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2000 Progress Report: Endocrine Disruption in Fish and Birds
EPA Grant Number: R825433C003Subproject: this is subproject number 003 , 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: Endocrine Disruption in Fish and Birds
Investigators: Fry, D. Michael , Hinton, David E. , Wilson, Barry W.
Institution: University of California - Davis
EPA Project Officer: Levinson, Barbara
Project Period: October 1, 1996 through September 30, 2000
Project Period Covered by this Report: October 1, 1999 through September 30, 2000
RFA: Exploratory Environmental Research Centers (1992)
Research Category: Center for Ecological Health Research , Targeted Research
Description:
Objective:This project evaluates the endocrine sensitive development of fish and birds using both in vitro approaches and whole animal field studies. The development of the central nervous system and reproductive tracts of embryos and newly hatched chicks exposed to endocrine active xenobiotics is an expanded focus of this subproject. Bird studies include Zebra Finches dosed as neonates with estradiol benzoate, octylphenol, methoxychlor and dicofol. We have evaluated the developmental changes to reproductive behavior by testing birds when they become adults. Field studies with Cliff Swallows (Petrochelidon pyrrhonota) are being conducted to evaluate the ecosystem effects of pesticides and endocrine disruptors in Yolo County. Methods are being developed to quantify alkylphenols and alkylphenol ethoxylates in food samples of wild swallows and from water samples taken from agricultural drains to evaluate the magnitude of estrogenic chemicals entering the watershed.
Progress Summary:Endocrine Disruptors and Effects on Development of Birds.
Andrea Erichsen's thesis data on the estrogenic exposure of newly hatched Zebra Finches was presented last year. The data showed significant developmental changes in adult reproductive behavior following neonatal dosing with environmentally relevant amounts of octylphenol and methoxychlor.
Oral dosing of ZF chicks on Days 5-11 after hatching with 3.8-380 µg/g body wt /day E2B produced altered courtship and singing behaviors in both males and females, expressed when the birds became adults. Female finches developed the ability to sing, and male finches exhibited reduced courtship and increased nesting behaviors. Male birds exhibited significantly increased latency in copulatory behavior compared to oil dosed controls.
Andrea's data indicate that adult male birds are not developmentally prevented from singing after oral dosing with estradiol benzoate or xenobiotics, although the types of songs they produce (courtship vs. general song) appear to be affected by treatment. Estrogen dosed male finches have reduced interest in females, and the males court females less than controls. Copulatory behavior is suppressed developmentally by estrogens, expressed quantitatively as an increase in latency of singing when a control stimulus female finch is introduced into the dosed male's test cage. Males also exhibit nest perching, or broodiness, a behavior similar to broody female behavior during incubation in the nest. It is unclear at this point why estrogen-treated males seem more broody and fond of making nests and perching in their nests, but it is clear that dosed males often perform this activity to the exclusion of courtship (invitation, bill wipe, dance, singing). Reduced numbers of mounting attempts also indicate demasculinization by neonatal exposure to estrogens.
Dosed females become capable of singing after treatment with estrogen or octylphenol for 6 days after hatching. Female Zebra Finches do not normally sing, and the ability to learn songs, remember them and to sing only occurs following a change in brain morphology to the male pattern, with increased size of the nuclei associated with song memory and the motor pathways for singing.
Zebra Finches have proven to be a particularly good model for monitoring estrogenic dependent changes in brain morphology and behavior, but the ability to take the laboratory information into the field has proven extremely difficult. Some of the behaviors should be able to be observed in the field, either from a distance, or with video surveillance, but Andrea's data indicate that many of the changes are subtle, or require observation under controlled conditions where vocalizations of the individual bird can be recorded accurately. Many of the behaviors could not be monitored in a colony-breeding situation, making field observation of Cliff Swallows a qualitative tool at best. For these reasons, we will concentrate on determining exposure risks to birds in the field, and compare exposures with the doses found in the lab to cause developmental changes in Zebra Finches. A long-term goal for his work is to compare the sensitivity of Zebra Finches with other species of passerines easily kept in captivity, to determine the range of doses which cause adverse effects.
