Ecotoxicological characterisation of sedimentation in the Kis-Balaton Water Protection System

The main function of the Kis-Balaton Water Protection System is to retain nutrients and total suspended solids, thus protecting the water quality of Lake Balaton. In this paper, the toxic nature of the sediment in the 2nd reservoir of the KBWPS has been characterised, using a battery of tests: Vibrio fischeri acute bioassay on whole sediment samples, and V. fischeri bioassay on pore water and elutriate samples. The latest version of the V. fischeri bioluminescence inhibition was applied, the Flash assay which uses a kinetic mode and is able to detect the toxicity of solid, turbid/coloured samples. Whole sediment toxicity showed a clear spatial distribution of toxicity, in parallel with elutriate toxicity. However, no pore water toxicity was detected, leading to the conclusion that contaminants are not water soluble.     

Construction of a modified mini-Tn5 luxCDABE transposon for the development of bacterial biosensors for ecotoxicity testing

A mini-Tn5 transposon was modified to introduce a promoterless luxCDABE cassette from Vibrio fischeri into environmentally relevant bacterial strains in order to develop bioluminescence-based biosensors for toxicity testing. The mini-Tn5 luxCDABE transposon was chromosomally integrated downstream from an active promoter into two Pseudomonas strains (Pseudomonas fluorescens 8866 and Pseudomonas putida F1). Characterisation of the bioluminescent transconjugants demonstrated that the transposon integration was stable and had no effect on growth rate. Both P. fluorescens 8866 Tn5 luxCDABE and P. putida F1 Tn5 luxCDABE were used to assess the toxicity of standard solutions (Cu, Zn and 3,5-DCP) as well as Cu- and 3,5-DCP-spiked groundwater samples. They were successfully used for bioluminescence-based bioassays and the potential value of using different bacterial biosensors for ecotoxicity testing was shown.     

Attenuation of groundwater contamination caused by cattle slurry: a plot-scale experimental study

Infiltration of contaminants was investigated in a flat pasture plot Lolium perenne L. which received 250 m3/ha of cattle slurry. Lysimeters and piezometers had previously been installed in the plot to sample groundwater at different depths. Water samples were analysed for pH, conductivity, NH4(+), NO3-, orthophosphate, Cl-, Na+, K+, Ca2+, Mg2+ and chemical oxygen demand (COD), and for faecal coliforms and faecal streptococci. Contaminant concentrations in water samples taken in lysimeters at a depth of 5 cm (2 h after slurry application) were already from 22% to 83%, of raw slurry. After slurry application and after 150 mm of rainfall, contaminant concentrations in groundwater were in all depths less than 95% of those initially measured in the slurry. For all contaminants except Cl-, NO3-, K+ and COD, concentrations in groundwater measured before application were reached within 15 days. Mechanical retention was the principal mechanism of attenuation of microorganism and COD levels, whereas cations were attenuated by sorption to soil matrix. Dilution by rain water had less significant effects, accounting for about a tenfold reduction in contaminant levels.     

Methodology for using contaminated soil leachability testing to determine soil cleanup levels at contaminated petroleum underground storage tank (UST) sites

The costs of environmental remediation at leaking petroleum underground storage tank (UST) sites are influenced significantly by soil cleanup levels. The use of conservative generic soil cleanup levels may be inappropriate at some sites contaminated by leaking petroleum USTs. At many contaminated sites, a primary objective of site remediation is long‐term protection of water resources (e.g., groundwater) from pollution. Leaching of pollutants from residual soil contamination to groundwater is a primary consideration in establishing site‐specific soil cleanup levels at fuel‐contaminated sites. The use of laboratory soil leachability testing methods may be useful in objectively evaluating the leaching potential of contaminants from residual soil contamination and estimating potential groundwater impacts. Developing soil cleanup levels that are protective of water resources must include a technically sound integration of site‐specific soil leachability data and contaminant attenuation factors. Evaluation of the leaching potentials of soil contaminants may also provide essential supplementary information for other site characterization methods that may be used to evaluate risks to human health. Contaminant leachability testing of soils may provide a cost‐effective and technically based method for determining soil cleanup levels that are protective of groundwater resources at contaminated petroleum UST sites.     

