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The nitrification inhibition test and the luminescent bacteria test were used to determine the inhibiting effect of different phenolic compounds. The chlorinated phenols demonstrated an increased toxicity in the nitrification inhibition test, which was much more sensitive to these compounds than other bacterial toxicity tests. In practice, the nitrification inhibition test was very useful for estimating the toxicity of chemical compounds to nitrifying bacteria, but the test is not recommended for the assessment of general bacterial toxicity. In comparison to other bacterial toxicity tests, the luminescent bacteria test also proved to be very sensitive. Due to its character, which measures a certain physiological parameter, this test system could be used as a screening tool for possible inhibitory effects to bacteria, although positive results should be confirmed by other bacterial toxicity tests.

The article describes the influence of wood preservatives on water bioceonosis. The “battery” of toxicology tests was used to cover luminescent bacteria test, algae growth inhibition test, crustacean and fish lethal tests. The test samples used were pure wood preservative and water after the wood preservative leaching from wood sample. It was observed that the wood preservative is leaching from the wood sample in 10% within 1 month of exposition. Achieved results indicate the necessity of toxic assessment of wood preservative after wood element protection. Keywords: Toxicity assessment, Wood preservative, Leaching

For a number of years, luminescent bacteria have been used as an indicator species for acute toxicity of substances in aquatic marine environments. Various other higher organisms have been used for this exact same purpose. Many of the higher organism test evaluations have been used successfully, but they have a few limitations, such as a low number of organisms exposed to toxicant during a test, less than desirable precision and the need for highly trained individuals to run the tests. One luminescent bacterial bioassay includes many organisms, can be run rapidly and requires training in sample dilution techniques. ASTM Subcommittee D02.12 has been working to develop an ecotoxicity method to evaluate the effects of lubricant formulations on luminescent bacteria. The theory describing this method, as well as the D02.12 method development activities, will be discussed.

Bacteria undergoing environmental effects is extremely interesting for structural, mechanistic, and evolutionary implications. Luminescent bacteria that have evolved in a specific ambient have developed particular responses and their behavior can give us new suggestions on the task and production of luciferina proteins. To analyze the UV interaction under controlled laboratory conditions, we used photoluminescent bacterial strains belonging to a new species evolutionarily close to Vibrio harveyi sampled from a coastal cave with a high radon content that generates ionizing radiation. The survival of the bacterial strains was analyzed, in the light and in the dark, following a variety of genotoxic treatments including UV radiation exposure. The strains were irradiated by a germicide lamp. The results demonstrated that most of the strains exhibited a low rate of survival after the UV exposure. After irradiation by visible light following the UV exposure, all strains showed a high capability of photoreactivation when grown. This capability was quite unexpected because these bacteria were sampled from a dark ambient without UV radiation. This leads us to hypothesize that the photoreactivation in these bacteria might have been evolved to repair DNA lesions also induced by different radiation sources other than UV (e.g., x-ray) and that the luminescent bacteria might use their own light emission to carry out the photoreactivation. The high capability of photoreactivation of these bacteria was also justified by the results of deconvolution. The deconvolution was applied to the emission spectra and it was able to show evidence of different light peaks. The presence of the visible peak could control the photolysis enzyme.

Here we describe a catalytic oxidation process for decomposing concentrated dye solutions, as a model for the treatment of concentrated industrial effluent streams. The prototype Fe-TAML/H2O2 oxidizing system rapidly and extensively degrades the recalcitrant azo dye, tartrazine, in water at ambient temperatures and at dye concentrations that are industrially important. Nearly complete dye removal can be obtained with performance-optimized dosing and pH control. At higher, but still remarkably low catalyst and oxidant concentrations, the dye is removed to below detection limits. 3D surface plots reveal that optimum decolorization at high dye concentration (16.5 g L−1, 30.9 mM) requires 50 times less catalyst and 5 times less oxidant per substrate than at low dye concentration (16.5 mg L−1, 30.9 μM), demonstrating a clear process benefit of higher substrate concentrations. The Fe-TAML/H2O2 system generates environmentally benign and/or biodegradable products from tartrazine: small organic acids, 4-phenolsulfonic acid, and a small amount of 4-nitrobenzenesulfonic acid. The acute toxicity of the Fe-TAML/H2O2/tartrazine reaction mixture toward luminescent bacteria is approximately half that of tartrazine as determined by the Microtox® assay. The results suggest a potential utility of the Fe-TAML/H2O2 technology for treating wastewaters containing high substrate concentrations from the dyeing industry in a straightforward manner to ameliorate environmental impacts, and because of the degradation resistance of tartrazine, that this potential utility might extend to other industries.

