Journal Article Citations and Abstracts

Perdido Bay is a shallow estuarine system approximately 130 km2 with three major freshwater inputs. On a seasonal basis the productivity and chlorophyll a concentration of phytoplankton in Perdido Bay are controlled by temperature. One input, Eleven Mile Creek, is influenced by a paper mill discharge. Eleven Mile Creek exhibits high levels of light attenuation, high concentrations of dissolved nutrients, and low rates of carbon fixation that are significantly different from the other inputs or areas of Perdido Bay and productivity in Eleven Mile Creek is light limited. Upper Perdido Bay had slightly elevated concentrations of dissolved nutrients which correlate with significantly higher rates of carbon fixation and phytoplankton biomass. Nutrients and color from Eleven Mile Creek are diluted by the Perdido River inflow, restricting nutrient and light limitations to the area at the mouth of Eleven Mile Creek.

Summers, J. Kevin, John F. Paul and Andrew Robertson. 1995. Monitoring the Ecological Condition of Estuaries in the United States. EPA/600/J-96/071. Toxicol. Environ. Chem. 49(1-2):93-108. (ERL,GB 813).

The purpose of the Environmental Monitoring and Assessment Program/Estuaries component (EMAP-E) is to determine the current status, extent, changes, and trends in ecological indicators of the condition of the nation's estuarine resources on a regional and national basis. Monitoring activities in the Virginian (Mid-Atlantic) and Louisianian (Gulf of Mexico) Provinces focus on measurements that describe the benthic community, fish community, water quality, levels of sediment and tissue contamination, sediment toxicity, wetlands extent/condition, and seagrasses extent/condition. Estuarine monitoring is based on an EMAP-E probability-based sampling design conducted over a 60-day period during July-September of each year. Monitoring activities in the Virginian Province began in 1990 and have continued annually while monitoring in the Louisianian Province was initiated in 1991. The results of 1990 monitoring in the Virginian Province show that 20% of the sediments of the Mid-Atlantic region suffered from poor biological conditions while 15% of the area showed undesirable conditions in relation to poor water clarity and the presence of marine debris. In 1991, 31% of Gulf of Mexico estuarine sediments in the Louisianian Province displayed poor biological conditions, as measured by benthic community structure, and 32% of the area was characterized by poor water clarity, the presence of marine debris, and elevated levels of fish tissue contaminants. Efforts are presently underway to begin to assess degradation "causes" using statistical associations among exposure and stressor data for degraded areas in the two provinces. Monitoring will continue in these provinces as well as be initiated in the Great Lakes in 1992 and monitoring in other areas (Carolinian Province, Southeast) is scheduled to begin in 1994.

Lee, Gunhee, Mark R. Ellersieck, Gary F. Krause and Foster L. Mayer. 1995. Predicting Chronic Lethality of Chemicals to Fishes from Acute Toxicity Test Data: Multifactor Probit Analysis. Environ. Toxicol. Chem. 14(2):345-349. (ERL,GB 827).

New methods for predicting chronic toxicity (lethality) from acute lethality data with fishes were developed and assessed. Typically, acute toxicity tests with aquatic organisms provide lethality estimates for a series of toxicant concentrations at 24, 48, 72, and 96 h of exposure. Statistical models (multiple regression) were developed that utilize acute toxicity data to establish the relation of lethality to toxicant concentration and exposure time for predicting chronic lethality. The models provide estimates of toxicant concentrations that result in a low probability of death as a function of extended exposure times. Results from 28 data sets having lethality data for both acute and chronic exposures were used to evaluate the method. It is posited that the proposed methods are highly accurate when acute lethality data meeting stated quality requirements are available.

Weis, P., J.S. Weis, J. Couch, C. Daniels and T. Chen. 1995. Pathological and Genotoxicological Observations in Oysters (Crassostrea virginica) Living on Chromated Copper Arsenate (CCA)-Treated Wood. EPA/600/J-95/373. Mar. Environ. Res. 39(1/4):275-278. (ERL,GB 836).

Oysters living on chromated copper arsenate (CCA)-treated wood in a residential canal were compared with oysters from a reference site 1.2 km distant. Canal oysters were frequently green in color and had 15 times more copper (nearly equal to 200 µg/g wet wt.) and 2-3 times more arsenic (nearly equal to 3µg/g) than reference oysters. Histological examination of soft tissues showed significantly more metaplastic degeneration in digestive gland diverticula of canal oysters. Giemsa-stained canal oyster gill cells had twice as many micronuclei as did reference oyster gill cells. Reference oysters caged by the CCA-treated wood in the canal had Cu tissue accumulations approaching that of native canal oysters by three months. These did not show increased digestive gland metaplasia but did have a nearly doubled incidence of micronuclei. Cu has been shown to cause digestive gland pathology in bivalves; thus, the treated wood could be responsible for the observed pathology. The Cr and As from the wood, although not accumulated to the extent to which Cu is, could influence the non-specific response which we observed; Cr may also be responsible for the observed genotoxicity. Other possible stressors in the canal e.g., boat exhaust, gardening chemicals) could also be involved.

Sun, Kai, Gary F. Krause, Foster L. Mayer, Mark R. Ellersieck and Asit P. Basu. 1995. Estimation of Acute Toxicity by Fitting a Dose-Time-Response Surface. EPA/600/J-95/374. Risk Anal. 15(2):247-252. (ERL,GB 838).

In acute toxicity testing, organisms are continuously exposed to progressively increasing concentrations of a chemical and deaths of test organisms are recorded at several selected times. The results of the test are traditionally summarized by a dose-response curve, and the time course is usually ignored for lack of a suitable model. A model which integrates the combined effects of dose and exposure duration on response is derived from the biological mechanisms of aquatic toxicity, and a statistically efficient approach for estimating acute toxicity by fitting the proposed model is developed in this paper. The proposed procedure has been computerized as software and a typical data set is used to illustrate the theory and procedure. The new statistical technique is also tested by a data base of a variety of chemical and fish species.

Folmar, L.C., J. Harshbarger, P.C. Baumann, G. Gardner and S. Bonomelli. 1995. Pathological and Serum Chemistry Profiles of Brown Bullheads (Ameiurus nebulosus) from the Black River and Old Woman Creek, Ohio. Bull. Environ. Contam. Toxicol. 54(1):50-59. (ERL,GB 842).

This study compares serum chemistry values for brown bullheads (Ameiurus nebulosus) from the industrially contaminated Black River, Ohio, with the rural and relatively uncontaminated Old Woman Creek, Ohio. Fifty-two percent (24 of 46) brown bullheads over 3-years-old had liver neoplasms: 30% (14) were of hepatocellular origin, not including 17% (8) that had foci of hepatocellular alteration and 48% (22) of cholangiocellular origin. Eleven fish had both types of liver tumors. In addition to liver tumors, four fish had squamous cell carcinomas, one had an epidermal papilloma and one had a mesothelioma. Only 1 of the 30 reference fish had a neoplasm of any type, a low grade cholangiocellular carcinoma. Of the 20 serum parameters measured, the mean values of nine were significantly different between the two populations. Seven of the nine were higher in the Black River fish (alanine and aspartate aminotransferase, lactate dehydrogenase, total protein, blood urea nitrogen, triglycerides and calcium). In contrast, manganese and inorganic phosphate were significantly lower. However, in individual fish, none of the serum parameters were significantly correlated with either type of liver neoplasm or either parasitosis.

Lewis, Michael A. 1995. Use of Freshwater Plants in Phytotoxicity Testing: A Review. Environ. Pollut. 87(3):319-336. (ERL,GB 848).

Phytotoxicity data for aquatic plants have served a relatively minor role in regulatory decisions concerning the environmental hazard of most potential contaminants. A variety of phytotoxicity tests have been conducted with freshwater green algae, duckweed, blue-green algae, diatoms and rooted macrophytes (whole plants and seeds). Several test methods have been standardized for microalgae which are used primarily with chemicals, effluents, contaminated sediment elutriates and hazardous waste leachates. Current scientific understanding concerning the phytotoxic effects of these contaminants is based mostly on results for a few green algae. The greatest limitation of these results is their uncertain environmental relevance due to the large interspecific variation in response of standard algal test species and the unrealistic experimental test conditions. Results of the few field validation toxicity tests conducted to resolve this uncertainty have been chemical-specific and unpredictable. Aquatic vascular plants have been used less frequently than algae as test species. Duckweeds have been used more often than rooted submersed species but the uncertain nature of their sensitivities relative to animal and other plant species has limited their use. Regulatory interest in wetland protection, contaminated sediment evaluations and sediment quality criteria development will result in increased use of whole rooted plants and their seeds as test species. Overall, regardless of the test species, if phytotoxicity data are to be more available and effective in the hazard assessment process, additional information concerning species sensitivity and environmental relevance of the results will be needed.

