Journal Article Citations and Abstracts

We examined the biodegradation of varying amounts of artificially weathered Alaskan North Slope crude oil in laboratory microcosm test systems that use natural seawater and simulate spills in open water and on sandy beaches. The model bioremediation treatment consisted of periodic applications of marine bacteria, selected to degrade n-alkanes and a range of aromatic compounds, suspended in a salts solution that supplied inorganic nitrogen and phosphorous. Beach microcosms dosed with low and high oiling lost an average of 22.5% and 11.3% oil weight, respectively. Open-water microcosms dosed with high and low oiling lost 19.1% and 2.9% oil weight, respectively. thus, the lower doses of oil were more efficiently degraded. The model bioremediation treatment also affected a greater number of selected analytical endpoints in the lower-oil-dose than higher-dose experiments and the former showed more substantial degradation of recalcitrant components. Above a certain threshold oil concentration, bioremediation did not effectively remove oil. Below this threshold the distinction between active bioremediation treatment and intrinsic biodegradation of the controls was less prominent; i.e., fewer of the oil components were statistically depleted by remediation treatment relative to controls. Furthermore, the oil-dose range over which bioremediation was realized in these systems occurred at very low oiling levels. Thus, under the environmental conditions simulated in these microcosms, the effectiveness of bioremediation peaked over a rather narrow low-dose oiling range.

Fisher, William S., Thomas C. Malone and James D. Giattina. 2003. Pilot Project to Detect and Forecast Harmful Algal Blooms in the Northern Gulf of Mexico. Environ. Monit. Assess. 81(1-3):373-381. (ERL,GB 1141).

More timely access to data and information on the initiation, evolution and effects of harmful algal blooms can reduce adverse impacts on valued natural resources and human health. To achieve this in the northern Gulf of Mexico, a pilot project was initiated to develop a user-driven, end-to-end (measurements to applications) observing system. A key strategy of the project is to coordinate existing state, federal and academic programs at an unprecedented level of collaboration and partnership. Resource managers charged with protection of public health and aquatic resources require immediate notice of algae events and a forecast of when, where and what adverse effects will likely occur. Further, managers require integrated analyses and interpretations, rather than raw data, to make effective decisions. Consequently, a functional observing system must collect and transform diverse measurements into usable forecasts. Data needed to support development of forecasts will include such properties as sea surface temperature, winds, currents and waves; precipitation and freshwater flows with related discharges of sediment and nutrients; salinity, dissolved oxygen, and chlorophyll concentrations (in vivo fluorescence); and remotely-sensed spatial images of sea surface chlorophyll concentrations. These data will be provided via a mixture of discrete and autonomous in situ sensing with near real-time data telemetry, and remote sensing from space (SeaWiFS), aircraft (hyperspectral imagery) or land (high-frequency radar). With calibration across these platforms, the project will ultimately provide a 4-dimensional visualization of harmful algae events in a time frame suitablel to resource managers.

DiDonato, Guy T., J. Kevin Summers and Thomas H. Roush. 2003. Assessing the Ecological Condition of a Coastal Plain Watershed Using a Probabilistic Survey Design. Environ. Monit. Assess. 85(1):1-21. (ERL,GB 1157).

Using a probabilistic survey design, we assessed the ecological condition of the Florida (U.S.A.) portion of the Escambia Riverwatershed using selected environmental and benthic macroinvertebrate data. Macroinvertebrates were sampled at 28 sites during July-August 1996, and 3414 individuals were identified. Taxonomic data were distilled into the 8 m of the Stream Condition Index (SCI), developed specifically for Florida waterways, and sites were classified on an ordinal scale as very good, good, poor, and very poor. The weighted cumulative distribution function of the SCI showed that 8% (± 7) of the stream and river miles in this landscape were in very good condition, while 25% (± 7), 51% (± 16), and 16%(±15) were in good, poor, and very poor condition, respectively.The only environmental parameter significantly correlated with SCI was dissolved oxygen (DO), and two sites classified as very poor had oxygen levels around or below 2 mg L-1. However, other sites exhibited similarly low SCI values without the attendant low DO, implying that factors determining site condition were complex and multivariate. The results of this survey corroborate Federal and state assessments demonstrating that many locations within this watershed exhibit significant degradation.

Leblond, Jeffrey D., Terence J. Evens and Peter J. Chapman. 2003. Biochemistry of Dinoflagellate Lipids, with Particular Reference to the Fatty Acid and Sterol Composition of a Karenia brevis Bloom. EPA/600/J-03/491. Phycologia. 42(4):324-331. (ERL,GB 1160).