Evolutionary perspectives in a mutualism of sepiolid squid and bioluminescent bacteria: combined usage of microbial experimental evolution and temporal population genetics

The symbiosis between marine bioluminescent Vibrio bacteria and the sepiolid squid Euprymna is a model for studying animal-bacterial Interactions. Vibrio symbionts native to particular Euprymna species are competitively dominant, capable of outcompeting foreign Vibrio strains from other Euprymna host species. Despite competitive dominance, secondary colonization events by invading nonnative Vibrio fischeri have occurred. Competitive dominance can be offset through superior nonnative numbers and advantage of early start host colonization by nonnatives, granting nonnative vibrios an opportunity to establish beachheads in foreign Euprymna hosts. Here, we show that nonnative V. fischeri are capable of rapid adaptation to novel sepiolid squid hosts by serially passaging V. fischeri JRM200 (native to Hawaiian Euprymna scolopes) lines through the novel Australian squid host E. tasmanica for 500 generations. These experiments were complemented by a temporal population genetics survey of V. fischeri, collected from E. tasmanica over a decade, which provided a perspective from the natural history of V. fischeri evolution over 15,000-20,000 generations in E. tasmanica. No symbiont anagenic evolution within squids was observed, as competitive dominance does not purge V. fischeri genetic diversity through time. Instead, abiotic factors affecting abundance of V. fischeri variants in the planktonic phase sustain temporal symbiont diversity, a property itself of ecological constraints imposed by V. fischeri host adaptation.     

Quantifying of bactericide properties of medicinal plants

Extended research has been carried out to clarify the ecological role of plant secondary metabolites (SMs). Although their primary ecological function is self-defense, bioactive compounds have long been used in alternative medicine or in biological control of pests. Several members of the family Labiatae are known to have strong antimicrobial capacity. For testing and quantifying antibacterial activity, most often standard microbial protocols are used, assessing inhibitory activity on a selected strain. In this study, the applicability of a microbial ecotoxtest was evaluated to quantify the aggregate bactericide capacity of Labiatae species, based on the bioluminescence inhibition of the bacterium Vibrio fischeri. Striking differences were found amongst herbs, reaching even 10-fold toxicity. Glechoma hederacea L. proved to be the most toxic, with the EC₅₀ of 0.4073 g dried plant/l. LC₅₀ values generated by the standard bioassay seem to be a good indicator of the bactericide property of herbs. Traditional use of the selected herbs shows a good correlation with bioactivity expressed as bioluminescence inhibition, leading to the conclusion that the Vibrio fischeri bioassay can be a good indicator of the overall antibacterial capacity of herbs, at least on a screening level.Key words: Labiatae, antibacterial property, bioactivity, Vibrio fischeri     

Dimensionless parameters to summarize the influence of microbial growth and inhibition on the bioremediation of groundwater contaminants

Monod expressions are preferred over zero- and first-order decay expressions in modeling contaminants biotransformation in groundwater because they better represent complex conditions. However, the wide-range of values reported for Monod parameters suggests each case-study is unique. Such uniqueness restricts the usefulness of modeling, complicates an interpretation of natural attenuation and limits the utility of a bioattenuation assessment to a small number of similar cases. In this paper, four Monod-based dimensionless parameters are developed that summarize the effects of microbial growth and inhibition on groundwater contaminants. The four parameters represent the normalized effective microbial growth rate (η), the normalized critical contaminant/substrate concentration (S*), the critical contaminant/substrate inhibition factor (N), and the bioremediation efficacy (η*). These parameters enable contaminated site managers to assess natural attenuation or augmented bioremediation at multiple sites and then draw comparisons between disparate remediation activities, sites and target contaminants. Simulations results are presented that reveal the sensitivity of these dimensionless parameters to Monod parameters and varying electron donor/acceptor loads. These simulations also show the efficacy of attenuation (η*) varying over space and time. Results suggest electron donor/acceptor amendments maintained at relative concentrations S* between 0.5 and 1.5 produce the highest remediation efficiencies. Implementation of the developed parameters in a case study proves their usefulness.     

Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN-oxidoreductase.

The bioluminescent bacterial enzyme system NAD(P)H:FMN-oxidoreductase-luciferase has been used as a test system for ecological monitoring. One of the modes to quench bioluminescence is the interaction of xenobiotics with the enzymes, which inhibit their activity. The use of endogenous flavin fluorescence for investigation of the interactions of non-fluorescent compounds with the bacterial luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri has been proposed. Fluorescence spectroscopy methods have been used to study characteristics of endogenous flavin fluorescence (fluorophore lifetime, the rotational correlation time). The fluorescence anisotropy behaviour of FMN has been analysed and compared to that of the enzyme-bound flavin. The fluorescence characteristics of endogenous flavin of luciferase and NAD(P)H:FMN-oxidoreductase have been shown to be applicable in studying enzymes' interactions with non-fluorescent compounds.     

Vapor Pressure Characterization to Predict Contaminant Releases from MGP Site Source Area Soils

Risk-based management of contaminated sites often requires timely screening and identification of source areas that release contaminants to groundwater and other reception of concern. Over-reliance on total concentrations often results in costly site characterization, delays in remediation, and overestimation of the soil volumes targeted for removal or treatment. Advances in diagnostic tools to accelerate source area characterization will improve the reliability of management decisions. The objective of this work was to determine the potential of using vapor pressure measurements as a rapid indicator of the mass release potential of suspected source area soils.

The current work has focused on determining the relationship between partial pressure and mobility for monoaromatic and polycyclic aromatic hydrocarbon (PAH) contaminants in source area soils. This work ascertained that for a wide spectrum of non-ionized, aromatic contaminants in MGP soils, total vapor pressure and benzene partial pressure could be measured with good repeatability. Under controlled conditions, photoionization detector (PID) measurements on 16 soil samples from two MGP sites were found to correlate well to laboratory measurements of equilibrium partitioning and resin-mediated soil contaminant desorption with R² values of over 0.9. Results indicate that vapor pressure correlates well to contaminant mobility in soils.     

Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis.

Genes encoding NAD(P)H-flavin oxidoreductases (flavin reductases) similar in both size and sequence to Fre, the most abundant flavin reductase in Escherichia coli, were identified in four species of luminous bacteria, Photorhabdus luminescens (ATCC 29999), Vibrio fischeri (ATCC 7744), Vibrio harveyi (ATCC 33843), and Vibrio orientalis (ATCC 33934). Nucleotide sequence analysis showed Fre-like flavin reductases in P. luminescens and V. fischeri to consist of 233 and 236 amino acids, respectively. As in E. coli Fre, Fre-like enzymes in luminous bacteria preferably used riboflavin as an electron acceptor when NADPH was used as an electron donor. These enzymes also were good suppliers of reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. In V. fischeri, the Fre-like enzyme is a minor flavin reductase representing < 10% of the total FMN reductase. That the V. fischeri Fre-like enzyme has no appreciable homology in amino acid sequence to the major flavin reductase in V. fischeri, FRase I, indicates that at least two different types of flavin reductases supply FMNH2 to the luminescence system in V. fischeri. Although Fre-like flavin reductases are highly similar in sequence to luxG gene products (LuxGs), Fre-like flavin reductases and LuxGs appear to constitute two separate groups of flavin-associated proteins.     

