Journal Article Citations and Abstracts

Fenthion (Baytex), an organophosphate insecticide, is frequently applied to salt-marsh environments to control mosquitoes. Shake-flask tests were used to study rates of abiotic and biotic degradation of fenthion and the environmental parameters that affect these rates. Water or water-sediment (500 mg dry weight/L) slurries from salt marshes located along the Northwest Florida Gulf Coast were used. Flasks contained 200 µg fenthion/L, and degradation rates were determined by following decrease of fenthion over time. Hydrolysis and biodegradation in water were relatively insignificant fate processes; fenthion disappeared from flasks containing water, formalin-sterilized water, or formalin-sterilized sediment very slowly (half-life >= 2 weeks). The presence of nonsterile sediment resulted in a rapid exponential disappearance of fenthion (half-life >= 3.8 days). Biodegradation was assumed since sterile sediment systems showed a much slower decrease of fenthion, and the production of polar compounds (hexane-unextractable) from radiolabeled fenthion was greater in the presence of sediment than sterilized sediment. No biotic degradation occurred at low oxygen concentration. An 8° C decrease in incubation temperature decreased the rate of sediment biodegradation 2.5-fold. Light caused a slight but significant increase in the biotic and abiotic degradation rates of fenthion in water. A two-fold variation in sediment-mediated degradation was noted among sediment samples from three stations within one field site. Inclusion of whole marsh plants or plant parts increased the disappearance rate of fenthion in test systems.

Clark, James R., Larry R. Goodman, Patrick W. Borthwick, James M. Patrick, Jr., Geraldine M. Cripe, Paul H. Moody, James C. Moore and Emile M. Lores. 1989. Toxicity of Pyrethroids to Marine Invertebrates and Fish: A Literature Review and Test Results with Sediment-Sorbed Chemicals. EPA/600/J-89/040. Environ. Toxicol. Chem. 8(5):393-401. (ERL,GB 618). (Avail. from NTIS, Springfield, VA: PB90-103599)

Data on the acute and chronic toxicities of permethrin, cypermethrin and flucythrinate to marine invertebrates and fish are reviewed. Generally, crustaceans are more sensitive than fish; oysters are comparatively insensitive. The mysid Mysidopsis bahia consistently is among the most sensitive crustaceans tested, with 96-h LC50s of less than 0.02 µg/L for permethrin and of less than 0.01 µg/L for fenvalerate, cypermethrin and flucythrinate. The potential for chronic toxicity to fish is minimal for permethrin, moderate for fenvalerate and relatively great for flucythrinate. Laboratory toxicity tests were conducted with sediment-source fenvalerate and cypermethrin under static and flow-through conditions to determine the degree of contamination necessary to achieve acute lethal effects on mysids, grass shrimp (Palaemonetes pugio) and pink shrimp (Penaeus duorarum). Mortality was observed in test animals only in systems where the concentrations of sediment-source pyrethroids were sufficient to establish lethal concentrations in the overlying water through sediment/water partitioning. For fenvalerate, lethal effects occurred at nominal sediment concentrations of 0.1 mg/kg (static and flow-through) for mysids and grass shrimp and at 10 mg/kg for pink shrimp. Nominal sediment concentrations of cypermethrin of 0.1 mg/kg (static) or 0.1 mg/kg (flow-through) resulted in mortality in mysids and grass shrimp, whereas 1.0 mg/kg was the only test concentration that caused mortality in pink shrimp in the static and flow-through test systems. The correspondence between aqueous concentrations and LC50s for test animals demonstrated the importance of quantitating the bioavailable portion of pyrethroids in field samples to characterize accurately the environmental risk associated with pyrethroid runoff after agricultural applications.

Cripe, Geraldine, Anne Ingley-Guezou, Larry R. Goodman and Jerrold Forester. 1989. Effect of Food Availability on the Acute Toxicity of Four Chemicals to Mysidopsis bahia (Mysidacea) in Static Exposures. EPA/600/J-89/037. Environ. Toxicol. Chem. 8(4):333-338. (ERL,GB 637). (Avail. from NTIS, Springfield, VA: PB90-103581)

The effect of nutritionally deficient Artemia nauplii on the growth of the mysid, Mysidopsis bahia, was evaluated in static systems. When Artemia nauplii with or without highly unsaturated fatty acid 20:5 w 3 were fed to 24-h-old M. bahia there was no difference in growth of mysids on either ration after 96 h of feeding. A study comparing amount of available food in static systems necessary for good survival and growth indicated that rations of 5, 10 or 30 Artemia per mysid per day (A/m/d) were different from 50, 70, 90 and 110 A/m/d as measured by dry weight. Static acute 96-h tests were conducted with <= to 24-h-old Mysidopsis bahia using either carbophenothion, cypermethrin, malathion, or 4-(tert-octyl)phenol. For each chemical, two replicate tests were conducted simultaneously with each of three rations of food provided. The rations chosen were 10 A/m/d, providing survival with minimal growth, a midpoint ration (60 A/m/d), and 110 A/m/d, clearly in excess of that necessary for good survival and growth. Only LC50s obtained in tests using 10 A/m/d were significantly different from other test results. These tests indicate that lack of food does adversely affect results of mysid static tests. In addition, excess food has the potential to reduce dissolved oxygen to nearly unacceptable concentrations.

Walsh, Gerald E. 1989. Bostrichobranchus digonas: Confirmation of Its Presence in the Gulf of Mexico. EPA/600/J-89/036. Fla. Sci. 52(1):100-103. (ERL,GB 643). (Avail. from NTIS, Springfield, VA: PB90-100678)

Although Bostrichobranchus pilularis has been reported from the Gulf of Mexico, descriptions are similar to those of B. digonas. Developmental stages of B. digonas are described and shown to be distinct from those of B. pilularis. Published literature and information presented here indicate that B. digonas is present in shallow water between central and northwestern Florida.

Walsh, Gerald E., Patrice M. Bohannon and Paul B. Wessinger-Duvall. 1989. Microwave Irradiation for Rapid Killing and Fixing of Plant Tissue. EPA/600/J-89/204. Can. J. Bot. 67(4):1272-1274. (ERL,GB 644). (Avail. from NTIS, Springfield, VA: PB90-147802)

Irradiation by microwaves allows for rapid killing and fixing of plant tissue, with excellent cellular integrity for histological examination. Two exposures to microwaves for three seconds in formalin/acetic acid/alcohol gave good preservation of nuclei, chloroplasts, and other plant structures. The microwave method offers a considerable saving of time over traditional methods for killing and fixing plant tissue.