The harmful marine dinoflagellate, Karenia brevis (Dinophyceae), frequently forms large toxic blooms in the waters off of the west coast of Florida (USA) and is responsible for massive fish kills and public health concerns. Despite decades of field studies on this organism, no investigation has yet characterized the lipid composition of a K. brevis bloom. To address this lack of information, samples from a 1999 K. brevis bloom from the northwest Florida coast were analyzed for their fatty acid and sterol composition. Fatty acids found in lipid fractions containing membrane phospholipids, chloroplast-associated glycolipids, and storage triglycerides differed significantly. The glycolipid fraction was found to contain octadecapentaenoic acid [18:5(n-3)], a fatty acid commonly associated with dinoflagellates. The phospholipid fraction was found to contain small amounts of two recently described, highly unsaturated fatty acids, octacosaoctaenoic acid [28:8(n-3)] and octacosaheptaenoic acid [28:7(n-6)]. Fatty acids from the triglyceride fraction were more abundant than those associated with glycolipid or phospholipids. Sterols were found mainly as free sterols and were dominated by two compounds (24S)-4a-methyl-5a-ergosta-8(14),22-dien-3b-ol (ED) and its 27-nor derivative (NED). The lipid composition of these samples very closely resembles laboratory-grown cultures of K. brevis and serves to provide an in situ field validation of past laboratory examinations of this organism. The implications of our data are discussed in the context of the physiological autecology of K. brevis, in the form of a minireview on the biochemistry of dinoflagellate lipids, as studied in both the laboratory and the environment.

Fisher, William S., Leah M. Oliver, James T. Winstead and Aswani K. Volety. 2003. Stimulation of Defense Factors for Oysters Deployed to Contaminated Sites in Pensacola Bay, Florida. EPA/600/J-03/405. Aquat. Toxicol. 64(4):375-391. (ERL,GB 1161).

A positive association between chemical contaminants and defense factors has been established for eastern oysters (Crassostrea virginica) from Florida, but it is unknown whether such factors can be stimulated through short-term exposure to contaminants in the field. Hatchery oysters were deployed at two contaminated and one reference site near Pensacola, Florida, during spring and summer 1998. Putative defense measurements, notably hemocyte count and bactericidal activity, were significantly elevated after 12-week deployment during summer at the most contaminated site. This site exhibited a dramatic increase in chemical concentrations in oyster tissue relative to both the initial concentrations in hatchery oysters and to oysters deployed at the reference site. Hemocyte activity was not stimulated after 16-week deployment of hatchery oysters in spring, despite similar increases in tissue chemical concentrations, so defense activation by short-term exposure may covary with other unmeasured environmental or physiological parameters. Using the converse approach, Pensacola Bay oysters were collected from two contaminated sites and deployed at the reference site for 16 week during spring. Results from this converse deployment were ambiguous; serum lysozyme concentrations were reduced for oysters transplanted from both sites, but hemocyte activities were not significantly changed. The principal outcome from this study was the demonstration of enhanced defense activities for oysters upon short-term summer deployment at a contaminated site.

Oliver, Leah M., William S. Fisher, Aswani K. Volety and Ziad Malaeb. 2003. Greater Hemocyte Bactericidal Activity in Oysters (Crassostrea virginica) from a Relatively Contaminated Site in Pensacola Bay, Florida. Aquat. Toxicol. 64(4):363-373. (ERL,GB 1162).

Bivalve mollusks such as Crassostrea virginica inhabiting polluted estuaries and coastal areas may bioaccumulate high concentrations of contaminants without apparent ill effects. However, changes in putative internal defense activities have been associated with contaminant accumulation in both experimental and long-term field exposures. In an effort to elucidate these relationships, 40 oysters were collected from Bayou Chico (BC) and East Bay (EB) in Pensacola Bay, FL, two estuaries known to differ in the type and magnitude of chemical contaminants present. Oyster tissue concentrations of metals, tri- and di-butyltim (TBT, DBT), polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were measured in individual oysters, as were hemocyte counts (HC), hemocyte bacterial killing indices (KI), serum lysozyme (LYS) and serum protein (PRO) levels. Average HC, KI, LYS and PRO were significantly higher in BC oysters, which also had significantly higher tissue concentrations of total trace metals, butyltins (BTs), PAHs, PCBs, pesticides, and Mn, Cu, Zn, and Sn. EB oysters had low organic contaminant levels and no detectable BTs, but significantly higher concentrations of Al, Cr, Fe, Ag, Cd, and Hg. Simple correlation analysis between specific defense measurements and specific chemical analytes showed specific positive relationships that corroborated previous findings in other FL estuaries. Canonical correlation analysis was used to examine relationships between defense measurements and tissue metals using linearly combined sets of variables. Results were also consistent with previous findings: the highest possible canonical correlations was positive: r = .864, P < 0.0019 among canonical variables composed of HC, KI and LYS for defense, and Fe, Cu, Ag, Cd, Sb, Sn, Ni, Pb and Hg for metals.

Cripe, G.M., C.L. McKenney, Jr., M.D. Hoglund and P.S. Harris. 2003. Effects of Fenoxycarb Exposure on Complete Larval Development of the Xanthid Crab, Rhithropanopeus harrisii. EPA/600/J-03/407. Environ. Pollut. 125(2):295-299. (ERL,GB 1163).

Pest control agents, such as juvenile hormone analogues (JHA), have been developed to limit effects on non-target organisms that co-inhabit insect pest habitats. Rhithropanopeus harrisii, an estuarine xanthid crab, was used to observe the impacts of the JHA, fenoxycarb, on the pattern of complete larval development as well as survival of larvae and successful metamorphosis to first crab stage. Significant mortality occurred in the first of four zoeal stages (after 2-3 days of exposure) at the highest treatment of 240 µg fenoxycarb/L and in megalopae exposed to 48 µg fenoxycarb/L. The time required to metamorphose to the first crab stage was significantly increased for megalopae in all treatments greater than or equal to 48 µg/L. This delay in development was sufficient to significantly prolong the entire developmental period from zoea to crabs. Unexposed larvae developed to crabs in an average of 16 days; larvae exposed to greater than or equal to 48 µg/L required 19-20 days. Reduced survival and extended duration of developing larval stages in the life history of a benthic invertebrate may alter the population dynamics of these organisms in the estuary.