Temperature affects species distribution in symbiotic populations of Vibrio spp

The genus Sepiola (Cephalopoda: Sepiolidae) contains 10 known species that occur in the Mediterranean Sea today. All Sepiola species have a light organ that contains at least one of two species of luminous bacteria, Vibrio fischeri and Vibrio logei. The two Vibrio species coexist in at least four Sepiola species (S. affinis, S. intermedia, S. ligulata, and S. robusta), and their concentrations in the light organ depend on changes in certain abiotic factors, including temperature. Strains of V. fischeri grew faster in vitro and in Sepiola juveniles when they were incubated at 26 degrees C. In contrast, strains of V. logei grew faster at 18 degrees C in culture and in Sepiola juveniles. When aposymbiotic S. affinis or S. ligulata juveniles were inoculated with one Vibrio species, all strains of V. fischeri and V. logei were capable of infecting both squid species at the optimum growth temperatures, regardless of the squid host from which the bacteria were initially isolated. However, when two different strains of V. fischeri and V. logei were placed in direct competition with each other at either 18 or 26 degrees C, strains of V. fischeri were present in sepiolid light organs in greater concentrations at 26 degrees C, whereas strains of V. logei were present in greater concentrations at 18 degrees C. In addition to the competition experiments, the ratios of the two bacterial species in adult Sepiola specimens caught throughout the season at various depths differed, and these differences were correlated with the temperature in the surrounding environment. My findings contribute additional data concerning the ecological and environmental factors that affect host-symbiont recognition and may provide insight into the evolution of animal-bacterium specificity.     

Ecological Effects of Metals in Streams on a Defense Materials Processing Site in South Carolina,USA

The Savannah River Site (SRS) is a 780 km² U.S. Department of Energy facility near Aiken, South Carolina, established in 1950 to produce nuclear materials. SRS streams are “integrators” that potentially receive water transportable contaminants from all sources within their drainage basins, necessitating a watershed approach to organize contaminant distribution data and characterize the effects of multiple contaminants on aquatic organisms. This study used several lines-of-evidence to assess the ecological effects of metals in SRS streams, including contaminant exposure models for apex predators and bioassessments of fish and macroinvertebrate assemblages. Concentrations of metals in sediments, fish, and water were elevated in streams affected by SRS operations, but contaminant exposure models for the river otter Lontra Canadensis and belted kingfisher Ceryle alcyon indicated that toxicological reference values were exceeded only by Hg and Al. Macroinvertebrate assemblage structure was unrelated to sediment metal concentrations. Fish assemblage data were inconclusive. This study indicated that (1) modeling studies and field bioassessments provide a complementary basis for addressing the individual and cumulative effects of contaminants, (2) habitat effects must be controlled when assessing contaminant impacts, (3) sensitivity analyses of contaminant exposure models can help to apportion sampling effort, and (4) most individual metals in SRS streams are unlikely to have significant ecological effects.     

Direct addition of cultures of tester bacteria into semi-permeable membrane devices (SPMDs) as a modified procedure for preliminary detection of mutagenic pollution of the marine environment by use of microbiological mutagenicity assays

Mutagenic pollution of the natural environment, including marine waters, is a very serious ecological problem. However, since chemical mutagens usually occur and act at low concentrations, their detection and identification is technically difficult, laborious and time-consuming. Therefore, preliminary detection of mutagenic pollution is commonly based on biological mutagenicity assays. On the other hand, triolein-containing semi-permeable membrane devices (SPMDs) provide a method for concentration of hydrophobic organic contaminants, including a large fraction of the mutagens. Combinations of SPMDs with microbiological toxicity and mutagenicity assays have already been described, but only SPMD-derived extracts, prepared with various organic solvents, were tested in such a way to date. We found that the presence of these solvents could interfere with the Vibrio harveyi bioluminescence-based mutagenicity assay. Moreover, preparation of the extracts from SPMD takes usually at least 48h. Here, we propose a modified procedure, based on direct addition of tester bacteria cultures into SPMD. We found that this procedure is significantly (at least two times) more rapid and several times more sensitive than that based on testing the extracts. This optimization is presented in this report. Moreover, we have performed preliminary studies on samples of marine waters. Positive results (i.e. detection of mutagenic activity) were obtained when test samples came from a region known to be highly contaminated by industrial pollution, while negative results were observed in the case of samples from a region supposed to be of low risk for mutagenic pollution.     

