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The term bioluminescence refers to a conspicuous light emission displayed by numerous aquatic and terrestrial organisms. This phenomenon has so far not been observed in several taxonomic groups like archaea, protista, platyhelminthes, chelicerata, cephalochordata, amphibians, reptiles, birds, plants, and mammals. However, some luminescent bacteria, fungi and microalgae like dinoflagellates are known. Bioluminescence has become a powerful biological tool and revolutionized several medical, physiological and biotechnological approaches, such as, for example, the study of metabolic pathways of bacteria to mammals. In 2008 O. Shimomura, M. Chalfie and R. Tsien were awarded the Chemistry Nobel prize for their discovery of the protein GFP, which is co-expressed with aequorin, the calcium-activated photoprotein involved in the bioluminescence reaction of the jellyfish Aequorea victoria. Some organisms are known to display bioluminescence only under certain kinds of stimulation and stress conditions; others appear to produce their light without prior stimulation either continuously or intermittently. Despite the discovery of more than 700 genera of bioluminescent organisms from marine, terrestrial, and freshwater environments, there is often still insufficient knowledge about their bioluminescence emission patterns under natural and stress conditions. Furthermore, there are no detailed reviews on stimulation techniques that can be used to test whether organisms previously not having been recognized to be luminescent are luminescent or not. This paper reviews various stimulants, such as chemical, mechanical, photic, thermal, magnetic and electrical ones, used in tests to elicit the emission of light in known bioluminescent organisms. This account should help researchers to extend their investigations to identify organisms hitherto not deemed to be luminescent.

With the growing requirements on environmental protection in drilling fluid additives, the traditional anti-collapsing agents are unable to meet the demand. Therefore, improving the environmental protection performance of anti-collapse agents has drawn great attention from scientists. Organic amines are commonly used as anti-collapse agents due to their good anti-collapse properties. In this paper, alkali diamines have been selected as research objects to evaluated the anti-collapse abilities by immersion tests and linear expansion experiments. The experimental results showed that alkyl diamines demonstrate good anti-collapse abilities. However, with increase in the number of C atoms, the solubility of alkyl diamines decreases, and the inhibition by the potassium dichromate method, dilution and inoculation method, and luminescent bacteria method. The result show that the biodegradability of the alkyl diamine anti-collapse agents increase gradually increase in the number of carbon atoms in the chain, while the solubility and biotoxicity decrease. Therefore, the longer the alkyl diamine carbon chain length, the better the environmental performance of anti-collapse agents. Hence, this research provides a guideline for synthesizing new organic amine anti-collapse agents.

The composition of atmospheric particulate matter, including particle-bound polyaromatic hydrocarbons, generally shows a clear seasonal pattern which is reflected in its ecotoxicity as well. This study aimed at characterising seasonal differences in the ecotoxicity of rural aerosol samples applying both luminescent bacteria and higher plants as test organisms. Higher plant phytotoxicity was assessed by the Sinapis alba root growth inhibition test and the Vegetative Vigour Test. Different bioassays and end-points showed different sensitivity: while the Sinapis alba assay showed no toxic effect, luminescent bacteria proved an excellent screening tool, detecting no toxicity in the summer sample and the highest inhibition in the winter sample, with EC20 = 9.87%. In the case of Vegetative Vigour Test, parallel application of different end-points revealed that atmospheric particulate matter might have a Janus-faced effect: stimulation of photosynthetic pigments due to nutrient content and growth impairment due to toxic components.

The intensive development of medical science has led to an increase in the availability and use of pharmaceutical products. However, nowadays, most of scientific attention has been paid to the native forms of pharmaceuticals, while the transformation products (TPs) of these substances, understood herein as metabolites, degradation products, and selected enantiomers, remain largely unexplored in terms of their characterization, presence, fate and effects within the natural environment. Therefore, the main aim of this study was to evaluate the toxicity of seven native compounds belonging to different therapeutic groups (non-steroidal anti-inflammatory drugs, opioid analgesics, beta-blockers, antibacterial and anti-epileptic drugs), along with the toxicity of their 13 most important TPs. For this purpose, an ecotoxicological test battery, consisting of five organisms of different biological organization was used. The obtained data shows that, in general, the toxicity of TPs to the tested organisms was similar or lower compared to their parent compounds. However, for example, significantly higher toxicity of the R form of ibuprofen to algae and duckweed, as well as a higher toxicity of the R form of naproxen to luminescent bacteria, was observed, proving that the risk associated with the presence of drug TPs in the environment should not be neglected. Electronic supplementary material The online version of this article (10.1007/s11356-020-08881-3) contains supplementary material, which is available to authorized users.

Organic ligands are commonly believed to decrease the toxicity of metal ions, but there is few experimental evidence, especially for chromium (Cr(III)), which often coexists with organic compounds in industrial effluents. Here, the complexation of Cr(III) with acetate, lactate, l-tartrate, biphthalate and oxalate was tested under the conditions of a toxicity test, with high ion strength, by spectroscopic techniques. The stability constants of the complexes were found to follow the order Cr(III) oxalate > Cr(III) lactate > Cr(III) biphthalate > Cr(III) L-tartrate > Cr(III) acetate. Then, aquatic toxicity of Cr(III) to Photobacterium phosphoreum for a 15-min exposure period was tested in the absence and presence of organic ligands. Results unexpectedly show that the complexation of Cr(III) with acetic, lactate, l-tartrate and biphthalate resulted in enhanced toxicity to luminescent bacteria, whereas the coordination of Cr(III) with oxalate sharply alleviated the toxicity of individual oxalate and inorganic Cr(III), which was further confirmed by the scanning electron microscopy (SEM). Our findings show thus that organics do not always mitigate the toxicity of Cr(III) in acidic water.

