2003 Progress Report: Development and Evaluation of Chemical Indicators for Monitoring Ecological Risk

EPA Grant Number: R828675C005
Subproject: this is subproject number 005 , established and managed by the Center Director under grant R828675
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: EAGLES - Great Lakes Environmental Indicators Project
Center Director: Niemi, Gerald J.
Title: Development and Evaluation of Chemical Indicators for Monitoring Ecological Risk
Investigators: Swackhamer, Deborah L. , Mount, David , Ankley, Gary , Burkhard, Lawrence , Simcik, Matthew , Cook, Philip , Erickson, Russell , Diamond, Steven
Institution: U.S. Environmental Protection Agency , University of Minnesota
Current Institution: University of Minnesota , U.S. Environmental Protection Agency
EPA Project Officer: Packard, Benjamin H
Project Period: January 10, 2001 through January 9, 2005 (Extended to January 9, 2006)
Project Period Covered by this Report: January 10, 2002 through January 9, 2003
RFA: Environmental Indicators in the Estuarine Environment Research Program (2000) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Water , Ecosystems


The overall objective of this subproject is to identify and validate effective contaminant indicators of adverse impacts on estuarine ecosystem health. Indicators will be developed in the Great Lakes but also will be applicable to both marine and freshwater ecosystems. These contaminant indicators will be used to evaluate ecological condition. Specifically, we will focus on the evaluation of (1) indicator polycyclic aromatic hydrocarbons (PAHs) of photo-induced toxicity to fish and benthic organisms; and (2) organic chemical indicators of xenoestrogenic exposure to fishes.

The effective assessment of ecological condition is best accomplished using integrative indicators of condition. These indicators should be cost-effective, be applicable across multiple scales, and provide useful information for environmental managers. Within the omnibus project, this contaminants subproject focuses on contaminant indicators that will provide a measure of condition of the estuarine ecosystem. These indicators also will serve as diagnostic indicators that will identify the primary stressors affecting the specific ecological endpoint of concern. We have focused on PAH compounds and environmental estrogens because they are widespread in the environment and have existing sources; therefore, they are of current concern.

The specific hypotheses we are testing are: (1) specific PAHs in combination with UV penetration are indicators of potential loss of vulnerable species within coastal fish and or benthic communities; and (2) specific chemicals are indicators of endocrine disruption in fish via the estrogen receptor. Data collected to test these hypotheses will be used to demonstrate the degree of usefulness of these two groups of indicator compounds as diagnostic indicators for estuarine ecosystems.

Our overall approach to this project is as follows. For both indicators, we will compare contaminant concentrations to a biological endpoint or condition across a gradient of nondegraded to highly degraded sites in approximately 20 locations studied by the other indicator project groups in the program. For the PAH photo-induced toxicity indicator, we will collect the necessary field data to test the model developed in the laboratory by the collaborators at the U.S. Environmental Protection Agency’s Mid-Continent Ecology Division (EPA-MED) (Diamond, Mount, and Erickson). These data include the concentrations of PAHs in sediment, larval fish, and oligochaetes (to determine the bioaccumulation factors and to provide the doses for the model); sediment photo-induced toxicity potential (assayed in the laboratory using the aquatic annelid Lumbriculus [laboratory test organism] and field sediments); and UV dose (obtained from field measurements). The toxicity that is predicted from the model will be compared to toxicity measured in the laboratory assay. Results will be used to calibrate the model, and independent field data will be used to validate the model. Although photo-induced toxicity has been studied extensively and its acute toxicity demonstrated in the laboratory, this will be the first field test of such an indicator.

The xenoestrogen indicators will be identified in an analogous manner. A suite of potential xenoestrogens will be measured in fish tissue, sediment, and/or water, and compared to vitellogenin induction in wild and caged male fish (a bioindicator of individual estrogen exposure) at the same gradient of sites. Using correlative statistical techniques, we will identify specific indicator xenoestrogens that are associated with vitellogenin induction. This would represent the first link of vitellogenin induction and chemical exposure in field sites other than near sewage treatment plants.

Progress Summary:

Our efforts in Year 1 of the project were focused largely on establishing and validating our field methods and assumptions.

Photo-Induced PAH Toxicity

Exposure of organisms to UV light and PAHs has the potential to excite certain PAHs that are internal to the organism (bioaccumulated) and cause acute cell damage and death. Larval fish are considered to be sensitive organisms because their transparency and behavior result in significant light exposure during this early life stage and because they have the potential for high PAH exposure as a result of high lipid content and incomplete enzyme systems that are not yet capable of metabolizing fully and/or depurating PAHs.

