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Dry weight (W), carbon (C), nitrogen (N) and energy (E) (calculated) accumulation were measured in the estuarine grass shrimp, Palaemonetes pugio, throughout larval development and during the first 2 weeks as postlarvae in seawater over sediment containing the pyrethroid insecticide fenvalerate (SCF; nominal concentrations of 1, 10, and 100 µg fenvalerate kg-1 sediment). The influence of fenvalerate-laden sediment on shrimp growth and utilization patterns of C,N, and E was dependent on fenvalerate concentration, age of shrimp, and whether shrimp were premetamorphic or postmetamorphic in development. The fenvalerate concentration in the sediment, which ultimately inhibited larval metamorphosis (100 µg fenvalerate kg-1 sediment), significantly reduced W accumulation in developing larvae and in postlarvae growing on the sediment for an equivalent time. Accumulation of C,N, and E varied not only with concentration of SCF, but differed between pelagic larvae developing in water above SCF and newly settled postlarvae growing in direct contact with SCF. Larvae developing above >=10 µg kg-1 SCF contained significantly less N, while postlarval shrimp settling onto >=10 µg kg-1 SCF accumulated significantly less C and E. Measurable variations in growth and energy reserves of toxicant-sensitive life stages in response to environmentally realistic insecticide exposures have a direct link to ecological consequences of toxic stress and may be useful as biomarkers to diagnose early damage in estuarine populations.

Folmar, Leroy C. 1999. Assays for Endocrine Disrupting Chemicals: Beyond Environmental Estrogens. In: Environmental Toxicology and Risk Assessment: Standardization of Biomarkers for Endocrine Disruption and Environmental Assessment. 8th Volume, ASTM STP 1364. D.S. Henshel, M.C. Black, and M.C. Harrass, Editors. American Society for Testing and Materials, West Conshohocken, PA. Pp. 59-94. (ERL,GB 1043).

Recent popular and scientific articles have reported the presence of estrogenic and other hormone mimicking chemicals in the environment and their potential for causing reproductive dysfunction in humans and wildlife. The purpose of this session was to present the best available, if not standard, analytical methods to assay for the effects of xenobiotic chemicals on a broad range of endocrine-mediated events, including reproduction, growth, development and stress responses in aquatic vertebrate and invertebrate animals.

Oliver, Leah M. and William S. Fisher. 1999. Appraisal of Prospective Bivalve Immunomarkers. Biomarkers. 4(6):510-530. (ERL,GB 1045).

Worldwide concern over threats to natural resources and public health has led to increased efforts to monitor and assess environmental condition. This has stimulated the need for development and application of select biological and ecological measurements, or indictors, that are responsive to environmental stress. Measures of bivalve mollusk defense activities, such as hemocyte density, phagocytic activity, locomotion and production of cytotoxic molecules, and hemolymph constituents, such as agglutinins and lysozyme, have potential as indicators and appear to be responsive to xenobiotic chemical insults in the aquatic environment. However, basic research on the relevance of these measurements in inferring resistance to disease or enhanced survival is currently insufficient, reducing their value as potential biomarkers to address environmental objectives. In addition, variation in defense activities caused by seasonal temperature and reproductive cycling, salinity changes, nutritional status, diseases and parasites, and genetic stocks is high and may limit applicability of bivalve defense-related measurements as indicators. This review examines these sources of variability and their possible implications of interpreting changes in bivalve defense activity as an indicator of stress. Examples of contaminant-induced changes in bivalve defense functions are described.

McKenney, Charles L., Jr. 1999. Hormonal Processes in Decapod Crustacean Larvae as Biomarkers of Endocrine Disrupting Chemicals in the Marine Environment. In: Environmental Toxicology and Risk Assessment: Standardization of Biomarkers for Endocrine Disruption and Environmental Assessment. 8th Volume, ASTM STP 1364. D.S. Henshel, M.C. Black, and M.C. Harrass, Editors. American Society for Testing and Materials, West Conshohocken, PA. Pp. 119-135. (ERL,GB 1057).

Knowledge of endocrine control of the complex larval developmental processes in insects (metamorphosis) has led to the introduction of insect hormones and their analogues as insecticides known as insect growth regulators (IGRs) with the largest group being juvenile hormone analogues (JHAs). Developmental and metabolic alterations in estuarine crustacean larvae induced by JHAs suggest that these compounds may be interfering with an endocrine system using JH-like compounds. These responses of crustacean larvae during the metamorphic process can be used in the development of biomarkers for the environmental impact of these types of compounds and other potential endocrine disrupting chemicals on estuarine biota.