Background A European inter-laboratory test with 29 participating laboratories investigated whether a battery of four ecotoxicological tests is suitable for assessing the environmental compatibility of construction products. For this purpose, a construction product was investigated with the dynamic surface leaching test (DIN CEN/TS 16637-2) and the percolation test (DIN CEN/TS 16637-3). The eluates were produced centrally by one laboratory and were tested by the participants using the following biotests: algae test (ISO 8692), acute daphnia test (ISO 6341), luminescent bacteria test (DIN EN ISO 11348), and fish egg test (DIN EN ISO 15088). As toxicity measures, EC50 and LID values were calculated. Results Toxic effects of the eluates were detected by all four biotests. The bacteria test was by far the most sensitive, followed by the algae test and the daphnia test; the fish egg test was the least sensitive for eluates of both leaching tests. The toxicity level of the eluates was very high in the bacteria, daphnia, and algae test, with lowest ineffective dilution values of LID = 70 to LID = 13,000 and corresponding EC50 values around or even below 1 volume percent. The reproducibility (approximated by interlaboratory variability) of the biotests was good ( Conclusion It can be confirmed that the combination of leaching and ecotoxicity tests is suitable to characterize with sufficient reproducibility the environmental impact posed by the release of hazardous substances from construction products.

Fifteen native luminescent bacteria were isolated from the Gulf of Thailand, and their sensitivity for the detection of toxicity of crude oil and its aromatic components was investigated. Of these isolates, Vibrio campbellii strain FS5 was one of the two most highly inhibited bacteria at all crude oil concentrations. This bacterium showed a decrease in luminescence intensity of between 10.7 and 80.2% after a 15-min exposure to 0.0001–10 mg/L of crude oil. The degree of bioluminescence inhibition increased with increasing concentrations of crude oil. The presence of crude oil at all concentrations had negative effects on the log bioluminescence per log number of viable cells after 15- to 105-min exposure. About 10 to 100 times, lower half maximal effective concentration (EC50) values were observed for polycyclic aromatic hydrocarbons (PAHs) than those for benzene, toluene, ethylbenzene, and xylene (BTEX). In the presence of each individual BTEX and PAH, the bioluminescence inhibition increased with increasing exposure time (1–32 h). This indigenous bacterium can be used as a simple and general indicator of oil contamination and its impact on coastal waters as well as for assessing potential toxicity during oil bioremediation.

Protecting the quality of lake watersheds by preventing and reducing their contamination is an effective approach to ensure the sustainability of the drinking water supply. In this study, acute toxicity assessment was conducted on the basis of acute bioluminescence inhibition assay using the marine bacterium Vibrio fischeri as the test organism and Luoma Lake drinking water source in East China as the research target. The suitable ranges of environmental factors, including pH value, organic matter, turbidity, hardness, and dissolved oxygen of water samples were evaluated for the toxicity testing of bioluminescent bacteria. The physicochemical characteristics of water samples at the selected 43 sites of Luoma Lake watershed were measured. Results showed that the variations in pH value (7.31–8.41), hardness (5–20 °d) and dissolved oxygen (4.44–11.03 mg/L) of Luoma Lake and its main inflow and outflow rivers had negligible impacts on the acute toxicity testing of V. fischeri. The luminescence inhibition rates ranged from −11.21% to 10.80% at the 43 sites. Pearson’s correlation analysis in the experiment revealed that temperature, pH value, hardness, and turbidity had no correlation with luminescence inhibition rate, whereas dissolved oxygen showed a weak statistically positive correlation with a Pearson correlation coefficient of 0.455 (p < 0.05).

Explosive-contaminated soil is harmful to people’s health and the local ecosystem. The acute toxicity of its extracting solution was tested by bacterial luminescence assay using three kinds of luminescent bacteria to characterize the toxicity of the soil. An orthogonal test L 16 (45) was designed to optimize the soil extracting conditions. The optimum extracting conditions were obtained when the ultrasonic extraction time, ultrasonic extraction temperature, and the extraction repeat times were 6 h, 40 °C, and three, respectively. Fourier transform infrared spectroscopy (FTIR) results showed that the main components of the contaminated soil’s extracting solution were 2,4-dinitrotoluene-3-sulfonate (2,4-DNT-3-SO3 −); 2,4-dinitrotoluene-5-sulfonate (2,4-DNT-5-SO3 −); and 2,6-dinitrotoluene (2,6-DNT). Compared with Photobacterium phosphoreum and Vibrio fischeri, Vibrio qinghaiensis sp. Nov. is more suitable for assessing the soil extracting solution’s acute toxicity. Soil washing can remove most of the contaminants toxic to luminescent bacterium Vibrio qinghaiensis sp. Nov., suggesting that it may be a potential effective remediation method for explosive-contaminated soil.

