Journal Article Citations and Abstracts

Topsmelt, Atherinops affinis, were spawned repeatedly in the laboratory during May-July, 1989. A periodic "temperature spike" from the holding temperature of approximately 18°C, up to approximately 20.5°C, introduced at 7 to 9 day intervals, resulted in maximum production of viable embryos on the fourth morning after the spike. Examination of embryonic stages and comparison to known developmental rates for A. affinis embryos revealed that spawning was generally nocturnal, occurring between 1900 and 0500 hrs. Survival of embryonic and larval A. affinis cultured at 21 + or - 1°C and 20 + or - 2 o/oo salinity was excellent (>80%). A larval growth curve was developed for the first 24 days post-hatch.

Turner, B.J., W.P. Davis and D.S. Taylor. 1992. Abundant Males in Populations of a Selfing Hermaphrodite Fish, Rivulus marmoratus, from Some Belize Cays. EPA/600/J-92/221. J. Fish Biol. 40(2):307-310. (ERL,GB 705). (Avail. from NTIS, Springfield, VA: PB92-195783)

The killifish Rivulus marmoratus is the only known selfing hermaphroditic vertebrate, and males of the species are usually exceedingly rare or non-existent in nature. Collections on several Belize cays in 1988 and 1989 yielded 13.5-24% males. Factors responsible for this unusually high proportion of males are currently not understood. Likewise, the biological significance of males in populations which otherwise consist of selfing hermaphrodites (with internal fertilization) is problematic and awaits further study.

Kroer, Niels and Richard B. Coffin. 1992. Microbial Trophic Interactions in Aquatic Microcosms Developed for Testing Genetically Engineered Microorganisms: a Field Comparison. EPA/600/J-93/385. Microb. Ecol. 23(2):143-157. (ERL,GB 708). (Avail. from NTIS, Springfield, VA: PB93-236248)

Microcosms may potentially be used as tools for evaluating the fate and effects of genetically engineered microorganisms released into the environment. Extrapolation of data to the field, however, requires that the correspondence between microcosm and field is known. Microbial trophic interactions within the microbial loop were compared quantitatively and qualitatively between field and microcosms containing estuarine water with and without intact sediment cores. The comparison showed that whereas proportions between trophic levels in microcosms were qualitatively similar to those in the field, rates of microbial processes were from 25 to 40% lower in microcosms. Nitrogen cycling was disrupted in microcosms incubated in the dark to eliminate primary production. Examination of the microbial parameters further suggests that sediment in microcosms may be an important factor regulating the bacterial trophic level. These results demonstrate that analysis of microbial trophic interactions is a sensitive method for the field comparison of aquatic microcosms and a potentially useful tool in the risk assessment of genetically engineered microorganisms.

Walsh, G., D. Weber, L. Esry, M. Nguyen, J. Noles and B. Albrecht. 1992. Synthetic Substrata for Propagation and Testing of Soil and Sediment Organisms. EPA/600/J-92/210. Pedobiologia. 36:1-10. (ERL,GB 713). (Avail. from NTIS, Springfield, VA: PB92-195676)

A method for formulation of synthetic substrata (soils and sediments) is given. Submersed, wetland, and terrestrial plants, earthworms, crustaceans, and vertebrates were maintained on synthetic substrata composed of various amounts of commercially available sand, clay, silt, and particulate and dissolved organic matter. Organic contents of the synthetic substrata were 3, 5, 7.5, and 10% by weight. All test species survived and grew well in the substrata. It is suggested that synthetic substrata have some advantages over natural substrata in tests with plants and animals. Among the advantages are: synthetic sediments may be formulated for specific studies, differences in texture and chemical characteristics between batches are minimized, and the substrata are not contaminated by anthropogenic substances as are many natural soils and sediments.

Hemmer, Michael J., Douglas P. Middaugh and Valerie Comparetta. 1992. Comparative Acute Sensitivity of Larval Topsmelt, Atherinops affinis, and Inland Silversides, Menidia beryllina to 11 Chemicals. EPA/600/J-92/209. Environ. Toxicol. Chem. 11(3):401-408. (ERL,GB 718). (Avail. from NTIS, Springfield, VA: PB92-195668)

Larval topsmelt (Atherinops affinis) and inland silversides (Menidia beryllina) were exposed in 96-hr static acute toxicity tests to eleven chemicals to determine the relative sensitivity to the two atherinid species. High to low LC50 ratios for endosulfan, methoxychlor, carbophenothion, chlorpyrifos, terbufos, fenvalerate, permethrin, 4-nitrophenol, and sodium lauryl sulfate were within a factor of < 2 for the two species. A. affinis was more sensitive to both azinphos-methyl and 2,4-dinitrophenol by factors of 6.7 and 4.4, respectively. Comparison of the relative sensitivity of A. affinis with three freshwater fish species (Lepomis macrochirus, Oncorhynchus mykiss, Pimephales promelas) and one estuarine fish species (Cyprinodon variegatus) are also presented. Sensitivities were similar between A. affinis and the two most sensitive freshwater species, L. macrochirus and O. mykiss. A. affinis is easily transported, cultured and maintained in the laboratory, and readily adaptable for use in toxicological studies.

Goodman, Larry R., Michael J. Hemmer, Douglas P. Middaugh and James C. Moore. 1992. Effects of Fenvalerate on the Early Life Stages of Topsmelt (Atherinops affinis). EPA/600/J-92/217. Environ. Toxicol. Chem. 11(3):409-414. (ERL,GB 719). (Avail. from NTIS, Springfield, VA: PB92-195742)

Flow-through acute and early life-stage (ELS) toxicity tests were conducted with topsmelt (Atherinops affinis), a Pacific Coast saltwater fish, and fenvalerate, a synthetic pyrethroid insecticide. The 96-h LC50 for juvenile fish was 0.66 µg/L. In the 30-d ELS test with laboratory-spawned embryos, average measured fenvalerate concentrations were nondetectable (< 0.075 µg/L) in two control treatments, 0.14, 0.34, 0.82, 1.5, and 3.2 µg/L. Survival of embryos to hatching ranged from 94% to 100%, with no statistically significant difference among treatments. No fry survived exposure to fenvalerate concentrations > 0.82 µg/L; overall survival in lower concentrations and control treatments ranged from 86% to 97%. There were no consistent concentration-dependent differences in weight between fish in the carrier-control treatment and fish exposed to fenvalerate. Mean wet weights of surviving fish ranged from 16.9 mg in 0.34 µg/L to 20.3 mg in 0.14 µg/L. The average bioconcentration factor for fish exposed to 0.14 and 0.34 µg fenvalerate/L was 315.