Myers, Mark S. and John W. Fournie. 2002. Histopathological Biomarkers as Integrators of Anthropogenic and Environmental Stressors. In: Biological Indicators of Aquatic Ecosystem Stress. S. Marshall Adams, Editor. American Fisheries Society, Bethesda, MD. Pp. 221-287. (ERL,GB 1139).

Histopathology is an extremely useful tool for assessing effects of exposure to stressors at the level of the individual. Even though the histopathological approach is somewhat qualitative, it is very valuable because the observed lesions represent an integration of cumulative effects of biochemical and physiological changes as well as representing actual injury to the organism. This approach also allows identification of specific cells, tissues, and organs that have been affected. We have presented information in the chapter on a number of lesions in various tissues and organs that may at some point in time have the potential as reliable histopathological biomarkers of specific environmental stressors, but require further validation by epidemiological and laboratory studies. Examples include lesions of the excretory kidney, gonad, gills, central nervous system, neurosensory organs, and hemopoietic tissues in head kidney. In our opinion, there are only a small number of histopathological biomarkers that have been shown via rigorous epidemiological methods and experimental validation to be reliable indicators of exposure to various stressors. These include liver lesions, splenic macrophage aggregates, some skin lesions, and certain musculoskeletal abnormalities. Some are relatively specific while others are more nonspecific, and all require the investigator to account for seasonal, physiological, age and sex-related variation. For example, an epizootic of liver neoplasms and other liver lesions involved in the stepwise histogenesis of liver neoplasia in a population of fish indicates that they have been exposed to hepatotoxic and pepatocarcinogenic contaminants. Elevated numbers of splenic macrophage aggregates is a less specific indicator, but still indicates that the affected fish probably have been exposed to contaminated sediments, low dissolved oxygen, or some other stressor. Because histopathology is a biologically meaningful method of evaluating the effects of stressors on animals, it should be an essential component of all environmental assessments. In order to assess the health status of a body of water it is necessary to evaluate the health of the organisms inhabiting the particular body of water, which requires a histopathological examination of representative tissues and organs from those organisms.

Leblond, Jeffrey D. and Peter J. Chapman. 2002. Survey of the Sterol Composition of the Marine Dinoflagellates Karenia brevis, Karenia mikimotoi, and Karlodinium micrum: Distribution of Sterols Within Other Members of the Class Dinophyceae. EPA/600/J-01/420. J. Phycol. 38(8):670-682. (ERL,GB 1149).

The sterol composition of different marine microalgae was examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4-methyl sterols, such as dinosterol, which are rarely found in other classes of algae. The ability to use sterol biomarkers to distinguish certain dinoflagellates such as the toxic species Karenia brevisHansen and Moestrup, responsible for red tide events in the Gulf of Mexico, from other species within the same class would be of considerable scientific and economic value. Karenia brevis has been shown by others to possess two majors sterols, (24S)-4a-methyl-5a -ergosta-8(14),22-dien-3b-ol (ED) and its 27-nor derivative (NED), having novel structures not previously known to be present in other dinoflagellates. This prompted the present study of the sterol signatures of more than 40 dinoflagellates. In this survey, sterols with the properties of ED and NED were found in cultures of K. brevis and shown also to be the principal sterols of Karenia mikimotoi Hansen and Moestrup and Karlodinium micrum Larsen,two dinoflagellates closely related to K. brevis. They are also found as minor components of the more complex sterol profiles of other members of the Gymnodinium/Peridinium/Prorocentrum (GPP) taxonomic group. The distribution of these sterols is consistent with the known close relationship between K. brevis, K. mikimotoi,and K. micrum,and and serves to limit the use of these sterols as lipid biomarkers to a few related species of dinoflagellates.

Leblond, Jeffrey D. and Peter J. Chapman. Unpublished. Polyunsaturated C27 Hydrocarbons in the Marine Dinoflagellate, Pyrocystis lunula (Dinophyceae): Preliminary Characterization. J. Phycol. 33 p. (ERL,GB 1179).

