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A bioluminescent enzyme inhibition-based assay was applied to predict the potential toxicity of carbon nanomaterials (CNM) presented by single- and multi-walled nanotubes (SWCNT and MWCNT) and aqueous solutions of hydrated fullerene С60 (C60HyFn). This assay specifically detects the influence of substances on parameters of the soluble or immobilised coupled enzyme system of luminescent bacteria: NAD(P)Н:FMN-oxidoreductase+luciferase (Red+Luc). A protocol based on the optical properties of CNM for correcting the results of the bioluminescent assay was also developed. It was shown that the inhibitory activity of CNM on Red+Luc decreased in the following order: MWCNT>SWCNT>C60HyFn. The soluble enzyme system Red+Luc had high sensitivity to MWCNT and SWCNT, with values of the inhibition parameter IC50 equal to 0.012 and 0.16mg/L, respectively. The immobilised enzyme system was more vulnerable to C60HyFn than its soluble form, with an IC50 equal to 1.4mg/L. Due to its technical simplicity, rapid response time and high sensitivity, this bioluminescent method has the potential to be developed as a general enzyme inhibition-based assay for a wide variety of nanomaterials.

The European Construction Products Regulation allows Member States to adopt rules for evaluating the environmental impact of their buildings. The aim of the project was to develop recommendations for a test battery for the ecotoxicological assessment of the environmental impact of construction products for outdoor use and contribute to the European harmonization of test methods. From a shortlist of 39 products 20 products were included in the ecotoxicological testing program. Monolithic and plate-like construction products were eluted in the Dynamic Surface Leaching test (DSLT) in accordance with CEN/TS 16637-2, granular products were eluted in a one stage batch test in accordance with DIN EN 12457-1. The eluates were examined in four aquatic toxicity tests (algae, daphnia, luminescent bacteria, fish eggs), a genotoxicity test (umu test) and in the respirometer test (OECD 301 F). Here, low to very high ecotoxicity was observed (up to a dilution factor of 1536). Six out of 8 eluates, whose TOC exceeded 10 mg L −1 showed a good biodegradability above 75%. The intra-laboratory repeatability of the Lowest Ineffective Dilution (LID) usually was within ±1 dilution steps (ecotoxicity tests) and ±2 dilution steps (leaching and ecotoxicity tests). This is acceptable, when considering that the overall variability of sample preparation, leaching test, and bioassays add up. The conclusions lead to practical recommendations for a suitable combination of leaching and ecotoxicity tests.

Potential of luminescent bacteria in production of metal nanoparticles has not been well evaluated up until now. These bacteria contain lux operon which is included of reductase enzymes, by increasing bacterial cell density, the expression of aldehyde synthetic enzymes elevate and enhance the yields of nanoparticles synthesis. Therefore, in this study extracellular synthesis of gold nanoparticles using natural occurring luminescent bacteria, VLA, VLB, VLC and genetically engineered luminescent bacteria, Pseudomonas putida KT2440 and Pseudomonas fluorescence OS8 has been successfully conducted. Nanoparticles were characterized and their antibacterial activity evaluated using microtiter plates at different concentrations against some hospital pathogenic bacteria .Biosynthetic gold nanoparticles produced in this study had maximum absorption at the ranges of 500 to 550 nanometer wavelengths. TEM images showed particle sizes between 10 to 50 nanometers and confirmed the success of purification process. The NPs were spherical and the FTIR analysis showed the existence of biomolecules on surface of purified nanoparticles that could be most probably related to reductase enzymes that is stabilized on nanoparticles surfaces. Further investigation on antibacterial properties of these novel nanoparticles which coated by reductase enzymes showed that any increase or decrease in antibacterial activity is depended on nanoparticles concentration.

Traditionally, the toxicity of river contaminants is analyzed chemically or physically through river bed sediments. The biotoxicity of polluted sediment leachates has not caught our attention. This study aims to overcome this deficiency through a battery of biotests which were conducted to monitor comprehensive toxicity of sediment leachates for the Yaogang River in East Jiangsu Province of China, which is in close proximity to former pesticide plants. The general physical and chemical parameters of major pollutants were analyzed from river bed sediments collected at five strategic locations. The ecotoxicity analyses undertaken include overall fish (adult zebrafish) acute toxicity, luminescent bacteria (Vibrio fischeri) bioassay, and zebrafish embryo toxicity assay. Compared with the control group, sediment leachates increased the lethality, inhibited the embryos hatching and induced development abnormalities of zebrafish embryos, and inhibited the luminescence of V. fischeri. The results show that sediment leachates may assume various toxic effects, depending on the test organism. This diverse toxicity to aquatic organisms reflects their different sensitivity to sediment leachates. It is found clearly that V. fischeri was the organism which was characterized by the highest sensitivity to the sediment leachates. The complicated toxicity of leachates was not caused by one single factor but by multiple pollutants together. This indicates the need of estimations of sediment leachate not only taking into account chemical detection but also of applying the biotests to the problem. Thus, multigroup bioassays are necessary to realistically evaluate river ecological risks imposed by leachates.

Drinking water treatment residue (DWTR), a byproduct generated during potable water production, exhibits a high potential for recycling to control eutrophication. However, this beneficial recycling is hampered by unclear metal/metalloid pollution risks related to DWTR. In this study, the pollution risks of Al, As, Ba, Be, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, and Zn due to DWTR application were first evaluated for lake water based on human health risk assessment models and comparison of regulatory standards. The risks of DWTR were also evaluated for sediments on the basis of toxicity characteristics leaching procedure and fractionation in relation to risk assessment code. Variations in the biological behaviors of metal/metalloid in sediments caused by DWTR were assessed using Chironomus plumosus larvae and Hydrilla verticillata. Kinetic luminescent bacteria test (using Aliivibrio fischeri) was conducted to analyze the possibility of acute and chronic detrimental effects of sediment with DWTR application. According to the obtained results, we identify a potential undesirable effect of DWTR related to Fe and Mn (typically under anaerobic conditions); roughly present a dosage threshold calculation model; and recommend a procedure for DWTR prescreening to ensure safe application. Overall, managed DWTR application is necessary for successful eutrophication control.