Winstead, James T. and Lee A. Courtney. 2003. Ovacystis-like Condition in the Eastern Oyster Crassostrea virginica from the Northeastern Gulf of Mexico. EPA/600/J-03/147. Dis. Aquat. Org. 53(1):89-90. (ERL,GB 1167).

Histological examination of the eastern oyster, Crassostrea virginica, from a study in Pensacola Bay, Florida, revealed 2 cases of abnormally large, basophilic ova that resembled cells characteristic of ovacystis disease previously reported in oysters from Maine and Long Island. The hypertrophied gametes measured up to 250 µm in diameter, had scant cytoplasm and contained granular nuclear-masses of Feulgen-positive material. Electron microscopy of reclaimed tissue revealed these masses to consist of virus-like particles (average 46 nm) similar to those reported in cases of ovacystis.

Kurtz, Janis C., Diane F. Yates, John M. Macauley, Robert L. Quarles, Fred J. Genthner, Cynthia A. Chancy and Richard Devereux. 2003. Effects of Light Reduction on Growth of the Submerged Macrophyte Vallisneria americana and the Community of Root-Associated Heterotrophic Bacteria. EPA/600/J-03/373. J. Exp. Mar. Biol. Ecol. 291(2):199-218. (ERL,GB 1169).

A shading experiment was conducted over a growing season to measure the effects of light reduction on Vallisneria americana in Perdido Bay on the Florida-Alabama border, and to determine the response of heterotrophic bacteria in the rhizosphere. Plants subjected to 92% light reduction showed the most pronounced effects in chlorophyll a concentration, above and below ground biomass, and leaf dimensions. The results and field observations further suggested the V. americanalife cycle, as exhibited in temperate waters, was impaired. Heterotrophic bacteria were enumerated and identified (i) from the roots and sediments of fully illuminated plants and from unvegetated sediments at three intervals and (ii) from the roots of plants that had been subjected to 92% light reduction for three months. Up to two orders of magnitude greater numbers of bacteria were enumerated from root samples than sediment samples on a dry weight basis. Bacteria enumerated from the roots of plants subjected to light reduction (1.3 ± 1.1 x 108 CFU g-1) were significantly higher than numbers of bacteria enumerated from the roots of fully illuminated plants (4.8 ± 1.8 x 107 g-1 in the summer) or sediment samples (1.4 ± 0.03 x 106 g-1). This suggests the roots of seagrasses stressed by light reduction provided more nutrients for bacterial growth. Higher percentages of Gram-negative bacteria were isolated from roots (up to 85% in the fall) than sediments (0-15%). Examination of isolates for traits characteristic of rhizosphere bacteria (siderophore production, formation of the phytohormone indole-3-acetic acid, and antifungal activity) did not show a clear distinction between root-associated and sediment isolates. Taxonomic identifications of root associated bacteria based on MIDI analysis of fatty acid methyl esters were consistent with bacteria known to be associated with other plants or found at oxic-anoxic interfaces. In addition, the bacterial identifications showed most species were found only with roots or only in sediments. Relative to the sediment samples, the higher numbers of bacteria and the higher incidence of Gram-negative bacteria associated with V. americana roots, together with the bacterial identifications, suggests selection of a rhizosphere bacterial community.

Larkin, Patrick, Leroy C. Folmar, Michael J. Hemmer, Arianna J. Poston and Nancy D. Denslow. 2003. Expression Profiling of Estrogenic Compounds Using a Sheepshead Minnow cDNA Macroarray. EPA/600/J-04/229. Environ. Health Perspect. 111(6):839-846. (ERL,GB 1171).

A variety of anthropogenic compounds are capable of binding to the estrogen receptor (ER) of vertebrate species. Binding of these chemicals to the ER can interfere with homeostasis by altering normal gene expression patterns. The purpose of this study was to characterize the expression of 30 genes using a sheepshead minnow (Cyprinodon variegatus) cDNA macroarray. Many of the genes on the array were previously identified by differential display reverse transcriptase-polymerase chain reaction to be upregulated or downregulated in sheepshead minnows treated through aqueous exposure to known or suspected estrogenic chemicals. The results of this study show that 17b-estradiol (E2), 17a-ethinyl estradiol (EE2), diethylstilbestrol (DES), and methoxychlor (MXC) have similar genetic signature for the 30 genes examined. The genetic signature of fish treated with p-nonlyphenol (pNP) was identical in pattern to fish treated with E2, EE2, DES, and MXC except for the additional upregulation of a cDNA clone that shares similarity to ubiquitin-conjugating enzyme 9. Endosulfan produced results that resembled the gene expression patterns of untreated control fish with exception of the upregulation of estrogen receptor a and the downregulation of a cDNA clone that shares similarity to 3-hydroxy-3-methylglutaryl-coenzyme A reductase. We show that our estrogen-responsive cDNA macroarray can detect dose-dependent changes in gene expression patterns in fish treated with EE2.