The lipids of different algal species have revealed a diversity of fatty acids, sterols, and hydrocarbons, several of which are considered of chemotaxonomic value and candidates for useful chemical biomarkers with the potential for characterizing phytoplankton community composition. Neutral fractions obtained from the lipids of two strains of Pyrocystis lunula, in the course of characterizing over forty dinoflagellates, were found to contain an abundant quantity of long-chain polyunsaturated hydrocarbons, along with previously reported keto-steranes. Gas chromatographic examination showed a retention time of the predominant hydrocarbon, relative to standards, which together with its molecular weight (m/z = 364) and mass spectrum suggested a multiply unsaturated C27 compound (C27H40) with eight double bonds. Reduction (Adams catalyst) gave a product identical to authentic n-heptacosane indicating a straight chain hydrocarbon. While the positions and stereochemistry of double bonds have not been established, the carbon number of this hydrocarbon and the number of double bonds strongly suggest a relationship to, and formation by decarboxylation of, the recently described, long-chain polyunsaturated C28 fatty acid, [28:8(n-3)], shown to be a constituent of dinoflagellate phospholipids. This hydrocarbon was not found in any other genus of the over forty examined dinoflagellates, nor was it found in isolates of two other species of Pyrocystis, P. fusiformis and P. noctiluca. The function(s) of this compound in P. lunula is currently unclear.

Leblond, Jeffrey D. and Peter J. Chapman. 2004. Sterols of the Heterotrophic Dinoflagellate, Pfiesteria piscicida (Dinophyceae): Is There a Lipid Biomarker?. J. Phycol. 40(1):104-111. (ERL,GB 1191).

Within U.S. waters, blooms of the dinoflagellate, Pfiesteria piscicida, have been recorded on an almost regular basis in the Chesapeake Bay and surrounding mid-Atlantic regions for the last two decades. Despite the apparent significance of such blooms to the environment and human health, and the attendant economic consequences, little work has addressed the physiology and biochemistry, particularly that of sterol composition, of P. piscicida. GC-MS characterization of trimethylsilyl ether derivatives of sterols from free sterol and sterol ester fractions was performed in an effort to determine whether P. piscicida produces unique sterols that may serve as potential biomarkers. This characterization revealed that, like most dinoflagellates, the majority of sterols was present as free sterols. Furthermore, the profile of free sterols was found to resemble those of photosynthetic dinoflagellates, with the dominant compound being the previously reported dinoflagellate sterol, dinosterol. A number of other 4a-methyl-substituted sterols and steroidal ketones common to other dinoflagellates were also identified. No strong candidate(s) for a unique sterol biomarker was present.

Walker, Calvin C., Kimberly A. Salinas, Peggy S. Harris, Sherry S. Wilkinson, James D. Watts and Michael J. Hemmer. 2007. A Proteomic (SELDI-TOF-MS) Approach to Estrogenic Activity Screening. Toxicol. Sci. 95(1):74-81. (ERL,GB 1266).

*A small fish model and surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI) were used to investigate plasma protein expression as a means to screen chemicals for estrogenic activity. Adult male sheepshead minnows (Cyprinodon variegatus) were placed into aquaria for seawater control, solvent control, and treatments of 1 71B-estradiol (E2), methoxychior, bisphenol-A, 4-tert-pentylphenol, endosulfan and chlorpyriphos. Fish plasma was applied to weak cation exchange (CM1 0) ProteinChip® arrays , processed and analyzed. The array produced approximately 42 peaks for E2 plasma and 30 peaks for solvent control plasma. Estrogen responsive mass spectral biomarker peaks were identified by comparison of E2 treated and control plasma spectra. Thirteen potential protein biomarkers with a range from 1-13 kDa were up- or down regulated in E2 treated fish and their performance as estrogenic effects markers was evaluated by comparing spectra from control, estrogen agonist and non-agonist stressor treated males and normal female fish plasma. One of the biomarkers, m/z 3025.5, was identified by MS/MS as C. variegatus zona radiata protein, fragment 2. The weak environmental estrogens methoxychlor, bisphenol-A and 4-tort-pentylphenol elicited protein expression profiles consistent with the estrogen expression model. Estrogen responsive peaks were not detected in plasma from fish in the seawater, vehicle, endosulfan or chlorpyriphos treatments. No difference was found between plasma protein expression of seawater control and solvent control fish. We show that water exposure of fish to estrogen agonists produces distinct plasma protein biomarkers that can be reproducibly detected at low levels using protein chips and mass spectrometry.

Salinas, Kimberly A., Michael J. Hemmer, Peggy S. Harris and Calvin C. Walker. 2008. A Simple and Rapid Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry Method to Screen Fish Plasma Samples for Estrogen-Responsive Biomarkers. Environ. Toxicol. Chem. 27(5):1175-1183. (ERL,GB 1295).