O'Neill, Ellen J., Claude R. Cripe, Leonard H. Mueller, John P. Connolly and Parmely H. Pritchard. 1989. Fate of Fenthion in Salt-Marsh Environments: II. Transport and Biodegradation in Microcosms. EPA/600/J-89/161. Environ. Toxicol. Chem. 8(9):759-768. (ERL,GB 647). (Avail. from NTIS, Springfield, VA: PB90-129479)

The fate of fenthion was examined in microcosms to define the possible interaction between sediment and biodegradation in the field. A mathematical model was also calibrated to calculate the distribution of fenthion in microcosms. Intact sediment cores, both with and without Juncus roemerianus, were removed from a salt marsh and placed into microcosm vessels to simulate the undisturbed sediment bed of a salt marsh and the areas containing Juncus. In formalin-sterilized microcosms without plants, fenthion disappeared exponentially from the water column with a half-life of 105.0 h. Fenthion had a half-life of 35.5 h in the microcosm without plants. In the microcosm with plants, the half-life was slightly faster (33.2 h). In fractionated sediment cores, fenthion was found at greater depths in nonsterile systems than predicted by diffusion and sorption in sterile microcosms, possibly because of bioturbation. Distribution of fenthion in sediment was not appreciably different between microcosms with and without plants. Fenthion appeared to be biodegraded in upper (1 to 7 mm) sediment layers.

Bortone, Stephen A., William P. Davis and Charles M. Bundrick. 1989. Morphological and Behavioral Characters in Mosquitofish as Potential Bioindication of Exposure to Kraft Mill Effluent. EPA/600/J-89/537. Bull. Environ. Contam. Toxicol. 43(3):370-377. (ERL,GB 659). (Avail. from NTIS, Springfield, VA: PB91-199893)

Although the specific chemicals or factors actually responsible for induction of arrhenoidy among mosquitofish have not yet been identified, it is known that a wide variety of potential compounds occur as by-products from the processing of wood pulp (Keith 1976). Purpose of study was to investigate the morphological and behavioral responses of mosquitofish environmentally exposed to kraft mill effluent (KME) and to evaluate the potential of these responses as bioassay endpoints. A method to quantify the morphological or behavioral responses of mosquitofish should provide an in situ bioindicator to assess impact of KME discharge on receiving water biota.

Barkay, Tamar, Cynthia Liebert and Mark Gillman. 1989. Environmental Significance of the Potential for mer(Tn21)-Mediated Reduction of Hg2+ and Hg0 in Natural Waters. EPA/600/J-89/166. Appl. Environ. Microbiol. 55(5):1196-1202. (ERL,GB 660). (Avail. from NTIS, Springfield, VA: PB90-129511)

The role of mer(Tn21) in adaptation of aquatic microbial communities to Hg2+ was investigated. Elemental mercury was the sole product of Hg2+ volatilization by freshwater and saline microbial communities. Bacterial activity was responsible for biotransformation because most microeukaryotes did not survive the exposure conditions, and removal of larger microbes (>1 µm) from adapted communities did not significantly (P >0.01) reduce Hg2+ volatilization rates. DNA sequences homologous to mer(Tn21) were found in 50% of Hg2+ resistant bacterial strains representing two fresh water communities, but in only 12% of strains representing two saline communities (the difference was highly significant; P < 0.001). Thus, mer(Tn21) played a significant role in Hg2+ resistance among strains isolated from freshwaters where microbial activity had a limited role in Hg2+ volatilization. In saline environments where microbially mediated volatilization was the major mechanism of Hg2+ loss, other bacterial genes coded for this biotransformation.

Genthner, Barbara R. Sharak, W. Allen Price, II and P.H. Pritchard. 1989. Anaerobic Degradation of Chloroaromatic Compounds in Aquatic Sediments under a Variety of Enrichment Conditions. EPA/600/J-89/162. Appl. Environ. Microbiol. 55(6):1466-1471. (ERL,GB 661). (Avail. from NTIS, Springfield, VA: PB90-128018)

Anaerobic degradation of monochlorophenols and monochlorobenzoates in a variety of aquatic sediments was compared under four enrichment conditions. A broader range of compounds was degraded in enrichments inoculated with sediment exposed to industrial effluents. Degradation of chloroaromatic compounds was observed most often in methanogenic enrichments and in enrichments amended with 1 mM bromoethane sulfonic acid. Degradation was observed least often in enrichments with added nitrate or sulfate. The presence of 10 mM bromoethane sulfonic acid prevented or inhibited degradation of most compounds tested. Primary enrichments in which KNO3 was periodically replenished to maintain enrichment characteristics degraded chlorobenzoates, but not chlorophenols. In contrast, primary enrichments in which Na2SO4 was periodically replenished failed to degrade any chloroaromatic compounds. Upon transfer to fresh medium, none of the sulfate enrichments required the presence of Na2SO4 for degradation, while only two nitrate enrichments required the presence of KNO3 for degradation. As a class of compounds, chlorophenols were degraded more readily than chlorobenzoates. However, as individual compounds 3-chlorobenzoate, 2-chlorophenol, and 3-chlorophenol degradation was observed most often and with an equal frequency. Within the chlorophenol class, the relative order of degradability was ortho > meta > para, while that of chlorobenzoates was meta > ortho > para. In laboratory transfers, 2-chlorobenzoate, 3-chlorobenzoate, and 2-chlorophenol degradation was most easily maintained, while degradation of para-chlorinated compounds was very difficult to maintain.

Genthner, Barbara R. Sharak, W. Allen Price, II and P.H. Pritchard. 1989. Characterization of Anaerobic Dechlorinating Consortia Derived from Aquatic Sediments. EPA/600/J-89/163. Appl. Environ. Microbiol. 55(6):1472-1476. (ERL,GB 662). (Avail. from NTIS, Springfield, VA: PB90-129487)

Four methanogenic consortia, which degraded 2-chlorophenol, 3-chlorophenol, 2-chlorobenzoate, and 3-chlorobenzoate, respectively, and one nitrate-reducing consortium which degraded 3-chlorobenzoate were characterized. Degradative activity in these consortia has been maintained in laboratory transfer for over two years. In the methanogenic consortia, the aromatic ring was dechlorinated before mineralization to methane and carbon dioxide. After dechlorination, the chlorophenol consortia converted phenol to benzoate before mineralization. All methanogenic consortia degraded both phenol and benzoate. The 3-chlorophenol and 3-chlorobenzoate consortia also degraded 2-chlorophenol. No other cross acclimation to monochlorophenols or monochlorobenzoates was detected in the methanogenic consortia. The consortium which required nitrate for the degradation of 3-chlorobenzoate degraded benzoate and 4-chlorobenzoate anaerobically in the presence of KNO3, but not in its absence. This consortium also degraded benzoate, but not 3-chlorobenzoate, aerobically.