In 2013, the “Qingdao oil pipeline explosion” released an estimated 2000 tons of oil into the environment. Sediment samples were collected from ten sites in Jiaozhou Bay and Shilaoren Beach to evaluate the influence of the spilled oil on the benthic environment 3 years after the oil spill accident. The compositions of oil, bacterial diversity and biotoxicity were examined in this study. The results showed that the concentration of total petroleum hydrocarbons (TPHs) peaked near the oil leak point and gradually decreased along the coastline, ranging from 21.5 to 133.2 μg/g. The distribution of polycyclic aromatic hydrocarbons (PAHs) was correlated with TPH, and naphthalenes were dominant in the 20 detected PAHs. The bacterial diversities in seriously polluted and slightly polluted sediments were completely different. As degrading bacteria, Alcanivorax and Lutibacter were the main genera at the oil-polluted sites. The analysis of biotoxicity by the luminescent bacteria method showed great differences among the polluted sites, the control site in Jiaozhou Bay, and the non-polluted site outside of Jiaozhou Bay. The biotoxicity also peaked at the site near the oil leak point. These results indicate that the oil spill that occurred 3 years ago still affects the environment and impacts the bacterial communities in the sediments. Electronic supplementary material The online version of this article (10.1186/s13568-018-0603-6) contains supplementary material, which is available to authorized users.

One of the major problems of airport operation is the impact of pollution caused by runoff waters. Runoff waters at an airport may contain high concentrations of different contaminants resulting from various activities of its operation. High quantities of aircraft de-icing/anti-icing fluids are used annually at airports worldwide. Aircraft de-icers and anti-icers may have negative environmental impacts, but their effects on aquatic organisms are virtually unknown. In order to address this issue, aircraft de-icers, pavement de-icers and wastewater samples were obtained from a regional airport. To evaluate the toxicity of wastewater samples and aircraft de-icing/anti-icing fluids (ADAFs), two bio-tests were performed: the Lemna growth inhibition test according to OECD guideline 221 and the luminescent bacteria test according to ISO guideline 11348-2. In the Lemna growth inhibition test, phytotoxicity was assessed using the endpoints frond number and frond area. The luminescent bacteria test involved the marine bacterium Vibrio fischeri. The estimates of effective concentrations (EC50) values were determined using the free software R and the "drc" library. Aquatic plants and marine bacteria showed a higher sensitivity towards ADAFs than to wastewater samples. Experiments showed that aircraft de-icing/anti-icing fluids and wastewater samples were relatively more toxic towards Lemna gibba L. in comparison to V. fischeri.

Crassostrea virginica (the Eastern or American oyster) bioaccumulates pollutants from the water column, and therefore, its tissues can be used as bioindicators of past and present estuarine health. In this pilot project, we decided to investigate whether its tissues would be a suitable medium for toxicity testing using tissues from a variety of southern Texas locations of known and suspected anthropogenically impacted and unimpacted areas. We also conducted toxicity tests on sediments adjacent to oyster reefs using standard protocols for sediment toxicity. We tested the toxicity of tissues and sediments on the luminescent bacteria Vibrio fischeri, whose bioassays are commonly referred to by the trade name Microtox®. Microtox tests are quick, relatively inexpensive and sensitive to a range of contaminants. Evidence from this preliminary study suggests that conducting toxicity tests on oyster tissues may predict localized contamination better than when conducting toxicity tests on subtidal sediment. The refinement of these methods to use oyster tissues to detect contamination may be especially useful for environmental impact studies and/or studies where rapid and inexpensive information is needed.

Following urbanization, a large number of urban rivers were contaminated and turned to black-odor rivers. The traditional approach for detecting water quality is based on chemical or physical analysis. However, biological toxicity of black-odor water has been less addressed. As two typical black-odor rivers, Jiushanwai River (JS) and Shanxia River (SX) are tributaries of Wen-Rui Tang River in Wenzhou (south of China). The eco-safety of the urban rivers was evaluated by bioassay for water toxicity in this study. Ten and 5 sampling sites were respectively set along JS and SX. Water samples were collected monthly from October 2010 to October 2011. The general physical and chemical parameters of river water were monitored. In order to investigate the ecotoxicological effects of black-odor water, the following bioassays were used: (1) Fish acute toxicity test (Danio rerio, comprehensive toxicity), (2) luminescent bacteria bioassay (Qinghaiensis vibrio, toxicity to bacteria), and (3) tropical claw embryo assay (Xenopus tropicalis, embryo toxicity). Biotoxicity of black-odor rivers water was demonstrated by D. rerio, Q. vibrio, and X. tropicalis embryos. Toxicological effects of black-odor water were respectively shown by mortality of zebrafish, and by the relative inhibitory light rate of luminescent bacteria. However, luminescent bacteria were more sensitive to inspect biotoxicity than zebrafish. In X. tropicalis embryos test, toxicological effects of black-odor water were mostly shown by embryos’ survival rate and teratogenic rate. Bioassay results showed that toxicity of SX water was higher than that of JS water, especially in summer. Statistical analysis of luminescent bacteria toxicity test showed that biotoxicity of SX and JS was high in summer, but low in winter and spring. The seasonal changes of water toxicity of the black-odor river were positively correlative with changes of water temperature (p