The present study demonstrates development of a rapid testing protocol based on a small portable luminometer using flash kinetic assessment of bacterial bioluminescence. The laboratory comparisons based on six model organic toxicants and two metals showed significant correlations between responses of freshwater bacteria Photorhabdus luminescens and standard marine bacterial species Vibrio fisheri. While P. luminescens was less sensitive in standard arrangements, the responses of both organisms were comparable in the newly introduced portable luminometer setup. The applicability and reproducibility of the portable luminometer protocol was further demonstrated in the assessment of 43 European wastewater effluents that were simultaneously tested for toxicity and analysed for 150 organic and 20 inorganic contaminants grouped into 13 major chemical classes. Clear association between the toxic responses in both compared bacterial species and the elevated levels of inorganic compounds (toxic metals), chlorophenols and benzotriazole anticorrosives was observed. The new protocol with a portable luminometer provides a fast (30 s) response and may be used as a tool for rapid in situ toxicity evaluation of freshwater environmental samples such as effluents.

Drinking water treatment residue (DWTR) seems to be very promising for controlling lake sediment pollution. Logically, acquisition of the potential toxicity of DWTR will be beneficial for its applications. In this study, the toxicity of DWTR and sediments amended with DWTR to Aliivibrio fischeri was evaluated based on the Microtox(®) solid and leachate phase assays, in combination with flow cytometry analyses and the kinetic luminescent bacteria test. The results showed that both solid particles and aqueous/organic extracts of DWTR exhibited no toxicity to the bacterial luminescence and growth. The solid particles of DWTR even promoted bacterial luminescence, possibly because DWTR particles could act as a microbial carrier and provide nutrients for bacteria growth. Bacterial toxicity (either luminescence or growth) was observed from the solid phase and aqueous/organic extracts of sediments with or without DWTR addition. Further analysis showed that the solid phase toxicity was determined to be related mainly to the fixation of bacteria to fine particles and/or organic matter, and all of the observed inhibition resulting from aqueous/organic extracts was identified as non-significant. Moreover, DWTR addition not only had no adverse effect on the aqueous/organic extract toxicity of the sediment but also reduced the solid phase toxicity of the sediment. Overall, in practical application, the solid particles, the water-soluble substances transferred to surface water or the organic substances in DWTR had no toxicity or any delayed effect on bacteria in lakes, and DWTR can therefore be considered as a non-hazardous material.

Modern analytical test methods increasingly detect anthropogenic organic substances and their transformation products in water samples and in the environment. The presence of these compounds might pose a risk to the aquatic environment. To determine a possible (eco)toxicological risk, aquatic samples are tested using various bioassays, including sub-organismic assays such as the luminescent bacteria inhibition test, the acetylcholinesterase inhibition test, and the umu-test. The effect-directed analysis (EDA) combines physicochemical separation methods with biological (in vitro) tests. High-performance thin-layer chromatography (HPTLC) has proved to be particularly well suited for the separation of organic compounds and the subsequent analysis of effects by the application of the biotests directly on the surface of the HPTLC plate. The advantage of using HPTLC in comparison to high-performance liquid chromatography (HPLC) for EDA is that the solvent which is used as a mobile phase during chromatography is completely evaporated after the separation and therefore can no longer influence the applied bioassays.

In situ biological purification technology has been widely used in the engineering of environmental pollution treatment, since it is a more convenient and effective approach, with low cost. Therefore, the potential risk to the safety of biological agents should be seriously taken into consideration. The efficient aerobic denitrifying bacteria strain HF3, which is isolated and cultured in a low-nitrogen medium in order to remove nitrogen under oligotrophic conditions, has been used for bioremediation treatment, with effective and reliable ability in removing the nutrients of aquatic environments. Different to other biological agents, the efficient strain used for biological purification was cultured from raw water, and was isolated among indigenous bacteria. It is, therefore, the inferior species, and pathogenic bacteria had been excluded during the isolation. In addition, non-native application of the efficient strains which had a high environmental risk could be avoided during the application. In this chapter, we discuss the safety of the efficient strain HF3 by three aspects: biological safety on drinking water quality, ecological safety on indigenous microorganisms, and toxicological safety on aquatic animals. The results showed that the efficient aerobic denitrifying bacteria strain HF3 had no significant effect on the microbial community, and had no toxicity on mice, luminescent bacteria, or zebrafish. The efficient strain HF3 could be inactivated without any influence on the inactivation efficiency. The results suggest that the biological agent used for bioremediation treatment is safe and poses no risks to the urban drinking water supply, which could provide theoretical guarantees for the security of a wider range of application. It is important to reveal the effect and ecological assessment of microbial remediation. Therefore, in this chapter, we describe the effect and ecological assessment of the microbial remediation process.