Oliver, Leah M. and William S. Fisher. 1995. Comparative Form and Function of Oyster Crassostrea virginica Hemocytes from Chesapeake Bay (Virginia) and Apalachicola Bay (Florida). EPA/600/J-95/461. Dis. Aquat. Org. 22(3):217-225. (ERL,GB 855).

Oysters Crassostrea virginica from Chesapeake Bay, Virginia, and Apalachicola Bay, Florida, USA, were collected in March and October 1992 to investigate possible differences in defense-related hemocyte activities between individuals from geographically separate populations. In March, hemolymph drawn from Chesapeake Bay oysters contained an average of 1.08 x 106 hemocytes ml-1 hemolymph, significantly lower than the average 1.63 x 106 hemocytes ml-1 hemolymph obtained from Apalachicola Bay oysters. Hemocyte number did not differ significantly in the October comparison. At both times of year, Chesapeake Bay oyster hemolymph samples contained significantly greater proportions of granular hemocytes compared to Apalachicola Bay hemolymph samples. Hemocyte samples from Chesapeake Bay oysters demonstrated a higher percentage of mobile hemocytes and greater particle binding ability than Apalachicola Bay oyster hemocytes when tested in March, but the reverse was found in the October experiments. Chesapeake Bay oyster hemocytes produced significantly more superoxide anion as measured by nitroblue tetrazolium reduction than did Apalachicola Bay oyster hemocytes in both March and October. Oyster hemolymph levels of the protozoan parasite Perkinsus marinus did not differ significantly between the 2 sites at either time of year. These results demonstrate the importance of background studies to characterize site-specific differences in oyster hemocyte defense-related functions.

Flemer, David A., Roman S. Stanley, Barbara F. Ruth, Charles M. Bundrick, Paul H. Moody and James C. Moore. 1995. Recolonization of Estuarine Organisms: Effects of Microcosm Size and Pesticides. EPA/600/J-95/375. Hydrobiologia. 304:85-101. (ERL,GB 857).

Two six-week laboratory experiments were conducted to evaluate effects of pesticides and microcosm size on benthic estuarine macroinvertebrate recolonization. Sediments fortified with the pesticides (fenvalerate: controls, 5 (low) and 50 mg-1 wet sediment (high); endosulfan: controls, 1 (low) and 10 mg g-1 wet sediment (high) were fined-grained, organically rich (approximately 3.5% organic carbon and 22% dry weight) material. Relative dominance of the four most abundant taxa in both experiments was consistent amongtreatments with few exceptions. The amphipod, Corophium acherusicum, dominated abundance in both experiments. In the fenvalerate experiment, large trays (400 cm2) contained significantly (p<0.05) more total number of taxa (TNT) than small microcosms (144 cm2) but tray size was not significantly related to total number of organisms (TNO). When size was adjusted to a common unit area, small trays was 2.5 times that of large containers; a ratio close to that of microcosm sizes (i.e., 2.8). This result suggests that larval supply may have been inadequate to 'saturate' the available sediment in large containers. Fenvalerate significantly reduced abundance in the high treatment compared to both controls and low treatment but low treatment was not significantly different from controls. The amphipod; Corophium acherusicum, accounted for most of the decrease in abundance in response to fenvalerate. The holothruroid, Leptosynpta sp. and the polychaete, Mediomastus ambiseta, increased in abundance significantly with increased concentration of fenvalerate. Combined effects of actual microcosm size and concentration of endosulfan were not significant for TNO or TNT. As in the fenvalerate experiment, adjusted abundance of small microcosms was 2.6 times that of large trays which approximated the ratio of unit area between microcosm sizes. Abundance of a few taxa responded significantly to adjusted and unadjusted area. Abundance of the tunicate, Molgula manhattensis, increased significantly with increased concentration of endosulfan. Abundance was affected by sample location (e.g., interior vs exterior cores) within microcosms. Abundance adjusted to unit area resulted in significantly greater TNO in external vs internal cores. This has improtance for sequential sub-sampling of microcosms to determine temporal dynamics. Statistically significant effects were measured in benthic community structure associated with microcosm size; however, the magnitude was relatively small. There appears to be no major biological reason to select one microcosm size over the other for screening for contaminant effects. Where feasible, the small trays provide savings in sample preparation and analysis, allow more replicates where laboratory space is limiting and generate less chemical waste. These benefits may be off-set by less 'artifacts' associated with edge effects of larger microcosms and the need for a larger mass of sediment to accommodate additional analytical requirements (e.g., thin vertical surficial samples to refine contaminant exposure at the sediment/water interface.

Connolly, John P. and Richard B. Coffin. 1995. Model of Carbon Cycling in the Planktonic Food Web. EPA/600/J-96/081. J. Environ. Engineering. 121(10):682-690. (ERL,GB 865).

A mathematical model of carbon fluxes through the heterotrophic microbial food web is developed from a synthesis of laboratory and field research. The basis of the model is the segregation of organic carbon into lability classes that are defined by bioassay experiments. Bacteria, phytoplankton, three trophic levels of zooplankton, and dissolved organic carbon (DOC) and particulate organic carbon (POC) are modeled. The descriptions of bacterial growth and utilization of the various classes of substrate were treated as "universal constants" in the application of the model to three distinct ecosystems, ranging from oligotrophic to highly eutrophic. The successful application of the model to these diverse ecosystems supports the basic validity of the description of the microbial food web and the dynamics of carbon flux. The model indicates that the dynamics of bacteria and protozoan zooplankton production govern the rates of oxidiation of carbon entering the water column. Explicit consideration of these groups would improve the capability of eutrophication models to predict dissolved oxygen dynamics, particularly when projecting responses to loading changes.

Saouter, Erwan, Mark Gillman, Ralph Turner and Tamar Barkay. 1995. Development and Field Validation of a Microcosm to Simulate the Mercury Cycle in a Contaminated Pond. Environ. Toxicol. Chem. 14(1):69-77. (ERL,GB 870).

A microcosm consisting of water, sediment and air compartments was used to simulate mercury geochemical cycling in a mercury-contaminated (mg L-1) stream-pond system at Oak Ridge, Tennessee. Total and dissolved mercury and total methylmercury were analyzed in water and sediment; total gaseous mercury and head-space mercury were analyzed in the water and in the head space, respectively. The production of gaseous mercury was correlated to dissolved mercury (0.2-mm filtration) and methylmercury was mainly produced in the sediment compartment. Addition of mercuric chloride to the system increased the production of head-space mercury by a factor of 10 but did not affect the methylation rate. Saturation of gaseous mercury in microcosm water varied from 500 to 1,500% above solubility limits and was controlled by unidentified factors. The microcosm maintained stable conditions for up to 3 weeks, and a mass balance analysis indicated that it reasonably simulated the cycling of mercury in the pond. This microcosm could be used to test remedial treatments aimed at decreasing the amount of mercury that is available for accumulation by biota.

Chapman, P.J., M. Shelton, Magda Grifoll and S. Selifonov. 1995. Fossil Fuel Biodegradation: Laboratory Studies. EPA/600/J-95/434. Environ. Health Perspect. 103(Suppl 5):79-83. (ERL,GB 871).

Biodegradation of the polycyclic aromatic hydrocarbons of creosote by undefined bacterial cultures was shown to be accompanied by the accumulation of neutral and acidic oxidation products. Formation of a number of identified neutral products is accounted for by demonstration of anomalous actions of an arene dioxygenase on the benzylic methylene and methylene carbons of napthenoaromatic hydrocarbons. Both neutral and acidic water-soluble fractions are also formed when various mixed bacterial cultures degrade weathered crude oil. While constituents of these fractions are not yet identified, the neutral materials have been shown to be toxic to developing embryos of invertebrates. These observations are discussed in relation to chemical and toxicological assessments of biodegradation of the complex chemical mixtures of fossil fuels.

Folmar, L.C., N.D. Denslow, R.A. Wallace, G. LaFleur, S. Bonomelli and C.V. Sullivan. 1995. Highly Conserved N-terminal Sequence for Teleost Vitellogenin with Potential Value to the Biochemistry, Molecular Biology and Pathology of Vitellogenesis. EPA/600/J-95/370. J. Fish Biol. 46(2):255-263. (ERL,GB 875).