In this study, we describe and evaluate the performance of a simple and rapid mass spectral method for screening fish plasma for estrogen-responsive biomarkers using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) coupled with a short term fish assay. Adult male sheepshead minnows (Cyprinodon variegatus) were placed into aquaria consisting of duplicate tanks for vehicle control and the following estrogen agonist treatments: 0.00713, 0.0163, 0.04, 0.05, 0.1305 0.222, 0.399 and 0.906u g 17B-estradiol/l; 100 ug 4-tert-pentylphenol /1; 6 and 12 ug methoxycor/l; and 120.5 and 1079.5 ug bisphenol-Al. Treatments with 80 ug chlorpyris/l and 0.6 ug endosulfan/l served as non-estrogenic negative controls. Test concentrations were maintained using an intermittent flow-through dosing apparatus, and fish were sampled at seven or 10 days. Plasma was obtained from individuals, diluted and applied to an inert gold array and analyzed. Multiple protein peaks, ranging from 2.9 to 12.9 KDa, were identified as markers of estrogeniC effects when comparing estrogen-treated and control fish using interpercentile reference values. A binary classification tree model was constructed from plasma protein profiles of the vehicle control and 0.222 ug/l 17B-estradioI treatments and used to evaluate all samples. Treatments with estrogen agonists 17B-estradiol, 4-tert-pentylphenol, methoxychlor and bisphenol-A generated reproducible diagnostic biomarkers based on the presence of specific estrogen-responsive plasma proteins. The controls and non-estrogenic compounds chlorpyrifos and endosulfan did not produce this estrogen responsive protein profile. An average measured NOEC for 17B-estradiol at 0.04 ug/l was estimated from concentration response exposures. MALDI-TOF-MS is a sensitive and specific tool for protein expression profiling and can be used to screen chemicals for mode of action in ecotoxicological studies.

Schlenk, Daniel, Richard Handy, Scott Steinert, Michael H. Depledge and William Benson. 2008. Biomarkers. Chapter 16. In: The Toxicology of Fishes. Richard T. Di Giulio and David E. Hinton, editors. CRC, Boca Raton, FL. Pp. 683-731. (ERL,GB 1315).

Describes the biomarkers of exposure, effect and susceptibility in fishes

Hemmer, Michael J., Geraldine M. Cripe, Becky L. Hemmer, Larry R. Goodman, Kimberly A. Salinas, John W. Fournie and Calvin C. Walker. 2008. Comparison of Estrogen-Responsive Plasma Protein Biomarkers and Reproductive Endpoints in Sheepshead Minnows Exposed to 17B-Trenbolone. Aquat. Toxicol. 88(2):128-136. (ERL,GB 1321).

Protein profiling can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In previous studies, mass spectral analysis revealed four peptides (2950.5, 2972.5, 3003.4, 3025.5 m/z) in the plasma of estrogen agonist-treated male and gravid female sheepshead minnows (Cyprinodon variegatus, SHM), which served as distinct estrogenic biomarkers. In this study, a 21 day reproductive assay with adult SHM was conducted to investigate possible dose-related effects of the synthetic androgen, 17$-trenbolone, on expression of these four estrogen-responsive peptides. In addition, the response of the peptide biomarkers were compared to traditional reproductive endpoints of fecundity, histopathology, secondary sex characteristics, length, weight, hepatosomatic index, female gonadosomatic index and plasma vitellogenin (VTG) levels. Fish were continuously exposed to 0.005, 0.05, and 5.0 :g/l, a solvent control (triethylene glycol, TEG), and a seawater control (SW) using an intermittent flow-through dosing system. Plasma was analyzed for the presence of the four peptide biomarkers by MALDI-TOF MS and VTG protein by quantitative ELISA. Male fish from the trenbolone treatments and controls showed no expression of the four peptide biomarkers or measurable levels of VTG. The estrogen-responsive biomarkers and plasma VTG were constitutively expressed in females from the SW, TEG, 0.005 and 0.05 :g/l exposures. All four peptide biomarkers were significantly reduced (p<0.0002 to p<0.005) at the 5.0 :g/l treatment level which correlated with significant reductions in fecundity and changes in ovarian morphology. A distinct but non-significant reduction in VTG was also observed in female fish from the 5.0 :g/l treatment. Results of this study suggest application of these estrogen-responsive protein biomarkers may be a cost effective alternative to fecundity measures which are labor intensive and expensive to conduct.

