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Final Report: Biomarkers of Exposure and Deleterious Effect: A Laboratory and Field Investigation
EPA Grant Number: R825433C004Subproject: this is subproject number 004 , 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: Biomarkers of Exposure and Deleterious Effect: A Laboratory and Field Investigation
Investigators: Hinton, David E.
Institution: University of California - Davis
EPA Project Officer: Levinson, Barbara
Project Period: October 1, 1996 through September 30, 2000
RFA: Exploratory Environmental Research Centers (1992)
Research Category: Center for Ecological Health Research , Targeted Research
Description:
Objective:Biomarkers can be useful tools in understanding the complex interactions that govern organism responses to environmental stressors and their sublethal effects on organism health. The objective of this research project was to investigate two types of biomarkers—stress proteins and histopathologic alterations—in the euryhaline clam (Potamocorbula amurensis). We tested the utility of these biomarkers as indicators of exposure and sublethal effects of environmental stressors in northern San Francisco Bay and the delta, formed by the confluence of the Sacramento and San Joaquin Rivers. Our field investigations included long-term monthly sampling of resident clams from four sites in northern San Francisco Bay and the delta, as well as in situ exposures of caged clams at sites within the upper San Francisco Bay. We also included laboratory exposure studies using individual xenobiotic compounds that had been identified as important toxicants in our field studies. In addition, we investigated the influence of physical stressors, such as salinity, on our biomarkers.
Summary/Accomplishments (Outputs/Outcomes):In our first field study, we collected clams to assess stress proteins (hsp70) and histological alterations of the reproductive system, digestive gland (DG), gill, and kidney. Contrary to our expectations, stress protein levels, which increased with rising cadmium concentrations in our laboratory experiment, were lowest in clams from the station with high cadmium and low salinity, and were highest at the station with low cadmium and high salinity. The results from laboratory exposures to a range of salinities showed that the ability to raise cellular hsp70 levels in response to heat shock was impaired in P. amurensis when exposed to very low salinity.
In our second field study, clams were outplanted for 7 days at two sites offshore of Mare Island Naval Shipyard, San Pablo Bay: a reference site and a site at which previous sediment bioassays had demonstrated toxicity to Eohaustorius estuarius (Amphipoda). Tissue levels of hsp70 and hsp76 were significantly reduced in clams exposed for 7 days at the reference site than in both controls and clams from site PP. Control clams showed significantly elevated stress protein levels after 7 days when compared to clams from the starting date of the field exposure.
Our first laboratory study was designed to assess and validate potential sublethal toxicity of hexavalent chromium (Cr-VI) in clams under controlled laboratory conditions. For 7 days, three replicates of clam were exposed to solutions of various Cr-VI concentrations. Mortality reached 100 percent in the highest concentration group within 7 days. There was no significant difference in mortality among the other groups. Western blot analyses revealed significantly elevated stress protein hsp70 levels in the second highest concentration group. Histopathologic analyses revealed mild DG atrophy in the control group. Clams exposed to 0.92 ppm Cr-VI showed moderate DG atrophy, moderate granulomatous inflammation, and necrosis in DG, ovary, and testis. Lesions observed in the 8.40 ppm treatment group included severe DG atrophy, severe granulomatous inflammation, and necrosis in the byssal gland, DG, gill, kidney, ovary, and testis. Our studies support a cause-effect relationship between contaminants and reduced health in Asian clams, and indicate that the DGs, gills, and reproductive organs are principal targets of Cr-VI toxicity at sublethal concentrations. The results from this study suggest that Cr-VI may have played a critical role in the increased incidence of diseased clams seen in our field studies, and that these adverse effects may be contributing to a decrease in clam population size at sites with the highest metallic contamination in the San Francisco Bay Estuary.
In our second laboratory study, cadmium (one of the main xenobiotic compounds detected in clam tissues from our field site) induced hsp70 expression in P. amurensis in a 2-week exposure, suggesting that our hsp70 field results did not reflect a response to cadmium exposure. In addition, a histological assessment of cadmium-exposed clams indicated an increased occurrence of focal necrosis of the ovary and mucus cell swelling in the gill as cadmium concentrations increased.
Finally, we conducted a laboratory experiment on the influence of salinity on stress protein expression. Resident clams collected from a site in northern San Francisco Bay at a salinity of 0.5 ppt had very low levels of hsp70, and were unable to increase cellular hsp70 concentrations in response to heat shock. Clams collected from sites with salinities of 5.6 and 14.9 ppt had increased significantly hsp70 levels in response to heat shock.