Methods for sediment collection and UV dose measurements are established and were tested successfully during the 2001 pilot studies. The diatom/water quality and fish/macroinvertebrate subprojects are collecting these samples and data. Our efforts focused on methods for collecting larval fish in the field. In pilot studies in Green Bay and southern Lake Superior, light traps were inefficient. Dip nets with mesh size of 500 µm or less were the most efficient method tested, but require significant manual sorting of the sample. The methodology for indexing photo-induced toxicity potential using Lumbriculus exposed to field-collected sediments has been well established at EPA-MED. Summer research at MED included developing methods for exposing larval fish in situ at contaminated sites, which will be an integral part of establishing the connectivity of sediment chemistry, Lumbriculus response, and effects on larval fish.

Endocrine Disruption From Environmental Estrogen Exposure

The development of this indicator requires that we establish a link between the occurrence of specific chemicals in coastal regions and environmental estrogen exposure to fish as indicated by a serum protein, vitellogenin (Vtg). Vtg is a precursor to egg-yolk formation, and can be induced in both males and females in response to activation of the estrogen receptor. Vtg induction in males is an unambiguous indicator of exposure to exogenous and environmental estrogens. Our efforts focused on establishing methods to collect sufficient numbers of wild bullheads to demonstrate environmental estrogen exposure. Pilot studies revealed that Fyke nets were the most effective method for collections at sites where bullheads were plentiful. We also designed the necessary experiments needed to develop and validate the Vtg assay for the coming field season.

Year 2 of the project has been devoted to site selection and the collection of field samples. Some additional laboratory work at EPA-MED also was accomplished.

Site Selection

The process of selecting sites using a random process that would test our indicators in a representative fashion proved to be challenging, time-consuming, and successful. The site selection for contaminants was slightly different from the other subprojects. We have the fewest number of sites (20), and we have specific gradients of condition that we wanted to capture in our sites that were not necessarily the dominant variables of interest to the other groups. We worked closely with Nick Danz and Ron Regal of the Natural Resources Research Institute/University of Minnesota at Duluth, who developed a process that was a model for the others. More than 700 potential sites were identified (called segment sheds, which are defined as the watershed and shoreline length corresponding to the area draining second-order tributaries and greater). The associated stressor data for more than 200 variables were collected into a database. A principal components analysis of key variables relating to agricultural activity and industrial activity was completed, resulting in a two-dimensional space accounting for about 70 percent of the variance. Sites were selected randomly from within given percentiles of the distributions, with a weighting of more contaminated sites relative to less contaminated sites. This resulted in sites containing high industry and high agricultural influence, those with high industry and low agricultural influence, those with low industry and high agricultural influence, and those with low industry and low agricultural influence. Sites also were stratified to be divided equally between the two ecoprovinces, and to include all five Great Lakes. A few sites were handpicked to ensure the inclusion of a given type of pollution, or to overlap with other research in the area. A total of 22 sites were eventually compiled.

Sites were visited on two occasions: (1) in the spring to collect larval fish; and (2) again in mid-summer to collect wild fish and to deploy caged male fathead minnows.

PAH Photo-Induced Toxicity Indicator

We successfully collected larval fish at 17 sites using dip nets from a small boat or by wading. The largest larval biomass in the nearshore region is thought to be yellow perch, so we concentrated our efforts during the post-spawning periods in each lake. Samples were protected from sunlight while processing and frozen for transport back to the UMN campus. Samples are currently being extracted and analyzed for a suite of 37 photoactive and nonphotoactive PAHs. Some additional time has been spent on optimizing the PAH analytical methods.

Sediments were collected from approximately 50 sites (sample collections were done by the fish/macroinvertebrate subproject) for the purpose of screening them for their PAH photo-induced toxicity potency. This was to help bound the extent of this potential response and to identify any interesting sites for more intensive investigation. These sediments currently are being assayed using the Lumbriculus assay at MED.

Water samples (a total of 28) were collected at all 22 contaminant sites, and the UV extinction coefficients were calculated from the absorbance readings of these samples. These data then were used to model the UV exposure at all sites for future use in the toxicity model. They were compared to the measured UV dose obtained from field measurements at five of the sites, and correlated well.