Significant advances have been made in the characterization of luciferases and other lux-specific proteins as well as the lux genes from a number of different species of marine and terrestrial luminescent bacteria. A common lux gene organization (luxCDAB..E) modulated by the presence of specific genes involved in regulation and flavin binding and metabolism (luxF-I,L,R,Y) has been found with the luciferase genes (luxAB) flanked by the genes involved in synthesis of its fatty aldehyde substrate (luxCDE). For many species, light intensity per cell is highly dependent on cellular growth resulting in a spectacular autoinduction of luminescence at high cell density. Consequently, the bacterial lux system is of particular interest as it can serve as an excellent model for more general signal transduction systems involved in developmental processes, intercellular communication, and even symbioses. Identification of the lux autoinducers and regulatory proteins of Vibrio harveyi and Vibrio fischeri has provided the biochemical and genetic basis for dissection of the luminescent system. Isolation of the lux genes and the ability to transfer these genes into prokaryotic and eukaryotic organisms have greatly expanded the scope and potential uses of bacterial bioluminescence as a safe, rapid, and sensitive sensor for a wide variety of compounds and metabolic processes.

Publisher Summary The bioluminescent bacteria comprise one of several groups of luminous organisms. Significant differences exist between the bioluminescence reactions of different organisms, including the structure and properties of the luciferases and substrates. Molecular oxygen is the only common feature of bioluminescence reactions, indicating that the luminescent systems in most organisms may have evolved independently. Luminescent bacteria are present in marine environment, freshwater, and terrestrial habitats. They can occur as free-living forms, saprophytes, commensal symbionts, parasites of animals, and specific light-organ symbionts. The luminescence produced by these bacteria, because of its inherent beauty and ease of detection, has attracted scientific attention. With the use of molecular approaches to study the luminescence systems of these bacteria, population biology, ecology, and molecular mechanisms of luminescence (lux) gene regulation can be studied. This chapter describes the current status of bioluminescent systems of luminous bacteria, emphasizing the biochemistry, lux gene organization, and the physiological and genetic regulation of lux gene expression. The effects of oxygen on luminescence illustrate the application of bacterial luminescence system as a sensor of specific molecules that affect metabolic function and gene expression. Knowledge of the basic biochemistry, molecular biology, and physiology of luminescent bacteria is thus not only of interest but of importance for future scientific endeavors.

The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.

During the past several years, the use of animals for toxicity testing has come under critical surveillance. For ethical and economic reasons, various techniques have been developed and proposed as potential alternatives for some of the whole animal toxicity assays. One assay proposed as an alternative to animal testing is the luminescent bacteria toxicity test (LBT), provided under the trade name of Microtox®. The sensitivity and specificity of the LBT was compared with two commonly used toxicity tests–-the L-929 Minimal Eugle's Medium (MEM) elution cytotoxicity test and the Draize test. Cytotoxicity and LBT test data from 709 medical device and biomaterial extracts were compared using a positive/negative ranking system which provided a measurement of false positive and false negative results. These data were compiled from nine separate laboratories producing or using a wide variety of biomaterials and medical device products. The LBT was more sensitive than the tissue culture assay and displayed few false negatives. LBT EC50 values were compared with eye irritancy categories for a group of 34 chemicals and 27 personal care products. As with tissue culture, the LBT was more sensitive and produced minimal false negatives. The data from this study indicate the LBT has potential as a rapid, simple method to screen biomaterials and personal care products for toxicity and irritancy.

The effects of hydrogen sulfide (H2S) were tested in three ecotoxicological tests in order to evaluate its confounding potential in assessment of pore water and groundwater toxicity. The luminescent bacteria Vibrio fischeri, the water flea Daphnia magna, and the microalgae Scenedesmus vacuolatus often are part of a biotest battery. A new technique for the synthesis of hydrogen sulfide solutions of defined concentrations using an electrochemical generator instead of sodium sulfide solutions was used. Because hydrogen sulfide is volatile, the loss rate of H2S was studied over time to enable estimation of the mean test concentrations over the whole test duration. Loss rates were calculated to be 13 +/- 6% after 30 min, and 39 +/- 11% and 43 +/- 16% after a 24- and 48-h exposure time, respectively. Sensitivities of the test organisms in terms of median effective concentration (EC50), corrected for the above loss rates, varied from 0.28 to 0.0036 and 0.055 mM for the luminescent bacteria, the crustacea, and the algae, respectively. A species-sensitivity distribution using EC and mean lethal concentration literature data for marine and freshwater crustaceans and phytoplankton showed a medium sensitivity of the water flea D. magna, though the bacteria V. fischeri and the algae S. vacuolatus were among the least-sensitive group of organisms. This demonstrates that only the algae and the bacteria are easy to use in the assessment of toxicity of matrices with H2S concentrations above 0.06 mM.