Purpose During the late 1950s and early 1960s, industrial waste material highly enriched with various contaminants (e.g., heavy metals, polycyclic aromatic hydrocarbons (PAHs)) was dumped in the inner Bay of Mecklenburg, western Baltic Sea. Between 2002 and 2004, a research program was initiated using chemical analysis in combination with bioanalytical techniques to assess the extent and variability in contamination at this dump site (DS). The data were compared to a reference area (RS) with similar environmental conditions, which is representative of the western Baltic Sea. Materials and methods Twelve PAHs were investigated to assess their ecological hazard, as theywere identifiedas major pollutants in the dumped material. In addition to analyzing the actual PAH contamination status in the sediments, PAHs measured in the soft tissue of Arctica islandica were also used as an indicator of contaminant bioaccumulation. A biotest battery was applied to determine the toxic effects of contaminants in the sediment. Results and discussion Significantly elevated PAH concentrations (sum of 12 PAHs) of ∼3,000 ng g −1 dw and higher bioaccumulation factors (BAFs) were determined in the soft body tissue of A. islandica collected at DS (t test, p=0.025). The results also showed that the sediment PAH contamination was significantly higher at DS (1,952–5,466 ng g −1 dw) than at RS (1,384– 2,315 ng g −1 dw). The results revealed a major heterogeneity in the PAH concentration at DS due to an attempt to cover the toxic material with clean clay. This resulted in a more heterogeneous distribution of the dump material rather than covering it up completely. However, not all relevant contaminants were included in this study, not only because it is too costly to determine them all but also because unidentified contaminants present at concentrations below the limit of detection cannot be measured. Bioassays were used to fill this gap in the hazard assessment in a cost-effective way by investigating the possible effects of sediment contamination on benthic organisms. The results showed a high variability and magnitude of growth and luminescence inhibition. Bacterialcontact tests withmarineorganisms showeda high toxicity response (>80 % inhibition) from DS sediments. In contrast, the luminescent bacteria test (Vibrio fischeri) showed equivalent effects of sediments from both DS and RS. Conclusions The spatial distribution of toxicity in DS, the bioaccumulation in mussels and the analytical evidence of

Luminescence intensity of recombinant Escherichia coli and Bacillus subtilis strains with cloned luxCD(AB)E genes of the natural luminescent microorganism Photobacterium leiognathi was studied under the influence of 30 individual samples of human blood serum of different component composition. A relationship was found between the level of residual bioluminescence and degree of the bactericidal effect. Moreover, the inhibition of E. coli lux + luminescence was shown to be related to activity of the complement-lysozyme system. The reaction of B. subtilis lux + primarily depended on the presence of β-lysin in the blood serum. These data provide an experimental substantiation of a new method of differential analysis of humoral factors of nonspecific innate immunity with recombinant luminescent bacteria.

The fate of canola oil in water under aerobic conditions was studied in respirometry tests. First-order biodegradation rates were estimated for a refined oil (commercial canola oil, CCO), 0.0037 h−1, and for two synthetic oils, one prepared with mono-acid triacylglycerols (TAO) 0.0048 h−1, and the other with free fatty acids and glycerol (FAO) 0.0038 h−1. Two abiotic factors limited substrate bioavailability: the autoxidation of the oleoyl chains and the insolubility of fully saturated triacylglycerols. In addition, the ecotoxic impact of the oils was assessed by Microtox® toxicity. For the acylglycerol oils, luminescent bacteria inhibition was not detected in the water phase, whereas solid-phase EC50 values below 1.8% sample volume were measured. Toxicity was observed in the aqueous phase (EC50

Microfibrillated cellulose (MFC), also referred to as nanocellulose, is one of the most promising innovations for forest sector. MFC is produced by fibrillating the fibres under high compression and shear forces. In this study we evaluated the worker exposures to particles in air during grinding and spray drying of birch cellulose. Processing of MFC with either a friction grinder or a spray dryer did not cause significant exposure to particles during normal operation. Grinding generated small amount of particles, which were mostly removed by fume hood. Spray dryer leaked particles when duct valve was closed, but when correctly operated the exposure to particles was low or nonexistent. To assess the health effects of the produced MFC, mouse macrophages and human monocyte derived macrophages were exposed to MFC and the viability and cytokine profile of the cells were studied thereafter. No evidence of inflammatory effects or cytotoxicity on mouse and human macrophages was observed after 6 and 24 h exposure to the materials studied. The results of toxicity studies suggest that the friction ground MFC is not cytotoxic and does not cause any effects on inflammatory system in macrophages. In addition, environmental safety of MFC was studied with ecotoxicity test. Acute environmental toxicity assessed with kinetic luminescent bacteria test showed high NOEC values (>100 mg/l) for studied MFC. However, MFC disturbed Daphnia magna mobility mechanically when the test was performed according to the standard procedure.

Biosensors based on luminescent bacteria may be valuable tools to monitor the chemical quality and safety of surface and drinking water. In this review, an overview is presented of the recombinant strains available that harbour the bacterial luciferase genes luxCDABE, and which may be used in an online biosensor for water quality monitoring. Many bacterial strains have been described for the detection of a broad range of toxicity parameters, including DNA damage, protein damage, membrane damage, oxidative stress, organic pollutants, and heavy metals. Most lux strains have sensitivities with detection limits ranging from milligrams per litre to micrograms per litre, usually with higher sensitivities in compound-specific strains. Although the sensitivity of lux strains can be enhanced by various molecular manipulations, most reported detection thresholds are still too high to detect levels of individual contaminants as they occur nowadays in European drinking waters. However, lux strains sensing specific toxic effects have the advantage of being able to respond to mixtures of contaminants inducing the same effect, and thus could be used as a sensor for the sum effect, including the effect of compounds that are as yet not identified by chemical analysis. An evaluation of the suitability of lux strains for monitoring surface and drinking water is therefore provided.