This study was conducted to assess the microbial toxicity of ionic silver solution () and silver nanoparticle suspension () based on the Microtox bioassay. In this test, the light inhibition of luminescent bacteria was measured after 15 and 30 min exposure to aqueous solutions and soils spiked with a dilution series of and . The resulting dose-response curves were used to derive effective concentration (EC25, , EC75) and effective dose (, , ) that caused a 25, 50 and 75% inhibition of luminescence. In aqueous solutions, value of after 15 min exposure was determined to be and remarkably lower than value of with . This revealed that was more toxic to luminescent bacteria than . In soil extracts, however, value of with 196 mg kg-1 was higher than value of with , indicating less toxicity of in soils. The reduced toxicity of in soils can be attributed to a partial adsorption of ionic on soil colloids and humic acid as well as a partial formation of insoluble AgCl with NaCl of Microtox diluent. This resulted in lower concentration of active Ag in soil extracts obtained after 1 hour shaking with than that spiked with . With longer exposure time, EC and ED values of both and decreased, so their toxicity increased. The toxic characteristics of silver nanomaterials were different depending on existing form of Ag (, ), reaction medium (aqueous solution, soil), and exposure time.

This study was focused on photodegradation of acetaminophen (AA) exposed to ultraviolet (UV) irradiation at 254 nm delivered by a system similar to that used for sterilization in sewage treatment plants. The degradation of AA during irradiation for up to 96 h was monitored by means of high-performance liquid chromatography (HPLC). Based on the mass/charge ratio ( m / z ) of the protonated ion and the mass fragmentation pattern in electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS/MS), the photoproduct was identified as 1-(2-amino-5-hydroxyphenyl)ethanone ( 1 ). Examination of toxicity by means of a luminescent bacteria test (ISO11348) indicated that AA solution was nontoxic, whereas photo-exposed AA solution was toxic (EC 50 : 29.46 mg/L). The toxicity of synthetic compound 1 was shown to account for a substantial part of the toxicity of photo-exposed AA. These results indicate the importance of investigating not only parent compounds, but also photoproducts the risk assessment of pharmaceuticals in aquatic environments.

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.

When selecting a landfill leachate treatment method the contaminant composition of the leachate should be considered in order to obtain the most cost-effective treatment option. In this study the filter material pine bark was evaluated as a treatment for five landfill leachates originating from different cells of the same landfill in Sweden. The objective of the study was to determine the uptake, or release, of metals and dissolved organic carbon (DOC) during a leaching test using the pine bark filter material with the five different landfill leachates. Furthermore the change of toxicity after treatment was studied using a battery of aquatic bioassays assessing luminescent bacteria (Vibrio fischeri) acute toxicity (30-min Microtox®), immobility of the crustacean Daphnia magna, growth inhibition of the algae Pseudokirchneriella subcapitata and the aquatic plant Lemna minor; and genotoxicity with the bacterial Umu-C assay. The results from the toxicity tests and the chemical analysis were analyzed in a Principal Component Analysis and the toxicity of the samples before and after treatment was evaluated in a toxicity classification. The pine bark filter material reduced the concentrations of metal contaminants from the landfill leachates in the study, with some exceptions for Cu and Cd. The Zn uptake of the filter was high for heavily contaminated leachates (≥73%), although some desorption of zinc occurred in less contaminated waters. Some of the leachates may require further treatment due to discharge into a natural recipient in order to reduce the risk of possible biological effects. The difference in pH changes between the different leachates was probably due to variations in buffering capacity, affected by physicochemical properties of the leachate. The greatest desorption of phenol during filtration occurred in leachates with high conductivity or elevated levels of metals or salts. Generally, the toxicity classification of the leachates implies that although filter treatment with pine bark removes metal contaminants from the leachates effectively, it does not alter leachate toxicity noticeably. The leachates with the highest conductivity, pH and metal concentrations are most strongly correlated with an increased toxic response in the score plots of both untreated and treated leachates. This is in line with the toxicity classification of the leachate samples. The results from this study highlight the importance of evaluating treatment efficiency from the perspective of potential recipient effects, rather than in terms of residual concentrations of individual contaminants when treating waters with a complex contamination matrix, such as landfill leachates.

Bioluminescence is emitted by various living organisms, including bacteria. While the induction mechanism in marine luminescent bacteria, such as Vibrio fischeri and V. harveyi, has been well characterized, this mechanism has not been studied in detail in the non-marine luminescent bacterium Photorhabdus luminescens. Therefore, we investigated the effect of cations and anions on the induction of luminescence by P. luminescens. Cultivation of cells in an inorganic salts solution (ISS) containing KCl, CaCl2, MgCl2, NaHCO3, and MgSO4 resulted in a rapid increase in luminescence intensity. Moreover, the induction of luminescence in the ISS medium was not dependent on cell density, since cell densities remained unchanged during 48 h. Furthermore, we found that compounds containing K+, Mg2+, and HCO3– were necessary to induce cell density-independent luminescence. The intensity of luminescence per cell cultured in medium containing KCl, MgCl2, and NaHCO3 was approximately 100-fold higher than that cultured in NB. In contrast, when cells actively grew in normal growth condition, the intensity of luminescence per cell was not increased even in the presence of K+, Mg2+, and HCO3–. Thus, these results suggest that the luminescence of P. luminescens is regulated by 2 independent cell density-dependent and -independent mechanisms.

The environmental impact of two biocide-free antifouling paints, fluoropolymer and silicone types, painted on a test cylinder was assessed using a battery of ecotoxicity test and chemical analyses for organic micro-pollutants such as perfluoroalkyl substances (PFAS). A biocide paint containing zinc pyrithione (ZnPT2) and cuprous oxide was assessed as a positive control. A standardized laboratory rotating-cylinder method using each test cylinder with artificial seawater was performed for 45 days. After 1 h rotation, the leaked seawater was subjected for bioassay and chemical analyses twice weekly. The seawater extracts from the biocide paint showed adverse effects on bacteria, algae, and crustaceans, but those from the biocide-free paints did not. The leakage seawater from biocide-free paints, after 7-day continuous rotation, contained the same concentration levels of PFAS as blank seawater used to conduct the tests. Thus, no significant toxicities of the biocide-free paints were found under the conditions...