Hazard assessment of industrial sites contaminated with coal tar and its products usually focuses on selected pollutants such as the 16 polycyclic aromatic hydrocarbons (PAHs) prioritized by the U.S. Environmental Protection Agency (U.S. EPA). The aim of this study was to investigate to which extent these 16 PAHs contribute to the Vibrio fischeri bioluminescence inhibition measured by the acute Lumistox® luminescent bacteria test. Five of the 16 PAHs-naphthalene (NAP), acenaphthylene (ACY), acenaphthene (ACE), fluorene (FLU), and phenanthrene (PHE)-revealed inhibiting effects when measuring saturated aqueous solutions of these compounds. However, in elutriates of PAH-contaminated soils, the amount of leached PAHs was very low, and the 16 PAHs did not considerably contribute to the observed bioluminescence inhibition. Nevertheless, bioluminescence inhibition was higher for elutriates with increased PAH concentration indicating the presence of other toxicants that co-occur with the 16 PAHs. No evidence was observed for increased bioluminescence inhibition due to synergistic effects among PAHs as calculated on the basis of toxic units for an aqueous solution containing all 16 priority PAHs. Data suggest that the U.S. EPA PAHs play only a minor role in causing acute toxicity to V. fischeri when exposed to aqueous elutriates of PAH-contaminated soils.

Dispersants are a preapproved chemical response agent for oil spills off portions of the U.S. coastline, including the Texas-Louisiana coast. However, questions persist regarding potential environmental risks of dispersant applications in nearshore regions (within three nautical miles of the shoreline) that support dense populations of marine organisms and are prone to spills resulting from human activities. To address these questions, a study was conducted to evaluate the relative toxicity of test media prepared with dispersant, weathered crude oil, and weathered crude oil plus dispersant. Two fish species, Cyprinodon variegatus and Menidia beryllina, and one shrimp species, Americamysis bahia (formerly Mysidopsis bahia), were used to evaluate the relative toxicity of the different media under declining and continuous exposure regimes. Microbial toxicity was evaluated using the luminescent bacteria Vibrio fisheri. The data suggested that oil media prepared with a chemical dispersant was equal to or less toxic than the oil-only test medium. Data also indicated that continuous exposures to the test media were generally more toxic than declining exposures. The toxicity of unweathered crude oil with and without dispersant was also evaluated using Menidia beryllina under declining exposure conditions. Unweathered oil-only media were dominated by soluble hydrocarbon fractions and found to be more toxic than weathered oil-only media in which colloidal oil fractions dominated. Total concentrations of petroleum hydrocarbons in oil-plus-dispersant media prepared with weathered and unweathered crude oil were both dominated by colloidal oil and showed no significant difference in toxicity. Analysis of the toxicity data suggests that the observed toxicity was a function of the soluble crude oil components and not the colloidal oil.

The effects of electromagnetic fields (EMFs) with frequencies varying from 36 to 55 GHz on the bioluminescent activity of bacteria were investigated. EMFs resulted in a decrease of bioluminescence which depended on frequency. The time of adaptation of cells to the electromagnetic field was longer than the intrinsic temporal parameters of the bioluminescent system. The effect had a nonthermal nature. It was found that disturbances of the geomagnetic field (magnetic storms) resulted in an increase of bioluminescence.

The Hudson-Raritan Estuary is one of several United States coastal areas where chemical data have suggested a potential for contaminant-related biological effects, and multiyear intensive bioeffects surveys have been conducted by the National Oceanic and Atmospheric Administration. The severity and spatial patterns in sediment toxicity were determined in an estuary-wide survey during spring 1991 using amphipods, bivalve larvae, and luminescent bacteria as test organisms. Spatial patterns in toxicity corresponded to the distributions of a number of toxic chemicals in the sediments. Areas that exhibited the greatest sediment toxicity included the upper East River, Arthur Kill, Newark Bay, and Sandy Hook Bay. The lower Hudson River adjacent to Manhattan Island, upper New York Harbor, lower New York Harbor off Staten Island, and parts of western Raritan Bay generally showed lower toxicity. Supporting chemical analyses of the sediments, including acid-volatile sulfide and simultaneously-extracted metals, suggested that metals were generally not the cause of the observed toxicity, with the possible exception of mercury. Among all contaminants analyzed, toxicity was most strongly associated with polynuclear aromatic hydrocarbons, which were substantially more concentrated in toxic samples than in nontoxic samples, and which frequently exceeded sediment quality criteria.