Barkay, Tamar, Cynthia Liebert and Mark Gillman. 1989. Hybridization of DNA Probes with Whole-Community Genome for Detection of Genes That Encode Microbial Responses to Pollutants: Mer Genes and Hg2+ Resistance. EPA/600/J-89/167. Appl. Environ. Microbiol. 55(6):1574-1577. (ERL,GB 664). (Avail. from NTIS, Springfield, VA: PB90-129529)

Nucleic acids extracted from microbial biomass were hybridized with probes representing four mer operons, to detect genes encoding adaptation to Hg2+. An enrichment in sequences similar to the mer genes of transposon Tn501 occurred during adaptation in a freshwater community. In an estuarine community, all four mer genes were only slightly enriched, suggesting that additional, yet uncharacterized, mer genes encoded adaptation to Hg2+.

Shields, Malcolm S., Stacy O. Montgomery, Peter J. Chapman, Stephen M. Cuskey and P.H. Pritchard. 1989. Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4. EPA/600/J-89/168. Appl. Environ. Microbiol. 55(6):1624-1629. (ERL,GB 668). (Avail. from NTIS, Springfield, VA: PB90-129537)

o-Cresol and 3-methylcatechol were identified as successive transitory intermediates of toluene catabolism by the trichloroethylene-degrading bacterium G4. The absence of a toluene dihydrodiol intermediate or toluene dioxygenase and toluene dihydrodiol dehydrogenase activities suggested that G4 catabolizes toluene by a unique pathway. Formation of a hybrid species of 18O- and 16O- labeled 3-methylcatechol from toluene in an atmosphere of 18O2 and 16O 2 established that G4 catabolizes toluene by successive monooxygenations at the ortho and meta positions. Detection of trace amounts of 4-methylcatechol from toluene catabolism suggested that the initial hydroxylation of toluene was not exclusively at the ortho position. Further catabolism of 3-methylcatechol was found to proceed via catechol-2,3-dioxygenase and hydroxymuconic semialdehyde hydrolase activities.

Couch, John A. 1989. Membranous Labyrinth in Baculovirus-Infected Crustacean Cells: Possible Roles in Viral Reproduction. EPA/600/J-89/423. Dis. Aquat. Org. 7(1):39-53. (ERL,GB 669). (Avail. from NTIS, Springfield, VA: PB90-245747)

The origins and morphogenesis of the membranous labyrinth (ML) in Baculovirus penaei (BP) infected cells of penaeid shrimps (Crustacea:Decapoda) are described. The ML is usually a highly ordered, complex system of membranes arranged as a labyrinth or in concentric form in the cytoplasm near or against the nuclear envelope. The ML originates from dilated Golgi and ER vesicles and from the outer nuclear envelope. It grows apparently from proliferation of the cellular membranes of these systems, and its development may be correlated with the stages of BP infection. It is hypothesized that, because of the close parallel and concurrent development of the ML and virus reproduction, and other evidence, the ML is virus induced and controlled and may play at least three roles in the virus reproductive strategy: 1) provides a conduit or transport system for viral or occlusion body precursors from cytoplasm to nucleus; 2) provides increased membrane surface and volume for increased ATPase activity (related to energy demand for virus reproduction and transport of viral products); and 3) provides a mechanism for cell collapse and release of virus and occlusion bodies at end of virus reproduction period. Possible experimental methods with which to determine the functional role of the ML are discussed and implications for such a system for nuclear, DNA, viruses are considered.

Mueller, James G., Peter J. Chapman and P. Hap Pritchard. 1989. Creosote-Contaminated Sites: Their Potential for Bioremediation. EPA/600/J-89/170. Environ. Sci. Technol. 23(10):1197-1201. (ERL,GB 671). (Avail. from NTIS, Springfield, VA: PB90-129552)

The demonstrated ability to biologically transform toxic organic chemicals into innocuous compounds under laboratory conditions has led to the view that microbial systems may be employed advantageously to remediate hazardous waste sites polluted with similar materials. Hence, bioremediation is a rapidly developing technology with the potential to provide efficient and economic means of removing organic pollutants from contaminated materials. However, bioremediation is currently restricted by a variety of factors. One of the most significant of these is the difficulty of effectively degrading complex mixtures of chemicals as often found at hazardous waste sites. Although preliminary evidence shows that such treatments are potentially effective in situ, additional data on the performance of bioremediation efforts in large scale field trials is also needed. Bioremediation of creosote-contaminated materials is reviewed here by characterizing coal-tar creosote, identifying techniques for assessing the biodegradability of its many chemical constitutents, examining known routes of microbial transformation of these chemicals, and reviewing the performance of previous bioremediation efforts. This approach is developed as a model system to project the potential application of bioremediation to ameliorate environments contaminated by complex mixtures of structurally diverse hazardous chemicals.

Mueller, James G., Peter J. Chapman and P. Hap Pritchard. 1989. Action of a Fluoranthene-Utilizing Bacterial Community on Polycyclic Aromatic Hydrocarbon Components of Creosote. EPA/600/J-89/425. Appl. Environ. Microbiol. 55(12):3085-3090. (ERL,GB 674). (Avail. from NTIS, Springfield, VA: PB90-245721)

Cultures enriched by serial transfer through a mineral salts medium containing fluoranthene were used to establish a stable, 7-membered bacterial community from a sandy soil highly contaminated with coal-tar creosote. This community exhibited an ability to utilize fluoranthene as sole carbon source for growth as demonstrated by increases in protein concentration and changes in absorption spectra when grown on fluoranthene in liquid culture. Biotransformation of other polycyclic aromatic hydrocarbons (PAHs) was verified by demonstrating their disappearance from an artificial PAH mixture using capillary gas chromatography. When grown on fluoranthene as sole carbon source and subsequently exposed to fluoranthene plus 16 additional PAHs typical of those found in creosote, this community transformed all PAHs present in this defined mixture. After 3 days of incubation, 13 of the original 17 PAH components were degraded to levels below the limit of detection (10 ng/L). Continued incubation resulted in extensive degradation of the remaining 4 compounds. The ability of this community to utilize a high molecular weight PAH as sole carbon source, in conjunction with its ability to transform a diverse array of PAHs, suggests that it may be of value in the bioremediation of environments contaminated with PAHs such as those impacted by creosote.

Genthner, Barbara R. Sharak, G.T. Townsend and P.J. Chapman. 1989. Anaerobic Transformation of Phenol to Benzoate via para-Carboxylation: Use of Fluorinated Analogues to Elucidate the Mechanism of Transformation. EPA/600/J-89/173. Biochem. Biophys. Res. Commun. 162(3):945-951. (ERL,GB 681). (Avail. from NTIS, Springfield, VA: PB90-140849)

Isomeric fluorophenols were used as phenol analogues to investigate the transformation of phenol to benzoate by an anaerobic, phenol-degrading consortium derived from freshwater sediment. Transformation of 2-fluorophenol and 3-fluorophenol occurred in the presence or absence of phenol and led to the accumulation of fluorobenzoic acids. Identification of the resulting fluorobenzoate products as 3-fluorobenzoate and 2-fluorobenzoate isomers, respectively, together with the nontransformation of 4-fluorophenol indicated that the carboxyl group was introduced para to the phenolic hydroxyl group.

