2002 Progress Report: Effects of Estrogen Pollution on the Reproductive Fitness of the Gulf Pipefish, Syngnathus scovelli

EPA Grant Number: R827072C017
Subproject: this is subproject number 017 , established and managed by the Center Director under grant R827072
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Alabama Center For Estuarine Studies (ACES)
Center Director: Shipp, Robert L.
Title: Effects of Estrogen Pollution on the Reproductive Fitness of the Gulf Pipefish, Syngnathus scovelli
Investigators: Boettcher, Anne , Hemming, Jon , Shardo, Judith
Institution: University of South Alabama , Middle Tennessee State University
EPA Project Officer: Packard, Benjamin H
Project Period: June 1, 2001 through June 30, 2003
Project Period Covered by this Report: June 1, 2001 through June 30, 2002
RFA: Alabama Center For Estuarine Studies (ACES) (1999) RFA Text |  Recipients Lists
Research Category: Targeted Research


Developmental and sex-related changes in fish resulting from environmental estrogen exposure have been implicated in causing adverse, population-level effects. Recent research has focused on the influence of environmental estrogens on adult fish, although it has long been known that juvenile fish will experience sex reversal with hormone treatment at critical stages of development. The influence of estrogens on oviparous fish fecundity and offspring sexual development has been assessed using sentinel species, including the fathead minnow, Pimephales promelas, the sheepshead minnow, Cyprinidon variegatus, and the rainbow trout, Oncorhynachus mykiss. The current research assesses the effects of xenoestrogenic exposure on the reproductive fitness of the male-brooding Gulf pipefish, Syngnathus scovelli. Such species possess adaptations that may limit the effect environmental estrogens have on their offspring. Baseline studies of untreated fish are used to establish benchmark conditions of various physiologic parameters and for comparison with subsequent exposure-derived data.

The specific objectives are to: (1) determine baseline blood and brood pouch fluid osmolality, total glucose, total protein, and plasma vitellogenin, and examine normal brood pouch development, gonad morphology, gonado-somatic and hepato-somatic indices for reference fish; (2) determine the sensitivity of S. scovelli to estrogenic exposure, utilizing vitellogenin induction levels; and (3) examine the effects of estrogen exposure on the formation of the male brood pouch, gonad morphology, gonado-somatic, and hepato-somatic indices.

Comparisons will be made with literature values for a sentinel species, Pimphales promelas, for which estrogen exposure data are available (Hemming, 2000; Hemming, et al., 2001; Nichols, et al., 2001).

Progress Summary:


The brood cycle of the Gulf pipefish lasts approximately 15 days. Four brood stages have been defined using the development of embryos as markers. These stages are termed: (1) no brood stage—the male has not received eggs from the female (0 day); (2) pharyngula stage—only embryonic eye pigmentation is visible through the brood pouch (approximately 5 days); (3) protruding snout stage—all embryonic fins developed, snout only slightly protruding, large amount of yolk still remaining (approximately 10 days); and (4) embryonic/juvenile stage–embryos fully developed, little or no yolk remaining, ready for release (approximately 15 days).

Baseline structural and physiological data for each stage were collected during Year 1 of the study. Histological data show that at the no-brood stage, the surface of the brood pouch is flat, smooth, and lined with pavement epithelial cells. During incubation, the pouch inner surface forms shallow depressions with low walls, arranged in longitudinal rows (pharyngula stage). From the pharyngula through the embryonic/juvenile stages, the walls increase in height, particularly the medial walls. The flap shows similar changes, and by the protruding snout stage or shortly thereafter, the pouch and flap walls meet, completely separating the embryos. Epithelial cells lining the floor of the depressions differ from those of the wall during incubation, and the floor pouch epithelium appears to be the site of attachment for the egg chorion through the protruding snout stage. These structural changes are paralleled by changes in specific reproductive markers and in the organic and inorganic makeup of the blood and brood pouch fluid of male pipefish. Gonadosomatic index values significantly decrease through the protruding snout stage, increasing thereafter. Neither hepatosomatic index nor hematocrit varies with brood stage, however. There are minor changes associated with the brood cycle affecting blood protein concentrations and brood pouch fluid glucose concentrations, with the greatest differences seen for the protruding snout stage. Blood glucose values do not vary with the brood cycle. Blood osmolality values stay constant throughout the brood cycle and are hyperosmotic relative to the environment. Brood pouch fluid osmolality initially is higher than blood osmolality, and then decreases just prior to juvenile release. Based on differences seen in baseline studies, the focus of xenoestrogen exposure studies will be on comparisons between the no-brood and protruding snout stages.