Here we provide a molecular description of a new psychrophilic strain, KCh11, of marine luminescent bacteria classified as Aliivibrio logei . We sequenced the entire lux operon of A. logei KCh1 and showed that it is substantially similar to the lux operon of Aliivibrio salmonicida . It was demonstrated that the reduced production of bioluminescence in A. salmonicida is most likely defined by a specific defect in its luxD gene.

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

Total petroleum hydrocarbons (TPH) contaminated soil samples were collected from Shengli Oilfield of China. Toxicity analysis was carried out based on earthworm acute toxicity, plant growth experiment and luminescent bacteria test. The soil was contaminated by-petroleum hydrogcarbons with TPH concentration of 10.57%. With lethal and sub-lethal rate as endpoint, earthworm test showed that the LD50 (lethal dose 50%) values in 4 and 7 days were 1.45% and 1.37% respectively, and the inhibition rate of earthworm body weight increased with higher oil concentration. TPH pollution in the soil inhibited seed germination in both wheat and maize experiment when the concentration of petroleum was higher than 0.1%. The EC50 (effective concentration 50%) for germination is 3.04% and 2.86% in maize and wheat, respectively. While lower value of EC50 for root elongation was to be 1.11% and 1.64% in maize and wheat, respectively, suggesting higher sensitivity of root elongation on petroleum contamination in the soil. The EC50 value in luminescent bacteria test was 0.47% for petroleum in the contaminated soil. From the experiment result, it was concluded that TPH content of 1.5% is considered to be a critical value for plant growth and living of earthworm and 0.5% will affect the activity of luminescent bacteria.

Degradation and toxicity reduction of perfluorooctanoic acid (PFOA) were investigated using TiO2 adsorption, vacuum ultraviolet (VUV) photolysis, and VUV/TiO2 photocatalysis in acidic and basic aqueous solutions. Chemical analyses of PFOA and its selected by-products and an acute toxicity assessment using the luminescent bacteria Vibrio fischeri (Microtox®) were conducted during and after the various treatment methods. PFOA was found to be best treated by VUV/TiO2 at pH 4 with HClO4, as illustrated by the almost complete degradation of PFOA within 360 min and rapid removal of acute microbial toxicity within 60 min. This difference in the efficiency may be attributed to the strong oxidation effectiveness of the radical species generated in acidic media and the electron scavenger effect of the addition of HClO4 in VUV/TiO2 photocatalysis. In addition, the proposed method could effectively decompose other perfluorocarboxylic acid (PFCA) species (C3–C7 perfluoroalkyl groups) if the initial intermediates forme...

In the phase transfer system solid calcium carbonate-acetonitrile, per acetate alpha-D-glucosaminilchloride glycosilate easily deprotoned isatine-3-oximes hydroxyl groups. It was found that the presence in the reaction mixture a catalytic amounts of 15-crown-5 accelerated the process twice. Obtained O-beta-D-glucosaminides were identified with 1H-NMR spectroscopy. Features of synthesized compound's NMR spectra are discussed in comparison with those of another N-acetylglucosamine 1-O-derivatives. The biological activity of the some oximes with different substituents in isatin residuum has been studied in a test of inhibition of bioluminescence of marine luminescent bacteria Photobacterium leiognathi Sh12. The nature of N-substituent of isatin fragment and 5-substituent of isatin main structure is compared with glycosides ability to suppress bacterial luminescence.

The compositions of explosive wastewater generated from TNT (2,4,6-trinitrotoluene) purification stage were characterized by using UV–vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatograph (HPLC) and gas chromatograph/mass spectroscopy (GC/MS). The acute toxicity was evaluated by bacterium bioluminescence assay using a freshwater luminescent bacterium (Vibrio qinghaiensis sp. Nov.) and a marine luminescent bacterium (Photobacterium phosphoreum). The results showed that the wastewater's biodegradability was poor due to the high amount of chemical oxygen demand (COD). The main organic components were dinitrotoluene sulfonates (DNTS) with small amount of TNT, dinitrotoluene (DNT), mononitrotoluene (MNT) and other derivatives of nitrobenzene. It was highly toxic to luminescent bacteria P. phosphoreum and V. qinghaiensis sp. Nov. After reaction time of 15 min, the relative concentration of toxic pollutants (expressed as reciprocal of dilution ratio of wastewater) at 50% of luminescence inhibition ratio was 5.32 × 10−4 for P. phosphoreu, while that was 4.34 × 10−4 for V. qinghaiensis. V. qinghaiensis is more sensitive and suitable for evaluating the wastewater's acute toxicity than P. phosphoreum. After adsorption by resin, the acute toxicity can be greatly reduced, which is helpful for further treatment by biological methods.

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.

A bacterial biosensor method for the selective determination of a bioavailable organomercurial compound, methylmercury, is presented. A recombinant luminescent whole-cell bacterial strain responding to total mercury content in samples was used. The bacterial cells were freeze-dried and used as robust, reagent-like compounds, without batch-to-batch variations. In this bacteria-based sensing method, luciferase is used as a reporter, which requires no substrate additions, therefore allowing homogenous, real-time monitoring of the reporter gene expression. A noninducible, constitutively light-producing control bacterial strain was included in parallel for determining the overall cytotoxicity of the samples. The specificity of the total mercury sensor Escherichia coli MC1061 (pmerRBlux) bacterial resistance system toward methylmercury is due to a coexpressed specific enzyme, organomercurial lyase. This enzyme mediates the cleavage of the carbon–mercury bond of methylmercury to yield mercury ions, which induce the reporter genes and produce a self-luminescent cell. The selective analysis of methylmercury with the total mercury strain is achieved by specifically chelating the inorganic mercury species from the sample using an optimized concentration of EDTA as a chelating agent. After the treatment with the chelating agent, a cross-reactivity of 0.2% with ionic mercury was observed at nonphysiological ionic mercury concentrations (100 nM). The assay was optimized to be performed in 3 h but results can already be read after 1 h incubation. Total mercury strain E. coli MC1061 (pmerRBlux) has been shown to be highly sensitive and capable of determining methylmercury at a subnanomolar level in optimized assay conditions with a very high dynamic range of two decades. The limit of detection of 75 ng/l (300 pM) allows measurement of methylmercury even from natural samples.