Henis, Yigal, K.R. Gurijala and Martin Alexander. 1989. Factors Involved in Multiplication and Survival of Escherichia coli in Lake Water. Microb. Ecol. 17(2):171-180. (ERL,GB X576).

The population of a strain of Escherichia coli that was resistant to nalidixic acid and streptomycin declined rapidly in samples of sterile and nonsterile Cayuga Lake water and reached an undetectable level in nonsterile water at 24 and 72 h when counted on cosin-methylene blue (EMB) agar and half-strength Trypticase soy agar, respectively. In sterile lake water amended with 10 ug of amino acids per ml or 0.1 M phosphate, E. coli multiplied exponentially for more than 24 h. The addition of Rhizobium leguminosarum biovar phaseoli to unamended sterile lake water prevented the decline of E. coli and its addition to amended sterile lake water prevented E. coli multiplication. The data suggest that E. coli cells grown on rich media suffer a shock when introduced into lake water because of low hypotonicity, the indigenous competing flora, or both. This shock is prevented by either phosphate buffer or by amino acids at low concentration. The shocked bacteria formed colonies on half-strength Trypticase soy agar. Depending on environmental conditions, the presence of a second organism either has no effect or results in an increase or decrease in E. coli numbers.

Baughman, Douglas S., David W. Moore and Geoffrey I. Scott. 1989. Comparison and Evaluation of Field and Laboratory Toxicity Tests with Fenvalerate on an Estuarine Crustacean. EPA/600/J-89/538. Environ. Toxicol. Chem. 8(5):417-429. (ERL,GB X594). (Avail. from NTIS, Springfield, VA: PB91-206839)

Field and laboratory toxicity tests were conducted on the grass shrimp, Palaemonetes pugio, to evaluate the usefulness of laboratory testing in estimating mortality from fenvalerate exposure associated with agricultural runoff. The study examined an integrated approach for assessing the impacts of fenvalerate on estuarine fauna, using 96-h static-renewal and 6-h pulsed-dose laboratory toxicity tests and in situ toxicity tests. The laboratory toxicity tests with fenvalerate gave 96-h LC50 values ranging from 0.007 to 0.071 ug/L and 6-h PDLC50 values ranging from 0.100 to 0.130 ug/L. Comparisons of the results of two field toxicity tests with laboratory-derived LC50 values showed good agreement between field and laboratory toxicity data. The variation between field and laboratory toxicity tests may have been due to the limitations of the water sampling regime used in characterizing the pesticide exposure during the field toxicity tests. These comparisons suggest that a combination of laboratory and field toxicity testing is required to estimate the actual field mortality from fenvalerate exposure associated with agricultural runoff. Future studies should include composite water sampling and more frequent discrete sampling methods to better characterize field exposure regimes.

Sinclair, James L. and Martin Alexander. 1989. Effect of Protozoan Predation on Relative Abundance of Fast- and Slow-Growing Bacteria. EPA/600/J-89/165. Can. J. Microbiol. 35(5):578-582. (ERL,GB X597). (Avail. from NTIS, Springfield, VA: PB90-129503)

The survival of six bacterial species that had different growth rates was tested in raw sewage and sewage that was rendered free of protozoa. When test bacteria were added to protozoa-free sewage at densities of approximately 10 to the fifth power to 10 to the sixth power cells/mL, five of the six species did not decline below 10 to the fifth power cells/mL. If protozoa were present, the population sizes of all test species were markedly reduced, but bacterial species able to grow faster in artifical media had the larger number of survivors. When the same bacteria were inoculated into protozoa-free sewage at densities of less than 10 to the third power cells/mL, only the three species able to grow quickly in artificial media increased in abundance. When the six species were inoculated at the same densities into sewage containing protozoa, the three slow-growing species were rapidly eliminated, and two of the three fast-growing species survived in detectable numbers. We suggest that in environments with intense protozoan predation, protozoa may alter the composition of the bacterial community by eliminating slow-growing bacteria.

Somerville, Charles C., Ivor T. Knight, William L. Straube and Rita R. Colwell. 1989. Simple, Rapid Method for the Direct Isolation of Nucleic Acids from Aquatic Environments. Appl. Environ. Microbiol. 55(3):548-554. (ERL,GB X604).

The direct isolation of nucleic acids from the environment may be useful in several respects, including the estimation of total biomass, detection of specific organisms and genes, estimations of species diversity, and cloning applications. We have developed a method which facilitates the concentration of microorganisms from aquatic samples and the extraction of their nucleic acids. Natural water samples of 350 ml to greater than 1000 ml are concentrated on a single, cylindrical filter membrane (Millipore SVGS01015), and cell lysis and proteolysis are carried out within the filter housing. Crude, high molecular weight nucleic acid solutions are then drawn off the filter. These solutions can be immediately analyzed, concentrated or purified depending on the intended application. The method is simple, rapid and economical and provides high molecular weight chromosomal DNA, plasmid DNA and speciated RNAs which co-migrate with 5S, 16S and 23S ribosomal RNAs. The methods presented here should prove useful in studying both the ecology and phylogeny of microbes which resist culture methods.

Sangodkar, U.M.X., T.L. Aldrich, R.A. Haugland, J. Johnson, R.K. Rothmel, P.J. Chapman and A.M. Chakrabarty. 1989. Molecular Basis of Biodegradation of Chloroaromatic Compounds. Acta Biotechnol. 9(4):301-316. (ERL,GB X608).

Chlorinated aromatic hydrocarbons are widely used in industry and agriculture, and comprise the bulk of environmental pollutants. Although simple aromatic compounds are biodegradable by a variety of degradative pathways, their halogenated counterparts are more resistant to bacterial attack and often necessitate evolution of novel pathways. An understanding of such evolutionary processes is essential for developing genetically improved strains capable of mineralizing highly chlorinated compounds. This article provides an overview of the genetic aspects of dissimilation of chloroaromatic compounds and discusses the potential of gene manipulation to promote enhanced evolution of the degradative pathways.

Harker, Alan R., Ronald H. Olsen and Ramon J. Seidler. 1989. Phenoxyacetic Acid Degradation by the 2,4,-Dichlorophenoxyacetic Acid (TFD) Pathway of Plasmid pJP4: Mapping and Characterization of the TFD Regulatory Gene, tfdR. EPA/600/J-89/129. J. Bacteriol. 171(1):314-320. (ERL,GB X611). (Avail. from NTIS, Springfield, VA: PB90-111170)

Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pRO101) and some Pseudomonas transconjugants grew on TFD. Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate. A mutant of one such phenol-degrading strain, Pseudomonas putida PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101. Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101. This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA. Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols.

