and outplanted Gillichthys and that our approach can detect a range of effects from none (reference sites) to intermediate (Carpinteria Salt Marsh) to severe (Stege Marsh).

These organismal responses (choriogenins and ovotestes) have been compared to sediment estrogenicity assessed using the estrogen-responsive luciferase reporter gene bioassay ( Rogers and Denison, 2000). Analysis of sediment extracts (ethanol) in BG1Luc4E2 cells revealed a distinct estrogenic response at specific stations at Stege Marsh that correlated with choriogenin expression. A comparison between station estrogenic potential (using the recombinant cell assay of sediment extracts) and choriogenin response in collected fish from stations R & S at Stege Marsh show increased choriogenin frequency and estrogenic potential.

Figure 2. Gillichthys Exhibit a Synchronous Development and Maturation of the Ovary Under Normal Conditions (A). Fish from EDC-impacted stations at Stege Marsh show distinct ovotestes (B), which is a classic indicator of sex reversal and/or endocrine disruption. Note the presence of testicular tissue (arrow) as well as oocytes. Stege and Carpinteria Salt Marsh had up to 20 percent of fish showing ovotestes, while Toms Point and Walker Creek had no fish with ovotestes.

Figure 3. Proportion of Male or Immature Gillichthys Collected at Specific Stations at Stege Marsh Exhibiting Choriogenin Expression (stations A-M and Q-S). Fish collected at the two reference sites (China Camp and Walker Creek) showed no choriogenin response. Male fish outplanted at specific stations also showed a choriogenin response within 2 months. Fish outplanted at the reference site (China Camp) showed no response.

The chemicals responsible for estrogenic activity in sediment and EDC responses in fish have, to date, not been directly identified. Likely candidates, however, include PCBs and phthalates. Both are elevated at Stege Marsh station R (Figure 4). The sediment PCBs are clearly bioavailable since outplanted Gillichthys accumulated high levels (> 1500 ng/g wet wt.) within 3 months. A listing of inorganic and organic contaminants at five marshes is presented in Table 1 (from BBC analyses) and provides the basis for future contaminant/bioeffects research using toxicity identification evaluation-like approaches. Clearly, Stege Marsh contains the highest contamination, with Carpinteria Salt Marsh containing some typical agrochemicals as well as metals.

Figure 4. Sediment PCBs and Phthalates (A) and Sediment and Tissue PCBs (B) from Outplanted Gillichthys. Pre-outplants showed low or no detectable PCB levels (not shown).

Apoptosis (Programmed Cell Death)

EDCs are known to induce estrogen receptor-responsive cancers in different reproductive tissues, including(breast, vaginal, cervical, and ovarian (Choi et al., 2004). In trout, estradiol has been shown to act as a promoter in hepatocytes rather than a complete carcinogen, and similar findings in Medaka have been made (Cooke and Hinton, 1999). Whereas the carcinogenic action of EDCs is established in mammalian tissues, little is known regarding their action upon lower vertebrates, including fish.

Recent studies have begun to examine apoptosis as a endpoint of toxicant exposure and effect in fishes and mammalian species (Gavrieli, et al., 1992). Using histological evaluations and biochemical techniques, a wide range of toxicants were shown to induce apoptosis, indicating that apoptosis and biochemical changes associated with this process may be useful biomarker responses. To our knowledge, however, apoptosis has not been rigorously applied to field investigations of toxicant-exposed organisms. Although some apoptosis may prevent cancerous cell proliferation, the inappropriate stimulation of apoptosis by toxicants may lead to developmental abnormalities, tissue dysfunction, or reproductive impairment (Raffray and Cohen, 1997). Thus, the development of apoptosis as a biomarker response is essential because it may be used as an early warning indicator for more severe toxicant effects, such as impaired fitness. Moreover, the assessment of apoptosis together with multilevel biomarkers may provide the key to determining linkages between molecular biomarker responses and higher level effects of toxicant exposure.