Middaugh, Douglas P. and Michael J. Hemmer. 1992. Reproductive Ecology of the Inland Silverside, Menidia beryllina (Pisces: Atherinidae) from Blackwater Bay, Florida. EPA/600/J-92/220. Copeia. 1992(1):53-61. (ERL,GB 724). (Avail. from NTIS, Springfield, VA: PB92-195775)

The reproductive ecology of the inland silverside, Menidia beryllina, was studied during Feb. 1988--March 1989 at Robinson Point, Blackwater Bay, Florida. Environmental variables including pH, rainfall, salinity, water temperature, and dissolved oxygen were measured weekly or biweekly. Fish were sampled weekly with a seine designed to catch adult, juvenile, and young-of-the year (YOY) individuals. Most reproductive activity occurred during Feb.--April 1988. The maximum mean weekly female gonadosomatic index (GSI) of 12.5 occurred in April. Fecundity ranged from 63 to 419 hydrated eggs/female. The maximum mean weekly male GSI of 6.1 occurred in early March. Catches of YOY individuals 7.6-37.5 mm SL were greatest in May. Some of these YOY individuals matured in July-Sept. and spawned. This reproductive activity resulted in recruitment of a second group of YOY fish into the population during Aug.-Oct. Growth rates of YOY in May-July, calculated by regression methods from weekly frequency distributions of standard length, was 0.34 mm/day for females and 0.31 mm/day for males. The reproductive pattern of M. beryllina from Blackwater Bay, Florida indicates that qualitatively it is an r-strategist with rapid growth of YOY, sexual maturation at an early age, relatively high fecundity, and multiple spawning within the first reproductive period for YOY fish in July-Sept. and again as 1- to 1-plus-year-old individuals.

Fisher, William S. 1992. Occurrence of Agglutinins in the Pallial Cavity Mucus of Oysters. EPA/600/J-93/070. J. Exp. Mar. Biol. Ecol. 162(1):1-13. (ERL,GB 737). (Avail. from NTIS, Springfield, VA: PB93-169043)

Mucus and fluid from the pallial (mantle) cavity of eastern oysters Crassostrea virginica Gmelin from Chesapeake Bay and Galveston Bay were found to agglutinate a variety of vertebrate erythrocytes (RBC) and bacteria. Agglutinating activity of pallial cavity fluid was probably due to dissociation of agglutinins from mucus on the external surface of organs. Agglutination titers of pallial cavity fluid from individual Galveston Bay oysters for six different RBC were positively correlated with high significance, indicating a strong interdependence regardless of RBC specificity. The relative agglutinating activity for different RBC by tissue explants and homogenized tissues (mantle, gill, digestive gland and adductor muscle) was similar to the relative activity of the pallial cavity fluid; mantle and gill tissue had the greatest agglutinating capacity over all RBC, whereas the adductor muscle had the least. Positive correlation was detected between agglutination titers of pallial cavity fluid (for different RBC) and titers of hemolymph agglutinins from the same oysters. It is hypothesized that agglutinins in the pallial cavity and hemolymph have a common source. The results of the explant and homogenate assays can be interpreted to suggest that the source is mantle and gill tissues.

Fournie, J.W., W.E. Hawkins and W.W. Walker. 1992. Adenocarcinoma of the Retinal Pigment Epithelium in the Guppy Poecilia reticulata Peters. EPA/600/J-92/387. J. Comp. Pathol. 106(4):429-434. (ERL,GB 738). (Avail. from NTIS, Springfield, VA: PB93-121192)

A single case of adenocarcinoma of the retinal pigment epithelium occurred in a guppy, Poecilia reticulata Peters. This is the first such tumor reported from fishes. The left eye of the affected fish was severely exophthalmic because of a large intraocular tumor mass. The tumor, which displaced normal retina anteriorly, consisted mainly of melanin-containing epithelial cells. Neoplastic cells were bilayered and arranged in a tubular pattern. The tumor appeared to be confined to the orbit. Although the specimen was from a group exposed to a mixture of halogenated organic compounds, we do not consider the lesion to have been chemically induced because of its rare occurrence.

Weber, David E., David A. Flemer and Charles M. Bundrick. 1992. Comparison of the Effects of Drilling Fluid on Macrobenthic Invertebrates Associated with the Seagrass, Thalassia testudinum, in the Laboratory and Field. EPA/600/J-92/408. Estuarine Coastal Shelf Sci. 35(3):315-330. (ERL,GB 753). (Avail. from NTIS, Springfield, VA: PB93-131837)

The structure of a macrobenthic invertebrate community associated with the seagrass, Thalassia testudinum, was evaluated under laboratory and field conditions. The research focused on: (1) the effects of pollution stress from a representative drilling fluid used in off-shore oil and gas operations, and (2) a comparison of responses of the seagrass-invertebrate community in the laboratory and field. A series of 15.3 cm diameter cores of the seagrass-invertebrate community was collected from field sites for establishment and sampling of microcosms and in the sampling of field plots over time. Weekly exposures to drilling fluid were conducted in the laboratory microcosms at a mean total suspended matter concentration of 110.7 mg1-1 (± 17.7 SD), and in field plots by usage of acrylic exposure chambers at a mean concentration of 132.8 mg1-1 (± 33.3 SD). Standing crop of T. testudinum was not affected by drilling fluid in the laboratory or field when measured after 6 and 12 week exposure periods. The numbers of macrobenthic invertebrates were suppressed by drilling fluid at both exposure periods in the laboratory, but inhibitory effects were absent in the field. Invertebrate densities in the field were similar among control and treated plots, and were much lower than densities occurring in the laboratory control. In most instances, species richness values were similar in the field and laboratory at the end of each 6 and 12 week period.

Yousten, Allan A., Fred J. Genthner and Ernest F. Benfield. 1992. Fate of Bacillus sphaericus and Bacillus thuringiensis Serovar Israeliensis in the Aquatic Environment. EPA/600/J-92/382. J. Am. Mosq. Control Assoc. 8(2):143-148. (ERL,GB 754). (Avail. from NTIS, Springfield, VA: PB93-121143)

Bacillus sphaericus spores were suspended in bottles of filtered (0.45 µm) freshwater and seawater under various conditions of temperature, pH and salinity. Heat resistant culturable counts (spores) slowly decreased with time. Spores suspended in dialysis bags submerged in a freshwater pond or in flowing seawater underwent a more rapid drop in heat resistant spore counts than did spores held in bottles. Thus, laboratory studies may overestimate spore longevity in the environment. Spore settling rate was related to the nature of particulate material in the water column. Paraspores (or perhaps spores and toxin) of B. thuringiensis serovar israelensis (B.t.i.) had a greater tendency to adhere to and settle with suspended sediment and fine particulates than did paraspores of B. sphaericus. These observations may at least partially explain the greater persistence of B. sphaericus larvicidal activity in field tests than that of B.t.i.

Mueller, James G., Sol. M. Resnick, Michael E. Shelton and Parmely H. Pritchard. 1992. Effect of Inoculation on the Biodegradation of Weathered Prudhoe Bay Crude Oil. EPA/600/J-92/384. J. Ind. Microbiol. 10:95-102. (ERL,GB 765). (Avail. from NTIS, Springfield, VA: PB93-121168)

Enrichment cultures from oil-contaminated beach material from Prince William Sound, Alaska generated both a mixed bacterial community of indigenous, oil-degrading marine microorganisms and a pure culture oil-degrader, strain E12V. The mixed and axenic cultures were used in comparative shake flask studies of inoculation on biodegradation of Prudhoe Bay crude oil. Within 12 h following inoculation of homogenized, oiled beach material with the mixed culture, total CO2 production was increased 2-fold relative to a noninoculated control. Moreover, measurements of phenanthrene degradation (as determined by the release of 14CO2 from [9-14C] phenanthrene) showed a 2- or 3-fold greater degradation when inoculated with either strain E12V or with the mixed culture, respectively. However, as medium, was replaced by a simulated tidal cycle, the observed stimulation of C02 production decreased, and the addition of strain E12V had no greater effect on total C02 production than the addition of inorganic nutrients alone. Chemical analysis of oil recovered after 7 days incubation also suggested that, while these cultures are capable of efficient biodegradation of Prudhoe Bay crude in liquid culture, inoculation of beach material with high numbers of these microorganisms had little effect on the rate and extent of biodegradation of weathered crude oil. Overall, the sustained stimulatory effect was no greater than that observed with the addition of inorganic nutrients alone.