N-terminal amino acid sequences for vitellogenin (Vtg) from six species of teleost fish (striped bass, mummichog, pinfish, brown bullhead, medaka, yellow perch and the sturgeon) are compared with published N-terminal Vtg sequences for the lamprey, clawed frog and domestic chicken. Striped bass and mummichog had 100% identical amino acids between positions 7 and 21, while pinfish, brown bullhead, sturgeon, lamprey, Xenopus and chicken had 87%, 93%, 60% 47%, 47-60% for four transcripts and had 40% identical, respectively, with striped bass for the same positions. Partial sequences obtained for medaka and yellow perch were 100% identical between positions 5 to 10. The potential utility of this conserved sequence for studies on the biochemistry, molecular biology and pathology of vitellogenesis is discussed.

McKenney, Charles L., Jr. and David M. Celestial. 1995. Interactions Among Salinity, Temperature, and Age on Growth of the Estuarine Mysid, Mysidopsis bahia, Reared in the Laboratory Through a Complete Life Cycle. I. Biomass and Age-Specific Growth Size. J. Crustacean Biol. 15(1):169-178. (ERL,GB 883).

A broad range of salinity-temperature conditions (salinities from 3 to 31 o/oo and temperatures from 19 to 31° C) significantly influence growth rates and subsequent biomass of the estuarine mysid, Mysidopsis bahia, reared in the laboratory from the first free juvenile stage through first brood production by the mature adult. Not only did salinity and temperature significantly interact to affect mysid growth, but a highly significant three-factor salinity-temperature-age interaction modified growth in this species. Response surfaces depict that maximum biomass was obtained after four weeks at temperatures between 24 and 29° C and in salinities (S) above 19 o/oo. Optimal salinity-temperature conditions for growth of M. bahia are correlated with both its resistance patterns to these dominant environmental factors and its distribution pattern in estuarine waters. Canonical analysis of the empirical data produced an absolute maximum dry weight at 26 o/oo S and 27° C after four weeks of growth. Salinity conditions accounting for optimal growth are in close agreement with the isosmotic point (24 o/oo S) for this species suggesting reduced growth efficiency concurrent with osmotic stress, particularly hypoosmotic stress. Maximum growth rates of mysids reared under a broad salinity-temperature range occurred during the second week, just prior to maturation, suggesting that changing levels of reproduction are correlated with modifications in mysid growth over time.

McErlean, Andrew J., Eleanor Williams and Frank Wittwer. 1995. Environmental Education: A Blueprint for Serious Achievement?. Environ. Prof. 17(3):263-270. (ERL,GB 885).

The present national effort devoted to environmental education (EE), particularly as it relates to K-12 education, is examined and indexed to other current events and their support levels. For the most part, EE efforts are embedded in science, mathematics, and engineering programs (SME), and the relationships to these other areas are discussed. In the present context, many aspects such as social, ethical, and religious consideration of EE are not addressed. The relationships between EE are not addressed. The relationships between EE and the expectation for scientific literacy (SL) and improved environmental decisionmaking in both short- and long -term contexts and also examined. Under existing programs, the prognosis for serious, effective accomplishment, or credible impact on universal EE literacy or enhanced decisionmaking, is doubtful.

Barkay, T., N. Kroer, L.D. Rasmussen and S.J. Sorensen. 1995. Conjugal Transfer at Natural Population Densities in a Microcosm Simulating an Estuarine Environment. FEMS Microbiol. Ecol. 16(1):43-54. (ERL,GB 887).

Estuarine microcosms were used to follow conjugal transfer of a broad host range IncP1 plasmid from a Pseudomonas putida donor to indigenous bacteria. Donor cells were added at a concentration similar to the natural abundance of bacteria in the water column (106 cells ml-1). Transfer was not detected in any of the test microcosms (calculated limit of detection of 10-7 and 10-4 transconjugants donor-1 in water column and sediment, respectively), with the exception of transfer to an isogenic recipient (added at 105 cells ml-1) in sediments of controls that had been inoculated with both donors and recipients. The same plasmid was transferred with high efficiencies (10-1 to 10-3) to a variety of recipients in filter and broth matings. These results suggest that if conjugal gene transfer occurred, it was at efficiencies that were not detectable in estuarine microcosms simulating natural population densities.

Couch, John A. 1995. Invading and Metastasizing Cardiac Hemangioendothelial Neoplasms in a Cohort of the Fish Rivulus marmoratus: Unusually High Prevalence, Histopathology, and Possible Etiologies. EPA/600/J-95/371. Cancer Res. 55(11):2438-2447. (ERL,GB 893).

An unusually high, unprecedented prevalence of cardiac hemangioendotheliomata, including hemangiomas, hemangioendotheliomas, and hemangioendotheliosarcomas, was found in a laboratory cohort of the small, teleost fish Rivulus marmoratus. The neoplasms occurred in 51 of 204 fish (25%) used in a carcinogenicity study of butylated hydroxyanisole fed in a lyophilized chicken liver diet for up to 9 months. The cardiac neoplasms occurred in approximately equal numbers of both control (fed lyophilized chicken liver but not exposed to butylated hydroxyanisole) and exposed (fed 0.8% butylated hydroxyanisole in chicken liver) fish. The neoplasms occurred in the bulbus arteriosus and ventricle (accompanied by an intense epicarditis), and in some cases, in the gills. At least one case of hemangioma was characterized by cavernous vessels in the bulbar wall. Hemangioendothelioma cases consisted of pleomorphic endothelial cells that formed continuous tracts and anastomosing, typical vascular channels of varying sizes. The hemangioendotheliosarcomas consisted of atypical, spindle, polygonal, or round endothelial cells that formed solid tumor masses that contained abnormal vessels with atypical endothelium tufting into their lumina. Some fish had neoplasm cases containing varying regions with mixtures of the above features. Incipient neoplasms occurred on/in the semilunar valves, and their cells appeared to invade the adjacent walls of the bulbus and/or the ventricle. The gill lesions represented possible metastatic neoplasms, probably formed by atypical endothelial cells that exfoliated from the edges of cardiac neoplasms into the lumina of the ventricle or bulbus and were then pumped to the arterioles of the gills via the ventral aorta. Fish examined from the original colony in the laboratory and from the wild had no neoplasms. Possible causes are discussed, and studies are under way in an attempt to determine the etiology of the neoplasm and to evaluate Rivulus marmoratus as a possible model organism for study of these types of cardiac neoplasms.

Middaugh, D.P. and D.D. Whiting. 1995. Responses of Embryonic and Larval Inland Silversides, Menidia beryllina, to No. 2 Fuel Oil and Oil Dispersants in Seawater. Arch. Environ. Contam. Toxicol. 29(4):535-539. (ERL,GB 897).

Embryonic inland silversides, Menidia beryllina, in the early blastula stage were exposed to the water-soluble fraction (WSF) of No. 2 Fuel oil and the oil dispersants Corexit 7664® and 9527®, singly and in combination. An ordinal ranking system was used to score observed daily craniofacial, cardiovascular, and skeletal responses in control embryos and those exposed to 1%, 10%, and 100% concentrations of the WSF of No. 2 Fuel oil, the dispersants Corexit 7664® and 9527® applied at the recommended field application concentrations, and the combination of No. 2 Fuel oil and respective dispersants in seawater. The non-parametric Kruskal-Wallis analysis of variance (ANOVA) and post hoc analyses were used to identify stastically significant differences for control embryos and those exposed to No. 2 Fuel oil and dispersants. Embryos exposed to No. 2 Fuel oil in 20 o/oo salinity seawater showed significant (a less than or equal to 0.01) responses only at the 100% WSF concentration. Corexit 7664® tested singly elicited significant responses at 10% and 100% concentrations. When No. 2 Fuel oil and Corexit 7664® were combined at recommended field application concentrations of the dispersant, the oil and dispersant mixture resulted in significant (a less than or equal to 0.01) responses at 1%, 10%, and 100% exposure concentrations. In contrast, Corexit 9527® did not cause significant responses at the three test concentrations of 1%, 10%, and 100% of the recommended field application rate. However, when No. 2 Fuel oil and Corexit 9527® were combined in seawater, the 10% and 100% exposure concentrations resulted in statistically significant (a less than or equal to 0.01) embryonic responses, relative to controls. Chemical analyses indicated that both dispersants increased the total WSF of No. 2 Fuel oil in seawater.

Genthner, Fred J. and Douglas P. Middaugh. 1995. Nontarget Testing of an Insect Control Fungus: Effects of Metarhizium anisopliae on Developing Embryos of the Inland Silverside Fish Menidia beryllina. EPA/600/J-95/460. Dis. Aquat. Org. 22(3):163-171. (ERL,GB 898).