Mayer, Foster L., Donald J. Versteeg, Michael J. McKee, Leroy C. Folmar, Robert L. Graney, Delbert C. McCume and Barnett A. Rattner. 1992. Physiological and Nonspecific Biomarkers. In: Biomarkers: Biochemical, Physiological, and Histological Markers of Anthropogenic Stress. EPA/600/A-92/222. Robert J. Huggett et al., Editor. Lewis Publishers, Boca Raton, FL. Pp. 5-85. (ERL,GB 736). (Avail. from NTIS, Springfield, VA: PB93-119832)

This chapter summarizes the advantages and disadvantages of physiological and nonspecific biomarkers. It lists the criteria to be used in selecting biomarkers to address specific ecological questions. After a general discussion of this topic, several specific biomarkers of possible utility in environmental monitoring of exposure and effects are described individually. For each of these biomarkers, the chapter presents research needs for developing and validating ecologically meaningful biomarkers.

Denslow, Nancy D., Ming M. Chow and Leroy C. Folmar. 1994. Isoforms of Apolipoprotein A-I in the Serum of Brown Bullheads (Ameiurus nebulosus) with Liver Cancer. Can. J. Zool. 72(8):1522-1527. (ERL,GB 853).

Brown bullheads (Ameiurus nebulosus) express two different isoforms of apolipoprotein A-I in the serum, a predominant form characterized by a pI of 6.9 and a more acidic isoform with a pI of 6.65. The two proteins were identified by N-terminal amino acid sequencing, with 50 residues for the pI 6.9 isoform and 20 residues for the pI 6.65 isoform. Sequences for the two proteins were identical for the first 20 amino acids; however, differences in their primary sequences were deduced by analysis of peptide maps obtained through proteolytic digestion. The ratio of the isoforms changes from 75% pI 6.9/25% pI 6.65 (no tumors) to equal amounts of both isoforms in bullheads with hepato- or cholangio-cellular carcinomas. This is the first report of these serum isoforms in feral fish showing that the expression of these proteins is directly related to both cholangio- and hepato-cellular carcinomas. The potential use of these proteins as tumor biomarkers is discussed.

Denslow, N.D., M.M. Chow, L.C. Folmar, S. Bonomelli, S.A. Heppell and C.V. Sullivan. 1996. Development of Antibodies to Teleost Vitellogenins: Potential Biomarkers for Environmental Estrogens. In: Environmental Toxicology and Risk Assessment: Biomarkers and Risk Assessment. Fifth Volume, ASTM STP 1306. D.A. Bengston and D.S. Henschel, Editors. American Society for Testing and Materials, Philadelphia, PA. Pp. 23-36. (ERL,GB 946).

We describe 2 IgG class and an IgM class monoclonal antibodies and a polyclonal antiserum which recognize a wide variety of teleost vitellogenins. Based on these and previous results we discuss the potential for developing a "universal" ELISA to recognize all vertebrate vitellogenins.

Blazer, V.S., D.L. Higginbotham and J.W. Fournie. 1996. Serum Factors as Indicators of Environmental Stress: Optimization of Methodologies for Striped Bass Serum. In: Modulators of Immune Responses. Vol. II. Joanne S. Stolen, Editor. SOS Publications, Fair Haven, NJ. Pp. 443-457. (ERL,GB 950).

With the increasing interest in "ecosystem health", many federal and state agencies are routinely monitoring biological responses of various fish species. Numerous physiological, histopathological and biochemical changes have been investigated as biomarkers of contaminant exposure. The immune system of animals, including fish, is very sensitive to some contaminant exposures, hence there is interest in developing markers of immune function/disease resistance. Our laboratories have been involved in developing soluble and cellular assays to measure the effects of contaminant exposures in the laboratory and attempting to field validate these arrays. Striped bass were chosen because of their commercial value and previous indications that environmental contaminants are affecting wild populations. Serum lysozyme, spontaneous hemolytic activity and cortisol were identified as potential indicators. Methods were standardized for these assays and the effects of clotting time, use of plasma versus serum and freezing at either -20 or -70°C were evaluated.

Blazer, Vicki S., John W. Fournie and Beverly A. Weeks-Perkins. 1997. Macrophage Aggregates: Biomarker for Immune Function in Fishes?. In: Environmental Toxicology and Risk Assessment: Modeling and Risk Assessment. 6th Volume, ASTM STP 1317. F. James Dwyer, Thomas R. Doane, and Mark L. Hinman, Editors. American Society for Testing and Materials, West Conshohocken, PA. Pp. 360-375. (ERL,GB 967).