Middaugh, Douglas P. and Charles L. McKenney, Jr. 2003. Occurrence and Orientation of Flounders (Bothidae: Paralichthys) on an Intertidal Beach. J. North Carol. Acad. Sci. 119(4):157-171. (ERL,GB 1172).

The intertidal movement and burying pattern of paralichthid flounders was studied on DeVeaux Bank, a semi-permanent island at the mouth of the North Edisto River in South Carolina. A total of 1,366 flounders buried within the study site during the two yr study. The standard length of flounders burying ranged from 19 to 48 cm. The frequence of flounders digging into substrates in the upper intertidal zone to form "beds" was greater on nighttime high tides than during daytime high tides. Most flounders, 99.99%, buried in sand, the remainder in mud. There was no significant difference in the directional orientation of buried flounders and the direction of tidal currents. The orientation of flounder beds generally deviated from the orientation of tidal current flow by 10 degrees azimuth or less. Flounder beds were oriented into flood tide currents an average of 68% and 32% into ebb tide currents. Periodic washover events formed megaripples in the sand within the study site. There was a significant difference in the size distribution of flounder beds on megaripples and between megaripples. Smaller flounders were positioned on megaripples while larger individuals were found between the sand ripples. Tidal flow turbulence or water depth may have played a role in the spatial distribution of flounders in these two locations. There was a significant correlation between the standard length of flounder beds and the water depth where they occurred. Smaller flounders buried at shallower water depths than larger flounders. Avoidance of predators, including the bottlenose dolphin, could have influenced the location where flounders buried in the intertidal zone.

Murrell, Michael C. 2003. Bacterioplankton Dynamics in a Subtropical Estuary: Evidence for Substrate Limitation. Aquat. Microb. Ecol. 32(3):239-250. (ERL,GB 1173).

Bacterioplankton abundance and metabolic characteristics were measured along a transect in Pensacola Bay, Florida, USA, to examine the factors that control microbial water column processes in this subtropical estuary. The microbial measures included 3 H-L-leucine incorporation, ectoenzyme activity (aminopeptidase, a-D-glucosidase, b-D-glucosidase) and bacterial abundance. Bacterioplankton abundance ranged from 1.8 to 15.3 x 109 1-1 (average: 6.2 x 106); highest abundances occurred during summer months, particularly in the upper estuary. Bacterial secondary production ranged from 20 to 273 µg C 1-1 d-1 (average 115), aminopeptidase activities ranged from 34-356 nmol 1-1 d-1 (average 165), a-D-glucosidase ranged from 0.4 to 61 nmol 1-1d-1 (average: 8.3), and b-D-glucosidase ranged from 1.4 to 53.1 nmol 1-1d-1 (average 10.5). Bacterioplankton exhibited strong seasonality, suggesting that temperature was an important driver of the observed variability. When normalized for bacterial biomass, metabolic rates exhibited a striking inter-annual pattern with lower rates during summer 2000 than 2001. This pattern was consistent with freshwater flows, which were much lower during 2000 than 2001, consequently lowering nutrient and dissolved organic carbon (DOC) supply to the estuary. These results underscore the importance of riverine flux of materials to support bacterial metabolism and suggest that bacterioplankton were substrate-limited during the low flow period. The empirical relationships between bacterioplankton and phytoplankton variables were similar to those found in literature synthesis studies, with the notable exception that bacterial abundances and production were higher than predicted from chlorophyll a concentration. One explanation for this departure is that these relationships are drawn largely from cool temperate environments and warm subtropical systems are underrepresented in the literature.

Lewis, Michael A., Darrin D. Dantin, Calvin C. Walker, Janis C. Kurtz and Richard M. Greene. 2003. Toxicity of Clay Flocculation of the Toxic Dinoflagellate, Karenia brevis, to Estuarine Invertebrates and Fish. Harmful Algae. 2(4):235-246. (ERL,GB 1175).

The benthic environmental effects of proposed control procedures for red tide events are relatively unknown but important to understand. The objective of this study was to determine the laboratory-derived toxicities of a clay flocculation technique proposed for the Florida red tide dinoflagellate, Karenia brevis using four estuarine test species. Phosphatic clay in mixture with three concentrations (0.5, 5.0 and 50.0 mg/l of a chemical coagulant (polyaluminum hydroxy chloride) was not acutely or chronically toxic in most cases to benthic infaunal and epibenthic macroinvertebrates (Leptocheirus plumulosus, Amplesica abidita, Palemonetes pugio) and larval fish (Cyprinodon variegatus). K. brevis alone (density range = 3880 to 5060 cells/ml; brevetoxin range = 19.8 to 140.7 µg/l was very toxic to juvenile C. variegatus and, to a lesser extent, L. plumulosus. The addition of clay and coagulant did not usually mitigate this toxicity. The aggregates of clay, coagulant and K. brevis cells when settled over a natural sediment, with few exceptions, were as toxic as the red tide organism alone. This suggests that the use of this control procedure will not increase nor decrease toxicity to benthic organisms attributable to an untreated Florida red tide. Validation of this conclusion, however, is needed since it is based on laboratory-derived, single species toxicity data using media collected from a simulated red tide event. The determination of the environmental effects on indigenous biota in near-coastal areas during a natural red tide event, prior to and after treatment with clay flocculation, would provide needed perspective for a more realistic hazard assessment.