Significant differences in hsp70 levels also were found between clams exposed in the laboratory to different salinities, but these differences were not as dramatic as those in clams collected from different field sites. This discrepancy may be because of the constant salinities clams were exposed to in the laboratory experiment; clams in the field are subject to tidal fluctuations and must constantly adapt to changing environmental conditions.
The following activities were accomplished:
• Overall, our studies indicate that an understanding of the condition of the host is necessary to interpret stress protein induction, or lack thereof, in field-collected organisms. Our studies showed that the influence of physical stressors, such as low salinity, can reduce an organism's ability to induce stress proteins. This can lead to an increased susceptibility to other stressors, and it can impact the overall health of the organism.
• Results from laboratory exposures to a range of salinities showed that the ability to raise cellular hsp70 levels in response to heat shock was impaired in P. amurensis when exposed to osmotic stress. This is an important finding for: (1) evaluating stress in field-collected organisms; and (2) assessing the impact of multiple stressors on these organisms. The inability to raise stress protein levels in clams experiencing osmotic stress may render them more susceptible to additional chemical or physical stressors.
• We correlated histopathologic lesions with stress intensity in all experiments in which clams were exposed to chemical stressors (reflecting conditions in the field). Histopathology remains a strong tool in demonstrating exposure and adverse effect. This finding suggests that chemical stressors have a cause-and-effect relationship with histopathologic lesions in clams. Environmental protection managers can take these data into consideration when assessing the health of an ecosystem and its components.
• Analysis of cellular stress protein levels, in combination with other health indicators, can aid in understanding the physiological condition of the organism, as well as identify cellular stress caused by xenobiotic compounds (cadmium, Cr-VI) in laboratory studies. So far, however, complex response patterns preclude their application as stress indicators in field studies in which multiple stressors are present. Exposure of organisms to multiple xenobiotic and physical stressors can lead to upregulation as well as downregulation of heat shock proteins. The response is dependent on the nature of the stressor and the physiological condition of the organism. This information should be taken into account by other researchers investigating stress responses in field organisms; this "masking" of stress response in the field potentially could lead to false negatives.
• Our studies support a cause-effect relationship between contaminants and reduced health in Asian clams and indicate that the DG, gills, and reproductive organs are principal targets of hexavalent chromium (Cr-VI) toxicity at sublethal concentrations. The results from this study suggest that Cr-VI may have played a critical role in the increased incidence of diseased clams seen in our field studies. These adverse effects may be contributing to a decrease in clam population size at sites with highest metallic contamination in the San Francisco Bay Estuary. Our results should be carefully considered by environmental managers and policymakers in the San Francisco Bay Area and other aquatic environments suffering from heavy metal contamination.
• Linking histopathology to other biomarkers (biochemical and molecular), we enhanced the combined approaches' strengths—the ability to demonstrate adverse effect through histopathology and the ability to quantify response through biochemical and molecular-based markers. The ability to realistically interpret conditions at one or more sites depends on the use of a suite of biomarker reactions/responses. For example, the inability to induce stress proteins may enhance the susceptibility of cells to environmental stressors, leading to an increase in histopathological lesions.
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. , Ecosystem Protection/Environmental Exposure & Risk, Water, INTERNATIONAL COOPERATION, Geographic Area, Scientific Discipline, RFA, ECOSYSTEMS, Ecosystem/Assessment/Indicators, Water & Watershed, Restoration, Aquatic Ecosystem Restoration, Aquatic Ecosystems & Estuarine Research, Terrestrial Ecosystems, Ecological Monitoring, Aquatic Ecosystem, Ecological Indicators, Biochemistry, Watersheds, Environmental Chemistry, Ecological Effects - Environmental Exposure & Risk, Ecosystem Protection, Ecology and Ecosystems, Environmental Monitoring, State, water quality, California (CA), wildlife habitat, watershed modeling, watershed land use, watershed, watershed management, watershed restoration, Clear Lake, hydrologic modeling, ecology assessment models, Clear Lake , environmental stress indicators, anthropogenic effects, fish habitat, land use, wetlands, watershed development, aquatic habitat, agricultural watershed, aquatic ecosystems, environmental stress, lake ecosysyems, watershed sustainablility, ecological assessment, hydrology, water management options, ecosystem stress, ecosystem response, ecosystem monitoring, nutrient dynamics
Relevant Websites:
Progress and Final Reports:
2000 Progress Report
Original Abstract
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