Diamond, David W., Laura K. Scott, Richard B. Forward, Jr. and W. Kirby-Smith. 1989. Respiration and Osmoregulation of the Estuarine Crab, Rhithropanopeus harrisii (Gould): Effects of the Herbicide, Alachlor. EPA/600/J-89/169. Comp. Biochem. Physiol. A. Comp. Physiol. 93A(2):313-318. (ERL,GB X615). (Avail. from NTIS, Springfield, VA: PB90-129545)

1. The effects of a sudden decrease in salinity and exposure to sublethal concentrations of the herbicide, Alachlor, on osmoregulation and respiration of the crab, Rhithropanopeus harrisii, were studied. 2. Crabs were hyperosmotic regulators at salinities below 24 ppt and became hypoosmotic at higher salinities. Upon a salinity decrease from 20 to 1 ppt, crabs adjusted their haemolymph osmolality to a stable hyperosmotic level in 8 hr. Alachlor concentration to 50 ppm did not affect this adjustment. 3. A salinity decrease from 10 to 0 ppt elevated VO2 and the critical oxygen tension. This response was unaffected by Alachlor concentrations as high as 25 ppm.

Tomasek, P.H., B. Frantz, U.M.X. Sangodkar, R.A. Haugland and A.M. Chakrabarty. 1989. Characterization and Nucleotide Sequence Determination of a Repeat Element Isolated from a 2,4,5-T Degrading Strain of Pseudomonas cepacia. EPA/600/J-89/171. Gene. 76(2):227-238. (ERL,GB X622). (Avail. from NTIS, Springfield, VA: PB90-129560)

Pseudomonas cepacia strain AC1100, capable of growth on 2,3,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T- strain PT88 by a ColE1::Tn5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) was identified which was repeated several times on the wild-type chromosome and was also present on AC1100 plasmid DNA. Various chromosomal fragments containing this sequence were cloned and their nucleotide sequence was determined. Examination of RS1100 revealed the presence of 38-39-bp terminal inverted repeats immediately flanked by 8-bp direct repeats. The translated sequence of the single large open reading frame of RS1100 showed structural similarity to the phage Mu transposase and other DNA-binding proteins. Thus the AC1100 repeated sequence has several structural features in common with insertion sequence elements. Three copies of RS1100 were mapped near 2,4, 5-t genes encoding degradation of 5-chloro-1,2,4-trihydroxybenzene, an intermediate in 2,4,5-T degradation. Neither RS1100 nor the 2,4,5-t genes hybridized to DNA isolated from Pseudomonas strains, including P. cepacia, suggesting that both gene fragments may be of foreign origin recruited in strain AC1100. The origin of these two DNA segments as well as the role played by RS1100 in the recruitment of 2,4,5-t genes in AC1100 are presently under investigation.

Thurman, Robert B. and Donald V. Lightner. 1989. Alternative Method for Analyzing Membranes Hybridized with Radiolabeled Probes. Aust. Microbiol. 10(5):531-532. (ERL,GB X627).

Dot-blot membranes hybridized with a 32P-labeled probe were cut into sections corresponding to the wells of the dot-blot apparatus. The pieces of membrane were then placed in scintillation cocktail, vortexed and counted to a relative counting error of plus or minus 10% cpm.

Kent, M.L., M. Moser and J.W. Fournie. 1989. Coccidian Parasites (Apicomplexa: Eucoccidorida) in Hardy Head Fish, Atherinomorus capricornensis (Woodland). EPA/600/J-89/172. J. Fish Dis. 12(2):179-183. (ERL,GB X629). (Avail. from NTIS, Springfield, VA: PB90-140815)

Authors describe coccidian merozoites (asexual stages) in the exocrine pancreas and oocysts in the gut epithelilum of hardy heads, Atherinomorus capricornensis (Woodland) (family Atherinidae) collected at Heron Island , Queensland, Australia, during the pre-ICOPA (International Congress of Parasitology) workshop held 16-24 August 1986. Examination of the pancreatic merozoites by electron microscopy revealed conoids, micronemes and rhoptries which are unique to the phylum Apicomplexa. As many as four individuals were observed within a single host cell. Although it was usually not possible to determine the type of cell infected, some were intact enough to be identified as pancreatic acinar cells. No parasites undergoing multiple synchronous fission (schizogony) were observed, but dividing pairs attached at the posterior end were frequently seen. Because of inadequate fixation, detection of sporocyst sutures or Stieda bodies in the oocysts by electron microscopy were not possible.

DeFlaun, Mary F. and John H. Paul. 1989. Detection of Exogenous Gene Sequences in Dissolved DNA from Aquatic Environments. EPA/600/J-89/174. Microb. Ecol. 18(1):21-28. (ERL,GB X630). (Avail. from NTIS, Springfield, VA: PB90-140773)

A method for the concentration and detection of gene sequences in the dissolved DNA from freshwater and marine environments has been developed. The limit of detection in the dot blot format was 167 fg/ml (100 ml sample) for exogenous herpes simplex thymidine kinase (TK) gene that was added to artificial seawater or river water. This procedure has been used to determine the longevity and monitor progressive changes in molecular weight of a plasmid containing the TK gene added to eutrophic estuarine water. The onset of plasmid degradation as determined by change in molecular weight was rapid (within 5 min). Intact plasmid was detected for at least 4 hours and sequences hybridizable to the TK gene probe were present for up to 24 hours.

Paul, John H., Wade H. Jeffrey, Andrew W. David, Mary F. DeFlaun and Lisa H. Cazares. 1989. Turnover of Extracellular DNA in Eutrophic and Oligotrophic Freshwater Environments of Southwest Florida. EPA/600/J-89/175. Appl. Environ. Microbiol. 55(7):1823-1828. (ERL,GB X631). (Avail. from NTIS, Springfield, VA: PB90-140823)

The turnover of extracellular DNA was investigated in oligotrophic springs of the Crystal River and the eutrophic Medard Reservoir of southwest Florida. The Medard Reservoir possessed large populations of bacterioplankton and phytoplankton (6.8 X 10 to the ninth power cells per liter and 28.6 ug of chlorophyll a per liter, respectively), while the Crystal River springs only contained a fraction of the microbial biomass found in the Medard Reservoir. Although dissolved DNA values were greater in the Medard Reservoir, higher rates of DNA removal resulted in similar extracellular DNA turnover times in both environments (9.62 plus or minus 3.6 h in the Crystal River and 10.5 plus or minus 2.1 h in the Medard Reservoir). These results indicate that regardless of trophic status or microbial standing stock, extracellular DNA turns over rapidly in subtropical planktonic freshwater environments. Therefore, recombinant DNA sequences from released genetically engineered microorganisms might not be expected to survive for long periods of time in freshwater planktonic environments.