Hansen, Lara, Steven F. Hedtke and Wayne R. Munns, Jr. 2003. Integrated Human and Ecological Risk Assessment: A Case Study of Ultraviolet Radiation Effects on Amphibians, Coral, Humans, and Oceanic Primary Productivity. EPA/600/J-03/207. Hum. Ecol. Risk Assess. 9(1):359-377. (ERL,GB 1178).

Ultraviolet radiation (UVR) is a naturally occurring stressor to most forms of life. The sole relevant source of this stressor is the sun. The Earth's stratospheric ozone layer reduces the amount of UVR that reaches the Earth's surface. The potential for continued depletion of this ozone layer due to human activities and the subsequent increase in UVR at the Earth's surface is a global environmental concern for both humans and ecosystems. An integrated risk assessment provides efficiency in data gathering, analysis and reporting by enabling risk assessors to use the combined knowledge from many disciplines to evaluate overall risk. This report describes the steps and example information that could be used for an integrated risk assessment but is not an actual risk assessment with all its associated calculations and conclusions. It is intended to illustrate the advantages of the integrated risk assessment approach for evaluating adverse effects of a nonchemical stressor.

Edwards, Katherine R., Joe Eugene Lepo and Michael A. Lewis. 2003. Toxicity Comparison of Biosurfactants and Synthetic Surfactants Used in Oil Spill Remediation to Two Estuarine Species. Mar. Pollut. Bull. 46(10):1309-1316. (ERL,GB 1182).

The relative environmental toxicities of synthetic and biogenic surfactants used in oil spill remediation efforts are not well understood. Acute and chronic toxicities of three synthetic surfactants and three microbially produced surfactants were determined and compared in this study for the estuarine epibenthic invertebrate, Mysidopsis bahia and the inland silverside, Menidia beryllina. The toxicities of the surfactants were determined in standard laboratory static and static-renewal tests of 4 to 7 days duration. Results were specific to the surfactant, response parameter and test species. The LC50 values (nominal concentrations) for M. bahia ranged from 3.3 mg/L (Triton X-100) to > 1000 mg/L (PES-61) and 2.5 mg/L (Triton X-100) to 413.6 mg/L (PES-61) for M. beryllina. Chronic first-effect concentrations (mg/L) for the six surfactants ranged from 2.3 to 465.0 (M. beryllina) and 1.0 to > 1000.0 (M. bahia) based on reductions in growth and fecundity. Few generalizations could be made concerning the results due to their variability but M. bahia was generally the more sensitive species and the toxicities of the biosurfactants were intermediate to those of the synthetic surfactants.

Smith, C. Lavett, James C. Tyler, William P. Davis, Robert S. Jones, David G. Smith and Carole C. Baldwin. 2003. Fishes of the Pelican Cays, Belize. Atoll Res. Bull. 497(August):1-88. (ERL,GB 1204).

The fishes of the Pelican Cays, Belize, were sampled using a combination of small rotenone stations and visual censuses. Records from the Pelican Cays are compared with lists of species known from the adjacent Rhomboidal Cays, the inshore coastline of Belize, the outer margin of the Belize Barrier Reef and the off-shore banks. At least three species are known only from the Pelican Cays (and nearby Rhomboidal and Wee-Wee Cays).

Geter, David R., John W. Fournie, Marius H. Brouwer, Anthony B. DeAngelo and William E. Hawkins. 2003. p-Nitrophenol and Glutathione Response in Medaka (Oryzias latipes) Exposed to MX, a Drinking Water Carcinogen. EPA/600/J-03/208. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 134(3):353-364. (ERL,GB X1030). (Oryzias latipes)

When chlorine is introduced into public drinking water for disinfection, it can react with organic compounds in surface waters to form toxic by-products such as 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX). We investigated the effect of exposure to MX on cytochrome P4502E1 (CYP2E1)-like activity and total glutathione (GSH) in the liver of the small fish model, medaka (Oryzias latipes). The multi-site carcinogen methylazoxymethanol-acetate (MAMAc) was the positive control compound. Both medaka liver microsome preparations and S-9 fractions catalyzed the hydroxylation of p-nitrophenol (PNP), suggesting CYP2E1-like activity in the medaka. Male medaka exposed for 96 h to the CYP2E1 inducers ethanol and acetone under fasted conditions showed significant increases in PNP-hydroxylation activity. Furthermore, total reduced hepatic GSH was reduced in fish fasted for 96 h, indicating that normal feeding is a factor in maintaining xenobiotic defenses. Exposure to MX and MAMAc induced significant increases in hepatic CYP2E1-like activity, however MX exposure did not alter hepatic GSH levels. These data strengthen the role of the medaka as a suitable species for examining cytochrome P450 and GSH detoxification processes and the role these systems play in chemical carcinogenesis.

Gooding, Meredith P., Vickie S. Wilson, Leroy C. Folmar, Dragoslav T. Marcovich and Gerald A. LeBlanc. 2003. Biocide Tributyltin Reduces the Accumulation of Testosterone as Fatty Acid Esters in the Mud Snail (Ilyanassa obsoleta). EPA/600/J-03/409. Environ. Health Perspect. 111(4):426-430. (ERL,GB X1033).