Jeffrey, Wade H., Stephen M. Cuskey, Peter J. Chapman, Sol Resnick and Ronald H. Olsen. 1992. Characterization of Pseudomonas putida Mutants Unable to Catabolize Benzoate: Cloning and Characterization of Pseudomonas Genes Involved in Benzoate Catabolism and Isolation of a Chromosomal DNA Fragment Able to Substitute for xylS in Activation of the TOL Lower-Pathway Promoter. EPA/600/J-92/381. J. Bacteriol. 174(15):4986-4996. (ERL,GB 766). (Avail. from NTIS, Springfield, VA: PB93-121135)

Mutants of Pseudomonas putida mt-2 that are unable to convert benzoate to catechol were isolated and grouped into two classes: those that did not initiate attack on benzoate and those that accumulated 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (benzoate diol). The latter mutants, represented by strain PP0201, were shown to lack benzoate diol dehydrogenase (benD) activity. Mutants from the former class were presumed either to carry lesions in one or more subunit structural genes of benzoate dioxygenase (benABC) or the regulatory gene (benR) or to contain multiple mutations. Previous work in this laboratory suggested that benR can substitute for the TOL plasmid-encoded xylS regulatory gene, which promotes gene expression from the OP2 region of the lower or meta pathway operon. Accordingly, structural and regulatory gene mutations were distinguished by the ability of benzoate-grown mutant strains to induce expression frm OP2 without xylS by using the TOL plasmid xylE gene (encoding catechol 2,3-dioxygenase) as a reporter. A cloned 12-kb BamHI chromosomal DNA fragment from the P. aeruginosa PAO1 chromosome complemented all of the mutations, as shown by restoration of growth on benzoate minimal medium. Subcloning and deletion analyses allowed identification of DNA fragments carrying benD, benABC, and the region possessing xylS substitution activity, benR. Expression of these genes was examined in a strain devoid of benzoate-utilizing ability, Pseudomonas fluorescens PFO15. The disappearance of benzoate and the production of catechol were determined by chromatographic analysis of supernatants from cultures grown with casamino acids. When P. fluorescens PFO15 was transformed with plasmids containing only benABCD, no loss of benzoate was observed. When either benR or xylS was cloned into plasmids compatible with those plasmids containing only the benABCD regions, benzoate was removed from the medium and catechol was produced. Regulation of expression of the chromosomal structural genes by benR and xylS was quantified by benzoate diol dehydrogenase enzyme assays. The results obtained when xylS was substituted for benR strongly suggest an isofunctional regulatory mechanism between the TOL plasmid lower-pathway genes (via the OP2 promoter) and chromosomal benABC. Southern hybridizations demonstrated that DNA encoding the benzoate dioxygenase structural genes showed homology to DNA encoding toluate dioxygenase from the TOL plasmid pWW0, but benR did not show homology to xylS. Evolutionary relationships between the regulatory systems of chromosomal and plasmid-encoded genes for the catabolism of benzoate and related compounds are suggested.

Yousten, Allan A., E.F. Benfield and Fred J. Genthner. 1992. Fate of Bacillus sphaericus 2362 Spores in Nontarget Invertebrates. EPA/600/J-93/065. Microb. Releases. 1:161-164. (ERL,GB 767). (Avail. from NTIS, Springfield, VA: PB93-168995)

Predatory stonefly larvae (Paragnetina media) acquired Bacillus sphaericus spores by eating spore-laden midge larvae. Leaf shredding stonefly larvae (Pteronarcys proteus) and crane fly larvae (Tipula abdominalis) acquired spores by feeding on contaminated leaf discs. Upon switching to uncontaminated diets, both stonefly larvae eliminated the spores. The cranefly larvae, however, retained 18% of the spores in its posterior gut for up to 5 weeks although spores were eliminated from its highly alkaline foregut. Spores recovered in cranefly fecal material had lost toxicity to mosquito larvae.

Fisher, William S., Julie D. Gauthier and James T. Winstead. 1992. Infection Intensity of Perkinsus marinus Disease in Crassostrea virginica (Gmelin, 1791) from the Gulf of Mexico Maintained Under Different Laboratory Conditions. EPA/600/J-93/057. J. Shellfish Res. 11(2):363-369. (ERL,GB 769). (Avail. from NTIS, Springfield, VA: PB93-168912)

A protozoan parasite, Perkinsus marinus, has been responsible for infection and mortality of eastern oysters, Crassostrea virginica, since before 1950. Studies on the course of infection intensity in individual animals have been restricted by the need to sacrifice animals for diagnosis, so quantitative association of disease intensity with environmental conditions and individual survival has not been accomplished. A recently developed hemolymph assay provided the means to quantitate infection intensity from live oysters. Application of this technique demonstrated progression of P. marinus intensity in Gulf of Mexico oysters maintained in laboratory aquaria in fed and unfed conditions at different test temperatures (18° - 27°C) and salinities (6 - 36 ppt). In one experiment, the infection intensities over eight weekly samplings increased 100.09 mL-1 hemolymph week-1 for low temperature/low salinity conditions and 100.36 hypnospores mL-1 hemolymph week-1 for high temperature/high salinity conditions. Temperature was more influential than salinity in P. marinus intensity and oyster mortalities. Oysters containing 103 - 104 hypnospores mL-1 hemolymph survived in low temperatures, but not in high. Feeding did not affect the intensity of P. marinus, but may have been a factor in survival of infected oysters.

Genthner, Fred J., Robert P. Campbell and P.H. Pritchard. 1992. Use of a Novel Plasmid to Monitor the Fate of a Genetically Engineered Pseudomonas putida Strain. EPA/600/J-93/066. Mol. Ecol. 1:137-143. (ERL,GB 770). (Avail. from NTIS, Springfield, VA: PB93-169001)

Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered microorganism (GEM) and its recombinant DNA in environmental samples. This broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA. The clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC-4, to grow on 3-chlorobenzoate. This catabolic phenotype was exploited using enrichment procedures to detect RC-4 (pSI30) cells, freeliving in the water column or when irreversibly bound to surfaces. The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. In filter-sterilized water, numbers of viable P. putida cells declined by less than one log in 64 days. In natural freshwater, numbers of P. putida cells fell rapidly over the first 5 days and then slowly declined to cell densities of approximately 100 per ml. Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected. Persistence of RC-4 (pSI30) and its ability to multiply upon addition of 3-chlorobenzoate were demonstrated 78 days after its addition to natural freshwater. The addition of 3-chlorobenzoate to natural freshwater containing as few as 3 RC-4 (pSI30) cells per ml resulted in an increase in their numbers. In flow-through microcosms RC-4 (pSI30), undetectable as freeliving cells, was found by enrichment as irreversibly bound sessile forms. These experiments revealed the stability of pSI30 and its utility in a 'combination' detection system for tracking the survival of a GEM and its DNA in environmental samples.