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.

Tetracycline was exposed to ozone by bubbling at different pH levels, and the acute toxicity of ozonation byproducts was tested by luminescent bacteria (Vibrio qinghaiensis sp.-Q67). Residual tetracycline in the solutions was detected by high performance liquid chromatography (HPLC). The results indicated that tetracycline was immediately degraded by ozonation and the degradation was favored at higher pH. Tetracycline (20 mg L–1) was completely degraded after 5 min of ozonation. However, total organic carbon (TOC) hardly decreased even though the ozonation time was extended to 40 min, suggesting that the ozonation process could not mineralize tetracycline. The luminescent bacteria assay indicated that the toxicity of the tetracycline solution was changed after ozonation. Chemical oxygen demand (COD) dropped by 15%, and the BOD5/COD ratio increased from 0.09 to 0.26, suggesting that the biodegradability of tetracycline was enhanced after 20 min of ozonation treatment.

Sixteen strains of luminescent bacteria of Vibrio and Photobacterium genera were isolated from water of the Azov and Black seas. Two strains prospective for biotesting were genetically identified as Vibrio fisheri V-9579 and Vibrio fisheri V-9580 according to Russian Industrial Microorganism Collection (VKMP) classification and accepted for depositing. The isolated luminescent strains exhibited high individual sensitivity to oil derived products, heavy metal salts, sodium dodecyl sulfate (SDS) and phenol (up to the maximum concentration limit for fishery impoundments). According to EC50, they are ten times more sensitive to heavy metal salts and potassium dichromate and 2–6 times more sensitive to SDS and phenol compared to P. phosphoreum (Cohn) Ford and Escherichia coli C600 (pPLS-5) strains. Using Vibrio fisheri VKMP V-9579 and Vibrio fisheri VKMP V-9580 as biosensors, we have shown their high sensitivity and efficacy to marine ecosystem toxicity assessment.

Luminescent bacteria and nematodes associate in a strategy where the bacteria act as virulent pathogens of insects, used as their food supply, while the nematodes graze on them. Upon reaching high density, the bacteria produce light and metabolites that turn the nematodes into hosts permitting them to be carried over to further nematode preys. In this issue of Molecular Microbiology, Lango and Clarke show that the corresponding shift in lifestyle is triggered by a metabolic switch closely linked to the tricarboxylic acid cycle, but apparently not by the well-known acetate switch that monitors entry of bacteria into the stationary phase of growth.

Direct bioautography performed with luminescence gene-tagged bacteria enables almost real-time detection of antimicrobial compounds in plant extracts. This method for the detection of chamomile ( Matricaria recutita ) components with antibacterial effect against Bacillus subtilis soil bacteria was more sensitive than commonly used bioautographic visualization by staining with a tetrazolium salt. Some compounds had a strong inhibiting effect only via the bioluminescence measurement. Extraction of antibacterial components of chamomile flowers was most effective with 50% ethanol; slightly lower efficiency was achieved with acetone and methanol, and hexane was least effective. The results were confirmed by using luminescent Pseudomonas syringae pv. maculicola plant pathogen bacteria.

The ecotoxicological characterization of wastes according to the European Waste List (EWL) is part of their assessment as hazardous or non-hazardous. Despite inclusion in national laws no methodological details have been fixed concerning the hazard property HP 14 (“ecotoxic”).This paper intends to discuss the classification of wastes by ecotoxicological testing, using 24 representative samples of solid wastes (identified by their EWL number) with different properties. They were sampled according to standard methods, and, with one exception (galvanic sludge), ecotoxicologically tested. No chemical investigation of the samples was performed but they were characterized according to their properties in the ABANDA data base. Nearly all of these wastes were “mirror entries” in the EWL (i.e., they can be hazardous or not depending on the concentration of hazardous substances). For the ecotoxicological characterization three aquatic tests with eluates (genotoxicity, Algae, Daphnia) as well as three terrestrial tests with solid wastes (bacteria, plants, earthworms) were conducted. All investigations were performed as limit tests with three dilution steps. Algae, plants and terrestrial bacteria were the most sensitive organisms. Since no waste eluate showed any indication of genotoxicity, the genotoxicity test should be replaced by the luminescent bacteria test (ISO 11348-3). Proposals for toxicity criteria as well as hazard classifications were taken from the literature but they were modified according to own experiences. Using these concentration limits for the classification whether these wastes are ecotoxic or not, and using different versions of the hazard classification approach, 15-19 waste samples out of 23 waste samples were classified as ecotoxic (64-83%). It is proposed to perform a plausibility check of the respective HP 14-classification. The procedure used in this contribution (i.e. sampling of the wastes, their ecotoxicological testing as well as their hazard classification) could form the basis of a standardized hazard classification approach as proposed recently in the literature. In summary, this works confirms that ecotoxicological tests are practical and sensitive in order to be used for the ecotoxicological hazard classification of very different wastes.

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.