Imposex, the development of male sex characteristics by female gonochoristic snails, has been documented globally and is causally associated with exposure to the ubiquitous environmental contaminant tributyltin (TBT). Elevated testosterone levels in snails also are associated with TBT, and direct exposure to testosterone has been shown to cause imposex. We discovered previously that the mud snail (Ilyanassa obsoleta) biotransforms and retains excess testosterone primarily as fatty acid esters. The purpose of this study was to determine whether TBT interferes with the esterification of testosterone, resulting in the elevated free (unesterified) testosterone levels associated with imposex. Exposure of snails to environmentally relevant concentrations of TBT (greater than or equal to 1.0 ng/L as tin) significantly increased the incidence of imposex. Total (free + esterified) testosterone levels in snails were not altered by TBT; however, free testosterone levels increased with increasing exposure concentration of TBT. TBT-exposed snails were given [14 C]testosterone to measure the production of [14C]testosterone-fatty acid esters. The production of testosterone-fatty acid esters decreased with increasing exposure concentration of TBT. These results indicate that TBT elevates free testosterone levels in snails by decreasing the production of retention of testosterone-fatty acid esters. These findings were confirmed among field-sampled snails where individuals collected from a high-tin-affected site exhibited a greater incidence of imposex, higher free testosterone levels, and lower testosterone-fatty acid ester levels when compared with individuals sampled from a low-tin-affected site. Decreased testosterone-fatty acid esterification among TBT-treated snails was not caused by direct inhibition of the acyl coenzyme A:testosterone acyltransferase (ATAT) enzyme responsible for testosterone esterification, nor by suppressed ATAT protein expression. The target of TBT may be a co-contributor to the testosterone fatty esterification process or a factor in the enhanced hydrolysis of the testosterone-fatty acid pool.

Hawkins, William E., William W. Walker, John W. Fournie, C. Steve Manning and Rena M. Krol. 2003. Use of the Japanese Medaka (Oryzias latipes) and Guppy (Poecilia reticulata) in Carcinogenesis Testing Under National Toxicology Program Protocols. EPA/600/J-03/391. Toxicol. Pathol. 31(Suppl):88-91. (ERL,GB X1034).

A need exists for whole animal toxicity, mutagenesis, and carcinogenesis models that are alternative to the traditional rodent test models and that are economical, sensitive, and scientifically acceptable. Among small fish models, the Japanese medaka (Oryzias latipes) is preeminent for investigating effects of carcinogenic and/or toxic waterborne hazards to humans. The guppy (Poecilia reticulata), although less widely used, is valuable as a comparison species. Both species are easy to maintain and handle in the laboratory and there is a large body of background information on their responsiveness to a range of classes of carcinogens. There are considerable data on the occurrence of background diseases and on spontaneous neoplastic lesions, both of which occur relatively rarely. With few modifications, the medaka and guppy are amenable to carcinogenicity testing under the rigid standards established by the National Toxicology Program (NTP) for rodent tests. The advantages of the small fish in carcinogenesis studies are best realized in long-term studies that involve environmentally realistic exposures. Studies to identify chronic effects can be conducted in about 12 months, near the life span of medaka in our laboratory. Practically, 9-month studies are optimal but shorter study cycles and a variety of exposure/growout and initiation/promotion scenarios are available. Studies on 3 compounds tested in medaka under NTP protocols are under review and preliminary analysis indicates that chronic carcinogenicity bioassays with medaka, guppy, and potentially with other small fish species are feasible and scientifically valid.

Hale, Stephen S., Anne Hale Miglarese, M. Patricia Bradley, Thomas J. Belton, Larry D. Cooper, Michael T. Frame, Christopher A. Friel, Linda M. Harwell, Robert E. King, William K. Michener, David T. Nicolson and Bruce G. Peterjohn. 2003. Managing Troubled Data: Coastal Data Partnerships Smooth Data Integration. EPA/J-03/392. Environ. Monit. Assess. 81(1-3):133-148. (ERL,GB X1035).

Understanding the ecology, condition, and changes of coastal areas requires data from many sources. Broad-scale and long-term ecological questions, such as global climate change, biodiversity, and cumulative impacts of human activities, must be addressed with databases that integrate data from several different research and monitoring programs. Various barriers, including widely differing data formats, codes, directories, systems, and metadata used by individual programs, make such integration troublesome. Coastal data partnerships, by helping overcome technical, social, and organization barriers, can lead to a better understanding of environmental issues, and may enable better management decisions. Characteristics of successful data partnerships include a common need for shared data, strong collaborative leadership, committed partners willing to invest in the partnership, and clear agreements on data standards and data policy. Emerging data and metadata standards that become widely accepted are crucial. New information technology is making it easier to exchange and integrate data. Data partnerships allow us to create broader databases than would be possible for any one organization to create itself.

Hyland, Jeffrey L., W. Leonard Balthis, Virginia D. Engle, Edward R. Long, John F. Paul, J. Kevin Summers and Robert F. Van Dolah. 2003. Incidence of Stress in Benthic Communities Along the U.S. Atlantic and Gulf of Mexico Coasts Within Different Ranges of Sediment Contamination from Chemical Mixtures. EPA/600/J-03/393. Environ. Monit. Assess. 81(1-3):149-161. (ERL,GB X1036).