Cliff Swallow Field Studies:
In 1998 we initiated field studies with Cliff Swallows (Petrochelidon pyrrhonota)
nesting in agricultural areas of Yolo County. Colonial breeding swallows, nesting
under bridges adjacent to agricultural drains surrounded by farm fields appear
to be an "at-risk" species because of their nesting locations and
insectivorous feeding habits. Cliff Swallows are the most easily manipulated
passerine species nesting in colonies in agricultural areas in California, and
the colonies are of sufficient size to provide good statistical comparisons
of nesting success between colonies.
Myrnie Mayville located 38 cliff swallow colonies in 1998 for her thesis work in Yolo County, and she followed nesting success of 7 of these colonies in 1998 and 1999. We obtained pesticide use records from the County Agricultural Commissioner, and from the CA Department of Pesticide Regulation to quantify pesticide use by Township, Range, and Section. Digital land use data (field boundaries) for Yolo County were obtained from the California Department of Water Resources and subsequently imported into ArcView GIS. Maps with field boundaries and identification numbers for each grower/permittee in the four sections nearest each colony were acquired from the Yolo County Agricultural Commissioner's Office. These data, along with colony locations, were entered into ArcView GIS. Pesticide use data, reported as quantity of pesticide product used on a field of a given number of acres, for Yolo County were acquired from the Yolo County Agricultural Commissioner's Office. Because of the availability of pesticide use data on a per section basis, and because cliff swallows forage within a mile of their colony during the breeding season (Brown and Brown, 1991), the four sections closest to each colony were used as the estimated foraging arena. Active ingredients (a.i.'s) and number of pounds of a.i. per quantity of pesticide product were obtained from several sources (The Pesticide Manual, 9th and 11th ed., Pesticide Information Profiles, Herbicide Handbook, Pesticide Fact Handbook, Vol. 1 and 2).
Mayville devised a toxicity index to facilitate comparison of sites with diverse pesticide use. The total toxicity index (TTI) was based on the LD50 of each pesticide a.i. to bobwhite quail. If LD50 data were not available for bobwhite quail, the LD50's for mallard or pheasant were used. The number of pounds of each a.i. used at each site was multiplied by the reciprocal of the LD50 for that a.i.. Active ingredients with LD50's of greater than 2000 mg/kg were excluded from the analysis because these are considered practically non toxic to birds. In addition, the LD50's for many of these a.i.'s were reported only as greater than 2000 mg/kg. This toxicity index permitted integration of the acute oral toxicity to birds of various pesticide a.i.'s with the total amount of active ingredients used around each colony. A spatial analysis was performed to ascertain whether patterns of cliff swallow reproductive success varied with pesticide use.
Analysis of reproductive data using ANOVA indicated that clutch size did not differ between colonies in either 1998 (P=0.89) or 1999 (P=0.28). Hatching success, defined as proportion of eggs laid that hatched, varied between colonies in 1998 (P=0.0124) but not in 1999 (P=0.452). Fledging success was significantly different between colonies in 1998 (P=0.0001) and 1999 (P=0.0001). Overall reproductive success (proportion of eggs laid that resulted in fledged young) was significantly different between colonies in 1998 (P=0.0001) and 1999 (P<0.0001). Number of young fledged per active nest was significantly different in 1998 (P>0.0001) and 1999 (P>0.0001). Number of young fledged per successful nest was significantly different in 1998 (P>0.0001) and 1999 (P=0.0061).
Multiple regression analyses of the 1998 field data for total toxicity index on reproductive parameters indicated a significant trend for hatching success, fledging success, reproductive success and number of young fledged per active nest. No significant trend was detected for clutch size or number of young fledged per successful nest. Similar results were obtained using the method of Patnode and White (1991), cholinesterase inhibitors with LD50's <2000 mg/kg, insecticide toxicity only, and pounds of insecticide A.I. used.
In 1999, multiple regression analyses of the total toxicity index on reproductive parameters also indicated a significant positive effect of total toxicity within the foraging arena on fledging success (P>T = 0.02). No trend was detected for clutch size, hatching success, reproductive success, number of young per active nest, number of young per successful nest or survival to day six. Similar results were obtained using the ranking method of Patnode and White (1991), as well as pesticides with cholinesterase inhibitors with LD50's <2000 mg/kg, insecticide toxicity only and pounds of insecticide A.I. used.