In this study, the mixture of mono- and di-rhamnolipids produced by Pseudomonas aeruginosa DS10-129 was characterized for its toxicity and modulatory effects on Cd availability to different bacteria. Gram-negative naturally bioluminescent Vibrio fischeri and recombinant bioluminescent Pseudomonas fluorescens, P. aeruginosa, Escherichia coli, and Gram-positive Bacillus subtilis were used as model organisms. Rhamnolipids reduced the bioluminescence of these bacteria in less than a second of exposure even in relatively low concentrations (30-min EC(50) 45-167 mg l(-1)). Toxicity of Cd to Gram-negative bacteria (30-min EC(50) values 0.16 mg l(-1) for E. coli, 0.96 mg l(-1) for P. fluorescens, and 4.4 mg l(-1) for V. fischeri) was remarkably (up to 10-fold) reduced in the presence of 50 mg l(-1) rhamnolipids. Interestingly, the toxicity of Cd to Gram-positive B. subtilis (30-min EC(50) value 0.49 mg l(-1)) was not affected by rhamnolipids. Rhamnolipids had an effect on desorption of Cd from soil: 40 mg l(-1) rhamnolipids increased the water-extracted fraction of Cd twice compared with untreated control. However, this additionally desorbed fraction of Cd remained bound with rhamnolipids and was not available to bacteria. Hence, in carefully chosen concentrations (still effectively complexing heavy metals but not yet toxic to soil bacteria), rhamnolipids could be applied in remediation of polluted areas.

The increasing multi-resistance of pathogenic bacteria requires the development of novel classes of antibiotics. Antimicrobial host defense peptides represent one promising class. Here we describe a protocol for screening large numbers of peptides against any microbe of interest. Peptides synthesized on a cellulose support by peptide array technology can be added to a microbe that expresses the luxCDABE (luciferase) gene cassette. Any substance that decreases the energy level within the microbe will cause a quantifiable decrease in light production. The potency of the compound, at different concentrations, is reflected by the rate of decrease in luminescence. In conjunction with peptide array technology, the screening assay is rapid and high throughput and demonstrates good correlation with conventional killing or minimal inhibitory concentration assays performed with the same peptides synthesized by standard solid-phase peptide synthesis. The protocol can be completed in 3 d.

Intensity of luminescence quenching in recombinant strains of Escherichia coli with cloned lux-operones by human blood serum is directly proportional to the degree of bactericidal effect assessed by nephelometric and bacteriological methods. This correlation was most characteristic of E. coli with luminescence genes from Photobacterium leiognathi, which substantiates its use in the development of the kinetic bioluminescent method to determine of serum bactericidal activity. The possibility of using this method for evaluation of activity of classic and alternative pathways of compliment activation was demonstrated by using zymosan or EGTA-Mg(2+)-treated sera and C1-C5-deficient sera.

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.

Recent studies indicated that bioluminescence of the marine bacterium Vibrio harveyi may both stimulate DNA repair and contribute to detoxification of deleterious oxygen derivatives. Therefore, it was also proposed that these reactions can be considered biological roles of bacterial luminescence and might act as evolutionary drives in development of luminous systems. However, experimental evidence for the physiological role of luciferase in protection of cells against oxidative stress has been demonstrated only in one bacterial species, raising the question whether this is a specific or a more general phenomenon. Here we demonstrate that in the presence of various oxidants (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and ferrous ions) growth of dark mutants of different strains of Vibrio fischeri and Photobacterium leiognathi is impaired relative to wild-type bacteria, though to various extents. Deleterious effects of oxidants on the mutants could be reduced (with different efficiency) by addition of antioxidants, A-TEMPO or 4OH-TEMPO. These results support the hypotheses that (1) activities of bacterial luciferases may detoxify deleterious oxygen derivatives, and (2) significantly different efficiencies of this reaction are characteristic for various luciferases.

De-icers are essential to wintertime road maintenance in Nordic countries. The use of road salts (NaCl, CaCl2 and MgCl2) is having significant effects on groundwater. There has been a growing concern of environmental effects resulting from de-icing. Regulatory bodies have tried to find de-icers which have less harmful impacts on soil and groundwater. Alternative new organic de-icers are e.g. potassium formiate, potassium acetate, and calcium magnesium acetate. At present, the decision finding for soil and water quality assessment is mainly based on the results of chemical analyses. However, ecotoxicological characterization will provide more useful information for determining environmental effects of de-icers. Six different de-icers, NaCl, CaCl2, MgCl2, potassium formicate KFo, potassium acetate KAc and calcium magnesium acetate CMA were studied with different bioassays. These bioassays were two plant tests (onion Allium cepa root elongation test and duckweed Lemna growth inhibition test), an enzyme inhibition test (reverse electron transport test, RET) and a microbial test (luminescent bacteria Vibrio fischeri, BioTox test). Most of the responses of the de-icers tested were toxic to test organisms. CMA was unsuitable for RET and BioTox tests because it was not possible to analyze turbid samples with these bioassays. On the whole, organic chemicals were more toxic than the inorganic salts. According to the Lemna and Allium cepa tests KFo was the most toxic and the toxicity ranking of the de-icers was KFo > KAc > CMA > CaCl2 > NaCl > MgCl2. The sensitivity ranking for tests in decreasing order was Lemna > Allium cepa > RET > BioTox. The plant tests proved to be relevant, easy, cost-effective and sensitive screening methods of de-icers: Lemna test was 92 times more sensitive and Allium test 70 times more sensitive than BioTox to KFo. The tested de-icers had significant effects on plant growth, microbial luminescence and enzymatic activity. Toxicity could hardly be predicted from the chemical concentrations of these de-icers. In summary, these results demonstrate a need for including toxicity measurements in the evaluation of technologies used in soil treatment and illustrate the potential value of such measurements for ensuring that no or minimal environmental contamination would arise from de-icing process.