Binary mixtures of copper-pesticide (zineb, maneb, carbaryl) were tested on bacteria, Photobacterium phosphoreum (Microtox test), and on protozoa, Colpidium campylum . Factorial experiments were used, and results treated with multiple regression analysis. The toxic concentrations chosen were comprised between the NOEL (No Effect Level) and the IC50, when considering the inhibition of the bacterial luminescence and the growth rate of protozoa, induced by each toxicant tested alone. Interactions of copper with zineb or maneb resulted in dramatic synergistic effects in the two species: a total inhibition of the bacterial luminescence or of the growth of colpidium cultures occured at concentrations considered as non toxic when each compound was tested alone. A synergism could be observed on the luminescent bacteria with concentrations as low as 20 ug/l Cu++ and 9 ug/l zineb or 9 ug/l maneb. On the colpidium cultures, these threshold concentrations were 63 ug/l Cu++, 50 ug/l zineb, 400 ug/l maneb. Carbaryl differed from the two dithiocarbamates since only a slight interaction was registered with copper on the protozoa at high concentrations (0.2 mg/l Cu++, 8 mg/l carbaryl).

Toxicity studies are generally conducted with single chemicals under standardized conditions. Yet results with the same substances can be different if antagonistic or synergistic interactions occur with the test medium constituents. In fact the toxic response of various chemicals seems highly dependent on temperature, contact time, salinity, buffering substances and water hardness. As Microtox test has previously been used to assess the toxicity of pure chemicals and complex effuents, it is interesting to study how some physico-chemical interactions can modulate the response of the luminescent bacteria in this system. In the present investigation, the effects of the test temperature and the role of pH, buffers, hardness and salinity were studied for different times of exposure of the Microtox bacterial reagent to various inorganic and organic compounds including cadmium, zinc, pentachlorophenol and benzene. The results indicated that physicochemical parameters must be considered when analyzing Microtox data of complex effuents or mixtures of chemicals.

Fitchburg, Massachusetts sanitary landfill leachate was subjected to toxicity tests using: fathead minnows (Pimephales promelas), zooplankton (Daphnia magna), green algae (Selenastrum capricornutum) and aerobic luminescent bacteria (Photobacterium phosphorium). The leachate was highly toxic to the test bacteria, moderately toxic to daphnids, and slightly toxic to fathead minnows. Algal cells, unable to grow at the 10-percent leachate exposure level, recovered after centrifugation and reinnocuation into algal nutrient medium. Low-flow summer hydrological data indicated that the leachate contributed about 7% to the total flow of the receiving stream, Flagg Brook, and about 0.6% to Sawmill Pond water located further downstream from the leachate outfall. These data, together with observed toxicity values for the test organisms, indicate that the leachate concentration in Flagg Brook impacts the diversity of aquatic life in this system, but may be less severe in Sawmill Pond where increased dilution results in leachate levels below the acutely toxic level. The considerable variation between toxicity test results obtained with the four test organisms, demonstrates the importance of conducting several such toxicity tests using organisms from different trophic levels, to assess the potential impact of a pollutant discharge on an aquatic ecosystem.

Two short-term bacterial toxicity tests that have received a great deal of attention during the last few years are the Microtox and Spirillum volutans assays. The former involves monitoring changes in the light output from luminescent bacteria (Photobacterium phosphoreum) in a temperature controlled photometer when exposed to various concentrations of toxic substances. In the S. volutans assay, motility patterns of this large aquatic bacterium are used as the test endpoints. As part of the authors' program in assembling and establishing a battery of short-term bacterial toxicity tests, they examined the use of Microtox and S. volutans for determining the toxicity of a wide variety of environmental samples. Samples examined included potable and surface waters, industrial effluents, soil/sludge extracts and landfill leachates. Some of the results are presented in this paper.

The terrestrial nematode, Heterorhabitis bacteriophora contains symbiotic terrestrial bacteria which emit low but detectable luminescence in the symbiotic state. When the nematode is ingested by caterpillar larvae (or other insects), the bacteria are released into t he hemocoel, where they grow rapidly (doubling time of 3 h or less), reach high cell numbers (109 ml−1 greater), and are brightly luminous. The enzyme that catalyzes light emission is a typical bacterial luciferase as judged by its substrate requirements and the kinetics in vitro. Bacteria grown in synthetic media emit luminescence that is nearly identical in color with that of other luminescent bacteria, while the color of light from infected caterpillar is substantially red-shifted.

The toxicity assessment of chemicals on complex microbial communities are generally preferred to toxic tests conducted with pure cultures of bacteria. The purpose of this study is to compare the “Respiration” test studying metabolic criteria of mix bacterial populations and the “Microtox” test using lyophilized cultures of luminescent bacteria. The inhibition of oxygen consumption was compared with the inhibition of the bioluminescence of Photobacterium phosphoreum. In the first part of this study, the methodology of the “Respiration” test and the “Microtox” test was improved to optimize their performance in toxicity assessment. In the second part, toxicity of an organic chemical, the 3,5-Dichlorophenol, and an inorganic chemical, copper sulfate, was evaluated with the two tests. Results were compared and characteristics of the testing methods are discussed according to their sensitivity, reproducibility, representativity and ease of execution. The repeatability study for the “Respiration” test and the “Microtox” test gives variation coefficients less than 15% and 10% respectively. The variation coefficients concerning the reproducibility study are found to be 15% or 18% for the “Respiration” test, 5% or 28% for the “Microtox” test, depending on the tested toxicant. These two toxicity tests are easy to perform. But the “Microtox” test offers the advantage of being more sensitive and much faster. On the other hand, the “Respiration” test which was conducted on a microbial community is probably more representative of microflora in rivers.