Developing embryos of the inland silverside fish, Menidia beryllina, were exposed to conidiospores of the entomopathogenic fungus, Metarhizium anisopliae. Several adverse effects were observed in both embryos and newly hatched larvae. These included transitory effects on the heart resulting in decreased cardiac output or circulation velocity, rupture of the chorion, fungal growth on the mandibles of larvae, focal vetebral abnormalities in larvae and teratogenic expressions in embryos and larvae. An ordinal ranking system was used to enumerate responses to conidiospores. This ranking system allowed significance to be determined by nonparametric analysis of variance. Responses were highly variable with significant (p< or = to 0.05) adverse effects observed in five of the six experiments conducted. Heat-killed spores failed to cause significant adverse effects, indicating that viable spores were required for the adverse effects.

Hemmer, Michael J., Lee A. Courtney and Lisa S. Ortego. 1995. Immunohistochemical Detection of P-glycoprotein in Teleost Tissues Using Mammalian Polyclonal and Monoclonal Antibodies. EPA/600/J-95/372. J. Exp. Zool. 272(1):69-77. (ERL,GB 899).

Mammalian P-glycoprotein is a highly conserved 170-kD integral plasma membrane protein functioning as an energy dependent efflux pump of exogenous and endogenous lipophilic aromatic compounds entering the cell by diffusion. In this study, the tissue specificity of one polyclonal (pAb) and three monoclonal (mAbs) antibodies to mammalian P-glycoprotein were identified in paraffin-embedded, parasagittal whole body sections of the guppy, Poecilia reticulata. Pab mdr (Ab-1) and mAbs C219, C494 and JSB-1 demonstrated differential staining patterns in the following tissues: bile canaliculi in the liver, exocrine pancreas, lumenal surface of the intestinal epithelium, renal tubules, interrenal tissue, branchial blood vessels, gas gland, pseudobranch, and the gill transverse septa. Positive P-glycoprotein expression in P. reticulata correlates well with published results for homogogous mammalian tissues of secretory and execretory function. These data indicate that one or more highly conserved members of the P-glycoprotein transporter family exist in a teleost species which can be detected using commercially available mammalian antibodies.

Winstead, James T. 1995. Digestive Tubule Atrophy in Eastern Oysters, Crassostrea virginica (Gmelin 1791), Exposed to Salinity and Starvation Stress. EPA/600/J-95/435. J. Shellfish Res. 14(1):105-111. (ERL,GB 900).

Oysters sampled in February, 1992, from a low salinity site (3 ppt) in Apalachicola Bay, Florida, showed digestive tubule atrophy when compared with oysters from a higher salinity site (18 ppt) 16 kilometers away. Experiments designed to induce tubule atrophy in the laboratory consisted of two starvation and two salinity stress tests. To quantify tubule condition for each oyster, inside to outside diameter tubule ratios were calculated from 20 tubules per animal using an ocular micrometer. Higher tubule ratios indicated greater tubule atrophy. The experiments showed poor nutrition, perhaps due to low salinity, may have played a significant role in the tubule atrophy of Apalachicola Bay oysters.

Sun, Kai, Gary F. Krause, Foster L. Mayer, Jr., Mark R. Ellersieck and Asit P. Basu. 1995. Predicting Chronic Lethality of Chemicals to Fishes from Acute Toxicity Test Data: Theory of Accelerated Life Testing. Environ. Toxicol. Chem. 14(10):1745-1752. (ERL,GB 904).

A method for modeling aquatic toxicity data based on the theory of accelerated life testing and a procedure for maximum likelihood fitting the proposed model is presented. The procedure is computerized as software which can predict chronic lethality of chemicals using data from acute toxicity tests. A data base of a variety of chemicals and fish species was analyzed. When the calculated values of prediction were compared to the maximum acceptable toxicant concentrations obtained from actual chronic toxicity experiments, the new technique provided accurate predictions. Problems in using the "maximum acceptable toxicant concentration" and applications of the proposed method are discussed.

Saouter, Erwan, Mark Gillman and Tamar Barkay. 1995. Evaluation of mer-Specified Reduction of Ionic Mercury as a Remedial Tool of a Mercury-Contaminated Freshwater Pond. J. Ind. Microbiol. 14(3/4):343-348. (ERL,GB 906).

The potential for mer-mediated reduction/volatilization of ionic mercury as a tool in the decontamination of a freshwater pond was evaluated using laboratory incubations and a microcosm simulation. In flask assays inoculations with ionic mercury-resistant bacteria (105-107 cells ml-1) isolated from the pond, significantly increased the rate of mercury loss (MANOVA, p less than or equal to 0.05) relative to uninoculated controls. The effects of cell density, mercuric mercury concentration, addition of nutrients and supplementation with the sulfhydryl reagent Beta-mercaptoethanol on the rate of mercury loss, were investigated. Inoculation (by 105 cells ml-1) of a flow-through microcosm that simulated the cycling of mercury in the contaminated pond, stimulated by more than 4-fold the formation of volatile elemental mercury. Thus, biological formation of volatile mercury may hold a promise as a remedial tool of contaminated natural waters.

Genthner, F.J., D.P. Middaugh and S.S. Foss. 1995. Validation of Embryo Tests for Determining Effects of Fungal Pest Control Agents on Nontarget Aquatic Animals. Arch. Environ. Contam. Toxicol. 29(4):540-544. (ERL,GB 907).

Developing embryos of the inland silverside fish, Menidia beryllina, and grass shrimp, Palaemonetes pugio, were exposed to conidiospores of the fungal weed control agent, Colletotrichum gloeosporioides f. sp. aeschynomene and the entomopathogen, Metarhizium anisopliae. Only Metarhizium anisopliae caused significant (p < = 0.05) mortalities in the exposed embryos. Collectotrichum gloeosporioides did, however, cause fatal infections in adults when conidia were injected into the peritoneum of fish or the hemocoel of shrimp.

Grifoll, Magdalena, Sergey A. Selifonov and Peter J. Chapman. 1995. Transformation of Substituted Fluorenes and Fluorene Analogs by Pseudomonas sp. Strain F274. EPA/600/J-02/250. Appl. Environ. Microbiol. 61(9):3490-3493. (ERL,GB 908).

Pseudomonas sp. F274, previously shown to catabolize fluorene via fluorenone and its angular dioxygenation, 2',3'-dihydroxy-2-carboxybiphenyl, phthalate and protocatechuate, was examined for its abililty to transform substituted fluorenes and S- and N-heterocyclic analogues. Halogen- and methyl-substituted fluorenes were metabolized to the correspondingly substituted phthalates via attack on the unsubstituted ring. In the case of 1-methylfluorene, initial oxidation of the methyl group to carboxyl prevented all other transformations but 9-monooxygenation. The strain also oxidized S-heteroatoms and benzylic methylenic groups of fluorene analogues. No angular dioxygenation of S- and N-heterocycles was observed.

Grifoll, Magdalena, Sergey A. Selifonov, Charylene V. Gatlin and Peter J. Chapman. 1995. Actions of a Versatile Fluorene-Degrading Bacterial Isolate on Polycyclic Aromatic Compounds. Appl. Environ. Microbiol. 61(10):3711-3723. (ERL,GB 909).

Pseudomonas cepacia F297 showed the ability to grow with fluorene as a sole source of carbon and energy; its growth yield corresponded to assimulation of about 40% of fluorene carbon. Accumulation of a ring meta-cleavage product during growth and identification of 1-indanone in growth media and washed cell suspensions suggest that strain F297 metabolizes fluorene by mechanisms analogous to those of naphthalene degradation. In addition to fluorene, strain F297 utilized for growth a wide variety of polycyclic aromatic compounds (PACs), including naphthalene, 2,3-dimethylnaphthalene, phenanthrene, anthracene, and dibenzothiophene. Fluorene-induced cells of the strain also transformed 2,6-dimethylnaphthalene, biphenyl, dibenzofuran, acenaphthene and acenaphthylene. Identification of products formed from those substrates (GC-MS) in washed cell suspensions, indicates that Pseudomonas cepacia F297 carries out the following reactions: (i) aromatic ring oxidation and cleavage, apparently using the pyruvate released for growth, (ii) methyl group oxidations, (iii) methylenic oxidations, and (iv) S-oxidations of aromatic sulfur heterocycles. Strain F297 grew with a creosote-PAC mixture, producing almost complete removal of all aromatic compounds containing 2-3 rings in 14 days, as demonstrated by GC analysis of the remaining PACs recovered from cultures. Identification of key chemicals confirmed that not only are certain compounds depleted, but also the anticipated reaction products are found.