To shorten the time needed to measure biochemical oxygen demand (BOD) in water samples and to provide a rapid feedback of the real condition of water quality, we tested and evaluated the validity and reliability of luminescent bacteria Vibrio fischeri, Photobacterium phosphoreum, and recombinant Escherichia coli as potential indicators of BOD in the domestic wastewaters. The results indicate that the luminescence intensities of these strains are dependent on temperature, pH, and BOD concentration. In comparison to the standard BOD(5) method, the time needed for BOD measurement can be shortened by 90, 120, and 150 min when V. fischeri, P. phosphoreum, and recombinant E. coli, respectively, are used. Recombinant E. coli can be adapted to measure BOD in domestic wastewater containing a wide range of BOD concentrations, V. fischeri is not suitable for measuring diluted wastewater, and P. phosphoreum has only a limited application in measuring concentrated wastewater. To the best of our knowledge, this is the first report in which V. fischeri, P. phosphoreum, and recombinant luminescent E. coli are compared in terms of their potential in BOD measurement systems.

Growth and emission characteristics of the luminescent bacterium Photobacterium phosphoreum strain KM MGU 331 originating from the White Sea and isolated from the intestine of a bottom-dwelling fish, the European sculpin, Myoxocephalus scorpius, were analyzed. The strain is characterized by a high rate of colony formation and high intensity of light emission on agarized medium at 4° C as well as by highly efficient (5 × 105 quanta s−1 cell−1) and prolonged (over 100 h) light generation upon submerged cultivation at 20°C. The acidic shift of pH in the medium didn’t exceed 0.3 pH units. Effects of temperature, pH, and sodium chloride concentration on emission characteristics of intact photobacterium cells were studied. The optimal temperature for luminescence was found to be 15°C. The maximum luminescence activity was stable in a wide pH range from 7.0 to 9.0. Luminescence occurred within the range of 0.2–3.8% NaCl with the maximum at 2.5%. The results obtained confirm the literature data suggesting that luminescent bacteria adapted to low-temperature conditions possess a highly conjugated system of electron transfer to luciferase.

In the European Waste List (2000/532/EC as amended) the ash of municipal waste incineration is defined as a so called mirror entry. This waste can be classified as hazardous or non-hazardous depending on the content of hazardous substances and other risk properties. For the assignment of waste in mirror entries, 14 criteria are defined. One of the criteria is H14 "ecotoxic". In the presented study, the ecotoxicological potential of 12 ashes from different incineration plants has been assessed using biological test systems. The test battery included aquatic tests with eluates (algae, daphnids, and luminescent bacteria) and terrestrial tests with solid waste (plants, earthworms and bacteria). The test results revealed a clear ecotoxicological hazard potential for some of the MWI ashes. Despite the fact that fresh ashes were several times more toxic than aged ashes both groups did not differ consistently in terms of toxicity. The results show also that there is no correlation between the biological effects and the analyzed chemical compounds of the ash samples.

The toxicity of sediments in the Gulf of Gdansk is analyzed in relation to the chemical composition of interstitial and near-bottom waters, and sediment properties. The toxicity of sediments, pore waters and saline elutriates is determined by using the Microtox ® test based on changes in light production of the luminescent bacteria Vibrio fischeri. The results indicate considerable toxicity in the majority of examined sediments. Since the sediment elutriates and pore waters are toxic in some cases, the total toxicity of the sediments is likely to be due to both sediment-bound and water soluble substances. The sediment toxicity is related to the percentage contribution of the fine fraction of sediments. A significant correlation between the toxicity of the sediments and the black carbon content implies anthropogenic contamination. The toxicity of the sediments is seen to increase with the increase of hydrogen sulfide concentration in pore waters. The ammonia in pore waters was found not to be responsible for the toxicity of the sediments.

The present study presents the toxicity data of benzoic acid and its derivatives on Pseudokirchneriella subcapitata, in terms of EC50 and NOEC values. Median effective concentrations (EC50) range from 0.55 to 270.7 mg/L (based on final yield) and 1.93 to 726.3mg/L (based on algal growth rate). No-observed-effect concentration (NOEC) is within the range of

Electrochemical measurement of respiratory chain activity is a rapid and reliable screening for the toxicity on microorganisms. Here, we investigated in-vitro effects of toxin on Escherichia coli (E. coli) that was taken as a model microorganism incubated with ferricyanide. The current signal of ferrocyanide effectively amplified by ultramicroelectrode array (UMEA), which was proven to be directly related to the toxicity. Accordingly, a direct toxicity assessment (DTA) based on chronoamperometry was proposed to detect the effect of toxic chemicals on microorganisms. The electrochemical responses to 3,5-dichlorophenol (DCP) under the incubation times revealed that the toxicity reached a stable level at 60 min, and its 50% inhibiting concentration (IC50) was estimated to be 8.0 mg L−1. At 60 min incubation, the IC50 values for KCN and As2O3 in water samples were 4.9 mg L−1 and 18.3 mg L−1, respectively. But the heavy metal ions, such as Cu2+, Pb2+ and Ni2+, showed no obvious toxicity on E. coli. With the exception of Hg2+, it showed 40.0 mg L−1 IC50 value when E. coli was exposed to its solution for 60 min. The lower sensitivity of DTA for the heavy metal ions could be attributed to the toxicological endpoint and the experimental conditions used. All results suggest that the DTA is a sensitive, rapid and inexpensive alternative to on-site water and wastewater toxic analysis.

Quantitative toxicology methods with the use of the enzymic activity of luminescent bacteria and the reproductive function of Tetrahymena pyriformis infusoria as test functions were employed to study the kinetics of the detoxication of model toxicants during photolysis of nitrite and nitrate. The model objects were the para-nitrosodimethylaniline (p-NDA) dye and a 2000-fold diluted solution of black liquor, which is formed during the sulfate boiling of cellulose. It was established that, irrespective of the wavelength of photolyzing UV radiation, be it 220 or 365 nm, the reactive product of NO2− and NO3− photolysis is the hydroxyl radical, which causes a symbatic decrease in the concentration of the toxicant and its toxicity. It was demonstrated that nitrite and nitrate ions can be used as initiators of free radicals during the destructive UV-light-induced purification of wastewater.