Macrophage aggregates (MAs) are believed to be functional equivalents of germinal centers, active in storage of exogenous and endogenous waste products, the immune response, and iron storage and recycling. Numerous studies have shown an increase in their number, size or hemosiderin content in fish collected at contaminated sites. For this reason, MAs have been suggested as potentially sensitive biomarkers of contaminant exposure. Although they are structures observed histologically, it has also been suggested they may be immunotoxicologic biomarkers. To determine possible relationships between MA formation and macrophage function, we examined data from two field studies and one laboratory study. We found a significant correlation between a decreased chemotaxic response of macrophages and the formation of more numerous, smaller MAs in mummichog from a contaminated site. In laboratory-exposures to arsenic, macrophage function appeared to be a more sensitive indicator at the lower levels of dietary arsenic. However, MA appeared to provide a more dose-dependent and comprehensive indicator of toxicity.

Davis, William P. 1997. Evidence for Developmental and Skeletal Responses as Potential Signals of Endocrine-Disrupting Compounds in Fishes. In: Chemically Induced Alterations in Functional Development and Reproduction of Fishes. Rosalind M. Rolland, Michael Gilbertson, and Richard E. Peterson, Editors. SETAC Press, Pensacola, FL. Pp. 61-72. (ERL,GB 992).

Integrated sets of bioindicators and ecological signals would be desirable for rapid detection and assessment of endocrine disruptor syndrome in wild populations of fish and wildlife. Early warning signals would be especially desirable to enable identification before the onset of major reproductive dysfunction or population consequences. Comparison of various research reports in the literature reveals that many of the same compounds that induce developmental, morphological, and/or skeletal anomalies are later identified as endocrine disruptors. Kepone, mirex, polychlorinated biphenyls (PCBs), trifluralin, dibutylphthalate (DBP), kraft-mill effluents (KMEs), and others induce skeletal, developmental, and/or sex-linked morphological abnormalities that become manifest biomarkers among surviving mature fishes. This report examines several specific cases of skeletal and external morphological responses and suggests their relevance as markers signaling the probability of disruption of an endocrine modulated function.

Bossart, Gregory, David Busbee, Garet Lahvis and Graham A.J. Worthy. 1993. Research Perspectives for Dolphin Mortalities in North America. EPA/600/R-93/217. U.S. Environmental Protection Agency, Center for Marine and Estuarine Disease Research, Environmental Research Laboratory, Gulf Breeze, FL. 22 p. (Avail. from NTIS, Springfield, VA: PB94-155819)

The second Gulf Breeze Symposium sponsored by EPA's Center for Marine and Estuarine Disease Research, focused on scientific research related to dolphin mortalities. During the symposium, four groups were formed to discuss and evaluate current scientific information, research strategies and research needs related to deaths of bottlenose dolphins Tursiops truncatus. The four study groups addressed research questions related to (1) pathology and disease, (2) pollution analyses and biomarkers of exposure, (3) physiology and biomarkers of effects and (4) standing and sampling logistics for bottlenose dolphins.

Denslow, Nancy D., Majorie Chow, Ming M. Chow, Sherman Bonomelli, Leroy C. Folmar, Scott A. Heppell and Craig V. Sullivan. 1997. Development of Biomarkers for Environmental Contaminants Affecting Fish. In: Chemically Induced Alterations in Functional Development and Reproduction of Fishes. Rosalind M. Rolland, Michael Gilbertson, and Richard E. Peterson, Editors. SETAC Press, Pensacola, FL. Pp. 73-86. (ERL,GB X866).

A number of anthropogenic chemicals that have been introduced into the environment may compromise normal endocrine function and sexual development or lead to neoplastic lesions. It is therefore important to determine wildlife exposure to these chemicals and to begin to establish exposure-effect relationships. We used 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to identify biomarkers associated with exposure of fish to contaminants. Two proteins, an acidic isoform of apolipoprotein A1 and vitellogenin (VTG), were more highly expressed in brown bullhead fish exposed to highly contaminated waters. We concentrated on VTG, the estrogen-inducible egg yolk precursor protein, as an ideal biomarker for estrogen-mimicking chemicals, and we developed a panel of high-affinity, vitellogenin-specific monoclonal antibodies (mAbs) (immunoglobulin G [IgG] type). In this report, we partially characterize 4 of the panels' most cross-reactive antibodies, which bind VTG from fish in a wide phylogenetic spectrum (including orders Perciformes, Atheriniformes, Batrachoidiformes, Salmoniformes, Cypriniformes, and Anguilliformes). While none of these antibodies cross-reacts with VTG in the order Siluriformes, another mAb 2D8 (immunoglobulin M [igM] type) does bind to VTG from fishes in this group as well as to VTGs from chickens and snakes, but with lower affinity. The panel of high-affinity IgG-type antibodies can be used in Western blots and enzyme-linked immunosorbent assays (ELISAs) to measure the expression of VTG in plasma from oviparous wildlife exposed to estrogen or estrogen-like contaminants.