Data on the action of mono- and polyatomic phenol mixtures on hydrobionts are discussed. Luminescent bacteria, Daphnia, algae zoospores, higher aquatic plants–Elodea and duckweed – were used as test objects. It is established that the toxicity of a paired mixture of mono- and polyatomic phenols is lower than the toxicity of their components.

In this study bacterial luminescent toxicity tests (LTT) were used to monitor the residual toxicity concentration of a biocide-inhibitor after several months storage. The study was completed in two parts. The first study investigated the relationship between the chemical and toxicity concentrations of a biocide-inhibitor stored in mild steel containers and glass containers. After three months storage in the mild steel containers there was a significant difference (p < 0.01) between the chemically assayed level of the biocide-inhibitor and the toxicity level determined by the LTT. The results of the laboratory studies were confirmed in the second part of the study when the biocide-inhibitor was incorporated in the sea water used to flood a large undersea pipeline during the pressure testing programme. The disparity between the chemical and toxic assayed concentrations was not as great when the biocide-inhibitor was stored in glass containers. The influence of the container used for biocide testing plus the failure of the chemical analysis to reflect the toxicity level of this biocide-inhibitor demonstrates a different application of LTT. The results of this study also illustrate the usefulness and relevance of LTT in testing the effectiveness of some biocides and indicate that LTT could be useful in monitoring the efficiency of biocides during their industrial application.

Reports investigations on the response of the bioluminescent bacteria, Photobacterium phosphoreum, to radiation and the use of the bacteria as a biological radiation dosemeter. By the latter the authors mean a water-equivalent biological system that will compare beams not merely on the basis of absorbed dose, but also have intrinsic RBE values for different radiation beams. The authors studied the effect of low LET beams of different quality on the bioluminescent survival fraction and compare the RBE values obtained with those previously published. The results show that the smallest detectable dose at present is about 1 Sv. However, there are reasons to expect an improvement in sensitivity of the system by a factor of about two in the future. It is believed that the phenomenon of bacterial luminescence loss produced by radiation will contribute to clinical radiation dosimetry and research.

Direct calorimetric determinations of the rate of heat production along with simultaneous determinations of the rate of photon emission and the number of viable cells have provided insight into the growth of Beneckea harveyi and Photobacterium leiognathi. These experiments were performed with a Tronac isothermal microcalorimeter modified with a fiber optic light guide to allow in situ detection of light. Escherichia coli and a dark variant of P. leiognathi were also examined to provide points of reference. It is demonstrated that B. harveyi seems to pause in the rate of metabolic heat production at the same point in time that the enzyme luciferase begins to be synthesized. This effect is not removed if B. harveyi is grown in conditioned medium. The thermograms for all species are correlated with cell generation time. The heat production per cell indicates that uncrowded cultures produce more heat than older, more crowded cultures, supporting the original observation of Bayne-Jones and Rhees (1929). These observations reopen for examination the suggestion that living systems tend toward a state of minimum metabolism per unit mass.