Additional experiments have been conducted at EPA-MED to determine the kinetics and magnitude of PAH bioaccumulation in larval fish and to evaluate responses of fish larvae to ambient PAH and sunlight during in situ exposures at a PAH-contaminated site in the Great Lakes. Preliminary findings indicate that accumulation of three waterborne PAHs by fathead minnows reached equilibrium within 24-48 hours, regardless of life stage (embryo versus larval). Benzo[a]pyrene appeared to be metabolized, even in embryos. Based on these experiments, it appears that starting experiments with naive larvae will not appreciably reduce PAH exposure relative to experiments with embryos.  The in situ experiments showed reduced survival and growth in fish larvae exposed to ambient conditions, with a dose-related reduction in these effects with decreasing sunlight exposure.  These data and their interpretation are still being finalized.

Environmental Estrogen Indicator

Our pilot field activities indicated to us that we could not be certain of finding sufficient numbers of bullheads at all sites, so we added the exposure of caged male fathead minnows to our design. Our concerns were realized; Fyke netting at 22 sites did not collect sufficient numbers of animals at any of the sites. This is partially caused by the random site selection process (bullheads are not randomly distributed and prefer certain habitats), the fact that populations move with changing water temperatures, and that we were limited in the amount of effort we could devote to net deployments (24-48 hour sets). The caged fish were a more controlled alternative to assay the potency of each site. Three cages per site, containing 10 fish each, were deployed for 8-day periods at 10 sites (there were not enough minnows commercially available to do all sites). Preliminary assessments of the minnows from three of the most contaminated sites indicted that there was no discernible Vtg induction. Anticipating that exposures may be too short to detect Vtg production, we also invested considerable resources in developing the methods to detect the mRNA for Vtg production, which will be produced more quickly than the protein itself. This is a more complex technique, and we are working with Dr. Nancy Denslow at the University of Florida (of the Gulf EaGLe Center) to assess the applicability of this method to our study. To this end, we are conducting a series of exposure experiments to determine the best endpoint for measuring estrogenic exposures in fish in the field, and these endpoints include Vtg, mRNA for Vtg, zona radiata protein (Zrp, an eggshell protein), and the mRNA for Zrp.

We also have devoted time to developing and validating the analytical methods needed to measure approximately 12 classes of estrogenic compounds.

Future Activities:

Our future activities will focus on both laboratory and fieldwork. First, we will define the best endpoint for determining estrogenicity in our field samples using laboratory and some controlled field experiments. Second, we will deploy caged fish at all sites and determine their response. This will be followed up by intensive chemical characterization of the site for environmental estrogenic compounds.

In the coming year, we will focus on three areas. The first is to determine the PAH concentrations in all fish larvae and sediment samples from the last field season and to determine the toxicity potency of the sediments in the laboratory. Second, we will integrate and synthesize the PAH, UV, and sediment potency data into the toxicity model. Finally, additional field samples will be collected, as needed, for calibration and validation of the model.

Journal Articles:

No journal articles submitted with this report: View all 22 publications for this subproject

Supplemental Keywords:

polycyclic aromatic hydrocarbons, PAHs, Great Lakes, GLEI, EaGLes, contaminant indicators, toxicity, estuarine ecosystems, photo-induced toxicity indicator, xenoestrogens,, RFA, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, Geographic Area, ECOSYSTEMS, Ecosystem Protection/Environmental Exposure & Risk, exploratory research environmental biology, Ecosystem/Assessment/Indicators, Ecosystem Protection, Monitoring/Modeling, Ecological Effects - Environmental Exposure & Risk, Environmental Monitoring, Ecological Monitoring, Ecological Risk Assessment, Ecology and Ecosystems, Great Lakes, Ecological Indicators, Risk Assessment, ecological condition, coastal ecosystem, anthropogenic stress, biodiversity, ecosystem assessment, environmental measurement, coastal environments, PAH, ecological assessment, ecosystem indicators, analytical chemistry, estuarine ecoindicator, xenoestrogen indicators, aquatic ecosystems, ecological risk, environmental stress, water quality, environmental estrogens, ecological models, fish models, ecological response

Relevant Websites:

http://glei.nrri.umn.edu Exit

Progress and Final Reports:

Original Abstract
  • 2001
  • 2002
  • 2004 Progress Report
  • Final Report

  • Main Center Abstract and Reports:

    R828675    EAGLES - Great Lakes Environmental Indicators Project

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R828675C001 Great Lakes Diatom and Water Quality Indicators
    R828675C002 Vegetative Indicators of Condition, Integrity, and Sustainability of Great Lakes Coastal Wetlands
    R828675C003 Testing Indicators of Coastal Ecosystem Integrity Using Fish and Macroinvertebrates
    R828675C004 Development and Assessment of Environmental Indicators Based on Birds and Amphibians in the Great Lakes Basin
    R828675C005 Development and Evaluation of Chemical Indicators for Monitoring Ecological Risk