Folmar, L.C., T. Moody, S. Bonomelli and J. Gibson. 1992. Annual Cycle of Blood Chemistry Parameters in Striped Mullet (Mugil cephalus L.) and Pinfish (Lagodon rhomboides L.) from the Gulf of Mexico. EPA/600/J-93/061. J. Fish Biol. 41(6):999-1011. (ERL,GB 771). (Avail. from NTIS, Springfield, VA: PB93-168953)

Annual cycle measurements were made on serum sodium (Na), potassium (K), chloride (Cl), iron (Fe), magnesium (Mg), calcium (Ca), carbon dioxide (CO2), total protein (TP), albumin (Albg), cholesterol (Chol), triglycerides (Trig), inorganic phosphorous (Pi), uric acid (Uric), blood urea nitrogen (BUN), creatinine (Crea), glucose (Glu), lactate dehydrogenase (LD-L), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine kinase (CK) in the striped mullet (Mugil cephalus L.) and the pinfish (Lagodon rhomboides L.). For each parameter, mean, standard deviation and coefficient of variation (C.V.) are reported. The lowest C.V.s were associated with the electrolytes and the greatest C.V.s with serum enzymes. The lowest variability for most parameters was observed in the vitellogenic and prespawning period.

Genthner, Fred J. and Douglas P. Middaugh. 1992. Effects of Beauveria bassiana on Embryos of the Inland Silverside Fish (Menidia beryllina). EPA/600/J-92/406. Appl. Environ. Microbiol. 58(9):2840-2845. (ERL,GB 774). (Avail. from NTIS, Springfield, VA: PB93-131811)

A chemical toxicity/teratogenicity test was adapted to assess potential adverse effect of a microbial pest control agent on a nontarget fish. Developing embryos of inland silverside, Menidia beryllina, were exposed to conidiospores of the insect-pathogenic fungus, Beauveria bassiana. Adherence of spores to the chorion followed by germination and penetration by the germ tube caused the embryos to rupture, sometimes resulting in death. Conidiospores treated with a biological detergent showed significantly less binding (p < or = to 0.01) to embryos than did untreated spores. Both detergent-treated and heat-killed spores failed to cause significant pathogenic responses.

Kent, M.L., J. Ellis, J.W. Fournie, S.C. Dawe, J.W. Bagshaw and D.J. Whitaker. 1992. Systemic Hexamitid (Protozoa: Diplomonadida) Infection in Seawater Pen-Reared Chinook Salmon Oncorhynchus tshawytscha. EPA/600/J-93/069. Dis. Aquat. Org. 14(2):81-89. (ERL,GB 785). (Avail. from NTIS, Springfield, VA: PB93-169035)

A systemic infection with a hexamitid flagellate resembling Hexamita salmonis caused high mortality in chinook salmon, Oncorhynchus tshawytscha, reared at a seawater netpen farm in British Columbia, Canada. Affected fish were anemic and had swollen abdomens containing serosanguinous ascites and large blood clots. The most remarkable histological changes were found in the liver and kidney. Livers of affected fish showed edema, congestion, and inflammation. The renal interstitium was moderately hyperplastic, due to proliferation of hemoblasts. The systemic infection was transmitted in the laboratory to chinook by intraperitoneal injection, by gavage, and by waterborne exposure (in both fresh and sea water) to heavily infected ascites and tissue. The infection was also transmitted in fresh- and sea-water by cohabitation with infected chinook. Based on the ease of transmission of the parasite in both fresh- and sea-water, and the high mortality associated with the infection, this disease poses a potentially serious threat to aquaculture of chinook salmon.

Devereux, Richard, Matthew D. Kane, Janet Winfrey and David A. Stahl. 1992. Genus- and Group-Specific Hybridization Probes for Determinative and Environmental Studies of Sulfate-Reducing Bacteria. EPA/600/J-93/064. Syst. Appl. Microbiol. 15(4):601-609. (ERL,GB 787). (Avail. from NTIS, Springfield, VA: PB93-168987)

A set of six oligonucleotides, complementary to conserved tracts of 16S rRNA from phylogenetically-defined groups of sulfate-reducing bacteria, was characterized for use as hybridization probes in determinative and environmental microbiology. Four probes were genus specific and identified Desulfobacterium spp., Desulfobacter spp., Desulfobulbus spp., or Desulfovibrio spp. The other two probes encompassed more diverse assemblages. One probe was specific for the phylogenetic lineage composed of Desulfococcus multivorans, Desulfosarcina variabilis, and Desulfobotulus sapovorans. The remaining probe was specific for Desulfobacterium spp., Desulfobacter spp., D. multivorans, D. variabilis, and D. sapovorans. Temperature of dissociation was determined for each probe and the designed specificities of each were evaluated by hybridizations against closely related nontargeted species. In addition, each probe was screened by using a "phylogrid" membrane which consisted of nucleic acids from sixtyfour non-targeted organisms representing a diverse collection of eukarya, archaea, and bacteria. The value of these probes to studies in environmental microbiology was evaluated by hybridizations to 16S rRNAs of sulfate-reducing bacteria present in marine sediments.

Shields, Malcolm S. and Michael J. Reagin. 1992. Selection of a Pseudomonas cepacia Strain Constitutive for the Degradation of Trichloroethylene. EPA/600/J-93/068. Appl. Environ. Microbiol. 58(12):3977-3983. (ERL,GB 790). (Avail. from NTIS, Springfield, VA: PB93-169027)

Tn5 insertion mutants of Pseudomonas cepacia G4 that were unable to degrade trichloroethylene (TCE), toluene, or phenol or to transform m-trifluoromethyl phenol (TFMP) to 7,7,7-trifluoro-2-hydroxy-6-oxo-2,4-heptadienoic acid (TFHA) were produced. Spontaneous reversion to growth on phenol or toluene as the sole source of carbon was observed in one mutant strain, G4 5223, at a frequency of difference 1x10-4 per generation. One such revertant, G4 5223-PR1, metabolized TFMP to TFHA and degraded TCE. Unlike wild-type G4, G4 5223-PR1 constitutively metabolized both TFMP and TCE without aromatic induction. G4 5223-PR1 also degraded cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, and 1,1-dichloroethylene and oxidized naphthalene to alpha naphthol constitutively. G4 5223-PR1 exhibited a slight retardation in growth rate at TCE concentrations of greater than or equal to 530 uM, whereas G4 (which was unable to metabolize TCE under the same noninducing growth conditions) remained unaffected. The constitutive degradative phenotype of G4 5223-PR1 was completely stable through 100 generations of nonselective growth.