Synoptic data on concentrations of sediment-associated chemical contaminants and benthic macroinfaunal community structure were collected from 1,389 stations in estuaries along the U.S. Atlantic and Gulf of Mexico coasts as part of the nationwide Environmental Monitoring and Assessment Program (EMAP). These data were used to develop an empirical framework for evaluating risks of benthic community-level effects within different ranges of sediment contamination from mixtures of multiple chemicals present at varying concentrations. Sediment contamination was expressed as the mean ratio of individual chemical concentrations relative to corresponding sediment quality guidelines (SQGs), including Effects Range-Median (ERM) and Probable Effects Level (PEL) values. Benthic condition was assessed using diagnostic, multi-metric indices developed for each of three EMAP provinces (Virginian, Carolinian, and Louisianian). Cumulative percentages of stations with a degraded benthic community were plotted against ascending values of the mean ERM and PEL quotients. Based on the observed relationships, mean SQG quotients were divided into four ranges corresponding to either a low, moderate, high, or very high incidence of degraded benthic condition. Results showed that condition of the ambient benthic community provides a reliable and sensitive indicator for evaluating the biological significance of sediment-associated stressors. Mean SQG quotients marking the beginning of the contaminant range associated with the highest incidence of benthic impacts (73-100% of samples, depending on the province and type of SQG) were well below those linked to high risks of sediment toxicity as determined by short-term toxicity tests with single species. Measures of the ambient benthic community reflect the sensitivities of multiple species and life stages to persistent exposures under actual field conditions. Similar results were obtained with preliminary data from the west coast (Puget Sound).

Orlando, Edward F., Gerald A. Binczik, Peter Thomas and Louis J. Guillette, Jr. 2003. Reproductive Seasonality of the Male Florida Gar, Lepisosteus platyrhincus. EPA/600/J-03/395. Gen. Comp. Endocrinol. 131(3):365-371. (ERL,GB X1038).

The objective of this study was to characterize the reproductive seasonality of a wild population of male Florida gar, Lepisosteus platyrhincus. We measured the gonadosomatic index, reproductive stage of the testes, seminiferous tubule area, and plasma concentrations of testosterone and 11-ketotestosterone for each fish. A seasonal pattern was observed in the reproduction of male gar. Following a quiescent period in the summer, there was an increase in plasma testosterone concentration during the fall, which was associated with the onset of active spermatogenesis as indicated by an increase in seminiferous tubule area and gonadosomatic index. Plasma concentrations of 11-ketotestosterone remained basal during the summer and winter and then increased during the early spring. These data, taken together with data from female gar collected in a parallel study by our lab, suggest that spawning occurred during the early spring (February-March), followed by a general decrease in reproductive parameters to summer levels. Recognizing that peaks of hormone concentrations and spawning period could vary with annual seasonal variation, this study is important because it is the first characterization of the seasonal morphological and endocrinological reproductive pattern in a holostean species. Furthermore, it increases our understanding of the reproductive biology of semitropical fishes.

Gunderson, M.P., S.A.E. Kools, M.R. Milnes and L.J. Guillette, Jr. 2003. Effect of Acute Stress on Plasma B-Corticosterone, Estradiol-17B and Testosterone Concentrations in Juvenile American Alligators Collected from Three Sites Within the Kissimmee-Everglades Drainage Basin in Florida (USA). EPA/600/J-03/400. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 135(3):365-374. (ERL,GB X1043).

The effect of acute stress on plasma b-corticosterone (B), testosterone (T) and estradiol-17b (E2), concentrations in juvenile alligators collected from sites with varying sediment contaminants was examined in this study. Dramatic increases in plasma B concentrations were observed in alligators from all of the sites after 2 h of capture although females from the intermediate contaminant site exhibited a significantly lower percentage increase in B than females from the other two sites. Males from the site with the highest contaminant levels exhibited elevated initial B concentrations relative to the other sites. This pattern was not observed after 2 h of restraint. Females from the highest contaminant site exhibited depressed initial T when compared to the other sites although this pattern was not observed after 2 h of restraint. Neither E2 nor T decreased after 2 h in females, whereas T concentrations decreased in all males over the same time period. The variance associated with these endpoints was also examined to determine whether it could serve as a more sensitive marker for perturbations of the endocrine system and stress response. Females from the higher and intermediate contaminant sites exhibited the lowest and highest standard errors (respectively) associated with 2 h plasma B concentrations with no differences among mean concentrations suggesting a perturbation of the stress response in these animals that was not detected by examining the means. We concluded that the environmental contaminants could be acting as stressors, leading to the observed differences.

Toft, Gunnar, Thea M. Edwards, Erik Baatrup and Louis J. Guillette, Jr. 2003. Disturbed Sexual Characteristics in Male Mosquitofish (Gambusia holbrooki) from a Lake Contaminated with Endocrine Disruptors. EPA/600/J-03/404. Environ. Health Perspect. 111(5):695-701. (ERL,GB X1047).