Heppell, Scott A., Nancy D. Denslow, Leroy C. Folmar and Craig V. Sullivan. 1995. Universal Assay of Vitellogenin as a Biomarker for Environmental Estrogens. EPA/600/J-96/079. Environ. Health Perspect. 103(Suppl 7):9-15. (ERL,GB 913).

Vitellogenin (VTG), the serum phospholipoglycoprotein precursor to egg-yolk, is potentially an ideal biomarker for environmental estrogens. This study was undertaken to develop antibodies against conserved regions on the vitellogenin molecule, antibodies that could form the basis for establishing bioassays to detect estrogen exposure in any oviparous vertebrate. We developed monoclonal antibodies (mAbs) generated against purified rainbow trout (Oncorhynchus mykiss) VTG, and selected for the property of specifically recognizing VTG purified from two phylogenetically distant vetebrates, trout and striped bass (Morone saxatilis). Results of enzyme-linked immunosorbant assay and Western blotting indicated that these mAbs specifically recognize purified VTG and VTG or other estrogen-inducible proteins in plasma or serum from representative species of 4 vertebrate classes (fish, amphibians, reptiles and birds). All of the mAbs generated were IgM class. A polyclonal antiserum was raised against a synthetic consensus peptide representing the conserved N-terminal amino acid sequence of VTG. The results of Western blotting indicate that this antiserum specifically recognizes VTG in plasma or serum from teleost fish of diverse families. It was used to detect VTG in Western blots of serum from brown bullhead (Ameiurus nebulosus) with cancer (hepatocellular and cholangio-carcinoma) collected from a contaminated industrial site outside of their normal vitellogenic season. Our results indicate that it is feasible to generate antibodies capable of recognizing VTG without regard to species and that development of a "universal" VTG assay is an achievable goal.

Oremland, Ronald S., Laurence G. Miller, Philip Dowdle, Tracy Connell and Tamar Barkay. 1995. Methylmercury Oxidative Degradation Potentials in Contaminated and Pristine Sediments of the Carson River, Nevada. Appl. Environ. Microbiol. 61(7):2745-2753. (ERL,GB 915).

Sediments from mercury-contaminated and uncontaminated reaches of the Carson River, Nevada, were assayed for sulfate reduction, methanogenesis, denitrification, and monomethylmercury (MeHg) degradation. Demethylation of [14 C]MeHg was detected at all sites as indicated by the formation of 14CO2 and 14 CH4. Oxidative demethylation was indicated by the formation of 14CO2 and was present at signficant levels in all samples. Oxidized/Reduced Demethylation Product (ORDP) ratios (i.e., 14CO2/14CH4) generally ranged from 4.0 in surface layers to as low as 0.5 at depth. Production of 14CO2 was most pronounced at sediment surfaces which were zones of active denitrification and sulfate reduction but was also significant within zones of methanogenesis. In a core taken from an uncontaminated site having more oxidized, coarse-grained sediments, sulfate reduction and methanogenic activities were very low and 14CO 2 accounted for 98% of the product formed from [14C]MeHg. There was no apparent relationship between the degree of mercury contamination of the sediments and the occurence of oxidative demethylation. However, sediments from Fort Churchill, the most contaminated site, were most active in terms of demethylation potentials. Inhibition of sulfate reduction with molybdate resulted in significantly depressed ORDP ratios, but overall demethylation rates were comparable between inhibited and uninhibited samples. Addition of sulfate to sediment slurries stimulated production of 14CO2 from [14C]MeHg, while 2-bromoethanesulfonic acid blocked production of 14CH4. These results reveal the importance of sulfate reducing and methanogenic bacteria in oxidative demethylation of MeHg in anoxic environments.

Eaton, Richard W. and Peter J. Chapman. 1995. Formation of Indigo and Related Compounds from Indolecarboxylic Acids by Aromatic Acid-Degrading Bacteria: Chromogenic Reactions for Cloning Genes Encoding Dioxygenases That Act on Aromatic Acids. EPA/600/J-96/165. J. Bacteriol. 177(23):6983-6988. (ERL,GB 920).

The p-cumate-degrading strain Pseudomonas putida F1 and the m- and p -toluate-degrading strain P. putida mt-2 transform indole-2-carboxylate and indole-3-carboxylate to colored products identified here as indigo, indirubin, and isatin. A mechanism by which these products could be formed spontaneously following dioxy-genase-catalyzed dihydroxylation of the indolecarboxylates is proposed. Indolecarboxylates were employed as chromogenic substrates for identifying recombinant bacteria carrying genes encoding p-cumate dioxygenase and toluate dioxygenase. Dioxygenase gene carrying bacteria could be readily distinguished as dark green-blue colonies among other colorless recombinant Escherichia coli colonies on selective agar plates containing either indole-2-carboxylate or indole-3-carboxylate.

Lubinski, B.A., W.P. Davis, D.S. Taylor and B.J. Turner. 1995. Outcrossing in a Natural Population of a Self-Fertilizing Hermaphroditic Fish. J. Hered. 86(6):469-473. (ERL,GB 949).

Outcrossing has been documented in a natural population of the self-fertilizing hermaphroditic killifish, Rivulus marmoratus. All of the 24 hermaphrodites collected in 1991 on Twin Cays, a small island adjacent to the Belize barrier reef, proved, by direct assay of their progeny, to be multiply heterozygous for mini- and microsatellite loci detected by DNA fingerprinting. This implies high levels of outcrossing, possibly associated with heterosis. The results are strikingly different from those obtained previously with this species, for all other populations studied have consisted of arrays of homozygous clones. Males of the species are usually rare in nature, but were relatively common on Twin Cays. The outcrossing in the population presumably stems from x hermaphrodite matings. The males are of the type known to be induced by elevated temperature treatments of immature hermaphrodites in the laboratory. Outcrossing in the Twin Cays populations may therefore be the direct result of the environmental induction of males. If true, this would be an example of "phenotypic plasticity" of almost unprecedented impact. However, there is evidence that social factors, as yet unresolved, may also be important in both the requisites of outcrossing: the induction of males and the reduction of internal self-fertilization in hermaphrodites. This species is quite successful in homozygous form, and seems to lack intrapopulatioin variation in protein-encoding loci. The adaptive significance of outcrossing is therefore not immediately apparent.

Gaston, Gary R., Steven S. Brown, Chet F. Rakocinski, Richard W. Heard and J. Kevin Summers. 1995. Trophic Structure of Macrobenthic Communities in Northern Gulf of Mexico Estuaries. Gulf Res. Rep. 9(2):111-116. (ERL,GB X805).

Trophic structure of estuarine benthic communities in the northern Gulf of Mexico was characterized according to the functional roles and geographic distributions of the macrobenthos. Macrobenthic organisms collected during two years of study were assigned to trophic groups to assess the relative utilization of detritus and other resources. Three groups of detritivores (less than 3%) and othersurface-deposit feeders, subsurface-deposit feeders, and filter feeders) were numerically dominant among the benthos, each of which accounted for 25-30% of total abundance across regions. Carnivorous macrobenthos also comprised an appreciable portion (12%), while omnivores (less than 3%) and other groups (less than 4%) were poorly represented. Dominance by detritivores is consistent with current concepts regarding the role of macrobenthos in processing detritus of Gulf of Mexico estuaries.

Winkler, Jorg, Kenneth N. Timmis and Richard A. Snyder. 1995. Tracking the Response of Burkholderia cepacia G4 5223-PR1 in Aquifer Microcosms. EPA/600/J-95/437. Appl. Environ. Microbiol. 61(2):448-455. (ERL,GB X807).

The introduction of bacteria into the environment for bioremediation purposes (bioaugmentation) requires analysis and monitoring of microbial population dynamics to define persistence and activity from both efficacy and risk assessment perspectives. Burkholderia cepacia G4 5223-PR1 is a Tn5 insertion mutant which constitutively expresses a toluene ortho-monooxygenase that degrades trichloroethylene (TCE). This ability of G4 5223 PR1 to degrade TCE without aromatic induction may be useful for bioremediation of TCE-containing aquifers and groundwater. Thus, a simulated aquifer sediment system and groundwater microcosms were used to monitor the survival of G4 5223-PR1. The fate of G4 5223-PR1 in sediment was monitored by indirect immunofluorescence microscopy, a colony blot assay, and growth on selective medium. G4 5223-PR1 was detected immunologically by using a highly specific monoclonal antibody which reacted against the O-specific polysaccharide chain of the lipopolysaccharides of this organism. G4 5223-PR1 survived well in sterilized groundwater, although in nonsterile groundwater microcosms rapid decreases in the G4 5223-PR1 cell population were observed. Ten days after inoculation no G4 5223-PR1 cells could be detected by selective plating or immunofluorescence. G4 5223-PR1 survival was greater in a nonsterile aquifer sediment microcosm, although after 22 days of elution the number of G4 5223-PR1 cells was low. Our results demonstrate the utility of monoclonal antibody tracking methods and the importance of biotic interactions in determining the persistence of introduced microorganisms.