Pritchard, Parmely H. 1992. Use of Inoculation in Bioremediation. EPA/600/J-92/383. Curr. Opin. Biotechnol. 3:232-243. (ERL,GB 791). (Avail. from NTIS, Springfield, VA: PB93-121150)

There are many possible situations where inoculation of chemically-contaminated sites with microorganisms possessing unique and specialized metabolic capabilities could significantly enhance bioremediation. However, there have been very few successful uses of inoculation to date. By more careful attention to selection and application of the inoculants, however, it is quite reasonable to expect that inoculation could become a major and effective component of biological cleanup methods.

Eaton, Richard W. and Peter J. Chapman. 1992. Bacterial Metabolism of Naphthalene: Construction and Use of Recombinant Bacteria to Study the Ring Cleavage of 1,2-Dihydroxynaphthalene and Subsequent Reactions. EPA/600/J-93/059. J. Bacteriol. 174(23):7542-7554. (ERL,GB 795). (Avail. from NTIS, Springfield, VA: PB93-168938)

The reactions involved in the bacterial metabolism of naphthalene to salicylate have been reinvestigated using recombinant bacteria carrying genes cloned from the NAH7 plasmid. When intact cells of Pseudomonas aeruginosa PAO1 carrying DNA fragments encoding the first three enzymes of the pathway were incubated with naphthalene, they formed products of the dioxygenase-catalyzed ring cleavage of 1,2-dihydroxynaphthalene. These products were separated by chromatography on Sephadex G-25 and identified by 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as 2-hydroxychromene-2-carboxylate (HCCA) and trans-o-hydroxybenzylidenepyruvate (tHBPA). HCCA was detected as the first reaction product in these incubation mixtures by its characteristic UV spectrum, which slowly changed to a spectrum indicative of an equilibrium mixture of HCCA and tHBPA. Isomerization of either purified product occurred slowly and spontaneously to give an equilibrium mixture of essentially the same composition. tHBPA is also formed from HCCA by the action of an isomerase enzyme encoded by the NAH7 plasmid. The gene encoding this enzyme, nahD, was cloned on a 1.95 kb KpnI-BglII fragment. Extracts of Escherichia coli JM109 carrying this fragment catalyzed the rapid equilibrium of HCCA and tHBPA. Metabolism of tHBPA to salicylaldehyde by hydration and aldol cleavage is catalyzed by a single enzyme encoded by a 1 kb MluI-StuI restriction fragment. Mechanisms for both the isomerase- and hydratase/aldolase- catalyzed reactions are proposed. The salicylaldehyde dehydrogenase gene, nahF, was cloned on a 2.75 kb BamHI fragment which also carries the naphthalene dihydrodiol dehydrogenase gene, nahB. By identifying the enzymes encoded by various clones, the gene order for the nah operon was shown to be p, A, B, F, C, E, D.

Barkay, Tamar, Ralph Turner, Erwan Saouter and Joanne Horn. 1992. Mercury Biotransformations and Their Potential for Remediation of Mercury Contamination. Biodegradation. 3(2/3):147-159. (ERL,GB 802).

Bacterially mediated ionic mercury reduction to volatile Hg0 was shown to play an important role in the geochemical cycling of mercury in a contaminated freshwater pond. This process, and the degradation of methylmercury, could be stimulated to reduce the concentration of methylmercury that is available for accumulation by biota. A study testing the utility of this approach is described.

Turner, Bruce J., John F. Elder, Jr., Thomas F. Laughlin, William P. Davis and D. Scott Taylor. 1992. Extreme Clonal Diversity and Divergence in Populations of a Selfing Hermaphroditic Fish. EPA/600/J-93/067. Proc. Natl. Acad. Sci. U.S.A. 89(22):10643-10647. (ERL,GB 814). (Avail. from NTIS, Springfield, VA: PB93-169019)

Recombination is unknown in natural populations of Rivulus marmoratus, a selfing hermaphrodite, and genetic variation is likely due to mutation alone. DNA fingerprinting with an array of microsatellite [e.g., (CT)9] and minisatellite (e.g., the 33.15 core sequence) probes reveals very high clonal diversity within samples of seven Floridian populations; five contain about as many clones as there are individuals. There are 42 clones among 58 individuals surveyed (mean = 1.4 individuals/clone), a level of genetic diversity unprecedented among clonal animals. Moreover, all of the probes recognize the same clones even though, at high hybridization stringencies, there is little overlap in the fingerprint patterns they generate. This suggests that most sympatric clones differ by multiple and independent mutational steps. In one population studied in detail, the average number of mutational steps separating two clones is estimated at about 9-10 and may be substantially higher. The mutational discontinuities among sympatric clones make it unlikely that they evolved by the accumulation of neutral mutations in populations that are otherwise genetically uniform. The data argue that the mixing of unrelated individuals from different local populations occurs to an extent previously unappreciated and/or that divergence of clones is mediated by natural selection. If confirmed, the latter would be a serious challenge to current ideas on the predominant role of recombination in promoting the evolution of biological novelty.

Champ, Michael A., David A. Flemer, Dixon H. Landers, Christine Ribic and Ted DeLaca. 1992. Roles of Monitoring and Research in Polar Environments -- A Perspective. EPA/600/J-93/382. Mar. Pollut. Bull. 25(9-12):220-226. (ERL,GB 826). (Avail. from NTIS, Springfield, VA: PB93-236214)

Researchers have been studying environmental processes in polar regions for more than 30 years. Nevertheless, the information gained has not been sufficient to provide in-depth understanding of the regions studied or most of the processes that occur there. This lack of understanding renders projections of the effects of human activities on terrestrial and marine ecosystems extremely tenuous. Some of the unanswered questions are: (1) What constitutes a significant impact in environments that are relatively unperturbed by humans? (2) What methods will minimize the environmental, health, and safety-related risks of living and working in polar regions? (3) Would the polar regions, due to their pristine nature and remoteness, serve as early warning indicators of global climate change or global pollution?

Pritchard, P.H., J.G. Mueller, J.C. Rogers, F.V. Kremer and J.A. Glaser. 1992. Oil Spill Bioremediation: Experiences, Lessons and Results from the Exxon Valdez Oil Spill in Alaska. Biodegradation. 3(2/3):315-335. (ERL,GB 849).

The use of bioremediation as a supplemental cleanup technology in the Exxon Valdez oil spill, in Prince William Sound, Alaska, has proven to be a good example of the problems and successes associated with the practical application of this technology. Field studies conducted by scientists from the U.S. Environmental Protection Agency have demonstrated that oil degradation by indigenous microflora on the beaches of Prince William Sound was accelerated by adding fertilizer directly to the surfaces of oil-contaminated beaches. Although several types of fertilizers were used in the studies, only the results from the application of an oleophilic fertilizer are presented. The fertilizer enhanced biodegradation of the oil, as measured by changes in hydrocarbon composition and bulk oil weight per unit of beach material, by approximately two-fold relative to untreated controls. Laboratory studies verified the usefulness of the oleophilic fertilizer as a nutrient source, but the contribution of its oleophilic components towards enhancing biodegradation is still unclear. These studies supported bioremediation as a useful cleanup strategy that was subsequently used by Exxon on a large scale. The Exxon Valdez experience has also provided a number of informative lessons that have significant relevance to future oil bioremediation efforts. This paper discusses these lessons and the difficulties in assessing the effectiveness of bioremediation in the field.