Paul, John H. and Andrew W. David. 1989. Production of Extracellular Nucleic Acids by Genetically Altered Bacteria in Aquatic-Environment Microcosms. EPA/600/J-89/176. Appl. Environ. Microbiol. 55(8):1865-1869. (ERL,GB X635). (Avail. from NTIS, Springfield, VA: PB90-140781)

The factors which affect the production of extracellular DNA by genetically altered strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas cepacia, and Bradyrhizobium japonicum in aquatic environments were investigated. Cellular nucleic acids were labeled in vivo by incubation with [3H]thymidine or [3H]adenine, and production of extracellular DNA in marine waters, artificial seawater, or minimal salts media was determined by detecting radiolabeled macromolecules in incubation filtrates. The presence or absence of the ambient microbial community had little effect on the production of extracellular DNA. Three of four organisms produced the greatest amounts of extracellular nucleic acids when incubated in low-salinity media (2% artificial seawater) rather than high-salinity media (10 to 50% artificial seawater). The greatest production of extracellular nucleic acids by P. cepacia occurred at pH 7 and 37 degrees C, suggesting that extracellular-DNA production may be a normal physiologic function of the cell. Incubation of labeled P. cepacia cells in water from Bimini Harbor, Bahamas, resulted in labeling of macromolecules of the ambient microbial population. Collectively these results indicate that (i) extracellular-DNA production by genetically altered bacteria released into aquatic environments is more strongly influenced by physiochemical factors than biotic factors, (ii) extracellular-DNA production rates are usually greater for organisms released in freshwater than marine environments, and (iii) ambient microbial populations can readily utilize materials released by these organisms.

Stewart, Gregory J. and Christopher D. Sinigalliano. 1989. Detection and Characterization of Natural Transformation in the Marine Bacterium Pseudomonas stutzeri Strain ZoBell. Arch. Microbiol. 152(6):520-526. (ERL,GB X639).

Soil isolates of Pseudomonas stutzeri have been shown previously to acquire genes by natural transformation. In this study a marine isolate, Pseudomonas stutzeri strain ZoBell, formerly Pseudomonas perfectomarina, was also shown to transform naturally. Transformation was detected by the Juni plate method and frequencies of transformation were determined by filter transformation procedures. Maximum frequencies of transformation were detected for three independent antibiotic resistance loci. Transformation frequencies were on the order of 4 X 10-5 transformants per recipient, a frequency over 100 times that of spontaneous antibiotic resistance. Transfer of antibiotic resistance was inhibited by DNase I digestion. Marine isolates achieved maximum competence 14 hours after transfer of exponential cultures to filters on solid media, although lower levels of competence were detected immediately following filter immobilization. Like soil isolates, P. stutzeri strain ZoBell is capable of cell contact transformation, but unlike soil isolates where transformation frequencies are greater for cell contact transformation as compared to transformation with purified DNA, the maximum frequency of transformation achieved by cell contact in the marine strain was approximately 10-fold less than transformation frequencies with purified DNA. These studies establish the first marine model for the study of natural transformation.

Paul, John H. and Scott L. Pichard. 1989. Specificity of Cellular DNA-Binding Sites of Microbial Populations in a Florida Reservoir. EPA/600/J-89/553. Appl. Environ. Microbiol. 55(11):2798-2801. (ERL,GB X640). (Avail. from NTIS, Springfield, VA: PB92-129618)

The substrate specificity of the DNA binding mechanism(s) of bacteria in a Florida reservoir was investigated in short and long term uptake studies with radiolabelled DNA and unlabelled competitors. Thymine oligonucleotides ranging in size from 2 bp to 19-24 bp inhibited DNA binding in 20 min incubations by 43 to 77%. Deoxynucleoside monophosphates, thymidine, and thymine had litte effects on short term DNA binding, although several of these compounds inhibited the uptake of radiolabel from DNA in 4 h incubations. Inorganic phosphate and glucose-1-phosphate inhibited neither short or longterm binding of [3H] or [32PDNA], indicating that DNA was not utilized as a phosphorous source in this reservoir. RNA inhibited both short and long term radiolabelled DNA uptake equally as well as unlabelled DNA. Collectively these results indicate that aquatic bacteria possess a generalized nucleic acid uptake/binding mechanism specific for compounds containing phosphodiester bonds and capable of recognizing oligonucleotides as short as dinucleotides. This binding site is distinct from nucleoside, nucleotide, phosphomonoester, and inorganic phosphate binding sites. Such a nucleic acid binding mechanism may have evolved for the utilization of extracellular DNA (and perhaps RNA) which is abundant in many marine and freshwater environments.

Zylstra, Gerben J., R.H. Olsen and D.P. Ballou. 1989. Cloning, Expression, and Regulation of the Pseudomonas cepacia Protocatechuate 3,4-Dioxygenase Genes. EPA/600/J-89/435. J. Bacteriol. 171(11):5907-5914. (ERL,GB X641). (Avail. from NTIS, Springfield, VA: PB90-264771)

The genes for the alpha and beta subunits of the enzyme protocatechuate 3,4-dioxygenase were cloned from the Pseudomonas cepacia DBO1 chromosome on a 9.5 kilobase pair PstI fragment into the broad-host-range cloning vector pRO2317. The resultant clone was able to complement protocatechuate 3,4-dioxygenase mutations in P. cepacia, P. aeruginosa, and P. putida. Expression studies showed that the genes were constitutively expressed and subject to catabolite repression in the heterologous host. Since the cloned genes exhibited normal induction patterns when present in P. cepacia DBO1, it was concluded that induction was subject to negative control. Regulatory studies with P. cepacia wild type and mutant strains showed that protocatechuate 3,4-dioxygenase is induced either by protocatechuate or by beta-carboxymuconate. Further studies of P. cepacia DBO1 showed that p-hydroxybenzoate hydroxylase, the preceding enzyme in the pathway, is induced by p-hydroxybenzoate and that beta-carboxymuconate lactonizing enzyme, which catalyzes the reaction following protocatechuate 3,4-dioxygenase, is induced by both p-hycroxybenzoate and beta-ketoadipate.

Zylstra, Gerben J., R.H. Olsen and D.P. Ballou. 1989. Genetic Organization and Sequence of the Pseudomonas cepacia Genes for the Alpha and Beta Subunits of Protocatechuate 3,4-Dioxygenase. EPA/600/J-89/426. J. Bacteriol. 171(11):5915-5921. (ERL,GB X642). (Avail. from NTIS, Springfield, VA: PB90-264854)

The locations of the genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase on a 9.5 kilobase pair PstI fragment cloned from the Pseudomonas cepacia DBO1 chromosome were determined. This was accomplished through the construction of several subclones into the broad-host-range cloning vectors pRO2317, pRO2320, and pRO2321. The ability of each subclone to complement mutations in protocatechuate 3,4-dioxygenase (pcaA) was tested in mutant strains derived from P. cepacia, P. aeruginosa, and P. putida. These complementation studies also showed that the two subunits were expressed from the same promoter. The nucleotide sequence of the region encoding for protocatechuate 3,4-dioxygenase was determined. The deduced amino acid sequence matched that determined by N-terminal analysis of regions of the isolated enzyme. Although over 400 nucleotides were sequenced before the start of the genes, no homology was found to known promoters. However, a terminator stem-loop structure was found immediately after the genes. The deduced amino acid sequence shows extensive homology with the previously determined amino acid sequence of protocatechuate 3,4-dioxygenase from another Pseudomonas species.