The key to effective cleaning and disinfection of food plants is the understanding of the type of the soil to be removed from the surfaces. An efficient cleaning and disinfection procedure consists of a sequence of rinses using good quality water with application of detergents and disinfectants. Disinfection is required in food plant operations, where wet surfaces provide favourable conditions for the growth of microbes. The efficacy of disinfectants is usually determined in suspensions, which do not mimic the growth conditions on surfaces where the agents are required to inactivate the microbes. Therefore, the suspension tests do not give adequate information and reliable carrier tests, which mimic surface growth, are needed. In developing a proposal for the testing of disinfectants on surfaces to an analytical standard, it is important to identify the major sources of variation in the procedure. In response to the need for a relatively realistic, simple and reliable test for disinfectant efficacy a method for culturing laboratory model biofilms has developed. The use of artificial biofilms i.e. biofilm-constructs inoculated with process contaminants in disinfectant testing can also be used for screening the activity of various disinfectants on biofilm cells. Both biofilm carrier tests showed clearly that the biofilm protects the microbes against the disinfectants. The chemical cleanliness is also essential in food plants. The total cleanliness of the process lines is mainly based on measuring the microbial load using culturing techniques. These results can give an incorrect picture of the total cleanliness, because the viable microbes do not grow when disinfectants are left on the surface. The luminescent bacteria light inhibition method offers a useful alternative for testing chemical residue left on surfaces after cleaning and disinfection operations.

Petroleum products are widely used in various sections of industry and they are one of the most abundant sources of environmental contamination. These products are classified by their physico-chemical properties such as boiling point, density and viscosity. Oil contamination in the environment is primarily evaluated by measuring the chemical concentrations of petroleum products in the solid or water phase. The results of chemical analyses do not correspond directly with the harmful environmental effects of petroleum products on the soil flora and fauna, because the interactions between oil compounds and the production of their methabolites in soil are not measured in chemical assessments. These kinds of effects of complex chemical mixtures in soil can be estimated by bioassays. Therefore, ecotoxicological tests are important for estimating soil quality in the risk assessment of oil-contaminated soil sites. The objective of this study was to examine the oil-contaminated soil site of a closed petrol station with both chemical and ecotoxicological methods. The goals of this study were to compare the sensitivity of the terrestrial and aquatic bioassays and to compare the toxicity responses of aquatic bioassays determined from three different extraction procedures. In addition, our aim was to characterise a cost-effective battery of bioassays that could be applied to a comparison of oil-contaminated soils. It was in our interest to investigate oil-contaminated soil with oil concentrations of 2500–12000 mg/kg and to find out the possible differences between terrestrial and aquatic toxicity tests. Six soil samples from a closed petrol station were examined for toxicity with terrestrial and aquatic tests. Terrestrial tests includedEnchytraeus albidus survival and reproduction assays and seed germination assays using wheat, cress, lettuce, and red clover seeds and growth inhibition assays of onions. The toxicities of the water-extractable fractions of the soil samples obtained from three different extractions were tested with aquatic bioassays based on plants (onion and duckweed growth inhibition tests), microbes (luminescent bacteria test), and enzyme inhibition (reverse electron transport test, RET). Chemical analyses of the solid samples were carried out simultaneously. Results. Oil concentrations ranged from 2500 to 12000 mg/kg, BTEX varied from 300 to 2800 mg/kg, and fuel additives: MTBE and TAME from 0.0 1 to 260 mg/kg. Only the sample contain-ing 12000 mg/kg oil had a significant toxic impact on all test organisms. Soil samples with oil concentrations 2500–6200 mg/ kg had no or only slight adverse effects on the test organisms with one exception, theE. albidus reproduction test. TheE. albidus survival and reproduction tests were the most sensitive bioassays of the terrestrial tests, and the luminescent bacteria test of the aquatic tests.

