Grantee Research Project Results

Final Report: 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: UC Davis Center for Children's Environmental Health and Disease Prevention
Center Director: Van de Water, Judith
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

Objective:

The overall objective of this research project was to use benthic invertebrates as indicators of sediment deposition and changes in water quality. The specific objective was to investigate cadmium (Cd) and selenium (Se) stress indicators in aquatic invertebrates prevalent in the Sacramento and San Joaquin Watersheds. A Cd pollution gradient has been established in the Sacramento River/Delta/San Francisco Estuary by Dr. San Luoma's group at the U.S. Geological Survey (USGS) in Menlo Park, California. Se contamination has been a heated issue in the San Joaquin River and San Francisco Estuary because of waste discharge from oil refineries and agricultural drainage discharge via the San Luis drain in the western San Joaquin Valley. The multiple-stress situation in these environments has made it extremely difficult to discern the ecological impact of a given pollutant (such as Cd or Se). We developed a biochemical indicator assay with the aim of resolving the effect(s) of Cd and Se on resident macroinvertebrates in these environments.

Summary/Accomplishments (Outputs/Outcomes):

Our rapid and convenient assay for sulfhydryl (SH)-rich proteins and peptides (Fan, et al., in press, 2004) is useful for monitoring the Asian clam (Potamocorbula amurensis) in particular, but eventually could be applied to other organisms. Thiol-rich peptides such as phytochelatins and metallothioneins (MTs) are important cellular chelators, which function in metal detoxification and/or homeostasis. This electrophoresis-based method allows the broad screening of thiol-rich peptides and proteins. Sample throughput is substantially improved over chromatography-based methods because of parallel sample analysis in 1 hour of electrophoretic separation. This method is versatile in that peptides ranging from glutathione to large proteins can be analyzed by simple modification(s) of the extraction and electrophoretic conditions. Moreover, the broad-screen, higher throughput nature of the method supports serendipitous detection of unexpected or novel thiol metabolites.

We observed three MT-like proteins (upper, middle, and lower bands from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in clams. Amino acid analysis of these three proteins indicated that they were isoforms, and that they had amino acid compositions similar to that of MTs isolated from other aquatic invertebrate species. Therefore, it is reasonable to assume that these proteins are isoforms of MT. Our analysis indicated that two isoforms were associated with copper and zinc 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 applied this analysis to a number of clam samples obtained from 18 months (July 1996 to January 1998) of sampling at established USGS field sites: the mouth of the Sacramento River (site 4.1), Suisun Bay (site 6.1), Carquinez Strait (site 8.1), and San Pablo Bay (site 12.5). Both sediment and clam tissue Cd levels showed a statistically significant (p < 0.05; USGS data supplied by Dr. Cindy Brown) difference from site 4.1 (contaminated) to site 12.5 (relatively less contaminated). It is likely that one of these MTs represents a Cd-specific indicator, and that Cd pollution in the San Francisco Estuary has impacted resident clams.

Regarding Se indicators, we hypothesized that microphytes would harbor the Se analogue of dimethylsulfoniopropionate (DMSP), a prevalent osmoregulant in phytoplankton. We developed an approach for analyzing such compounds, and we used it to demonstrate the existence of the Se analogue for DMSP, dimethylselenoniumproprionate (DMSeP) (Larsen, et al., 2001).

Despite the possible prevalence of compounds such DMSeP in lower food chain items, we hypothesized that the likely candidates for bioaccumulation, bioavailability, and toxic effects would include proteinaceous Se or selenoamino acids such as selenomethionine (Se-Met). We based this hypothesis on dietary efficiency considerations up the food chain, and we supported it with our previous findings in Se biogeochemistry (Fan, et al., 1997; Fan and Higashi, 1998). It also was supported by the dominance of proteinaceous Se and Se-Met in the aquatic food chain—particularly in top predatory bird and fish species (e.g., Fan, et al., 1998; Fan and Higashi, 2000; Fan, et al., 2002a). Although low in free Se-Met, microphytes isolated from Se-laden waters also were abundant in proteinaceous Se-Met.

This prompted a more extensive survey of Se speciation in food chain organisms (including microphytes, macroinvertebrates, fish, and bird embryos) residing mainly in the agricultural drainage systems of San Joaquin Valley, California (Fan, et al., 2002a). We measured total Se in biomass, water-soluble fractions, and protein-rich fractions, along with gas chromatography-mass spectrometry (MS) analysis of proteinaceous Se-Met. We also correlated the adverse effect (histopathological abnormality) of selenium on the reproductive system with Se biochemical markers in selected top predator fish species collected in the field, in a collaborative effort with Drs. D. Hinton's and S. Teh's groups (Fan, et al., 2002a). Proteinaceous Se in the omnivorous carp (Cyprinus carpio) liver demonstrated a relation to ovarian lesions, but deformed stilt (Himantopus mexicanus) embryos were more abundant in proteinaceous Se-Met than normal embryos. Although limited in the number of organisms surveyed, these findings provide an impetus for further field and laboratory feeding studies to substantiate the hypothesis that proteinaceous selenomethionine has an adverse effect on the reproductive systems of aquatic organisms. Most recently, we found a very high Se burden (> 50 µg/g) in the ovaries of a threatened fish species (splittail) collected around the Sacramento/San Joaquin River Delta area.