Aims: The aim of this study was to compare the luminescent intensity of bioluminescence from marine luminous bacteria with different motility. Methods and Results: Luminescent bacteria were separated according to their motility using a microfluidic device. The cell densities of the separated samples were measured using a counting plate. The luminescent intensity of the separated samples was measured using a luminometer. The luminescent intensity per cell was calculated, and the values from the mobile (swimmers) and the nonmobile cells (nonswimmers) per single cell were compared; as a result, the former were proved to be larger than the latter. Conclusions: Microfluidics were shown to be effective for the separation of bioluminescent bacteria and the bioluminescent intensity difference per cell was recognized with this experiment. Significance and Impact of the Study: This study introduced for the first time a method to examine the individual cell function of Photobacterium kishitanii.

Sunscreen products are meant to protect people from damaging UVA and UVB radiation. However, in some formulations the UV filters they contain can react and form many photodegradation products. Their potential toxicity has not yet been investigated. In this study effect-specific analysis has been used to evaluate the bioactivity of photodegradation products in sunscreens. HPTLC-bioluminescence coupling with the luminescent bacterium Vibrio fischeri was used. Problems in method development were because of the sensitivity of the bacteria and the wettability of HPTLC plates. A separation system using HPTLC LiChrospher plates and automated multiple development (AMD) with tert -butyl methyl ether- n -hexane was chosen. Detection was by UV in addition to Vibrio fischeri . First, biodetection was performed on pure standard solutions of the UV filters. UV filters with molecular weight >400 had no bioactivity; these included all newer UV filters (not in use before 1998). Five commercially available sunscreens with ...

The photo-induced transformation in aqueous solution of hexaconazole and dimethomorph over irradiated titanium dioxide was studied. The investigation involved monitoring pesticides decomposition, identifying intermediate compounds, assessing mineralization, and evaluating toxicity of pesticides derivatives. HPLC/UV and HPLC/MS were used to follow the disappearance of the initial pesticides and the formation of intermediate products, while the acute toxicity was evaluated by using the Vibrio fischeri luminescent bacteria assay. Hexaconazole photocatalytic transformation proceeds through the formation of highly persistent compounds. The formation of cyanuric acid, a non-toxic compound refractory to photocatalytic treatment, was recognized. Conversely, the toxicity assays prove that neither hexaconazole nor its intermediates exhibit acute toxicity. Dimethomorph under photocatalytic treatment is completely mineralized within 14 h of irradiation. However, its transformation proceeds through the formation of toxic intermediates. A correlation exists between the evolution of the intermediate compounds and the toxicity profile, as the highest toxicity is measured when the intermediates with lower EC50 (hydroquinone and 4-chlorophenol) are formed.

The toxicity of a number of commercial pesticides, such as Lontrel, Sencor, Glyphosate, and Hymexazol, as well as Lontrel complexes with Cu, Co, Mn, Mg, Mo, Ni, and Zn, which are present in substantial concentration in the environment, were studies using biotesting methods. The suppression of the enzymic activity of Benekea harvey luminescent bacteria and of the reproductive function of Tetrahymena pyriformis infusoria was studied; the corresponding EC50 values were determined. It was established that the toxicity of these compounds correlates with their ability to form stable complexes with adenosinetriphosphoric acid (characterized by the complexation constant), a process that make multicellular organisms energy deficient.

Computational coefficients for estimating the effectiveness of bioluminescence expression in natural luminescent bacteria Photobacterium leiognathi 54 and transgenic strain E. coli Z905/pPHL7 bearing lux-operon in a multicopy plasmid are suggested and their use at molecular, cell, and population levels was considered. It was shown that at the population level, all transgenic variants have an advantage over natural variants of P. leiognathi 54 irrespective of the type of lux-operon regulation. At the cell level, the effectiveness of bioluminescence expression in the bright and dim variants of the transgenic strain increased by several orders. At the level of one lux-operon, the effectiveness of expression in the bright variant of the transgenic strain is substantially higher than in the natural bright variant; in dim variants, the efficiency values are similar; the effectiveness of bioluminescence expression in the dark variant of E. coli Z905-2/pPHL7 is by two orders of magnitude lower than in the dark variant of P. leiognathi 54.

The ability of marine luminescent bacteria to synthesize polyesters of hydroxycarboxylic acids (polyhydroxyalkanoates, PHA) as reserve macromolecules was studied. Twenty strains from the collection of the luminescent bacteria CCIBSO (WDCM839) of the Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, assigned to different taxa (Photobacterium leiognathi, Ph. phosphoreum, Vibrio harveyi, and V. fischeri) were analyzed. The most productive strains were identified, and the conditions ensuring high polymer yields in batch culture (40–70% of the cell dry mass weight) were determined. The capacity for synthesizing two-and three-component polymers containing hydroxybutyric acid as the main monomer and hydroxyvaleric and hydroxyhexanoic acids was revealed in Ph. leiognathi and V. harveyi strains. The results allow luminescent microorganisms to be regarded as new producers of multicomponent polyhydroxyalkanoates.