A filter dust of a waste incineration plant provided for underground disposal was elutriated with three different elutriation media possibly be found in underground disposal sites. Two of these media were salt solutions being relevant for salt mines, when water comes into contact with the saltincrustation. The other one was a groundwater, relevant for non-salt mines, for example abandoned coal mines. The genotoxicity of these elutriates was tested with the MUTATOX™ test, using dark mutants of luminescent bacteria. In addition total organic carbon (TOC) and elementcontents of the elutriates were determined. The groundwaterelutriate was not genotoxic, but the two salt solution elutriates were. TOC of the elutriates was below detection limit (

Samples of urban dusts, road site dusts and car park dusts, were collected at two selected sites each in Hong Kong and London. Sequential extraction was used to characterise the chemical compositions of these urban dusts. Copper, lead, zinc, pH, electrical conductivity and organic content were measured. Biotoxicity tests of urban dusts were conducted on higher plants (Brassica chinensis and Lolium perenne), a dinoflagellate green alga (Dunaliella tertiolecta) and luminescent bacteria (Photobacterium phosphoreum). A significant correlation was found between total lead (r =; – 0.70, p < 0.01) and zinc (r =; – 0.74, p < 0.05), and the 20 min – EC50 using P. phosphoreum. In addition, there was a significant correlation (r = – 0.72, p < 0.01) between the exchangeable lead content in dust and the 48 h- EC30 using D. tertiolecta. No specific trend was obtained for higher plants. Total lead and zinc contents were higher in dusts from London while the percentage of exchangeable fraction of metal contents was higher in those from Hong Kong.

A new sensing system based on the immobilization of luminescent bacteria,Photobacterium phosphoreum, was proposed for continuous real-time monitoring of pollutants. The response curves demonstrate thatPhotobacterium phosphoreum immobilized on the strontium alginate was very sensitive to seven reference chemicals used. The significant inhibitory concentrations for bioluminescence emission were 5 ppm for Pb(NO3)2, NiCl2, CdCl2, 50 ppm for NaAsO2, 0.1 ppm for HgCl2, 0.5 ppm for pentachlorophenol and less than 5 ppm for SDS, respectively. The alginate mixed-cells (AMC) retained their luminescence during experimental period (29 days) under storage condition of − 80°C. The variables affecting performance of continuous flow through monitoring (CFTM) were optimized in order to ensure stability and efficiency. The flow through cell with strontium-alginate immobilized luminescent bacteria was tested with salicylate and 4-nitrophenol and a rapid response of luminescence was recorded by time drivemode in bioluminescence spectrometer after exposure to both toxicants.

Because luminescent microorganisms change their light emission in the presence of bioactive substances, they provide an effective way of monitoring toxicity. Here we report the direct coupling of bioluminescence to chromatography which is capable of detecting single toxic components in complex mixtures. Two approaches were investigated, utilizing thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The TLC-bioluminescence imaging technique was developed into a versatile and rugged method and proved to be superior to the HPLC-based design. Employing genetically engineered luminescent bacteria gives customized selectivity of bioactivity detection. The combination of analytical separation technology with the biospecific sensing ability of living cells provides a novel screening tool for targeting bioactive components in complex samples.

Photobacterium kishitanii is one species of luminescent bacteria. This bacterium is known to convert chemical energy into light; it glows in the dark with a visible peak wavelength (ca. 475 nm) that is easily recognized. Luminescent bacteria produce autoinducers and respond to this molecule to switch on the luciferase structural operon. Luminescence is, therefore, controlled by the cell-population density.If bacterial cells are viewed as enzyme bags, substrates, such as oxygen or autoinducers, diffuse into the bags through a semipermeable cell wall and are catalyzed by the enzyme. Oscillation in the product concentration is often observed in systems where a semipermeable membrane separates the substrate and the enzyme. Such behavior is simulated using a reaction-diffusion model.We have reported several characteristics of the luminescence from a bacterial suspension. For example, in a batch culture, higher initial bacteria density resulted in an earlier luminescence starting time [1]. Apart from such linear characteristics, we have reported an oscillation in the luminescence intensity both spatially and temporally [2]. We hypothesize that a group of bacteria behaves differently from a sum of single cells. The nonlinearity of the bacterial luminescence might be a key to understanding the phenomenon. In a previous experiment, we separated bacteria into small groups that showed similar characteristics, such as motility and adsorption activity. For example, bacteria separated according to their motility using a microfluidic device were proved to show different bioluminescent intensities for each cell [3]. On an agar plate, the luminescence intensity from actively dividing cells was less than that from mature cells [4]. In addition to this basic study to understand the oscillation in bacterial luminescence, reactors were designed to realize stable bacterial luminescence. In a PDMS cell, only the parts of the suspension that faced the wall were illuminated [5]. This result suggested that the geometrical symmetry of oxygen supply to the suspension helped maintain spatial stability without convection of the bacterial luminescence.Here, we report the luminescence behavior of bacteria that adsorbed on material surfaces. In this work, we show experimentally that the bacteria adsorb on the inner surfaces of a polyethylene terephthalate (PET) or polystyrene (PS) bottle and start to emit light when a liquid broth is added. We also show that the luminescence from the suspension oscillates. Experimentally, using a self-made luminescence detector, measurement of luminescence intensity from well-stirred bacterial suspensions in the bottles of different materials (PVC, PET, or PS) was performed. After the observation of oscillation in luminescence, the bottles were washed three times using the same broth, followed by the luminescence measurement. Such washing and measurements were repeated, and oscillation was repeatedly observed. The chemical condition of adsorption on the surface was investigated using surface analysis methods, such as AFM or FTIR, and the material characteristics with regard to cell adsorption and oscillation mode were discussed.