Mayville's data showed that agricultural chemical use is correlated with reduced fledging success of swallows, but the relationship was not clearly defined. She was not able to collect food brought to feed nestlings to determine exposure of chicks. Monitoring the exposure of swallows will be one priority in 2001. Peter Gibert has constructed artificial nest structures by collecting natural mud nests and firing them in a pottery kiln to make them into durable ceramic. He attached groups of five ceramic nests to wooden shelves with access holes to enable easy access to chicks, and will be placing the group nests on bridges in Yolo County this spring. The quasi-artificial nests will make sample collection much easier, and will enable Peter to collect food samples for analysis. The insects in the food samples will be stored frozen prior to identification and screening for pesticides and for alkylphenols with the immunoassay Peter is developing in Hammock's lab.
Extending the laboratory studies with Zebra Finches into a field study has proven to be extremely difficult. We have not discovered any behavioral marker for developmental changes before birds reach adulthood, and recovery of a sufficient number adult swallows after doing them as nestlings appears impossible. Peter has documented the breeding assessments and banding literature for Cliff Swallows, and while their fidelity to the colony and nesting location is good, we would have to dose and band at lease 100 nestlings at each dose to insure the return of enough birds after their winter migration to South America. The sexual dimorphism in singing behavior of Cliff Swallows also does not appear to be as clear as for Zebra Finches, and it is not likely that we would be able to document changes in male and female singing behavior without extensive lab confirmations of behavior.
The exposure of swallows and other species of insectivorous birds to estrogenic chemicals is, nonetheless, important, and the magnitude of the exposure should be determined to determine the hazards to birds nesting in the Sacramento Valley.
Determination of Pesticides and Endocrine Disruptors in the Watershed.
Peter Gibert has further adapted the pesticide data to a GIS format, and has begun mapping chemical use in the Sacramento watershed. This information will provide compound specific data for determining the hazard exposure risk for specific colonies, and allow us to monitor colonies with extensive or minimal agricultural chemical use. We are particularly interested in mapping the alkylphenol use, as octylphenol has proven to be a potent estrogen in the Zebra Finch studies, and is one of the primary alkylphenols used in the mixing agents for agricultural chemicals. The compounds used are generally the alkylphenol polyethoxylates, but these compounds become metabolized to the base alkylphenol under field conditions.
We have continued to work with the County Agricultural Commissioned to receive pesticide application data suitable for geographical analysis on a monthly basis. The active ingredient list is adequate for analysis of pesticides, but very difficult to evaluate for adjuvants such as alkylphenol polyethoxylates used as surfactants and mixing agents, as data from the County are reported by brand name only, and the inert ingredients are proprietary. Data submitted by the counties to the State Department of Pesticide Regulation is converted to actual constituents, and the data are accurate for both active and inert ingredients, but the data are 1-2 years delayed. Current data are needed to be able to assess exposure of insects and monitor movement of chemicals offsite into agricultural drains.
Development of an Alkylphenol Immunoassay for Monitoring Environmental Exposures.
Peter Gibert has been working in Bruce Hammock's Core facility lab to utilize an immunoassay for octyl- and nonylphenols, an assay developed by Hammock in 1980 using Triton X100™ as the immunogen. The antiserum, however, has apparently been in the freezer too long, as it is inactive, and Peter has been developing a new assay, conjugating haptens of tertiary octylphenol and technical nonylphenol, immunizing rabbits, and testing new antisera. It looks promising that we will have a working assay this spring, and will be able to assay insect prey and food brought to swallow nests in the colonies Peter will be monitoring. The quasi-artificial nests will enable him to collect samples on a routine basis. This immunoassay will also be extremely useful for monitoring alkylphenols in agricultural runoff and throughout the watershed in general.
Supplemental Keywords:Water, Scientific Discipline, Health, RFA, Endocrine Disruptors - Environmental Exposure & Risk, Water & Watershed, Biology, Health Risk Assessment, endocrine disruptors, Biochemistry, Watersheds, Ecology and Ecosystems, Endocrine Disruptors - Human Health, pesticide runoff, animal models, birds, endocrine disrupting chemicals, ecological research, EDCs, fish, developmental biology, avian development, pesticide exposure, reproductive processes, xenobiotic, Cliff Swallow, Zebra Finches, animal reporductive impairment
Progress and Final Reports:
Original Abstract
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