While endogenous estrogen is known generally to reduce apoptosis in the ovary, EDCs have been shown to induce apoptosis in fish and mammalian ovaries (reviewed by Andreu-Vieyra and Habibi, 2000). EDCs are known inducers in a variety of tissues. For example, the EDC 4-nonylphenol is a potent inducer of apoptosis in neural stem cells via a classic cascade mechanism (Kudo, et al., 2004). Apoptosis is important in limiting cancer cell proliferation and a defect in the apoptosis machinery often is linked to tumor formation. Estrogenic compounds are known to increase apoptosis in hepatocytes and kidney tissue, and carcinogens will induce apoptosis in a number of tissues. Our data from studies of Gillichthys show that fish from contaminated marshes (Stege Marsh and to a lesser extent at select stations at Carpinteria Salt Marsh) show increased ovarian tumors. Furthermore, fish from these sites also show increased apoptosis rates in ovaries and livers. Whether the increased frequency of tumors and/or cells exhibiting apoptosis is caused by EDCs or other contaminants is not known at present. Fish from Stege Marsh also showed elevated liver apoptosis as compared to the other sites.

Table 1. Average Concentrations of Selected Metals, Organic Chemicals, and Current-Use Pesticides in Sediment From Five California Tidal Marshes (ND = not detected; <MDL = below minimum detection limit)

Marsh

(sample size)

CC

CS

ST

TP

WC

(6)

(36)

(26)

(6)

(6)

Metals (mg/kg dry wt)

Ag

0.32

0.23

0.28

0.04

0.07

Al

35522.56 13649.93 31469.58 27786.18 27648.96

As

12.27

5.10

15.90

7.37

9.74

Cd

0.24

0.23

0.46

0.18

0.21

Cu

51.30

15.07

78.33

19.16

25.63

Pb

32.93

14.28

87.94

10.32

12.64

Se

0.65

0.30

0.75

0.34

0.48

Zn

109.39

81.51

272.67

67.21

68.09

Organics (µg/kg dry wt) Total PAHs1

1664.0

1377.0

4271.0

927.0

960.0

Total PCBs2

<MDL

<MDL

648.0

<MDL

<MDL

p,p'-DDD

1.4

3.7

25.0

ND

ND

Pesticides (µg/kg dry wt)

Eptam

ND

ND

19.9

ND

ND

Pebulate

ND

ND

4.3

ND

ND

Molinate

ND

ND

10.9

ND

ND

Cycloate

ND

ND

3.1

ND

ND

Promethryn

ND

ND

8.9

ND

ND

Chlorpyrifos

ND

1.0

ND

ND

ND

Methidathion

ND

4.1

4.6

ND

ND

Napropamide

ND

ND

5.9

ND

ND

Bifenthrin

ND

2.3

ND

ND

ND

Permethrin

ND

3.1

ND

ND

ND

1 Total PAHs = the sum of 33 parent and alkyl substituted PAHs

2 Total PCBs = the sum of 44 PCB congeners

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 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. Liver samples from Gillichthys collected at all sites have now been analyzed utilizing sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by Western blotting with the antibody to P4501A. The assay has been refined since 2003 to be highly specific and repeatable.

Analyses indicate that as a group neither field collected or outplant Gillichthys show dramatic differences in P450 levels in relationship to known contamination. Although significant increases occurred at several sites, correlation with organic contaminants is weak. We are presently analyzing individual stations and fish at each site with respect to contamination and P450 levels, respectively, to determine if this biomarker is useful for this species. A complete analysis by individual fish, including sex, sexual maturity, histology, and so forth, 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.

Analyses with crab embryos and gill are being finalized (see subproject R828676C001 annual report) using the same antibody.

Acetylcholine Esterase Activity

Acetylcholine esterase (ACHE) activity is critical for termination of the neurotransmitter signal from acetylcholine and is the mechanism of action by which organophosphate (OP) pesticides act as insecticides. Measurement of ACHE activity can, therefore, be used as a specific biomarker of exposure to OP pesticides. Brain tissue was collected from fish at five marshes and analyzed for total ACHE activity. Both Mugu Lagoon and China Camp fish exhibited ACHE activity in a similar range (~35 µmol/min/g), whereas Stege and Carpinteria Salt Marsh fish exhibited lower levels. Because of variability between fish, however, only Carpinteria Salt Marsh showed a statistically significant decrease in ACHE activity. Several pesticides have been detected at Carpinteria (primarily impacted by agrochemicals). These include chlorpyrifos, promethrin, and bifenthrin (Table 1).