Kukor, Jerome J., Ronald H. Olsen and June-Sang Siak. 1989. Recruitment of a Chromosomally Encoded Maleylacetate Reductase for Degradation of 2,4-Dichlorophenoxyacetic Acid by Plasmid pJP4. EPA/600/J-89/436. J. Bacteriol. 171(6):3385-3390. (ERL,GB X644). (Avail. from NTIS, Springfield, VA: PB90-264763)

When Pseudomonas aeruginosa PAO1c or P. putida PPO200 or PPO300 carry plasmid pJP4, which encodes enzymes for the degradation of 2,4-dichlorophenoxyacetic acid (TFD) to 2-chloromaleylacetate, cells do not grow on TFD and UV-absorbing material with spectral characteristics of chloromaleylacetate accumulates in the culture medium. Using plasmid pRO1727, we cloned from the chromosome of a nonfluorescent pseudomonad, Pseudomonas sp. strain PKO1, 6-and 0.5-kilobase BamHI DNA fragments which contain the gene for maleylacetate reductase. When carrying either of the recombinant plasmids, pRO1944 or pRO1945, together with pJP4, cells of P. aeruginosa or P. putida were able to utilize TFD as a sole carbon source for growth. A novel polypeptide with an estimated molecular weight of 18,000 was detected in cell extracts of P. aeruginosa carrying either plasmid pRO1944 or plasmid pRO1945. Maleylacetate reductase activity was induced in cells of P. aeruginosa or P. putida carrying plasmid pRO1945, as well as in cells of Pseudomonas strain PKO1, when grown on L-tyrosine, suggesting that the tyrosine catabolic pathway might be the source from which maleylacetate reductase is recruited for the degradation of TFD in pJP4-bearing cells of Pseudomonas sp. strain PKO1.

Steffan, R.J., A. Breen, R.M. Atlas and G.S. Sayler. 1989. Application of Gene Probe Methods for Monitoring Specific Microbial Populations in Freshwater Ecosystems. Can. J. Microbiol. 35(7):681-685. (ERL,GB X648).

Several gene probe methods were used to monitor specific microbial populations in freshwater microcosms. Detection methods included nonselective plating - colony hybridization, selective plating - colony hybridization, most probable number - filter hybridization, and community DNA extraction - dot blot hybridization. Tests were conducted in freshwater microcosms inoculated with a 4-chlorobiphenyl degrading Alcaligenes A5 or a 2,4,5-trichlorophenoxyacetic acid degrading Pseudomonas cepacia AC1100. Colony hybridization performed on colonies detected on a nonselective medium sometimes failed to detect both Alcaligenes A5 and P. cepacia AC1100 when the target populations comprised less than 0.1% of the total population, even though the target populations comprised less than 0.1% of the total population, even though the target populations were present at concentration greater than 10 to the fourth power viable cells/mL as indicated by other detection methods. Selective plating - colony hybridization, most probable number - filter hybridization, and dot blot hybridization using DNA extracted from the microbial community consistently indicated persistence of the added P. cepacia AC1100 in the microcosms. Although differing in their detection reliability and sensitivity, the various gene probe detection methods indicated persistence with a slow decline of both Alcaligenes A5 and P. cepacia AC1100 over an 8-week period.

Kent, M.L., L. Margolis and J.W. Fournie. 1989. New Eye Disease of Pen-Reared Chinook Salmon in British Columbia Caused by the Cestode Gilquinia squali. Am. Fish. Soc. Newsletter. 17(4):5. (ERL,GB X653).

Authors document first reported occurrence of eye infections by a juvenile state (metacestode) of a well-known tapeworm, Gilquinia squali to be diagnosed in young chinook salmon, Oncorhynchus tshawytscha held in seawater netpen sites in British Columbia.

Devereux, Richard, Mary Delaney, Friedrich Widdel and David A. Stahl. 1989. Natural Relationships Among Sulfate-Reducing Eubacteria. EPA/600/J-89/424. J. Bacteriol. 171(12):6689-6695. (ERL,GB X654). (Avail. from NTIS, Springfield, VA: PB90-245739)

Phylogenetic relationships among 20 nonsporeforming and two endospore-forming species of sulfate-reducing eubacteria were inferred from comparative 16S rRNA sequencing. All genera of mesophilic sulfate-reducing eubacteria except the new genus Desulfomicrobium and the gliding Desulfonema species were included. The sporeforming species Defulfotomaculum ruminis and Desulfotomaculum orientis were found to be gram-positive organisms sharing 83% 16S rRNA sequence similarity, indicating that this genus is diverse. The gram-negative nonsporforming species could be divided into seven natural groups; group 1, Desulfovibrio desulfuricans and other species of this genus that do not degrade fatty acids (this group also included 'Desulfomonas' pigra); group 2, the fatty acid-degrading 'Desulfovibrio' sapovorans; group 3, Desulfobulbus species; group 4, Desulfobacter species; group 5, Desulfobacterium species and 'Desulfococcus' niacini; group 6, Desulfococcus multivorans and Desulfosarcina variabilis; and group 7, the fatty acid-oxidizing 'Desulfovibrio' baarsii. (The quotation marks are used to indicate the need for taxonomic revision.) Groups 1 to 3 are incomplete oxidizers that form acetate as an end product; groups 4 to 7 are complete oxidizers. The data were consistent with and refined relationships previously inferred by oligonucleotide catalogs of 16S rRNA. Although the determined relationships are generally consistent with the existing classification based on physiology and other characteristics, the need for some taxonomic revision is indicated.

Coyne, V.E., C.J. Pillidge, D.D. Sledjeski, H. Hori, B.A. Ortiz-Conde, D.G. Muir, R.M. Weiner and R.R. Colwell. 1989. Reclassification of Alteromonas colwelliana to the Genus Shewanella by DNA-DNA Hybridization, Serology and 5S Ribosomal RNA Sequence Data. Syst. Appl. Microbiol. 12:275-279. (ERL,GB X660).

The taxonomic relationship between Alteromonas colwelliana and representative Alteromonas and Shewanella strains was determined employing molecular systematics. DNA:DNA hybridization did not reveal homology between A. colwelliana and the other bacterial strains. However, results of comparative serology and 5S rRNA sequence similarity analyses indicated that A. colwelliana is related to Shewanella putrefaciens, Shewanella hanedai Shewanella benthica and thus, is included in the genus Shewanella.