This study reports preliminary results on the concentration of marine heterotrophic bacteria (Apparent Bacterial Concentration, ABC), and their luminous fraction (Apparent Luminous Bacterial Concentration, ALBC), here expressed as percentage of luminous bacteria (LB%), in coastal waters along the Campania shoreline. The results show that the Campania coastal area includes sectors characterised by different levels of water quality, following a geographical gradient from north to south. LB% appears related to inputs of pollution, showing low values in contaminated areas (

In Europe hazardous wastes are classified by 14 criteria including ecotoxicity (H 14). Standardized methods originally developed for chemical and soil testing were adapted for the ecotoxicological characterization of wastes including leachate and solid phase tests. A consensus on which tests should be recommended as mandatory is still missing. Up to now, only a guidance on how to proceed with the preparation of waste materials has been standardized by CEN as EN 14735. In this study, tests including higher plants, earthworms, collembolans, microorganisms, duckweed and luminescent bacteria were selected to characterize the ecotoxicological potential of a boiler slag, a dried sewage sludge, a thin sludge and a waste petrol. In general, the instructions given in EN 14735 were suitable for all wastes used. The evaluation of the different test systems by determining the LC/EC50 or NOEC-values revealed that the collembolan reproduction and the duckweed frond numbers were the most sensitive endpoints. For a final classification and ranking of wastes the Toxicity Classification System (TCS) using EC/LC50 values seems to be appropriate.

Luminescent Vibrio harveyi is a natural microflora of marine and coastal water bodies and is associated with mortality of larval shrimp in penaeid shrimp hatcheries. It is also known that the bacteriophages occur virtually in all places where their hosts exist. In this study, distribution of luminescent V. harveyi and the bacteriophages affecting these hosts was examined in a commercial Penaeus monodon hatchery during three shrimp larval production cycles, including a cycle affected by luminescent bacterial (LB) disease outbreak. Out of a total of 1195 samples drawn from seawater source, sand-filtered water, nauplius, zoea, mysis and post larval rearing tanks, maturation and spawning tanks, Artemia hatching tank and algal culture tanks processed using conventional microbiological techniques, 21.4% of the samples harboured luminescent bacteria. During the larval production cycle affected by LB disease (LBD), luminescent V. harveyi could be recovered from 52% of the hatchery samples, whereas during luminescent bacterial disease-free larval production cycle (LBDF), these bacteria could be recovered from only about 9% of the samples. The predominant source of luminescent bacteria was the brood shrimp and their rearing tanks in maturation and spawning facilities. 73% of the maturation and 80% of the spawning tank water samples harbored LB during LBD, whereas, only 20% and 32% of the maturation and spawning tanks respectively harbored LB during LBDF. LB could be isolated from 17% of the water samples in tanks from nauplius stage onwards with increasing counts that subsequently lead to LB disease. Bacteriophages affecting the luminescent V. harveyi could be isolated from as many as 36% (21% and 43% of the samples analysed during LBDF and LBD respectively) of a total of 181 water samples drawn from various sources in the hatchery, using 27 luminescent V. harveyi hosts by agar overlay technique. The maturation tank water samples were found to be the predominant source of bacteriophages, followed by spawning tank water samples as observed with the LB. Sixty five bacteriophages, 13 during LBDF and 52 during LBD were isolated, which were grouped in to seven types based on their plaque morphology. The study has indicated that the brooders, maturation and spawning facilities in the shrimp hatchery are the main source of luminescent V. harveyi and their bacteriophages and that occurrence of LB even in low counts during early larval stages can possibly lead to development of LB disease despite presence of bacteriophages in the larval rearing tanks.

An improved microbial assay was developed for the detection of chloramphenicol (CAP) residues in shrimp muscle using Photobacterium leiognathi (L-2 strain) isolated from squid, as test organism, using a pad plate technique. CAP spiked in shrimp muscle tissues at concentrations ranging from 1 to 100 μg/kg was extracted using ethyl acetate and ammonium hydroxide. The assay showed a maximum inhibition zone of 23.0 mm for 100 μg/kg and a minimum inhibition zone of 9.3 mm for 1 μg/kg. The method had a good recovery of 95.63% with a minimum detection limit of 1 μg/kg. It can be performed within 18 h following simple extraction. The method therefore proved to be advantageous over chromatographic procedures as it was inexpensive, quite sensitive and can be adopted for rapid screening of CAP in shrimp tissues.

The aim of the work was to study the biodegradation process of biocomposites prepared from renewable resources and the ecotoxicological assessment of their biodegradation products. Biocomposites from modified starch reinforced with various cellulose fibers were prepared by the extrusion process. Biodegradation studies were carried out according to the ISO respirometic method. Ecotoxicity of biodegradation products was assessed by the luminescent bacteria test. It was found that biodegradation of biocomposites was above 60% within 24 days according to the results of respirometric test. Increase in the amount of natural fiber reinforcement, as well as smaller fiber size increased the biodegradability of biocomposites. On the basis of the preliminary results of the ecotoxicological test using luminescent bacteria it seems that the biodegradation products of the biocomposites studied are ecologically safe.

Luminescent bacteria in the family Vibrionaceae (Bacteria: c-Proteobacteria) are commonly found in complex, bilobed light organs of sepiolid and loliginid squids. Although morphology of these organs in both families of squid is similar, the species of bacteria that inhabit each host has yet to be verified. We utilized sequences of 16S ribosomal RNA, luciferase a-subunit (luxA) and the glyceraldehyde-3-phosphate dehydrogenase (gapA) genes to determine phylogenetic relationships between 63 strains of Vibrio bacteria, which included representatives from different environments as well as unidentified luminescent isolates from loliginid and sepiolid squid from Thailand. A combined phylogenetic analysis was used including biochemical data such as carbon use, growth and luminescence. Results demonstrated that certain symbiotic Thai isolates found in the same geographic area were included in a clade containing bacterial species phenotypically suitable to colonize light organs. Moreover, multiple strains isolated from a single squid host were identified as more than one bacteria species in our phylogeny. This research presents evidence of species of luminescent bacteria that have not been previously described as symbiotic strains colonizing light organs of Indo-West Pacific loliginid and sepiolid squids, and supports the hypothesis of a non-species-specific association between certain sepiolid and loliginid squids and marine luminescent bacteria. � The Willi Hennig Society 2007.