Williams, R.R., B. Dehdashti and D.V. Lightner. 1992. Performance of an Aquatic Multispecies System in Evaluating the Effects of a Model Microbial Pest Control Agent on Nontarget Organisms. EPA/600/J-92/405. J. Toxicol. Environ. Health. 37(2):247-264. (ERL,GB X683). (Avail. from NTIS, Springfield, VA: PB93-131803)

A recirculating multispecies test system was developed in conjunction with a study of the fate and persistence of a model microbial pest control agent on nontarget marine and freshwater organisms. The basic unit of the system was 113-l glass aquarium with vertical biological filters in the center of the aquarium, such that two compartments were formed. This allowed the sequestration of predator and prey species within the same system. Organisms from six phyletic groups were subjected to a genetically altered strain of Pseudomonas putida for 15-29 d in either artificial seawater or fresh water. The system was able to maintain the animals for these periods with a minimum of maintenance. Additionally, the system design lent itself to disinfection, dismantling, and rebuilding between experiments with a minimum of labor, and has potential for longer-term studies.

Warner-Bartnicki, Audrey L. and Robert V. Miller. 1992. Characterization of Stress-Responsive Behavior in Pseudomonas aeruginosa PAO: Isolation of Tn-lacZYA Fusions with Novel Damage-Inducible (din) Promoters. EPA/600/J-92/215. J. Bacteriol. 174(6):1862-1868. (ERL,GB X742). (Avail. from NTIS, Springfield, VA: PB92-195718)

Although the pervasive soil and water microorganism Pseudomonas aeruginosa demonstrates heightened sensitivity to UV radiation, this species possesses a recA gene that, based on structural and functional properties, could mediate a DNA damage-responsive regulon similar to the SOS regulon of Escherichia coli. To determine whether P. aeruginosa encodes such stress-inducible genes, the response of P. aeruginosa to DNA-damaging agents including far-UV radiation (UVC) and the quinolone antimicrobial agent norfloxacin was investigated by monitoring the expression of fusions linking P. aeruginosa promoters to a B-galactosidase reporter gene. These fusions were obtained by Tn3-HoHoI insertional mutagenesis of a P. aeruginosa genomic library. Eight different damage-inducible (din) gene fusions were isolated which lack homology to the P. aeruginosa recA gene. Expression of the three gene fusions studied, dinA::lacZYA, dinB::lacZYA, and dinC::lacZYA, increased following UVC and quinolone exposure but not following heat shock. Similar to E. coli SOS genes, the din genes were induced to different extents and with dissimilar kinetics following UVC irradiation.

Ogunseitan, O.A., G.S. Sayler and Robert V. Miller. 1992. Application of DNA Probes to Analysis of Bacteriophage Distribution Patterns in the Environment. EPA/600/J-92/388. Appl. Environ. Microbiol. 58(6):2046-2052. (ERL,GB X743). (Avail. from NTIS, Springfield, VA: PB93-121200)

Radiolabeled bacteriophage DNA probes have been used in this study to determine the distribution of Pseudomonas aeruginosa-infecting bacteriophages in natural samples of lake water, sediment, soil, and sewage. The sensitivity of detection of bacteriophage with the DNA probes was between 10 to the third power and 10 to the fourth power PFU and 10 to the sixth power and 10 to the seventh power CFU of lysogenized bacteria detectable with a homologous phage DNA probe. Analyses of environmental samples suggest that up to 40% of P. aeruginosa in natural ecosystems contain DNA sequences homologous to phage genomes. By using different bacteriophage DNA probes, the diversity of the bacteriophage population in sewage was estimated to be higher than that in other natural samples. The indication that transducing phages and prophages are widely distributed in the Pseudomonas populations investigated has considerable implications for the frequency of natural gene transfer by transduction and of lysogenic conversion of host bacteria in natural ecosystems.

Byrd, Jeffrey J., Joseph G. Leahy and Rita R. Colwell. 1992. Determination of Plasmid DNA Concentration Maintained by Nonculturable Escherichia coli in Marine Microcosms. EPA/600/J-92/385. Appl. Environ. Microbiol. 58(7):2266-2270. (ERL,GB X746). (Avail. from NTIS, Springfield, VA: PB93-121176)

The concentration of plasmid pBR322 DNA in nonculturable Escherichia coli JM83 was measured to deterine whether the plasmid concentration changed during survival of E. coli in marine and estuarine water. E. coli JM83 containing the plasmid pBR322 was placed in both sterile seawater and sterile estuarine water and analyzed for survival (i.e., culturability) and plasmid maintenance. The concentration of pBR322 DNA remained stable in E. coli JM83 for 28 days in an artificial seawater microcosm, even though nonculturability was achieved within 7 days. E. coli JM83 incubated in sterile natural seawater or sterile estuarine water did not reach nonculturability within 30 days. Under all three conditions, plasmid pBR322 DNA was maintained at approximately the initial concentration. Cloning of DNA into the plasmid pUC8 did not alter the ability of E. coli to maintain vector plasmid DNA, even when the culture was in the nonculturable state, but the concentration of plasmid DNA decreased with time in the microcosm. We conclude that E. coli is able to maintain plasmid DNA while in the nonculturable state and that the concentration at which the plasmid is maintained appears to be dependent upon the copy number of the plasmid and/or the presence of foreign DNA.

Hicks, Randall E., Rudolf I. Amann and David A. Stahl. 1992. Dual Staining of Natural Bacterioplankton with 4',6-Diamidino-2-Phenylindole and Fluorescent Oligonucleotide Probes Targeting Kingdom-Level 16S rRNA Sequences. EPA/600/J-92/386. Appl. Environ. Microbiol. 58(7):2158-2163. (ERL,GB X747). (Avail. from NTIS, Springfield, VA: PB93-121184)

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of tranfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.

Lewis, Michael A. 1992. Periphyton Photosynthesis as an Indicator of Effluent Toxicity: Relationship to Effects on Animal Test Species. EPA/600/J-92/407. Aquat. Toxicol. 23:279-288. (ERL,GB X748). (Avail. from NTIS, Springfield, VA: PB93-131829)

The use of aquatic plants in effluent toxicity evaluations is uncommon despite the availability of test methods and numerous recommendations for their use. It has been assumed that aquatic plants are less sensitive than animal test species and consequently, results from toxicity tests with invertebrates and fish have been used as a surrogate data base. This study evaluated the ability of such animal toxicity tests to provide 'safe' concentrations for in-stream periphyton. The toxicity of several samples of a treated municipal effluent were determined during 5-month tests to monitor short-term changes in periphyton photosynthesis (carbon-14 uptake) and observe effects on young production and survival of cultured daphnids and the fathead minnow. The effect levels from the various tests were compared. The effluent was seldom acutely toxic to Daphnia magna and the fathead minnow but it was consisently acutely and chronically toxic to Ceriodaphnia dubia. Significant inhibition and stimulation of periphyton photosynthetic activity occurred at concentrations of 6 to 39% effluent. Periphyton photosynthesis was a more sensitive effect parameter than animal survival and in some cases than Ceriodaphnia reproductive performance. Results indicate that effluent toxicity tests conducted routinely with daphnids and fish may not be sufficient to protect the aquatic flora in receiving waters. Therefore, estimates of phytotoxicity are needed for effective risk assessments for effluents.