Katsuwon, J. and A.J. Anderson. 1989. Response of Plant-Colonizing Pseudomonads to Hydrogen Peroxide. EPA/600/J-89/427. Appl. Environ. Microbiol. 55(11):2985-2989. (ERL,GB X699). (Avail. from NTIS, Springfield, VA: PB90-264847)

Colonization of plant root surfaces by Pseudomonas putida may require mechanisms that protect this bacterium against superoxide anion and hydrogen peroxide produced by the root. Catalase and superoxide dismutase may be important in this bacterial defense system. Stationary-phase cells of P. putida were not killed by hydrogen peroxide (H2O2) at concentrations up to 10mM, and extracts from these cells possessed three isozymic bands (A, B, and C) of catalase activity in native polyacrylamide gel electrophoresis. Logarithmic-phase cells exposed directly to hydrogen peroxide concentrations above 1mM were killed. Extracts of logarithmic-phase cells displayed only band A catalase activity. Protection against 5 mM H2O2 was apparent after previous exposure of the logarithmic-phase cells to nonlethal concentration (30 to 300 uM) of H2O2. Extracts of these protected cells possessed enhanced catalase activity of band A and small amounts of bands B and C. A single form of superoxide dismutase and isoforms of catalase were apparent in extracts from a foliar intercellular pathogen, Pseudomonas syringae pv. phaseolicola. The mobilities of these P. syringae enzymes were distinct from those of enzymes in P. putida extracts.

Lee, Byung Mu and Geoffrey I. Scott. 1989. Acute Toxicity of Temephos, Fenoxycarb, Diflubenzuron, and Methoprene and Bacillus thuringiensis var. Israelensis to the Mummichog (Fundulus heteroclitus). Bull. Environ. Contam. Toxicol. 43:827-832. (ERL,GB X704).

The southeast United States has the single largest concentration of mosquito control efforts in the United States primarily due to the large concentration of fresh, brackish, and salt water marshes in these areas (NAS 1976). Salt marsh mosquitoes (Aedes sollicitans and Aedes taeniorhynchus) are the major mosquito pests along the entire Atlantic and Gulf Coast of the United States. Salt marsh mosquito control involves the application of chemical insecticides into breeding grounds, near estuarine tidal creeks, in an attempt to kill and control larval mosquitoes. The headwaters of many estuarine tidal creeks serve as nursery grounds for many fish species. The mummichog, Fundulus heteroclitus, is one of the dominant fish species present in these creeks. The application of chemical larvicides for mosquito control into salt marsh breeding grounds may pose a potential toxicity hazard to nontarget aquatic organisms. The larvicides generally recommended for use in South Carolina include: Abate (temephos), Dursban (chloropyrifos), Malathion, Altosid (methoprene), Pyrethrins, and Vectobac (Bacillus thuringiensis var. israelensis, Bti). Altosid and abate are among the most widely used larvicides in South Carolina and may be potentially toxic to nontarget species.

Hinckley, D.A. and T.F. Bidleman. 1989. Analysis of Pesticides in Seawater after Enrichment onto C8 Bonded-Phase Cartridges. Environ. Sci. Technol. 23:995-1000. (ERL,GB X709).

We have examined the collection of organochlorine organophosphate, and pyrethroid insecticides from distilled water, seawater of 33-35 parts-per-thousand salinity, and river water containing a high concentration of dissolved organic carbon (DOC). Water volumes of 1-4 L were pulled at a flow rate of 15-40 mL.min through a 47-mm glass-fiber filter to trap particulate matter, followed by tandem 500-mg C8-bonded silica cartridges. The cartridges were extracted with diethyl ether-hexane, the extract was blown down under nitrogen, and the analytes were quantified by capillary gas chromatography with electron capture detection. The cartridges were easy to clean and average blanks of less than or equal to 0.2 ng/L were obtained. Breakthrough to back cartridges was 1-10% of the front cartridge quantity for most pesticides in the three types of water. Recoveries for a variety of pesticides at the 7-110 mg/L level ranged from 85% to 120%. The collection method was used to determine pesticide concentrations in a tidal creek, where shrimp were killed during a runoff event, and hexachlorocyclohexane concentrations in ocean surface water and snow from the Arctic.

TeBeest, D.O., C.W. Shilling, L. Hopkins Riley and G.J. Weidemann. 1989. Number of Nuclei in Spores of Three Species of Colletotrichum. Mycologia. 8(1):147-149. (ERL,GB X729).

Studies described were undertaken to determine the number of nuclei per spore of five Colletotrichum isolates when grown in liquid and agar culture. The results of the experiments showed that in all five isolates, 97.7% or more of the spores obtained from agar cultures were uninucleate, 04.-2.2% were binucleate, and fewer than 1% of the spores contained 3 nuclei (Table 1). None of the spores obtained from agar cultures contained more than 3 nuclei.

Summers, J. Kevin. 1989. Simulating the Indirect Effects of Power Plant Entrainment Losses on an Estuarine Ecosystem. Ecol. Modell. 49:31-47. (ERL,GB X755).

Entrainment caused by the operation of the Chalk Point Steam Electric Station has been shown to be a major source of mortality to the early life stages of forage fish populations in the Patuxent River, MD, USA. While direct losses to these populations are important as a source of reduction for population abundance, these losses also represent decreases in estuarine forage supplies and potential reductions in the abundances of estuarine predators. A simple estuarine trophic dynamics model was constructed to determine the magnitude of the potential losses to major estuarine consumers in the Patuxent River ecosytem due to the power plant-related losses of forage fish. Simulations were completed using two sets of feeding assumptions: feeding proportional to forage abundance, and feeding based on dietary preferences. The model demonstrates that striped bass, bluefish, and weakfish could experience significant losses (greater than 25%) to overall population production levels if they prefer to prey upon bay anchovy and silversides and entrainment losses to these forage populations is greater than or equal to 70% of juvenile recruitment. The model also shows that indirect predator losses would be expected to be low (less than 5) if the majority of their diets consisted of forage other than bay anchovy and silversides.

Gardner, George R., Richard J. Pruell and Leroy C. Folmar. 1989. Comparison of Both Neoplastic and Non-Neoplastic Disorders in Winter Flounder (Pseudopleuronectes americanus) from Eight Areas in New England. Mar. Environ. Res. 28(1-4):393-397. (ERL,GB X757).

Distribution patterns of liver disease observed in winter flounder indigenous to the northeastern USA indicated that hepatocytic neoplasms were absent in populations from uncontaminated offshore areas and endemic in populations from moderately to highly contaminated inshore areas. Liver neoplasms in winter flounder collected from eight different locations ranged from 0% in animals collected offshore from Cape Cod to 32% in the nearshore area of New Bedford, MA. Similarly, an array of other hepatic lesions ranged from 9% in Martha's Vineyard to 79% in Boston Harbor. Proliferate lesions in endocrine, exocrine, respiratory, sensory, excretory and digestive organs and alteration of plasma protein were also characteristic of winter flounder populations residing in the nearshore environment. The concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), other organic compounds and trace metals associated with marine sediment were elevated in urban embayments as compared with offshore locations. Degree of sediment chemical contamination and disease suggest a causal relationship.