Lastly, we developed an approach that can directly assess the Se load in high molecular weight proteins by combining denaturing gel electrophoresis and ultrasensitive laser ablation (LA)-inductively coupled plasma (ICP)-MS (Fan, et al., 2002b). Protein extracts of an avocet embryo and a largemouth bass ovary collected from Se-laden San Francisco Bay/Delta waters were examined, and they revealed eight and five distinct bands of Se-containing proteins, respectively. Quantitative Se analysis of these proteins using LA-ICP-MS seems promising, but awaits further development.

The following activities were accomplished:

• We developed a rapid and inexpensive electrophoresis-based assay for SH-rich proteins, a bioindicator of toxic metal exposure and toxicity compensation. This method is a substantial improvement over chromatography-based methods because of parallel sample analysis in 1 hour of electrophoretic separation. This method is versatile in that peptides ranging from glutathione to large proteins can be analyzed by simple modification(s) of the extraction and electrophoretic conditions. Moreover, the broad-screen, higher throughput nature of the method supports serendipitous detection of unexpected or novel thiol metabolites. This assay will allow investigators to quickly diagnose metal exposure and toxicity response in aquatic organisms and environments.

• Using our new assay, we observed three MT-like proteins (isoforms) in clams. One of these isoforms had an affinity for Cd and may be indicative of a detoxification mechanism in the Asian clam. This finding will be of interest to investigators studying cadmium toxicity abatement in aquatic organisms.

• We developed a method for trace Se organic form analysis based on liquid chromatography (LC)-ICP-MS and LC-MS, and we then showed that a major sulfur compound in algae has a selenium analogue, a previously unknown major organic form of Se with implications for both food chain accumulation and volatile dissipation (detoxification) of Se. The discovery of such potentially major unknown forms can alter the view of how Se is accumulated up the food chain.

• We published key Se data on food chain items and top predators, showing the possibility of proteinaceous Se—and in particular proteinaceous Se-Met—as the ecotoxic (accumulative) form of Se in aquatic wildlife. By identifying this specific form of Se as the accumulative form, investigators and resource managers can better target further studies and toxicity mitigation efforts for the most effective results.

• We developed a new method for directly assessing proteinaceous Se, particularly in terms of the protein profiles. This method will be used in future studies.

• Studies funded by the U.S. Environmental Protection Agency (EPA)/Center for Ecological Health Research (CEHR) led to a fully funded study on dietary Se and Se biochemical forms in a U.S. Fish and Wildlife Service-listed "threatened" fish (splittail) in the bay/delta. This will allow us to establish a cause-and-effect relationship between reproductive impairment and biotoxicity markers in a wild organism, for more definitive information about pollutant food chain effects.

• Studies supported by the U.S. EPA/CEHR helped to organize a special issue of Aquatic Toxicology dedicated to the ecotoxicity of Se (Fan, et al., 2002). This issue made information regarding Se ecotoxicity widely available to investigators, and will hopefully generate much collaboration.

Supplemental Keywords:

ecosystem, ecosystem protection, environmental exposure and risk, geographic area, international cooperation, water, terrestrial ecosystems, aquatic ecosystem, aquatic ecosystem restoration, aquatic ecosystems and estuarine research, biochemistry, ecological effects, ecological indicators, ecological monitoring, ecology and ecosystems, environmental chemistry, restoration, state, water and watershed, watershed, watershed development, watershed land use, watershed management, watershed modeling, watershed restoration, watershed sustainability, agricultural watershed, exploratory research environmental biology, California, CA, Clear Lake, Lake Tahoe, anthropogenic effects, aquatic habitat, biogeochemical cycling, ecological assessment, ecology assessment models, ecosystem monitoring, ecosystem response, ecosystem stress, environmental stress, environmental stress indicators, fish habitat, hydrologic modeling, hydrology, integrated watershed model, lake ecosystems, lakes, land use, nutrient dynamics, nutrient flux, water management options, water quality, wetlands., RFA, Scientific Discipline, INTERNATIONAL COOPERATION, ECOSYSTEMS, Geographic Area, Water, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, Water & Watershed, Ecosystem/Assessment/Indicators, Environmental Chemistry, State, Aquatic Ecosystem, Ecological Effects - Environmental Exposure & Risk, Terrestrial Ecosystems, Biochemistry, Environmental Monitoring, Ecological Monitoring, Ecology and Ecosystems, Aquatic Ecosystem Restoration, Ecological Indicators, Watersheds, 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

Relevant Websites:

http://ice.ucdavis.edu/cehr/ Exit

Progress and Final Reports:

Original Abstract

1997

1998

1999

Main Center Abstract and Reports:

R825433    UC Davis Center for Children's Environmental Health and Disease Prevention

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