Resolving Multiple Stressors by Biochemical Indicator Patterns and their Linkages to Adverse Effects on Benthic Invertebrate Patterns

EPA Grant Number: R825433C006
Subproject: this is subproject number 006 , established and managed by the Center Director under grant R825433
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: EERC - Center for Ecological Health Research (Cal Davis)
Center Director: Rolston, Dennis E.
Title: Resolving Multiple Stressors by Biochemical Indicator Patterns and their Linkages to Adverse Effects on Benthic Invertebrate Patterns
Investigators: Fan, Teresa W-M. , Higashi, Richard M.
Institution: University of California - Davis
EPA Project Officer: Packard, Benjamin H
Project Period: October 1, 1996 through September 30, 2000
RFA: Exploratory Environmental Research Centers (1992) RFA Text |  Recipients Lists
Research Category: Center for Ecological Health Research , Targeted Research


To use benthic invertebrates as indictors of sediment and water quality.


The investigators have studied the Cd and Se stress indicators in aquatic invertebrates prevalent in the Sacramento and San Joaquin Watersheds. Se contamination has been a heated issue in the San Joaquin River and San Franciso estuary due to waste discharge from oil refineries and agricultural drainage discharge via San Luis Drain in the western San Joaquin Valley. The multiple stress situation in these environments has made it extremely difficult to discern the ecologic impact of a given pollutant such as Cd or Se. They have taken the biochemical indicator approach with the aim of resolving the effect(s) of Cd and Se on resident macroinvertebrates in these environments.

The investigators have developed a rapid and convenient assay for sulfhydryl (SH) rich proteins and peptides from the Asian clam Potamocorbula amurensis. This assay involved fluorescence tagging of the SH rich peptides, followed by polyacrylamide gel electrophoretic (SDS-PAGE) separation, and fluorescence imaging to quantify the amount of each protein or peptide. Three metallothionein (MT)-like proteins (upper, middle, and lower bands from SDS-PAGE) were observed that demonstrated differential metal ion binding affinity (as analyzed by capillary electrophoresis/ICP-MS, CE/ICP-MS. Amino acid analysis of these three proteins indicated that they were isoforms and that they had amino acid composition similar to those of MTs isolated from other aquatic invertebrate species. Therefore, it is reasonable to assume that these proteins are isoforms of MT. The CE/ICP-MS analysis indicated that two isoforms were associated with Cu and Zn and may be related to homeostatic control of these essential metals. A third isoform had affinity for Cd and may be indicative of a detoxification mechanism in the Asian clam. We have applied the analysis to a number of clam samples obtained from 18 months (July. 96 to Jan. 98) of sampling at established USGS field sites: the mouth of Sacramento river (site 4.1), Suisun Bay (site 6.1), Carquinas Strait (site 8.1), and San Pablo Bay (site 12.5). Both sediment and clam tissue Cd levels showed statistically significant (p < 0.05) difference from site 4.1 (contaminated) to site 12.5. All three MTs exhibited seasonal variations, of which the upper and middle bands exhibited a similar pattern while the pattern for the lower band differed. The average tissue content of the lower band MT had a statistically significant (p <<< 0.05) positive correlation with the Cd tissue content of clams collected from the four sites, while no correlation was observed for the upper and middle bands. Therefore, it is most likely that the lower band MT represent a Cd-specific indicator and that the resident clam of the San Francisco estuary was impacted by Cd pollution.