The toxlcity of selected sediments in the Adriatic Sea and the effect of phytoplankton bloonl on sedlments in the vicinity of Rovln], Yugoslavia, using bacterial bloluminescence toxicity assay, have been investigated. Sedlments under the influence of urban and industrial wastes tend to be more contaminated than those in the open sea. The toxic effect of decayed material derived from sinlung inucus aggregates was higher at locations influenced by local pollution. The time course study at 1 station suggested that increases in toxicity of sediment extracts is mainly due to changes in organic matter derived from decayed products of mucus aggregates During summer 1988, an intense nonseasonal phytoplankton bloom occurred in the Northern Adriatic (Degobbis in press) forming large inucus aggregates. The Istrian coast of the Northern Adriatic was fouled with large quantities of this mucus material. The nature of these aggregates is unknown, but most have associated pelagic and benthic organisms, empty shells, much detritus and bacteria. This event has been previously described in the waters of Rijeka Bay (Zanon 1931) and the Gulf of Trieste (Herndl 1988). Sinking of these aggregates transfers nutrients and decayed material from the surface mixed layer to the seafloor, causing a drastic decrease in the number of benthic organisms caused by mechanical events or anoxic conditions (Zavodnik 1977, Stachowitsch 1984). Sediments in the vicinity of Rovinj were covered (80 to 90 % of whole area) with a 1 to 2cm thick layer of decayed material. Since 1987 our laboratory has dealt with a program for monitoring the local pollution. In order to determine any secondary effects of decayed material on sediments at 4 locations in the vicinity of Rovinj, the Addressee for correspondence O Inter-Research/Printed in F. R. Germany bioluminescence toxicity assay was used. It is a rapid method of comparing and ranking the toxicity of organic extracts of contaminated sediments (Schiewe et al. 1985) and is a reliable screening technique for a variety of pollutants in sediments (Williams et al. 1986). The results of our assay exhibited a high degree of similarity with other sediment toxicity tests (Giesy et al. 1988) and hence this technique can be used as an exploratory screening test. Materials and methods. The surface layer of sediment samples were collected by SCUBA divers from 11 locations (Fig. 1). About 500 g wet sediment was frozen at -20°C and transported to laboratory. Dry weight was determined by heating 10 g aliquots at 105 'C for 24 h. The presence of total organlc material (TOM) was estimated by weight loss after heating at 500°C. Extractable organic material (EOM) was determinated according to Meyers e t al. (1984). For the bioluminescence assay, l00 g wet wt of sediments were extracted with dichloromethane-methanol ( 2 : 1) as previously described by Schiewe et al. (1985). After several washings to remove methanol, dichloromethane extracts were evaporated to dryness and dissolved in dimethylsulfoxide (DMSO). Extraction blanks were prepared by the same procedure but without sediment. Water samples (21) were extracted with dichloromethane, evaporated to dryness and dissolved in l m1 DMSO. Extraction blanks were prepared with distilled water. Bacterial luminescence of a laboratory grown, healthy culture of the marine bacterium Photobacter phosphoreum will be inhibited upon exposure to toxic substances (Bulich & Isenberg 1981). The luminescence of cultures exposed to a series of dilutions of sediment and water extracts was measured in a Packard-Tri-Carb 308 Mar Ecol. Prog Ser. 57: 307-310, 1989 Fig. 1 Map of Rov~nj, Northern Adriatic, shoiving sarnpl~ng sites (Sl to S l l ) liquid scintillation spectrometer. Briefly, in 0.5 m1 saline suspensions containing 106 exponentially growing luminescent bacteria, 0.5ml of subsequent dilutions of sediment and water extracts in 2 O/O saline were added. After 15 min incubation at 15°C the bioluminescence was measured, compared with blank samples, and corrected for spontaneous decline in photoactivity. The results are expressed as mg of dry weight of extracted sediments or m1 of water causing a 50 % reduction of bioluminescence in 15min (ED50). Estimates of ED5, were obtained using linear regression analysis, as descnbed by Schiewe et al. (1985) and 95 O/O confidence intervals for each estimate of ED50 were calculated based on Fieller's theorem (Finney 1964). Results and discussion. Three of the 11 sample stations (Fig. l ) , i.e., S l , S-2, and S-3, are under the direct influence of industrial and/or urban runoff from a fish cannery, tobacco factory and urban waste, respectively. Comparison of toxicities of organic extracts of selected sediments and waters from the stations are presented in Table 1 Sediments influenced by urban sewage and industrial runoff tend to be more contaminated. The toxicity of waters directly influenced by industrial and urban wastes (S-I , S-2, S-3) were also higher than those of the open sea However, this correlation, between the toxicity of sediment and water extracts was not observed at all stations. Table 1. Comparison of toxicit~es of organic extracts of selected sediments (Sed., mg' dry wt) and waters (mg). ET),,: amount (rng dry wt or ml H20) causing 50 "is reduction of biolumn~escence in l5 min; S D . not determned; CI. confidence Interval at 95 significance in parentheses

Luminescent bacteria isolated from light organs of seven different species (3 genera) of fishes of the family Leiognathidae were subjected to taxonomic analysis. Of the 733 isolated all but seven were identified as Photobacterium leiognathi; the others are considered to be either chance contaminants of the sampling procedure or transients within the organ. In most fish, the luminous organ appeared to contain a single predominating strain of P. leiognathi with small numbers of one to three other strains of the same species, differing by only one or two characters.

Vibrio fischeri and Lucibacterium harveyi constituted 75 of the 83 luminous bacteria isolated from Sargasso Sea surface waters. Photobacterium leiognathi and Photobacterium phosphoreum constituted the remainder of the isolates. Luminescent bacteria were recovered at concentrations of 1 to 63 cells per 100 ml from water samples collected at depths of 160 to 320 m. Two water samples collected at the thermocline yielded larger numbers of viable, aerobic heterotrophic and luminous bacteria. Luminescent bacteria were not recovered from surface microlayer samples. The species distribution of the luminous bacteria reflected previously recognized growth patterns; i.e., L. harveyi and V. fischeri were predominant in the upper, warm waters (only one isolate of P. phosphoreum was obtained from surface tropical waters).