The database of luminescent bacteria stored in the IBSO collection is one of the metasections of BIOLUMBASE. A logical schema of the metasection “Natural luminescent organisms”, classification of entities, and methods of attribute presentation have been developed. The database of luminescent bacteria maintained in the IBSO collection is being widened by findings of the collection staff as well as by information from scientific literature. The expectant contents of the database will be useful for resolving various problems of microbial ecology and biotechnology which deal with luminescent bacteria, luminescent system derived from them, and lux-genes cloned to other organisms. A potential user would be able not only to access cataloged data on strains but also to get information on properties, functions, use, and bibliography and to perform an attribute-match search of a strain.

Cells of Photobacterium phosphoreum (strain 496) grow equally well in a complex medium with 1% and 3% NaCl, but luminescence occurs with 3% but not 1% NaCl in the medium. However, log phase cells growing in 1% NaCl will develop luminescence within 30 to 45 min following a shift to 3% NaCl. Unlike the situation in certain other species of luminescent bacteria, this increase is not paralleled by increases in luciferase, the key enzyme in the luminescent reaction. In the present study it has been found that nalidixic acid and novobiocin, inhibitors of DNA gyrase, block this development of bioluminescence at concentrations where growth is not completely inhibited, but without concomitant changes in extractable luciferase activity. In a nalidixic acid-resistant mutant, growth was normal, but luminescence was greater than in the wild type. Addition of nalidixic acid stimulated luminescence even more, but again without concomitant changes in extractable luciferase activity. These results suggest that transcription of the certain genes affecting bioluminescence in bacteria is differentially sensitive to gyrase activity, and thus to DNA structure and/or conformation. However, the proteins and/or factors responsible for affecting bioluminescence under these conditions have not been identified.

Organic concentrates of the River Rhine water were tested for toxicity with a 15 minute test (MicrotoxTM) on luminescent bacteria (Photobacterium phosphoreum, Bacteria). A total number of 18 stations in the Dutch lower Rhine area were sampled five times with an interval of one month (April 1988–August 1988), whereas two stations located the most apart (Lobith and Maassluis) were sampled with two-week intervals in the period between January and October. The river samples were concentrated by neutral absorption on XAD resins, followed by elution with acetone. Toxicity is expressed on a relative scale. The relative toxicity is in the range of approximately 3 to 18, which means that the organic toxicants present in Rhine water had to be concentrated between 55-and 330-fold. During transport in The Netherlands, the dissolved organic toxicity contained in the water of the River Rhine decreases gradually, either as a consequence of degradation, or by adsorption to newly formed particulates. In the industrialized areas of the lower Rhine in The Netherlands a slight, but temporary increase of toxicity is observed. Since water quality improved strongly with respect to inorganic (heavy metal) pollution, the organic toxicity reflects the actual pollutional status more closely than in former days. The speed and simplicity of the combination of XAD concentration and Microtox toxicity determination enables large-scale monitoring studies on organic toxicants in moderately polluted surface water.

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.

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.

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.

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.

After 3 weeks of starvation the bacterial light-organs of the bioluminescent shallow-water fishAnomalops katoptron cease to produce light. Because of a reduction of the number of symbionts in the cells of the light organ, it is concluded that the fish supplies its luminescent bacteria with nutrients out of its own metabolism. As a result of starving the fish, the luminescent bacteria decrease in number and finally cease to emit light.

The effect of temperature and salinity on numbers of luminescent bacteria present in waters of the Mystic (Conn.) River estuary was evaluated. Counts decreased with decreasing salinity; none were detected at freshwater stations. A population maximum of 35 per ml was noted at the highest salinity station (30±2‰). Highest counts were observed during winter and spring and lowest numbers occurred during summer and fall months. Isolates (111) were identified and compared with previously-described luminescent bacteria; i.e.,Beneckea (Vibrio) harveyi, Photobacterium (V.) fischeri, P. phosphoreum, andP. leiognathi. All species were isolated but distinct seasonal differences were noted.P. (V.) fischeri andB. (V.) harveyi represented 93% of the luminous population on an annual basis. Only the former was found during the period December through March (highest count 7 per ml) whileB. (V.) harveyi was the dominant species noted between May and October (maximum count 11 per ml).P. leiognathi andP. phosphoreum were found only during July and August as 7% of the total luminous population. All isolates grew at NaCl concentrations between 6 and 30‰; none grew below 6‰

WE have postulated previously that the catalytic decomposition by catalase of hydrogen peroxide to molecular oxygen and water is accompanied by changes in the valency of catalase iron1. This supposition is strongly supported by spectroscopic and manometric experiments carried out on azide-catalase. As additional evidence in support of this view, we brought forward the results of experiments carried out in Barcroft differential manometers, showing that under certain conditions even the activity of free catalase is inhibited when oxygen is completely removed from the surrounding medium and replaced by pure nitrogen. Our manometric experiments were repeated by Weiss and Weil-Malherbe2 who, using Warburg manometers, failed to obtain this inhibition. Johnson and van Schouvenburg3 also failed to confirm our results using luminescent bacteria as indicators of decomposition of hydrogen peroxide by catalase in complete absence of oxygen. However, we repeated our experiments from time to time, using different enzyme preparations, and invariably confirmed our previous results. The failure by other workers to obtain similar inhibition was explained by us4, 5 as being due probably either to some difficulty in complete elimination of oxygen from Warburg manometers or to some defects in their enzyme preparations.