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 from fish and crabs from the different marshes using the Comet assay that determines the percentage of DNA migrating from nuclei under electrophoretic condition.

We have found that sample preparation time (collection, centrifugation of cells, and freezing time) and method all appear to impact the background levels of DNA damage in Gillichthys blood cells. As a result, recent analyses suggest that there is too much variability between individuals from stations within sites to be able to detect a clear signal. While Stege Marsh fish show some elevation of DNA damage, we cannot detect a clear stressor-related signal with the Comet assay and Gillichthys to date. We plan to analyze the Comet data together with all other biomarkers on an individual fish basis to determine if we can “tease” out a stressor signal with this biomarker.

Organismal Biomarkers

A number of organismal-level biomarkers have been assessed in Gillichthys from different sites and also are described in the EIC (R828676C001) subproject annual report. Some of these biomarkers include histopathological markers (Figure 5), which are characteristic in fish under pollution and environmental stress, as well as parameters that use otolith structure for determining age and growth. Other metrics include liver-somatic and gonadal-somatic indices.

Figure 5. Example of Histopathology of Gillichthys Liver from Stege Marsh Fish (A). Severe MA vacuolar degeneration in liver. A basophilic focus is a pre-neoplastic lesion and is indicative of contaminant exposure. Glycogen depletion and lipidosis in Stege Marsh Gillichthys liver: lipid has replaced normal glycogen, and the basophilia corresponds to an increase in organelles (rough endoplasmic reticulum, mitochondria, etc.) (B).

Presently, extensive data analyses are being conducted in collaboration with EIC researchers, starting at the level of the individual fish. The approach is a “bottom-up” one that will enable Gillichthys to tell us about its habitat quality and proceeds in a step-wise manner. This involves the description of interrelationships among fish biomarkers and their responses in space using principal component analyses that are conducted independently of the environment. Then we will use spatial patterns to select environmental and chemical variables that will be conducted independent of the fish. Finally, we will examine relationships between fish and environmental indices. This will enable us to define the formal analyses in a comprehensive manner, which will allow indicators to be specified.

Developing A Larval Fish Species for Stressor Effects on Growth, Respiration, and Biomarker Responses

A manuscript describing experiments using larval topsmelt exposed to cadmium has been submitted for publication and describes the linkages between exposure to a metal (cadmium) and larval fish physiology and growth. These data are being used to develop a dynamic energy budget model for larval estuarine fish. These include growth (based on weight and otoliths), respiration, food consumption, and apoptosis in different tissues. In addition, the biomarker for metal exposure, metallothionein, was measured in larval tissue and was found to increase at the higher cadmium concentrations.

An additional manuscript, recently published in Environmental Toxicology and Chemistry, describes how otolith growth rate analyses developed for this work can be used to improve upon routine larval toxicity tests used by the U. S. Environmental Protection Agency to regulate municipal and industrial waste discharges.

Conclusions

We concluded that the fish indicator we have utilized is extremely useful for predicting the stress associated with wetland sites in California. Gillichthys is ubiquitous to salt marshes on the west coast and caused by its behavior and physiology (e.g., limited home range, sexually dimorphic, etc.), it is an excellent indicator for determining stressor levels on an endemic species. We have shown that perturbation of reproduction, including clear instances of endocrine disruption, can be observed in both wild collected fish, as well as in experimental outplants. Because the indicators we have employed with Gillichthys is conserved highly in the teleosts, we can transfer these approaches to most wetland fish species with limited modifications. Data analyses that will describe the inter-relationships between specific biomarkers and overall fish health are ongoing, as well as the relationship to environmental stressors at specific stations within the marshes. Lastly, we have completed a study on larval fish responses to a common stressor at our sites, cadmium, and are now using the key parameters to establish a dynamic energy budget model that will be applicable to other species as well. Our research will result in the development of new methodologies, single indicators, and complex multivariate indicator portfolios using the fish indicator species.