Warren, Tessie M., Valerie Williams and Madilyn Fletcher. 1992. Influence of Solid Surface, Adhesive Ability, and Inoculum Size on Bacterial Colonization in Microcosm Studies. EPA/600/J-93/218. Appl. Environ. Microbiol. 58(9):2954-2959. (ERL,GB X759). (Avail. from NTIS, Springfield, VA: PB93-205011)

Microcosm studies were performed to evaluate the effect of solid surfaces, bacterial adhesive ability, and inoculum size on colonization success and persistence of Pseudomonas fluorescens and Xanthomonas maltophilia, each with a Tn5 insertion that conferred resistance to kanamycin and streptomycin. Two types of microcosms were used: (i) a simple system that was colonized by Aeromonas hydrophila and a coryneform and (ii) a complex system produced from lake water enrichment cultures. Simple microcosms contained 100 ml of peptone- and yeast extract-supplemented artificial lake water or 60 ml of peptone- and yeast extract- supplemented artificial lake water with 70 g of 3-mm glass beads. Complex microcosms contained 100 ml of lake water with no nutrient additions or 100 ml of lake water with 70 g of glass beads. The microcosms were incubated for 35 days at 20 degrees C. In lake water enrichment microcosms, the presence of beads increased the abilities of P. fluorescens or X. maltophilia to colonize, but their numbers decreased with time in microcosms both with and without beads. The adhesiveness of the bacteria, measured in an in vitro assay, did not relate to colonization success. In simple microcosms, the inoculum size (10, 10 to the second power, or 10 to the third power) of P. fluorescens did not influence colonization success. However, in complex microcosms, an inoculum of 10 to the third power cells was insufficient to ensure colonization by P. fluorescens, while 10 to the sixth power cells resulted in colonization of liquid and beads. Simple microcosms studies, utilizing only a few species, were poor models for complex natural sytems. In complex enrichment systems, colonization of surfaces resulted in higher numbers of organisms but did not noticeably promote persistence. Adhesiveness of a particular organism may be a relatively minor factor influencing its ability to colonize solid surfaces in complex natural environments.

Weis, Judith S. and Peddrick Weis. 1992. Transfer of Contaminants from CCA-Treated Lumber to Aquatic Biota. J. Exp. Mar. Biol. Ecol. 161(2):189-199. (ERL,GB X761).

Green algae, Ulva lactuca (L.) and Enteromorpha intestinalis (L.), were collected from bulkheads made of wood treated with chromated copper arsenate (CCA). Control algae were collected from nearby rocks. Metal levels in the Ulva and Enteromorpha from the CCA dock were elevated substantially over control levels. Snails, Nassarius obsoletus (Say), collected from an area distant from CCA-wood, were placed with control or experimental Ulva or Enteromorpha. Snails feeding on experimental Ulva retracted into their shells and lay inactive on the bottom of the containers, a process that preceded death. Snails eating Enteromorpha followed. By 4 wk, all the experimental snails were retracted or dead, while all control snails remained active. Thus, metals in the treated wood are taken up by attached algae, and can be toxic to grazing herbivores. Oysters, Crassostrea virginica (Gmelin), were collected from a CCA dock, a bulkhead in a canal lined with CCA wood, and rocks (reference site). Animals from the single dock had elevated Cu, and those from the bulkhead had 12 times the reference levels of Cu, and significantly elevated As. Fiddler crabs, Uca pugilator (Bosc) and U. panacea (Salmon), were collected from burrows close to or distant from CCA-treated wood structures and were analysed for metal content. Those living near CCA wood had elevated metal content, as did the sediments in which they resided. This indicates that sediments, which can absorb contaminants leached from CCA wood, are a route of exposure of benthic biota to these contaminants.

Braunagel, S.C., K.D. Daniel, L.M. Reilly, L.A. Guarino, T. Hong and M.D. Summers. 1992. Sequence, Genomic Organization of the EcoRI-A Fragment of Autographa californica Nuclear Polyhedrosis Virus, and Identification of a Viral-Encoded Protein Resembling the Outer Capsid Protein VP8 of Rotavirus. EPA/600/J-93/060. Virology. 191:1003-1008. (ERL,GB X768). (Avail. from NTIS, Springfield, VA: PB93-168946)

We present the sequence and genomic organization of the EcoRI-A fragment of the Autographa californica multicapsid nuclear polyhedrosis virus, which represents 11% of the AcMNPV genome. Fifteen putative open reading frames and their respective amino acid sequences are described. One open reading frame is similar to the VP8 protein of rotavirus.

Vannelli, Todd and Alan B. Hooper. 1992. Oxidation of Nitrapyrin to 6-Chloropicolinic Acid by the Ammonia-Oxidizing Bacterium Nitrosomonas europaea. EPA/600/J-93/073. Appl. Environ. Microbiol. 58(7):2321-2325. (ERL,GB X769). (Avail. from NTIS, Springfield, VA: PB93-169076)

Suspensions of Nitrosomonas europaea catalyzed the oxidation of the commercial nitrification inhibitor nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine]. Rapid oxidation of nitrapyrin (at a concentration of 10 uM) required the concomitant oxidation of ammonia, hydroxylamine, or hydrazine. The turnover rate was highest in the presence of 10mM ammonia (0.8 nmol of nitrapyrin per min/mg of protein). The product of the reaction was 6-chloropicolinic acid. By the use of 18-mass isotope oxygen, it was shown that one of the oxygens in 6-chloropicolinic acid came from diatomic oxygen and that the other came from water. Approximately 13% of the radioactivity of [2,6-14C]nitrapyrin was shown to bind to cells. Most (94%) of the latter was bound indiscriminately to membrane proteins. The nitrapyrin bound to membrane proteins may account for the observed inactivation of ammonia oxidation.

Buell, C.R. and A.J. Anderson. 1992. Genetic Analysis of the aggA Locus Involved in Agglutination and Adherence of Pseudomonas putida, a Beneficial Fluorescent Pseudomonad. EPA/600/J-93/219. Mol. Plant-Microbe Interact. 5(2):154-162. (ERL,GB X772). (Avail. from NTIS, Springfield, VA: PB93-205029)

An isolate of Pseudomonas putida, which rapidly adheres to plant roots, is agglutinated by a glycoprotein from root surfaces. Agglutination is prevented and adherence to the root surface is diminished by Tn5 insertion in mutant 5123. Two cosmid clones from wild type P. putida and a 2.7-kbp EcoRI-HindIII subclone present in both cosmid clones restored agglutinability to wild type levels in transconujugants of the nonagglutinable (Agg negative) Tn5 mutant 5123. These three clones increased agglutinability in transconjugants of the parental Agg positive isolate. The 2.7-kbp EcoRI-HindIII subclone restored adherence to bean root surfaces of 5123 to wild type levels in a short-term binding assay. Deletion analysis of the 2.7-kbp fragment indicated only 1.45 kbp was necessary for complementation of agglutinability in 5123. This sequence, termed the aggA locus, contains an open reading frame of 1,356 nucleotides encoding a predicted 50,509-Da protein. The distribution of the aggA locus in plant-associated bacteria, as detected through Southern hybridization, is limited to bacteria that express the agglutination phenotype.