The synthetic estrogen, ethynyl estradiol (EE2) will be used for exposure experiments. Preliminary EE2 stability experiments have shown that exposure experiments can be run under the normal light:dark regime for the culture of pipefish, but will require daily EE2 renewals. In the original proposal, fish blood samples were to be sent out for vitellogenin analyses; however, Dr. Tim Sherman (University of South Alabama, Biology) offered his assistance in the development of an enzyme-linked immunosorbent assay for the pipefish samples, using antibodies available from Cayman Chemical Company. The results obtained thus far are comparable to those available through the Protein Chemistry Core Facility and the Molecular Biomarkers Core Facility, Interdisciplinary Center for Biotechnology Research, at the University of Florida. In the spring, this assay will be used to monitor vitellogenin levels in field-collected fish and in laboratory-cultured fish exposed to specific concentrations of EE2.


The Master of Science student, Charlyn Partridge, has been supported by this project. Several undergraduates (Jessica Rozelle, Christi Cazalas, Jeremy Roop, and Jeff Bolland) have been supported by and/or have conducted directed research projects associated with this project. These students have experienced hands-on research in both the laboratory and the field. Two of the undergraduates, Jessica Rozelle and Jeff Bolland, are conducting Honors Research related to the project. Components related to the current research also have been incorporated into the BLY 436 Animal Physiology course as both lecture and laboratory material.


Hemming JM. Assessment of the efficacy of a constructed wetland to reduce or remove wastewater effluent estrogenicity and toxicity using biomarkers in male fathead minnows (Pimephales promelas Rafinesque, 1820). Dissertation, Environmental Science, University of North Texas 2000.

Hemming JM, Waller WT, Chow MC, Denslow ND, Venables B. Assessment of the estrogenicity and toxicity of a domestic wastewater effluent flowing through a constructed wetland system using biomarkers in male fathead minnows (Pimephales promelas Rafinesque, 1820). Environmental Toxicology and Chemistry 2001;20(10):2268-2275.

Nichols KM, Snyder EM, Snyder SA, Pierens SL, Miles-Richardson SR, Giesy JP. Effects of nonylphenol ethoxylate exposure on reproductive output and bioindicators of environmental estrogen exposure in fathead minnows (Pimephales promelas). Environmental Toxicology and Chemistry 2001;20(3):510-522.

Future Activities:

Based on the results of the first year of study, which as described above, indicate that the greatest differences among brood stages are between the no brood stage (0 day) and the protruding snout stage (10 days), our focus for the estradiol exposure experiments will be on these two stages. Exposure experiments will be conducted during late spring 2003. Additional field samples also will be collected for comparison with the vitellogenin assays. As a companion study, Jeff Bolland has initiated a field survey designed to monitor seasonal changes in reproductive and life history stages of an estuarine population of Gulf pipefish. His study will be carried out from January through December 2003. This will expand our database to include a more extensive set of field values for vitellogenin levels, as well as GSI and HSI.

Journal Articles:

No journal articles submitted with this report: View all 5 publications for this subproject

Supplemental Keywords:

xenoestrogens, environmental estrogens, male-brooding Gulf pipefish, Syngnathus scovelli, sex reversal, gonadosomatic index, GSI, hepatosomatic index, HIS, ethynyl estradiol, EE2, exposure experiments, vitellogenin levels, ecosystem, ecosystem protection, environmental exposure, risk, geographic area, water, aquatic ecosystem, estuarine research, chemical engineering, chemistry, ecology, ecological effects, human health, ecological indicators, risk assessment, indicators, sensitive populations, environmental chemistry, Alabama, AL, coastal ecosystem, coastal environments, Gulf Coast, estuaries, estuarine waters, fishery sampling, human modifications, land use, water use., RFA, Scientific Discipline, ECOSYSTEMS, Water, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, Ecology, estuarine research, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Restoration, Aquatic Ecosystem, Aquatic Ecosystems, Ecological Effects - Environmental Exposure & Risk, Terrestrial Ecosystems, Ecological Monitoring, Ecology and Ecosystems, Aquatic Ecosystem Restoration, Ecological Indicators, coastal ecosystem, eutrophication, water use, nursery habitats, estuaries, fish, watersheds, nutrients, biomass, fisheries, algal blooms, submerged aquatic vegetation, ecosystem, environmental indicators, water quality, estuarine waters, human modifications

Relevant Websites:

http://www.southalabama.edu/aces/ Exit
http://www.ourstolenfuture.org/index.htm Exit

Progress and Final Reports:

Original Abstract
  • 2001
  • Final

  • Main Center Abstract and Reports:

    R827072    Alabama Center For Estuarine Studies (ACES)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R827072C001 Fluorescent Whitening Agents As Facile Pollution Markers In Shellfishing Waters
    R827072C002 Red Snapper Demographics on Artificial Reefs: The Effect of Nearest-Neighbor Dynamics
    R827072C003 Stabilization of Eroding Shorelines in Estuarine Wave Eliminates with Constructed Fringe Wetlands Incorporating Offshore Breakwaters
    R827072C004 Interaction Between Water Column Structure and Reproduction in Jellyfish Populations Of Mobile Bay (SGER)
    R827072C005 Effects of Variation in River Discharge and Wind-Driven Resuspension on Higher Trophic Levels in the Mobile Bay Ecosystem
    R827072C006 Results of Zooplankton Component
    R827072C007 Benthic Study Component
    R827072C008 A Preliminary Survey of Macroalgal and Aquatic Plant Distribution in the Mobile Tensaw Delta
    R827072C009 Fisheries-induced changes in the structure and function of shallow water "nursery habitats": an experimental assessment
    R827072C010 Effects Of Variation in River Discharge and Wind-Driven Resuspension on Lower Trophic Levels of the Mobile Bay Ecosystem
    R827072C011 Evaluation of Alabama Estuaries as Developmental Habitat for Juvenile Sea Turtles
    R827072C012 Effects of Salinity Stress on Natural and Anthropogenically-Derived Bacteria in Estuarine Environments
    R827072C013 The Role of Land-Use/Land-Cover and Sub-estuarine Ecosystem Nitrogen Cycling in the Regulation of Nitrogen Delivery to a River Dominated Estuary; Mobile Bay, Alabama
    R827072C014 Environmental Attitudes of Alabama Coastal Residents: Public Opinion Polls and Environmental Policy
    R827072C015 Synthesis and Characterization of an Electrochemical Peptide Nucleic Acid Probe
    R827072C016 Determinants of Small-Scale Variation in the Abundance of the Blue Crab Callinectes Sapidus
    R827072C017 Effects of Estrogen Pollution on the Reproductive Fitness of the Gulf Pipefish, Syngnathus scovelli
    R827072C019 A Model for Genetic Diversity Aquatic Insects of the Mobile/Tensaw River Delta
    R827072C020 Evaluating Trophic Processes as Indicators of Anthropogenic Eutrophication in Coastal Ecosystems: An Exploratory Analysis
    R827072C021 Effects of Anthropogenic Eutrophication on the Magnitude and Trophic Fate of Microphytobenthic Production in Estuaries
    R827072C022 Characteristics of Ship Waves and Wind Waves in Mobile Bay
    R827072C023 Methods Comparison Between Stripping Voltammetry and Plasma Emission Spectroscopy for Metals in Mobile Bay
    R827072C024 Changes in Water Conditions and Sedimentation Rates Associated With Construction of the Mobile Bay Causeway
    R827072C025 Cold-Induced Hibernation of Marine Vibrios in the Gulf of Mexico: A Study of Cell-Cell Communication and Dormancy in Vibrio vulnificus
    R827072C026 Holocene Sedimentary History of Weeks Bay, AL: Human and Natural Impacts on Deposition in a Gulf Coast Estuary
    R827072C027 Shelter Bottlenecks and Self-Regulation in Blue Crab Populations: Assessing the Roles of Nursery Habitats and Juvenile Interactions for Shelter Dependent Organisms
    R827072C028 Predicting Seagrass Survival in Nutrient Enriched Waters: Toward a New View of an Existing Paradigm
    R827072C029 DMSP and its Role as an Antioxidant in the Salt Marsh Macrophyte Spartina alterniflora
    R827072C030 A Preliminary Survey of Aerial and Ground-Dwelling Insects of the Mobile/Tensaw Delta
    R827072C031 Natural Biogeochemical Tags of Striped Mullet, Mugil cephalus, Estuarine Nursery Areas in the North Central Gulf of Mexico
    R827072C032 Resolution of Sedimentation Rates in Impacted Coastal Environments Using 137Cs and 210Pb Markers: Dog River and Fowl River Embayments
    R827072C033 Investigation of the Use of Pulse Amplitude Modulated (PAM) Fluorometry as an Indicator of Submerged Aquatic Vegetation Health in Mobile Bay
    R827072C034 Influence of Invasive Plant Species in Determining Diversity of Aquatic Vegetation in the Mobile-Tensaw Delta
    R827072C035 The Influence of Shallow Water Hydrodynamics on the Importance of Seagrass Detritus in Estuarine Food Webs
    R827072C036 Food Web Interactions, Spatial Subsidies and the Flow of Energy Between the Mobile Bay Delta and Offshore Waters: A SGER Proposal to the Alabama Center for Estuarine Studies
    R830651C001 Meteorological Modeling of Hurricanes and Coastal Interactions: A Stability Study For Vertical Pressure Levels
    R830651C002 Characterization of Glycoprotein Cues Used by the Parasitic Rhizocephalan Barnacle Loxothylacus texanus To Identify Its Blue Crab Host, Callinectes sapidus
    R830651C003 Survey of Diamondback Terrapin Populations in Alabama Estuaries
    R830651C004 An Assessment of Environmental Contaminant Levels in Water and Dragonfly Larvae Tissues from the Mobile/Tensaw Delta