Previous laboratory studies have demonstrated that estrogenic and antiandrogenic chemicals can alter several sexual characteristics in male poeciliid fishes. Whether similar disturbances occur under field conditions remains to be confirmed. Lake Apopka, Florida, is contaminated with numerous chemicals, some of which possess endocrine-disrupting activity. Male mosquitofish (Gambusia holbrooki) were collected monthly from December 2000 through May 2001 from Lake Apopka and two nearby reference lakes, Orange Lake and Lake Woodruff National Wildlife Refuge. Selected sexual characteristics were compared temporally and among lakes during the collection period. Male fish from Lake Apopka had slightly shorter gonopodia and on average 32 and 47% fewer sperm cells per milligram testis, when compared with the fish collected from Orange Lake and Lake Woodruff, respectively. The testes weights increased markedly during spring, with significantly smaller testes in fish from Lake Apopka than from Orange Lake, but surprisingly, the smallest testes occurred in males obtained from the Lake Woodruff population. The highest liver weights were found in the Lake Apopka population. Whole-body concentrations of testosterone and estradiol varied among months; the peak testosterone concentration occurred in January and was significantly lower in male fish from Lake Apopka compared with Orange Lake. The intensity of male courtship behavior was highly correlated to body testosterone concentration, but no statistically significant differences in sexual behavior among the lakes were found. We conclude that sexual characteristics of relevance to male reproductive capacity are altered in the Lake Apopka mosquitofish population, and we discuss the presence of chemicals with antiandrogenic effects in Lake Apopka as a possible cause of the observed alterations.

Tilton, Susan C., Christy M. Foran and William H. Benson. 2003. Effects of Cadmium on the Reproductive Axis of Japanese medaka (Oryzias latipes). EPA/600/J-03/413. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 136(3):265-276. (ERL,GB X1048).

Cadmium (Cd) is a ubiquitous element and a significant inorganic pollutant that has previously been found to bioaccumulate in reproductive organs of fish and disrupt important endocrine processes, especially those involved in synthesis, release and metabolism of hormones. Clearly, there is potential for reproductive effects in fish populations exposed to Cd, however few studies have investigated the non-lethal consequences of Cd in fish. To this extent, adult male and female Japanese medaka were exposed to 0-10 ppb Cd for 7 weeks. Reproductive endpoints were monitored during weeks 6 and 7 of exposure and compared to physiological responses along the hypothalamus-pituitary-gonadal (HPG) axis, including plasma vitellogenin (VTG), hepatic estrogen receptor (ER), plasma steroids, gonadal-somatic indices (GSI), and gonadal steroid release. There were no observed effects on VTG and ER by long-term Cd exposure. However, gonadal steroid release was significantly decreased in males and females at all exposure concentrations and female plasma estradiol levels were significantly altered at concentrations higher than 5 ppb Cd. Overall, responses along the HPG axis were more sensitive to Cd exposure than the reproductive and developmental endpoints, which were not affected in this study, indicating that higher level impairment in fish might be relatively protected.

Rooney, Andrew A., Dieldrich S. Bermudez and Louis J. Guillette, Jr. 2003. Altered Histology of the Thymus and Spleen in Contaminant-Exposed Juvenile American Alligators. J. Morphol. 256(3):349-359. (ERL,GB X1053).

Morphological difference in spleen and thymus are closely related to functional immune differences. Hormonal regulation of the immune system has been demonstrated in reptilian splenic and thymic tissue. Spleens and thymus were obtained from juvenile alligators at two reference sites in Florida, USA: Orange Lake and a National Wildlife Refuge, Lake Woodruff, as well as from a contaminated lake, Lake Apopka. Lake Apopka has been extensively polluted with agricultural pesticides. Tissues were prepared for histological analysis to determine if previously detected endocrine abnormalities associated with contaminant exposure might also be reflected in morphological differences in splenic and thymic structures important for immunological response. Similar tissues were taken from captive-raised juvenile female alligators (3 years old) that were hatched from eggs collected on Lake Woodruff and Lake Apopka. Difference in thymic ratios (medulla/cortex) were found among alligators collected from the two lakes (P= 0.0051). Alligators from Lake Apopka had smaller thymic ratios than animals from either reference lake. Males from Lake Woodruff had significantly smaller lymphocyte sheaths in the spleen than females (P=0.0009), indicative of a normal sexual dimorphism. Lymphocyte sheath width differed among females obtained from the three lakes, with females from Lake Apopka having the smallest sheath width and those from Orange Lake having the largest. Malpighian body area was largest in alligators from Orange Lake, intermediate in Lake Woodruff, and smallest in Lake Apopka. In contrast to that observed for wild-caught animals, no difference was found in the thymic medulla/cortex ratio of captive-raised female alligators (P=0.378). Captive-raised female alligators from Lake Apopka and Lake Woodruff displayed lake-associated differences in lymphocyte sheath width as observed in wild animals: Lake Apopka alligators had smaller lymphocyte sheath width compared to Woodruff alligators (P= 0.0396). In contrast to wild-caught animals, area of the Malpighian bodies did not differ by lake in the captive-raised female alligators (P=0.066). The enlarged thymic cortex suggests a change in T-lymphocyte maturation within the thymus of alligators from a contaminated environment, Lake Apopka. The results point to alterations in the histology of the thymus and spleen. Further studies are required to examine the functional significance of these observations.