Zdor, Robert E. and A.J. Anderson. 1992. Influence of Root Colonizing Bacteria on the Defense Responses of Bean. EPA/600/J-93/221. Plant Soil. 140:99-107. (ERL,GB X774). (Avail. from NTIS, Springfield, VA: PB93-205045)

Colonization of plant roots by fluorescent pseudomonads has been correlated with disease suppression. One mechanism may involve altered defense responses in the plant on colonization. Altered defense responses were observed in bean (Phaseolus vulgaris) inoculated with fluorescent pseudomonads. Systemic effects of root inoculation by Pseudomonas putida isolate Corvallis. P. tolaasii (P9A) and P. aureofaciens REW1-I-1 were observed in bean leaves from 14-day-old plants. SDS- polyacrylamide gel electrophoresis demonstrated that levels of certain acid-soluble proteins increased in the leaf extracts of inoculated plants. Plants inoculated with REW1-I-1 produced more of a 57Mr protein, and plants inoculated with isolates P9A and REW1-I-1 produced more of a 38Mr protein. Northern hybridization revealed enhanced accumulation of mRNAs, that encode the pathogenesis-related protein PR1a, in leaves of plants inoculated with P. putida and REW1-I-1. Only REW1-I-1, but not P9A or P. putida induced symptoms of an hypersensitive response on tobacco leaves, bean cotyledons, and in bean suspension cultures. Phenolics and phytoalexins accumulated in bean cotyledons exposed to REW1-I-1 for 24h but little change in levels of these compounds occurred in cotyledons inoculated with P9A and P. putida. Both suspension culture cells and roots treated with REW1-I-1 rapidly evolved more hydrogen peroxide than those exposed to P9A and P. putida. However, roots from 14-day-old plants colonized by P9A, P. putida or REW1-I-1 did not have higher levels of phenolics, phytoalexins or mRNAs for two enzymes involved in phenolic biosynthesis, phenylalanine-ammonia lyase and chalcone synthase. A selective induction of plant defense strategies upon root colonization by certain pseudomonads is apparent.

Katsuwon, Jirasak and A.J. Anderson. 1992. Characterization of Catalase Activities in a Root-Colonizing Isolate of Pseudomonas putida. EPA/600/J-93/222. Can. J. Microbiol. 38(10):1026-1032. (ERL,GB X775). (Avail. from NTIS, Springfield, VA: PB93-205052)

Pseudomonas putida, a saprophytic root-colonizing bacterium, produces multiple forms of catalase. Catalase A, which increases in specific activity during growth phase and after treatment with H2O2, is located in the cytoplasm and is inhibited by 3-amino-1,2,4-triazole, EDTA, and cyanide, but not by chloroform-methanol treatment. Catalase B, which is induced by external H2O2 or during stationary phase of growth, is membrane associated and is inhibited by chloroform-methanol, EDTA, and cyanide, but not by aminotriazole. Catalase A has a broad pH optimum, from pH 6.0 to 11.0, with two peaks, at pH 8.0 and 11.0. Catalase B is most active at pH 5.0-11.0. Mutant J-1, generated by ethyl methanesulfonate mutagenesis, lacked catalase A activity in extracts of cells harvested throughout lag to early stationary growth phase in liquid medium. Catalase B was produced by J-1 in stationary phase. Exposure of J-1 to H2O2 caused the production of both catalase A and catalase B. Mutant J-1 was more susceptible to cell death than the wild type upon direct exposure to 2.5 mM H2O2 but survived this treatment after exposure to lower (0.3 mM), nonlethal doses of H2O2. The ability to adapt to H2O2 may be related to the behaviour of J-1 on roots where active oxygen species are produced by root surface enzymes. J-1 colonized root surfaces at wild-type levels and produced catalases A and B after exposure to root surfaces for 12 h.

Lewis, Michael A. 1992. Effects of Mixtures and Other Environmental Modifying Factors on the Toxicities of Surfactants to Freshwater and Marine Life. Water Res. 26(8):1013-1023. (ERL,GB X844).

The effects of several environmental modifying factors on surfactant toxicity have been reported in the scientific literature during the past 40 years. The results from 58 reports describing, among other factors, the effects of mixtures, hardness and temperature are summarized in this report. The most common test compound used in the reviewed studies was the anionic linear alkylbenzene sulfonate. Most tests have been conducted in the laboratory with single species of freshwater fish, but a few studies have been conducted in ponds, lakes and rivers with natural biotic assemblages. Of the 58 reports reviewed, those describing the toxicities of mixtures containing surfactants are the most numerous (n=32). The impact of the presence of pesticides and metals on surfactant toxicity has been unpredictable but oil-surfactant mixtures are usually more toxic than expected. Increasing water hardness and temperature increases surfactant toxicity in some cases but the outcome has been compound and species-specific. The presence of suspended solids and naturally occurring dissolved substances decreases the bioavailability of cationic surfactants but not that of anionic and nonionic surfactants. The impact of other chemical and physical modifying factors on surfactant toxicity are too poorly understood to generalize. Therefore, additional research is needed to determine these effects particularly on the chronic toxicities of surfactants at the multispecies level. These data are necessary if environmental hazard assessments for most surfactants are to go beyond their current simplification and be more effective in predicting ecological risk.

Paul, John F., K. John Scott, A. Fred Holland, Steven B. Weisberg, J. Kevin Summers and Andrew Robertson. 1992. Estuarine Component of the US E.P.A.'s Environmental Monitoring and Assessment Program. Chem. Ecol. 7(1-4):93-116. (ERL,GB X931).

The U.S. Environmental Protection Agency's Office of Research and Development has initiated the Environmental Monitoring and Assessment Program (EMAP) to monitor status and trends in the condition of the nation's near coastal waters, forests, wetlands, agro-ecosystems, surface waters, deserts and rangelands. The programme is also intended to evaluate the effectiveness of Agency policies at protecting ecological resources occurring in these systems. Monitoring data collected for all ecosystems will be integrated for regional and national status and trends assessments. The near coastal component of EMAP consists of estuaries, coastal waters, and the Great Lakes. Near coastal ecosystems have been regionalized and classified, and an integrated sampling strategy has been developed. EPA and NOAA have agreed to coordinate and, to the extent possible, integrate the near coastal component of EMAP with the NOAA National Status and Trends Program. A demonstration project was conducted in estuaries of the mid-Atlantic region (Chesapeake Bay to Cape Cod) in the summer of 1990. In 1991, monitoring continued in mid-Atlantic estuaries and was initiated in estuaries of a portion of the Gulf of Mexico. Preliminary results indicate: there are no insurmountable logistical problems with sampling on a regional scale; several of the selected indicators are practical and sensitive on the regional scale; and an efficient effort in future years will provide valuable information on condition of estuarine resources at regional scales.