Characterization of 13 isolates of luminescent bacteria from specimens of Cleidopus gloriamaris has revealed differences between these organisms and previously described species of Photobacterium. Further studies on the biochemical and serological attributes of these and other luminescent bacteria are essential, before ecological studies on the fish-microbe symbiosis are possible.

The Leiognathid (pony) fish, which occur in the shallow coastal waters of the tropical and subtropical Indo-Pacific region, are capable of emitting a bright light from their ventral surface. In these small fish, as in several other (but not all) bioluminescent fish, the source of light is symbiotic luminous bacteria, maintained within a special organ (Harvey, 1952, 1958 ; Buchner, 1965). The evidence that bacteria are involved as symbionts has come from microscopic observations to gether with the fact that cultures of luminous bacteria have been obtained from the organs (Harms, 1928 ; Haneda, 1940, 1950) . In the present experiments addi tionai proof of the bacterial origin of the light is presented, together with evidence that the symbiotic bacteria are distinct from many of the free living luminescent bacteria which may be isolated directly from sea water in the same area. In different grOU@)5of fish there are very different and sometimes highly elabo rate types of organs and modes for display of the bacterial light. In pony fish the system involves several special and unusual elements. The organ itself, which surrounds the esopilagus like a donut, and communicates with it †? zA±a paired ducts (Haneda, 1940, 1950) is literally packed with bacteria. Upon dissection it is always fotlnd to be emitting light, brightly and continuously, irrespective of the tinle of day or other environmental factors. The light reaches the outside (ventral) surface via indirect and somewhat sophisticated optics. The gut tract makes a loop into the wall of the swim (air) bladder at the site of the light organ. Tile organ thus faces directly into the swim bladder, and is provided with an eyelid-like flap which can control the amount of light shining into the air-filled bladder. The swim bladder is internally reflecting, being lined with guanine crystals—the same material which is responsible for tue silvery skin of many fish (Denton, 1970, 1971). The ventral portion of the swim bladder is only partially reflective (“half silvered―), and to it attach specialized translucent ienticular muscle cells. The optical arrangement thus takes the light from a small source and causes it to be evenly diffused over a larger area, namely most of the ventral part of the body. As shown below, it was possible to estimate the total number of viable bacteria within an organ, and to compare this with the weight of the organ. Such counts indicate that a large percentage, if not all, of the bacteria which are crammed into the ducts of the organ are both viable and bioluminescent. The isolated symbiotic bacteria were compared with free living bacteria isolated directly from sea water in the same area where the fish were collected. Although tilebacteriafrom the two sourceshad similarcolonialmorphology, and were the same in certain other special respects, tile two appeared to possess distinctly differ ent types of luciferases, corresponding to types previously known from different strains (Hastings, Weber, Friedland, Eberhard, Mitchell and Gunsalus, 1969), hut not previouslycorrelatedwith symbioticand freelivinglifestyles.

Cells of a species of luminescent bacteria were treated with a gas stream containing products formed by the photochemical oxidation of cis -2-butene and NO. Luminescence and viability decreased with the time of irradiation of reactants. The rate of luminescence decrease was dependent on the ratio of the initial concentrations of cis -2-butene and NO with a ratio of 2 giving the most rapid loss. Known photochemical oxidation products, ozone, NO 2 , formaldehyde, acetaldehyde, and PAN were examined individually. Aldehydes did not appear to contribute to the decrease in luminescence but ozone and PAN did. Although NO 2 alone up to 0.5 ppm produced no decrease, it may contribute to the luminescence loss in the total irradiation mixture. The total oxidant concentration produced upon irradiation gave luminescence decreases which were matched by comparable concentrations of pure ozone. However, this does not imply that luminescence losses caused by photochemical oxidants are due only to ozone. A possible mechanism of the toxic effect and the interpretation of this bioassay in relation to other organisms are briefly discussed.

We assessed the applicability of multi-strain bacterial bioreporter bioassays to drug screening. To this end, we investigated the reactions of a panel of 15 luminescent recombinant Escherichia coli bacterial bioreporters to a library of 420 pharmaceuticals. The panel included bacterial bioreporters associated with oxidative stress, DNA damage, heat shock, and efflux of excess metals. Eighty nine drugs elicited a response from at least one of the panel members and formed distinctive clusters, some of which contained closely related drugs. In addition, we tested a group of selected nine drugs against a collection of about 2000 different fluorescent transcriptional reporters that covers the great majority of gene promoters in E. coli. The sets of induced genes were in accord with the in vitro toxicity of the tested drugs, as reflected by the response patterns of the 15-member panel, and provided more insights into their toxicity mechanisms. Facilitated by microplates and robotic systems, all assays were conducted in high-throughput. Our results thus suggest that multi-strain assemblages of bacterial bioreporters have the potential for playing a significant role in drug development alongside current in vitro toxicity tests.