Future Activities:

During Year 5 of the project, we will complete the two remaining primary goals. Our first objective is to complete the statistical analysis and models of linkages between physiological/biochemical responses to stress, then to body burdens and sediment loads of contaminants, and finally to organismal fitness parameters. This task will be accomplished as soon as remaining analyses of body burdens and biomarkers (physiological as well as organismal) are completed. Second, the majority of our effort will be focused on preparing manuscripts for publication. We will continue to present our results at scientific meetings and increase the level of outreach to potential user groups regarding the applicability of individual and aggregate indicators to their programs in conjunction with the integration component of PEEIR.

References:

Andreu-Vieyra CV, Habibi HR. Factors controlling ovarian apoptosis. Canadian Journal of Physiology and Pharmacology 2000;78(12):1003–1012.

Brooks AJ. Factors influencing the structure of an estuarine fish community: the role of interspecific competition (Gillichthys mirabilis, Leptocottus armatus). PhD Dissertation # AAT 9956163, University of California, Santa Barbara, CA, 1999, pp 212.

Choi SM, Yoo SD, Lee BM. Toxicological characteristics of endocrine-disrupting chemicals: Developmental toxicity, carcinogenicity, and mutagenicity. Journal of Toxicology and Environmental Health B-Critical Reviews 2004;7(1):1–32.

Cooke JB, Hinton DE. Promotion by 17 beta-estradiol and beta-hexachlorocyclohexane of hepatocellular tumors in medaka, Oryzias latipes. Aquatic Toxicology 1999;45(2-3):127-145.

Forrester GE, Fredericks BI, Gerdeman D, Evans B, Steele MA, Zayed K, Schweitzer LE, Suffet IH, Vance RR, Ambrose RF. Growth of estaurine fish is associated with the combined concentration of sediment contaminants and shows no adaption or acclimation to past conditions. Marine Environmental Research 2003;56(3):423-442.

Gavrieli Y, Sherman Y, Bensasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. Journal of Cell Biology 1992;119(3):493-501.

Kudo C, Wada K, Masuda T, Yonemura T, Shibuya A, Fujimoto Y, Nakajima A, Niwa H, Kamisaki Y. Nonylphenol induces the death of neural stem cells due to activation of the caspase cascade and regulation of the cell cycle. Journal of Neurochemistry 2004;88(6):1416–1423.

Nimrod AC, Bensen WH. Environmental estrogenic effects of alkylphenol ethoxylates. Critical Reviews in Toxicology 1996;26(3):335-364.

Raffray M, Cohen GM. Apoptosis and necrosis in toxicology: A continuum or distinct modes of cell death? Pharmacology & Therapeutics 1997;75(3):153-177.

Rogers JM, Denison MS. Recombinant cell bioassays for endocrine disruptors: development of a stably transfected human ovarian cell line for the detection of estrogenic and anti-estrogenic chemicals. In Vitro & Molecular Toxicology 2000;13(1):67-82.

Soto AM, Chung KL, Sonnenschein C. The pesticides endosulfan, toxaphene, and dieldrin have estrogenic effects on human estrogen-sensitive cells. Environmental Health Perspectives 1994;102(4):380-383.

White R, Jobling S, Hoar SA, Sumpter JP, et al. Environmentally persistent alkylphenolics compounds are estrogenic. Endocrinology 1994;135(1):175-182.

Yoklavich MM, Cailliet GM, Barry JP, Ambrose DA, et al. Temporal and spatial patterns in abundance and diversity of fish assemblages in Elkhorn Slough, California. Estuaries 1991;14(4):465–480.


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 133 publications 35 publications in selected types All 34 journal articles

Type Citation Sub Project Document Sources
Journal Article Rose WL, Hobbs JA, Nesbit RM, Green PG, Cherr GN, Anderson SL. Validation of otolith growth rate analysis using cadmium-exposed larval topsmelt (Atherinops affinis). Environmental Toxicology & Chemistry 2005;24(10):2612-2620. R828676 (Final)
R828676C002 (2004)
 not available
Supplemental Keywords:

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

http://www.bml.ucdavis.edu/peeir exit EPA

Progress and Final Reports:
2002 Progress Report
2003 Progress Report
Original Abstract
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

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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