Pelz, Oliver, Luis A. Cifuentes, Beth T. Hammer, Cheryl A. Kelley and Richard B. Coffin. 1998. Tracing the Assimilation of Organic Compounds Using delta13C Analysis of Unique Amino Acids in the Bacterial Peptidoglycan Cell Wall. 25(3):229-240. (ERL,GB X916).

Stable isotope analysis of bacterial nucleic acids can be used to trace carbon that is assimilated and respired by the bacterioplankton in aquatic ecoystems. However, in sediment and soil environments humic acids co-extract with the nucleic acids, resulting in inaccurate isotope analysis. In this study we have examined the use of amino acids found in bacterial cell walls as biomarkers to trace carbon sources that support growth. In the development of the method, peptidoglycan from laboratory grown Pseudomonas sp. was hydrolyzed to amino acids. Stable carbon isotope ratios (d13C) were analyzed with a dual mass spectrometer, ion trap and isotope ratio, equiped with a gas chromatograph sample inlet (GC/ITMS/IRMS). Comparisons of d13C values of whole cells, cell wall peptidoglycan and the amino acids D-alanine and diaminopimelic acid from the cell wall were made using different carbon substrates and through different stages of growth to determine isotopic fractionation of these compounds. The d 13C values of whole cells, peptidoglycan and D-alamine and the substrate sources (glucose, glutamic acid, isoleucine, lysine, phenylalanine) were similar. The d13C values of the D-alamine were within 0.5 o/ooof the substrate. In comparison, diaminopimelic acid was enriched in 13C by 10.3 o/oo relative to whole cells, peptidoglycan and substrate. Additional laboratory experiments also demonstated that the d13C of D-alamine did not vary significantly relative to the whole cell and substrate through different growth stages. Stable carbon isotope analysis of the bacterial amino acids was determined at two field locations, water from Santa Rosa Sound, Florida, a humic rich estuarine ecosystem, and jet-fuel contaminated soils of Tyndall Air Force Base, Florida. D-alamine and diaminopimelic acid were isolated from these water and soil samples and the amino acids were analyzed for purity after extraction and for their d13C values relative to organic matter in the environments. In the Santa Rosa Sound the d13C value of D-alamine was -27.6 ± 0.6 o/oo. This value is in the range of d13C values of bacteria and organic matter previously measured in the system, -24.0 to -27.0 o/oo. The d13C value of D-alamine in soil samples from Tyndall Air Force Base was -20.5 ± 1.7 o/oo (n = 4) similar to ranges of values measured for spilled jet-fuel and CO2 respired from the soil at this site. Results from this study demonstrate that D-alamine can be used as a biomarker for analysis of carbon sources that are assimilated by bacteria in soils and sediments.

Denslow, Nancy D., Christopher J. Bowman, Gillian Robinson, H. Stephen Lee, Ronald J. Ferguson, Michael J. Hemmer and Leroy C. Folmar. 1999. Biomarkers of Endocrine Disruption at the mRNA Level. In: Environmental Toxicology and Risk Assessment: Standardization of Biomarkers for Endocrine Disruption and Environmental Assessment. 8th Volume, ASTM STP 1364. D.S. Henshel, M.C. Black, and M.C. Harrass, Editors. American Society for Testing and Materials, West Conshohocken, PA. Pp. 24-35. (ERL,GB X978).

A large number of estrogen-mimicking, anthropogenic chemicals capable of disrupting normal reproductive function have been identified. The ubiquitous distribution of these compounds, many as components of complex industrial or municipal waste, has spurred an effort to develop methods to screen for chemicals which disrupt normal endocrine regulation of reproduction. We have developed assays that both allow exposure of animals in vivo and measure the response at the level of gene activation. We have developed a probe for measuring the induction of vitellogenin mRNA by Northern Blot in livers of sheepshead minnows treated with 17-b-estradiol. We have also developed a strategy for using Differential Display Polymerase Chain Reaction for determining gene induction profiles following exposure to estradiol. These methods should be adaptable to a variety of structurally diverse estrogen mimics.

Guillette, Louis J., Jr., Andrew A. Rooney, D. Andrew Crain and Edward F. Orlando. 1999. Steroid Hormones as Biomarkers of Endocrine Disruption in Wildlife. In: Environmental Toxicology and Risk Assessment: Standardization of Biomarkers for Endocrine Disruption and Environmental Assessment. 8th Volume, ASTM STP 1364. D.S. Henshel, M.C. Black, and M.C. Harrass, Editors. American Society for Testing and Materials, West Conshohocken, PA. Pp. 254-270. (ERL,GB X979).