Luciferase from the luminescent bacteria Photobacterium leiognathi, strain S-1, has been isolated and purified from lysed cells and shown to be a glycoprotein. A soluble, non-carbohydrate containing luciferase was also isolated from the lysate. This latter luciferase amounts to less than 20% of the total luciferase activity. The purified glycoprotein is (1) susceptible to inactivation by lysozyme, (2) gives positive color reactions to the phenol, resorcinol, and anthrone tests for sugars, and (3) gives overlapping bands when stained for carbohydrate and protein on gel electrophoresis. Neither the soluble luciferase from S-1 nor a soluble luciferase isolated from Beneckea harveyi, mutant MB-20, give positive results to any of these carbohydrate tests.

A feasibility study on the potential use of a luminescent bacteria bioassay in the toxicity screening of trichothecenes is presented. The toxic effects of four 12,13-epoxytrichothecenes to bioluminescent bacteria were determined for the first time using the Microtox™ Toxicity Analyzer (TM: Trademark of Microbics Corporation, Carlsbad, California). The influence of test temperature and exposure time on the toxic effect of trichothecenes to the luminescent bacteria are discussed. The toxicities measured are compared with toxic effects determined using other bioassays. Potential relationships between the toxicities found and variations in the chemical structure of these compounds are considered.

The toxicities of solutions of 10 mercury compounds to luminescent bacteria were measured using the Microtox Toxicity Bioassay. The aim of this study was to assess the influence that the counter-ions have on the aquatic toxicity of mercury salts. The toxicities of these mercury compounds were very similar, except for mercurous tannate and mercuric salicylate. This can be attributed to differences in the ionization and speciation patterns of these compounds relative to the other compounds tested. In general, the toxicity of the solutions at pH 5 was not significantly different from the toxicity of these solutions at pH 6, but a clear reduction in toxicity was observed when the pH of the solution was adjusted to pH 9. Significant differences were found between the toxicity of Hg(I) and Hg(II) salts of the same anion at pH 9. When cysteine was added to a mercuric nitrate solution (at pH 6), a reduction in the toxicity was observed. This can be explained in terms of the strong binding of mercury to cysteine, thus reducing the concentration of mercury species available to cause an observable toxic effect to the bioluminescent bacteria.

Luminous bacteria are a well-known group of (primarily marine) bacteria that are ecologically quite diverse (Nealson and Hastings, 1979). They participate in a wide variety of symbioses, including loose associations as gut symbionts, species-specific associations as extracellular symbionts of light organs of marine fishes and squids, and intracellular associations as symbionts of luminous tunicates (Nealson et al., 1981). Some examples of these associations are shown in Table 1.

Johann Heinrich von Thunen-Institute, Federal Research Institute for Rural Areas, Forestry and Fisheries began publishing the Informationen aus der Fischereiforschung – Information on Fishery research in 2010

A pheromone is a chemical excreted by an organism into the environment that acts to elicit a specific response from other organisms of the same species. The importance of pheromones in the life cycle of various species of mammals, insects, and fungi is well known. In the past decade, it has become apparent that pheromones influence the behavior and development of prokaryotes. Pheromones excreted by myxobacteria, actinomycetes, and cyanobacteria elicit specific developmental responses from these organisms. In addition, pheromones excreted by Streptococcus faecalis function in conjugation, and pheromones of luminescent bacteria regulate bioluminescence of these organisms.

The toxicity of cadmium compounds against luminescent bacteria has been measured using the Microtox(R) toxicity bioassay and has been related to the cadmium species. Since the Microtox(R) test is carried out in NaCl (2%) and cadmium forms stable chloro complexes, NaNO(3) and NaClO(4) have been tested successfully as alternative to sodium chloride to provide the adequate osmotic protection of the bacteria. The influence of medium and ionic strength as well as different exposure times on EC(50) values has been evaluated.

Arsenic is an abundant metalloid and a dangerous pollutant when in solution under the arsenate or arsenite forms—As(V) and As(III), respectively. Since its biological effects are expected to depend on the oxidation state and on speciation, effect of pH on either As(V) or As(III) speciation and resulting toxicity was investigated using the Microtox bioassay based on change in light emission by the luminescent bacteria Vibrio fischeri. Within a 5.0–8.0 pH range, EC50 values for As(V) were found to decrease as pH became basic, reflecting an increase in toxicity; whereas in the case of As(III), EC50 values were almost unchanged within a 6.0–8.0 pH range and lowered only at pH 9.0. HAsO42− and H2AsO3− were found to be the most toxic species. A statistical approach based on testing the null hypothesis of additive toxicity revealed an antagonistic effect between the arsenate chemical species. At low concentrations, As(V) was regularly found to be more toxic than As(III), independent of the pH value. Conversely, at high concentrations, the toxicity of both As(III) and As(V) was found to chiefly depend on pH, as a consequence of the strong influence of this parameter on the chemical speciation.