Yousten, Allan A., Ernest F. Benfield and Fred J. Genthner. 1995. Bacillus sphaericus Mosquito Pathogens in the Aquatic Environment. EPA/600/J-95/438. Mem. Inst. Oswaldo Cruz Rio. 90(1):125-129. (ERL,GB X808).

The fate of Bacillus sphaericus spores in the aquatic environment was investigated by suspending spores in dialysis bags in fresh and seawater. Spore viability was lost more rapidly in seawater. Neither B. sphaericus nor B. thuringiensis israelensis (B.t.i.) spores mixed with pond sediment appeared to attach to the sediment. However, rapid decrease in B.t.i. toxicity suggested attachment of parasporal bodies to sediment. B.sphaericus toxin settled more slowly and less completely. B. sphaericus spores fed to larvae of four aquatic invertebrates were mostly eliminated from the animal gut in less than one week. An exception was the cranefly (Tipula abdominalis) where spores persisted in the posterior gut for up to five weeks.

Davis, William P., D. Scott Taylor and Bruce J. Turner. 1995. Does the Autecology of the Mangrove Rivulus Fish (Rivulus marmoratus) Reflect a Paradigm for Mangrove Ecosystem Sensitivity?. EPA/600/J-95/458. Bull. Mar. Sci. 57(1):208-214. (ERL,GB X810).

The killifish Rivulus marmoratus, mangrove rivulus, represents the one of the two potentially truly 'mangrove dependent' fish species in western Atlantic mangrove ecosystems. The distribution of this species closely parallels the range of red mangroves. These plants and fish exhibit parallel ecological and physiological tolerances to the wide ranges of tropical temperatures and salinities, as well as substrate and hydrological conditions of mangrove habitats. The mangrove rivulus, R. marmoratus is, as well, the only truly marine representative of a speciose genus of otherwise freshwater fish species. Many of the biological specializations of this species characterize the specific challenges to survival in mangrove forest conditions. As recent studies report, this fish species, once considered 'rare,' has been shown to be very abundant in specific substrate microhabitats of the mangal. Among the unique specializations of this fish are amphibious emersion from water, survival in moist detrital substrate during periods of low water or drought, and reproduction through internal self-fertilization producing homozygous clones. The autecology of this species provides fascinating insights and generates a wealth of questions regarding evolution of specific adaptations for distribution, dispersal, colonization, population genetics and the interrelationships between adaptation and specialization. The Indian River Lagoon (IRL) represents both the site of rediscovery of the fish as well as the northern frontier of the species distribution range, habitat, and ecosystem. This suggests close interrelationships and parallels in the parameters to which the species has adapted, perhaps representing a commonage, of ecological association. These aspects are discussed with respect to details and insight needed to develop strategies for the management of unique communities and ecosystems, especially along their natural distributional borders. The topic raises such questions as: Are some 'exotic species' actually examples of newly arrived colonizers, representing dynamic biotic responses to climate change and/or anthropogenic habitat modification?

Taylor, D. Scott, William P. Davis and Bruce J. Turner. 1995. Rivulus marmoratus: Ecology of Distributional Patterns in Florida and the Central Indian River Lagoon. EPA/600/J-95/459. Bull. Mar. Sci. 57(1):202-207. (ERL,GB X811).

The Neotropical killifish Rivulus marmoratus is widely distributed, but locally rare, throughout much of coastal south and central Florida. Habitat alteration has affected the species throughout the state, especially on the east coast (Indian River Lagoon) where the destruction of mangroves and impounding of high marsh for mosquito control has altered and fragmented suitable habitat. Within tropical salt-marsh and mangrove forests, R. marmoratus seems best adapted to certain micro-habitats, specifically those precluding the survival/establishment of competing fishes. On the east coast of Florida, this microhabitat preference is the land crab (Cardisoma guanhumi) burrow. In south Florida and the west coast, R. marmoratus is most often captured in stagnant pools and old mosquito ditches in mangrove forests. Populations of R. marmoratus in Florida consist of arrays of homozygous clones. Can habitat fragmentation, with its possible effects on clonal diversity, affect the continued success of this species? Are certain clones better-adapted to specific environmental conditions? Isolated populations consisting of only a single clone could persist indefinitely. Association with habitat type may answer some of these questions. Some degree of plasticity is apparent, as northern clones 'transplanted' from crab burrows survive well and achive high population levels in isolated pools more typical of south Florida habitats. However, the appearance of male fish in these pools, a phenomenon unknown in natural populations from burrows, suggests aberrant population structure. While general questions remain about the adaptive significance of clonal diversity, the mere presence of this novel fish in salt marsh/mangrove habitats may indicate that other aspects of biodiversity are in good 'order.'

Shiba, Tsuneo, Russell T. Hill, William L. Straube and Rita R. Colwell. 1995. Decrease in Culturability of Vibrio cholerae Caused by Glucose. EPA/600/J-96/073. Appl. Environ. Microbiol. 61(7):2583-2588. (ERL,GB X812).

The culturability of Vibrio cholerae O1 serotype Inaba strain 569B was decreased by the addition of glucose to cell suspensions in starvation media. A similar effect was observed with sucrose, maltose, and fructose. We term this inhibitory effect glucose shock. It was not observed with arabinose or xylose or with carboxylates, such as acetate and pyruvate. No acidification of the medium occurred in the presence of these carbohydrates. Glucose shock was prevented by the addition of nitrogen or phosphorus sources. In the presence of phosphate, the bacterium produced formic acid from glucose. The phenomenon of glucose shock was also observed in V. cholerae O1 serotype Inaba strain RIMD 2203082 but not in strain RIMD 2203088 (O1 Inaba), IID 936 (O1 Ogawa), or RIMD 2214034 (non-O1). The culturability of Escherichia coli, Enterobacter aerogenes, and Listonella anguillarum did not decrease in starvation media with added glucose. Hence, the phenomenon should have ecological significance in determining the distribution of bacteria in marine ecosystems in situations where carbohydrates are abundant, but nitrogen and phosphorus are limiting.

Shields, M.S., M.J. Reagin, R.R. Gerger, R. Campbell and C Somerville. 1995. TOM, a New Aromatic Degradative Plasmid from Burkholderia (Pseudomonas) cepacia G4. EPA/600/J-96/072. Appl. Environ. Microbiol. 61(4):1352-1356. (ERL,GB X813).

Burkholderia (Pseudomonas) cepacia PR123 has been shown to constitutively express a toluene catabolic pathway distinguished by a unique toluene ortho-monooxygenase (Tom). This strain has also been shown to contain two extrachromosomal elements of

Knudsen, Steen, Peter Saadbye, Lars H. Hansen, April Collier, Bodil L. Jacobsen, Jorgen Schlundt and Olle H. Karlstrom. 1995. Development and Testing of Improved Suicide Functions for Biological Containment of Bacteria. EPA/600/J-96/077. Appl. Environ. Microbiol. 61(3):985-991. (ERL,GB X814).

We have developed very efficient suicide functions for biological containment based on the lethal Escherichia coli relF gene. The suicide functions are placed in duplicate within a plasmid and arranged to prevent inactivation by deletion, recombination, and insertional inactivation. The efficiency of this concept was tested in a plasmid containment system that prevents transfer of plasmids to wild-type bacteria. Protection against plasmid transfer was assayed in test tubes and in rat intestine. Protection was efficient and refractory to inactivation by mutation and transposons. The efficiency of the suicide system was also tested in soil and seawater. We show that unprecedented suicide efficiency can be achieved in soil and seawater after suicide induction by IPTG (isopropyl-B-D-thiogalactopyranoside). More than 7 orders of magnitude reduction in suicide bacteria was achieved.

Weis, Judith S. and Peddrick Weis. 1995. Effects of Chromated Copper Arsenate (CCA) Pressure-treated Wood in the Aquatic Environment. EPA/600/J-96/069. Ambio. 24(5):269-274. (ERL,GB X815).