Xenobiotic compounds introduced into the environment by human activity have been shown to adversely affect the endocrine system of wildlife. Various species exhibit abnormalities of (1) plasma sex steroid hormones, (2) altered steroid synthesis from the gonad in vitro and (3) altered steroidogenic enzyme function. These endpoints are sensitive and relatively easy to measure quantitatively with reliability and precision. These observations have led to the conclusion that sex steroid hormones could be markers of exposure to, and altered function from, endocrine disrupting contaminants (EDCs). However, there are serious limitations in the use of steroid hormones as generalized markers of EDC exposure. Steroid hormones exhibit seasonal, ontogenetic, gender and species-specific variation. Moreover, the regulation of sex steroid plasma concentrations is a relatively complex phenomenon capable of short-term (minutes - hours) alteration due to environmental inputs, such as acute stress -- an activational response. Alterations in steroid synthesis and degradation also can be a response to altered embryonic development due to EDC exposure - an organizational response. If steroid hormones are to be used as biomarkers, then closely controlled, well designed sampling has to be performed. Additionally, an appreciation of the variation possible in endocrine responses among the species to be studied must be obtained.

Foran, Christy M., Bethany N. Peterson and William H. Benson. 2002. Transgenerational and Developmental Exposure of Japanese Medaka (Oryzias latipes) to Ethinylestradiol Results in Endocrine and Reproductive Differences in the Response to Ethinylestradiol as Adults. EPA/600/J-02/379. Toxicol. Sci. 68(2):389-402. (ERL,GB X1028).

17a-Ethinylestradiol (EE), a synthetic estrogen found in birth control pills, has been detected in the effluent of municipal wastewater treatment plants in several countries. Because EE was designed to be extremely potent at the estrogen receptor (ER), environmental exposure to low concentrations has the potential to disrupt the development of normal endocrine and reproductive function when exposure occurs during critical periods in development. Japanese medaka, Oryzias latipes, were used to evaluate the effect of exposure to EE during development on adult reproduction and endocrine function and the sensitivity of these animals to estrogen exposure as adults. To determine if the response to exogenous estrogen stimulation was diminished or sensitized, adults resulting from the developmental exposure groups were reexposed to EE at respectively higher concentrations. Hatching exposure produced no changes in adult vitellogenin (VTG) content in the liver or circulating steroid concentrations, nor was reproduction affected. Reexposure of these adults inhibited reproduction, increased hepatic VTG and ER, and increased estrogen concentration measured in male plasma. Parental exposure produced permanent changes in hepatic content of ER and VTG in the adults resulting from exposure during gametogenesis and was related to a diminished response of males to subsequent estrogen exposure. The potential for this transgenerational exposure to decrease the responsiveness of males to EE is supported by comparing the concentration-response curves for hepatic VTG and ER in males exposed in ovo and as hatchlings. Our results indicate that the relationship between biomarkers and estrogen exposure will be altered by the timing and frequency of exposure.

Tilton, Susan C., Christy M. Foran and William H. Benson. 2005. Relationship Between Ethinylestradiol-Mediated Changes in Endocrine Function and Reproductive Impairment in Japanese Medaka (Oryzias latipes). Environ. Toxicol. Chem. 24(2):352-359. (ERL,GB X1072).

Many biochemical endpoints currently are used to describe endocrine function in fish; however, the sensitivity of these parameters as biomarkers of impaired reproduction or sexual development is not well understood. In the present study, adult Japanese medaka (Oryzias latipes) were assessed for reproductive output and endocrine function, including circulating steroid concentrations, ex vivo steroidogenesis from the gonads, aromatase activity, hepatic estrogen receptor (ER), and plasma vitellogenin (VTG) after exposure to 0, 0.2, 5,500, and 2,000 ng/L of 17 a-ethinylestradiol (EE) for 14 d. The EE altered these biochemical responses at various sites along the hypothalamus-pituitary-gonadal axis at concentrations as low as 0.2 ng/L, but it only depressed reproductive function at concentrations of 500 ng/L or greater. Offspring also had reduced ability to hatch at 500 ng/L of EE, but this concentration did not produce any other observed changes in development or sexual phenotype. The reproductive parameters correlated well with VTG, ER, and gonadosomatic index (GSI) in both sexes of adult medaka, which could be indicative of the ER-mediated mode of action for EE. Vitellogenin and ER were elevated at higher concentrations of EE in both sexes, whereas GSI was decreased. Overall, most biochemical endpoints were more sensitive than reproduction or development to exposure, indicating that reproductive function may be relatively protected.