Regarding Se indicators, they hypothesized that the likely candidates will include proteinaceous Se or selenoamino acids such as selenomethionine (Se-Met). This was largely based on previous findings in Se biogeochemistry and the dominance of proteinaceous Se and Se-Met in aquatic foodchain and top predator bird and fish species, in particular. To test this hypothesis, they are taking two approaches. One approach is to correlate the adverse effect (histopathological abnormality) on reproductive system with Se biochemical markers in selected top predator fish species collected in the field. Preliminary results indicated that histoplathological lesions in the ovaries of common carp collected from Se contaminated areas were evident and correlated with total Se load in the ovary. Most recently, we have found very high Se burden (> 50 µg/g) in the ovaries of threatened fish species (i.e. splittail) collected around the Sacramento/SJ river Delta area. Correlation of Se data with histopathology for this fish is underway. Moreover, preliminary speciation of selenoproteins by a combination of SDS-PAGE and laser ablation-ICPMS techniques indicated that only a handful of ovarian proteins extracted from field contaminated bird and fish species were enriched in Se. The other approach is to conduct laboratory feeding studies on resident invertebrate and fish species so that a cause-and-effect relationship between reproductive impairment and Se biochemical markers can be defined. The Se feeding experiment with crayfish (Procambarius clarkii) has been completed and tissue samples are being analyzed for total Se and selenoproteins. A similar feeding study on splittail has been initiated and samples will be taken periodically for Se and histopathological analyses.

Expected Results:

For this final year, the investigators will complete analyses of crayfish for the prevalent Se form(s) that are transferred from the diet to the ovary, exoskeleton, and other organs. They will continue to analyze for the abundance of proteinaceous Se-Met in ovaries and livers of field-collected fish species to correlate with histopathological lesions. Moreover, we will attempt to complete the analysis for the splittail feeding experiment and interpret the data as they are generated.

Supplemental Keywords:

wetlands, biomarkers, fish, multiple stressors, aquatic ecosystems, benthic invertebrates, water quality., RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Geographic Area, Water, ECOSYSTEMS, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, Water & Watershed, Environmental Chemistry, Ecosystem/Assessment/Indicators, State, Aquatic Ecosystem, Ecological Effects - Environmental Exposure & Risk, Biochemistry, Environmental Monitoring, Terrestrial Ecosystems, Ecological Monitoring, Ecology and Ecosystems, Aquatic Ecosystem Restoration, Watersheds, Ecological Indicators, watershed development, wetlands, ecosystem monitoring, watershed management, fish habitat, watershed, anthropogenic effects, agricultural watershed, biogeochemcial cycling, aquatic habitat, watershed land use, watershed modeling, ecological assessment, hydrology, integrated watershed model, lakes, aquatic ecosystems, environmental stress, biochemical indicator , lake ecosysyems, water quality, watershed sustainablility, ecosystem stress, hydrologic modeling, California (CA), benthic invertebrate patterns, ecology assessment models, environmental stress indicators, water management options, wildlife habitat, ecosystem response, land use

Progress and Final Reports:

  • 1997
  • 1998
  • 1999
  • Final Report

  • Main Center Abstract and Reports:

    R825433    EERC - Center for Ecological Health Research (Cal Davis)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R825433C001 Potential for Long-Term Degradation of Wetland Water Quality Due to Natural Discharge of Polluted Groundwater
    R825433C002 Sacramento River Watershed
    R825433C003 Endocrine Disruption in Fish and Birds
    R825433C004 Biomarkers of Exposure and Deleterious Effect: A Laboratory and Field Investigation
    R825433C005 Fish Developmental Toxicity/Recruitment
    R825433C006 Resolving Multiple Stressors by Biochemical Indicator Patterns and their Linkages to Adverse Effects on Benthic Invertebrate Patterns
    R825433C007 Environmental Chemistry of Bioavailability in Sediments and Water Column
    R825433C008 Reproduction of Birds and mammals in a terrestrial-aquatic interface
    R825433C009 Modeling Ecosystems Under Combined Stress
    R825433C010 Mercury Uptake by Fish
    R825433C011 Clear Lake Watershed
    R825433C012 The Role of Fishes as Transporters of Mercury
    R825433C013 Wetlands Restoration
    R825433C014 Wildlife Bioaccumulation and Effects
    R825433C015 Microbiology of Mercury Methylation in Sediments
    R825433C016 Hg and Fe Biogeochemistry
    R825433C017 Water Motions and Material Transport
    R825433C018 Economic Impacts of Multiple Stresses
    R825433C019 The History of Anthropogenic Effects
    R825433C020 Wetland Restoration
    R825433C021 Sierra Nevada Watershed Project
    R825433C022 Regional Transport of Air Pollutants and Exposure of Sierra Nevada Forests to Ozone
    R825433C023 Biomarkers of Ozone Damage to Sierra Nevada Vegetation
    R825433C024 Effects of Air Pollution on Water Quality: Emission of MTBE and Other Pollutants From Motorized Watercraft
    R825433C025 Regional Movement of Toxics
    R825433C026 Effect of Photochemical Reactions in Fog Drops and Aerosol Particles on the Fate of Atmospheric Chemicals in the Central Valley
    R825433C027 Source Load Modeling for Sediment in Mountainous Watersheds
    R825433C028 Stress of Increased Sediment Loading on Lake and Stream Function
    R825433C029 Watershed Response to Natural and Anthropogenic Stress: Lake Tahoe Nutrient Budget
    R825433C030 Mercury Distribution and Cycling in Sierra Nevada Waterbodies
    R825433C031 Pre-contact Forest Structure
    R825433C032 Identification and distribution of pest complexes in relation to late seral/old growth forest structure in the Lake Tahoe watershed
    R825433C033 Subalpine Marsh Plant Communities as Early Indicators of Ecosystem Stress
    R825433C034 Regional Hydrogeology and Contaminant Transport in a Sierra Nevada Ecosystem
    R825433C035 Border Rivers Watershed
    R825433C036 Toxicity Studies
    R825433C037 Watershed Assessment
    R825433C038 Microbiological Processes in Sediments
    R825433C039 Analytical and Biomarkers Core
    R825433C040 Organic Analysis
    R825433C041 Inorganic Analysis
    R825433C042 Immunoassay and Serum Markers
    R825433C043 Sensitive Biomarkers to Detect Biochemical Changes Indicating Multiple Stresses Including Chemically Induced Stresses
    R825433C044 Molecular, Cellular and Animal Biomarkers of Exposure and Effect
    R825433C045 Microbial Community Assays
    R825433C046 Cumulative and Integrative Biochemical Indicators
    R825433C047 Mercury and Iron Biogeochemistry
    R825433C048 Transport and Fate Core
    R825433C049 Role of Hydrogeologic Processes in Alpine Ecosystem Health
    R825433C050 Regional Hydrologic Modeling With Emphasis on Watershed-Scale Environmental Stresses
    R825433C051 Development of Pollutant Fate and Transport Models for Use in Terrestrial Ecosystem Exposure Assessment
    R825433C052 Pesticide Transport in Subsurface and Surface Water Systems
    R825433C053 Currents in Clear Lake
    R825433C054 Data Integration and Decision Support Core
    R825433C055 Spatial Patterns and Biodiversity
    R825433C056 Modeling Transport in Aquatic Systems
    R825433C057 Spatial and Temporal Trends in Water Quality
    R825433C058 Time Series Analysis and Modeling Ecological Risk
    R825433C059 WWW/Outreach
    R825433C060 Economic Effects of Multiple Stresses
    R825433C061 Effects of Nutrients on Algal Growth
    R825433C062 Nutrient Loading
    R825433C063 Subalpine Wetlands as Early Indicators of Ecosystem Stress
    R825433C064 Chlorinated Hydrocarbons
    R825433C065 Sierra Ozone Studies
    R825433C066 Assessment of Multiple Stresses on Soil Microbial Communities
    R825433C067 Terrestrial - Agriculture
    R825433C069 Molecular Epidemiology Core
    R825433C070 Serum Markers of Environmental Stress
    R825433C071 Development of Sensitive Biomarkers Based on Chemically Induced Changes in Expressions of Oncogenes
    R825433C072 Molecular Monitoring of Microbial Populations
    R825433C073 Aquatic - Rivers and Estuaries
    R825433C074 Border Rivers - Toxicity Studies