Uptake and deleterious effects of leachates from chromated copper arsenate (CCA) wood have been found. High levels of metals, particularly copper (Cu), and in some cases arsenic (As) accumulate in biota that live on the wood, and have reduced community diversity. The contaminants in these organisms may be transferred to their consumers, with deleterious effects. Metals leached from the wood can also be adsorbed onto fine sediment particles from which they can be accumulated by benthic organisms. Benthic organisms in sediments adjacent to the wood exhibit reduced community diversity and elevated metal concentrations. The extent and severity of effects in any particular area will depend on the amount of wood, its age water-quality parameters, and the degree of dilution by water movement.

Kelley, Cheryl A., Christopher S. Martens and William Ussler, III. 1995. Methane Dynamics Across a Tidally Flooded Riverbank Margin. EPA/600/J-96/078. Limnol. Oceanogr. 40(6):1112-1129. (ERL,GB X816).

The cycling of methane across the soils of the tidally flooded bank margins in the tidal freshwater portion of the White Oak River estuary, North Carolina, was investigated from October 1990 to November 1991. A comparison between these bank edges and adjacent submerged stations was made. The bank stations showed large amounts (up to 1,000 mg m-2 d-1) during summer. This seasonality seems to be linked to the cycle of growth and senescence of vascular plants growing in these soils. In contrast, the submerged stations produced smaller amounts of methane year-round, with moderate increases during summer. Vascular plants are not rooted in these submerged sediments. Seasonal variations in methane production rates at the tidally flooded bank stations led to significant changes in the dissolved methane pool sizes. Although production rates and pore-water concentrations increased with seasonal increases in soil temperatures, the flux of methane to the atmosphere remained fairly low and variable. Methane flux varied with stage of the semidiurnal tidal cycle and exhibited a bimodal distribution, with fluxes greatest when the water level was nearest the soil surface. During summer months, methane production was up to 10 times greater than its flux to the atmosphere. This large difference between production and flux is attributed mainly to bacterially mediated methane oxidation at the soil surface during low-tide exposure and(or) in the root zone of plants. However, in midsummer, lateral export of dissolved methane from the bank soils, driven by tidal flushing, may account for almost 30% of this discrepancy between production and flux.

Selifonov, Sergey A., Jerome E. Gurst and Lawrence P. Wackett. 1995. Regioselective Dioxygenation of ortho-Trifluoromethylbenzoate by Pseudomonas aeruginosa 142: Evidence for 1,2-Dioxygenation as a Mechanism in ortho-Halobenzoate Dehalogenation. EPA/600/J-96/074. Biochem. Biophys. Res. Commun. 213(3):759-767. (ERL,GB X817).

Pseudomonas aeruginosa strain 142 oxidizes 2-halobenzoates via a multicomponent oxygenase (V.Romanov and R.P. Hausinger, J. Bacteriol., 1994, 176(11). The intermediacy of a highly unstable cis-diol in the reaction has been proposed. Direct evidence for this is currently lacking and the stereochemical course of the reaction cannot be inferred from previous studies. In this study, 2-trifluoromethylbenzoate was stoichiometrically oxidized by P. aeruginosa 142 to a chiral product identified as (-)2-trifluoromethyl-cis-1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylic acid. These data rigorously establish a dioxygenative mechanism for 2-halobenzoate metabolism.

Daskalakis, Kostas D. and Thomas P. O'Connor. 1995. Normalization and Elemental Sediment Contamination in the Coastal United States. EPA/600/J-96/080. Environ. Sci. Technol. 29(2):470-477. (ERL,GB X819).

Elemental contamination has been examined in sediments collected from 350 sites along the coast of the United States by NOAA's National Status and Trends (NS&T) Program. To isolate natural from man-made factors, data from 60 sites with fewer than 10,000 people living within 20 km were used to test correlations between concentrations of trace elements and four candidate normalizing factors: iron, aluminum, grain size, and total organic carbon. At least 50% of the concentration variations for the elements As, Cr, Cu, Ni, Pb, Sn, and Zn can be accounted for by covariation with Fe or Al, and either of those major elements can be used for normalization. For Ag, Cd, Hg, Sb, and Se, on the other hand, no candidate normalizers accounted for more than even 30% of the variation. Based on these results, the spatial extent of contamination has been examined, and it was found that severe contamination is mostly limited to small areas near large population centers.

Upton, Todd, Steven Wiltshire, Stephen Francesconi and Shlomo Eisenberg. 1995. ABF1 Ser-720 Is a Predominant Phosphorylation Site for Casein Kinase II of Saccharomyces cerevisiae. EPA/600/J-96/070. J. Biol. Chem. 270(27):16153-16159. (ERL,GB X820).

ABF1 is a multifunctional phosphoprotein that binds specifically to yeast origins of replication and to transcriptional regulatory sites of a variety of genes. We isolated a protein kinase from extracts of Saccharomyces cerevisiae on the basis of its ability to specifically phosphorylate the ABF1 protein. Physical and biochemical properties of this kinase identify it as casein kinase II (CKII). The purified kinase has a high affinity for the ABF1 substrate as indicated by a relatively low Km value. Furthermore, when incubated with ABF1 and anti-ABF1 antibodies, the kinase forms an immunocomplex active in the phosphorylation of ABF1. Biochemical and genetic mapping localized a major site for phosphorylation at Ser-720 near the C terminus of the ABF1 protein. This serine is embedded within a domain enriched for acidic amino acid residues. A Ser-720 to Ala mutation abolishes phosphorylation by CKII in vitro. The same mutation also abolishes phosphorylation of this site in vivo, suggesting that CKII phosphorylates Ser-720 in vivo as well. Although three CKII enzymes, a yeast, sea star, and recombinant human, utilize casein as a substrate with similar efficiencies, only the yeast enzyme efficiently phosphorylates the ABF1 protein. This suggests that ABF1 is a specific substrate of the yeast CKII and that this specificity may reside within one of the B regulatory subunits of the enzyme. Thus, phosphorylation of ABF1 by yeast CKII may prove to be a useful system for studying targeting mechanisms of CKII to a physiological substrate.

Hoch, Matthew P. and David L. Kirchman. 1995. Ammonium Uptake by Heterotrophic Bacteria in the Delaware Estuary and Adjacent Coastal Waters. EPA/600/J-96/160. Limnol. Oceanogr. 40(5):886-897. (ERL,GB X829).

Uptake of NH4- by heterotrophic bacteria and the relative importance of NH4+ and dissolved free amino acids (DFAA) as nitrogen sources for bacterial production were examined in the Delaware estuary and adjacent coastal waters during 1988 and 1990. Although total uptake of NH4+ and bacterial production were approximately 4-fold higher in 1988 than in 1990, percent NH4+ uptake by bacteria in the upper and lower estuary was similar for both years. Bacterial uptake rates were highest at the mouth of the bay in summer and represented 10-25% of total uptake of NH4+. Less than 5% of NH4+ uptake was by bacteria at salinities less than 20 o/oo. In contrast to NH4- uptake, DFAA uptake was greatest in the upper estuary and often exceeded nitrogen requirements for bacterial growth. Bacteria accounted for 15-35% of total NH4+ uptake at coastal and offshore stations in 1990. About 50% of bacterial nitrogen demand was supported by NH4+ at the mouth of the bay and in coastal waters during summer, when DFAA concentrations were generally lowest. Although DFAA concentration and uptake did not explain all variability, they appeared to explain large-scale features in NH4+ uptake by heterotrophic bacteria. Ammonium uptake by bacteria was lowest in the estuary, where DFAA concentrations and uptake were highest; at an offshore station, where DFAA concentrations and uptake were low relative NH4- uptake by bacteria was highest. These and other results suggest that NH4+ uptake by bacteria is relatively high in oligotrophic water and low in eutrophic systems, which has important implications on the role of heterotrophic bacteria in the N cycling of marine environments.

Rayburn, J.R., Mendel Friedman and J.A. Bantle. 1995. Synergistic Interaction of Glycoalkaloids alpha-Chaconine and alpha-Solanine on Developmental Toxicity in Xenopus Embryos. EPA/600/J-96/159. Food Chem. Toxicol. 33(12):1013-1019. (ERL,GB X830).

The embryo toxicities of two major potato glycoalkaloids, alpha-chaconine and alpha-solanine, were examined individually and in mixtures using the frog embryo teratogenesis assay-Xenopus. Calculations of toxic units (TUs) were used to assess possible antagonism, synergism or response addition of several mixtures ranging from approximately 3:1 to 1:20 TUs of alpha-chaconine to alpha-solanine. Some combinations exhibited strong synergism in the following measures of developmental toxicity: (a) 96-hr LC50, defined as the medium concentration causing 50% embryo lethality; (b) 96-hr EC50 (malformation), defined as the concentration causing 50% malformation of the surviving embryos; and (c) teratogenic index which is equal to LC50/EC50 (malformation). The results indicated that each of the mixtures caused synergistic mortality or malformation. Furthermore, these studies suggested that the synergism observed for a specific mixture cannot be used to predict possible synergism of other mixtures with different ratios of the two glycoalkaloids; toxicities observed for individual glycoalkaloids may not be able to predict toxicities of mixtures; and specific combinations found in different potato varieties need to be tested to assess the safety of a particular cultivar.

Rayburn, J.R., J.A. Bantle, C.W. Qualls, Jr. and Mendel Friedman. 1995. Protective Effects of Glucose-6-Phosphate and NADP Against alpha-Chaconine-induced Developmental Toxicity in Xenopus Embryos. EPA/600/J-96/158. Food Chem. Toxicol. 33(12):1021-1025. (ERL,GB X831).

In previous studies a metabolic activation system (MAS) composed of Aroclor 1254-induced rat liver microsomes led to an apparent reduction of potato glycoalkaloid developmental toxicity in the frog embryo teratogenesis assay-Xenopus (FETAX). The reasons for this reduction were investigated in this study. The effect of the exogenous MAS on glycoalkaloid developmental toxicity was examined in two experiments in which a concentration series of alpha-chaconine was tested with a MAS with and without a reduced nicotinamide adenine dinucleotide (NADPH) generator system consisting of NADPH, oxidized nicotinamide adenine dinucleotide (NADP), glucose-6-phosphate (G6P) and glucose-6-phosphate dehydrogenase. The NADPH generator system and each of its individual components were tested at a single high concentration of alpha-chaconine to evaluate their potential effects on toxicity. The findings indicated that the protective effect of the MAS was not the result of detoxification by microsomal enzyme systems, but caused by two components of the NADPH generator system, namely NADP and G6P. G6P was more protective of alpha-chaconine-induced toxicity than NADP at the concentrations tested. Thus, FETAX with a MAS must be performed with appropriate controls that take into account the possible interactions with individual components of the system.

Wells, J. D., J.R. Fuxa and G. Henderson. 1995. Virulence of Four Fungal Pathogens to Coptotermes formosanus (Isoptera: Rhinotermitidae). J. Entomol. Sci. 30(2):208-215. (ERL,GB X834).

Four species and 10 isolates of entomogenous fungi were tested for virulence against the Formosan subterranean termite, Coptotermes formosanus Shiraki. Five isolates of Beauveria bassiana (Balsamo) Vuillemin and three of Metarhizium anisopliae (Metschnikoff) Sorokin were virulent to C. formosanus. The isolates of these two fungal species were chosen because they originated from different phylogenetic groups of host insects. The B. bassiana isolates originated from hosts in the Orthoptera, Lepidoptera, Homoptera, Dermaptera, and Isoptera; the M. anisopliae isolate from an isopteran was most virulent to C. formosanus, when LD50 and mean time until death were both considered. There was no other pattern in virulence of isolates relative to the phylogeny of their original insect hosts. All five B. bassiana isolates and one of the three M. anisopliae isolates produced full growth of external mycelium and conidia on the dead termites. Termites exposed to fungal doses killing >67% of the insects died more quickly than those exposed to doses killing

Forschler, Brian T. and Gregg Henderson. 1995. Subterranean Termite Behavioral Reaction to Water and Survival of Inundation: Implications for Field Populations. Environ. Entomol. 24(6):1592-1597. (ERL,GB X835).

Laboratory tests involving termite response to rising water levels demonstrated that subterranean termites (Reticulitermes spp.) do not try to escape being submerged. Studies in which termites were completely submerged in water provided LT50S of 19.6h for eastern subterranean termites. Reticulitermes flavipes (Kollar), 13.9h for R. virginicus (Banks), and 11.1h for, Formosan subterranean termites, Coptotermes formosanus Shiraki. These data suggest that subterranean termites, in the field, escape drowning not by seeking higher ground but by entering a state of quiescence when confronted with excessive amounts of water in their subterranean habitat. Under normal rainfall conditions, water should move through the soil profile within several hours. If this does not occur then high termite mortality could result. In the summer of 1992, we characterized the foraging populations of 5 subterranean termite colonies using the triple mark--recapture technique. These colonies averaged 99,000 (range, 157,000-14,000) foraging termites per colony. In the spring of 1993, these same colonies were recharacterized and their populations averaged 21,000 (range, 53,000-1000) foraging termites per colony. This represents a 77% reduction in the average foraging populations for these colonies. In this article, we provide empirical evidence that these population reductions were a result of heavy rainfall in west-central Georgia during the winter of 1992-1993.

Replicon, Jean, Allen Frankfater and Robert V. Miller. 1995. Continuous Culture Model to Examine Factors That Affect Transduction among Pseudomonas aeruginosa Strains in Freshwater Environments. Appl. Environ. Microbiol. 61(9):3359-3366. (ERL,GB X848).

Transduction among Pseudomonas aeruginosa strains was observed in continuous cultures operated under environmentally relevant generation times, cell densities, and phage-to-bacterium ratios, suggesting its importance as a natural mechanism of gene transfer. Transduction was quantified by the transfer of the Tra-Mob- plasmid Rms149 from a plasmid-bearing strain to an F116 lysogen that served as both the recipient and source of transducing phages. In control experiments in which transduction was prevented, there was a reduction in the phenotype of the mock transductant over time. However, in experiments in which transduction was permitted, the proportion of transductants in the population increased over time. These data suggest that transduction can maintain a phenotype for an extended period of time in a population from which it would otherwise be lost. Changes in the numbers of transductants were analyzed by a two-part mathematical model, which consisted of terms for the selection of the transductant's phenotype and for the formation of new transductants. Transduction rates ranged from 10-9 to 10-6 per total viable cell count per ml per generation and increased with both the recipient concentration and the phage-to-bacterium ratio. These observations indicate an increased opportunity for transduction to occur when the interacting components are in greater abundance.

Ripp, Steven and Robert V. Miller. 1995. Effects of Suspended Particulates on the Frequency of Transduction Among Pseudomonas aeruginosa in a Freshwater Environment. Appl. Environ. Microbiol. 61(4):1214-1219. (ERL,GB X889).

Transduction has been shown to play a significant role in the transfer of plasmid and chromosomal DNA in aquatic ecosystems. Such ecosystems contain a multitude of environmental factors, any one of which may influence the transduction process. It was the purpose of this study to show how one of these factors, particulate matter, affects the frequency of transduction. In situ transduction rates were measured in lake water microcosms containing either high or low concentrations of particulate matter. The microcosms were incubated in a fewshwater lake in central Oklahoma. Transduction frequencies were found to be enhanced as much as 100-fold in the presence of particulates. Our results suggest that aggregations of bacteriophages and bacterial cells are stimulated by the presence of these suspended particulates. This aggregation increases the probability of progeny phages and transducing particles finding and infecting new host cells. Consequently, both phage production and transdiction frequencies increase in the presence of particulate matter.

Kelly, Thomas J., Eun Ju Park, Carol A. Masler and John P. Burand. 1995. Characterization of the Glycosylated Ecdysteroids in the Hemolymph of Baculovirus-Infected Gypsy Moth Larvae and Cells in Culture. Eur. J. Entomol. 92(1):51-61. (ERL,GB X1001).

Fourth-instar gypsy moth (Lymantria dispar; Lepidoptera: Lymantriidae) larvae, infected with the gypsy moth baculovirus (LdNPV), show an elevated and prolonged extension of the hemolymph ecdysteroid titer peak associated with molting. The ecdysteroid immunoreactivity associated with this peak elutes as two peaks following HPLC on a C18 reverse-phase column. Both peaks elute in a region more polar than 20-hydroxyecdysone, but less polar than the highly polar ecdysteroid immunoreactivity associated with the apolysis peak of control animals. Glycosylated ecdysteroid standards, produced by in vitro incubation of UDP-glucose with ecdysone or 20-hydroxyecdysone and culture medium from LdNPV-infected gypsy moth cells, show elution times identical to the two immunoreactive peaks. Enzymatic hydrolysis studies verified this identity. The data suggest that the hemolymph of LdNPV-infected L. dispar larvae contains both glucose-conjugated ecdysone and glucose-conjugated 20-hydroxyecdysone which, by analogy with the literature, are presumably 22-O-b-D-glucopyranoside and 20-hydroxyecdysone 22-O-b-D-glucopyranoside