An Evaluation of Vapor Intrusion Into Buildings through a Study of Field Data

A systematic examination of cases on file with the Massachusetts Department of Environmental Protection was undertaken to identify a universe of sites with volatile organic compound (VOC) contamination in groundwater in close proximity to buildings. Such locations were grouped according to site variables, such as contaminants of concern and concentrations in various media; soil type; depth to groundwater; distance to building; and building construction. Indoor air, soil gas, and/or groundwater field data collected from these sites was then assembled and used to: (1) evaluate available transport models which describe the intrusion of vapors into buildings and predict indoor air contaminant concentrations resulting from the volatilization of VOC s in the subsurface; (2) examine the validity of established regulatory criteria; (3) identify specific trends and field conditions which appear to most influence vapor phase contaminant migration and intrusion processes; and (4) evaluate the possibility of vapor migration being inhibited by a "freshwater lens".     

Biodegradation of lactic acid based polymers under controlled composting conditions and evaluation of the ecotoxicological impact

The biodegradability of lactic acid based polymers was studied under controlled composting conditions (CEN prEN 14046), and the quality of the compost was evaluated. Poly(lactic acids), poly(ester-urethanes), and poly(ester-amide) were synthesized and the effects of different structure units were investigated. The ecotoxicological impact of compost samples was evaluated by biotests, i.e., by the Flash test, measuring the inhibition of light production of Vibrio fischeri, and by plant growth tests with cress, radish, and barley. All the polymers biodegraded to over 90% of the positive control in 6 months, which is the limit set by the CEN standard. Toxicity was detected in poly(ester-urethane) samples where chain linking of lactic acid oligomers had been carried out with 1,6-hexamethylene diisocyanate (HMDI). Both the Flash test and the plant growth tests indicated equal response to initial HMDI concentration in the polymer. All other polymers, including poly(ester-urethane) chain linked with 1,4-butane diisocyanate, showed no toxicological effect.     

"Quorum sensing", or how bacteria "talk" to each other

Recent advances in studying the quorum-sensing systems, which regulate gene expression depending on population density, are reviewed. Low-molecular-weight acyl derivatives of L-homoserine lactone (N-AHL) freely diffuse through cell membranes and determine cell-to-cell communications in bacteria. The quorum-sensing systems have first been found to regulate bioluminescence in marine bacteria Photobacterium (Vibrio) fischeri and Vibrio harveyi. Such systems are widespread and control expression of genes for virulence factors, proteases, antibiotics, etc., in various Gram-negative bacteria, including plant, animal, and human pathogens. Quorum sensing is a prominent example of social behavior in bacteria, as signal exchange among individual cells allows the entire population to choose an optimal way of interaction with the environment and with higher organisms.     

Evaluating Effects of Contaminants on Fish Health at Multiple Levels of Biological Organization: Extrapolating from Lower to Higher Levels

Effects of environmental stressors such as contaminants on the health of aquatic ecosystems usually involve a series of biological responses ranging from the biomolecular/biochemical to the population and community levels. To establish relationships and to determine the feasibility of extrapolating between higher and lower levels of biological organization, spatial patterns in fish responses to contaminant loading were investigated in a stream receiving point-source discharges of various contaminants near its headwaters. Relationships among fish responses at four major levels of biological organization (biochemical/physiological, individual, population, and community levels) were evaluated relative to patterns in contaminant loading along the spatial gradient of the stream. Both individual and integrated response analysis demonstrated that bioindicators at several levels of biological organization displayed similar downstream patterns in their response to contaminant loading within the stream. Some of the bioindicator responses at lower levels of organization appear to be useful for the ecological risk assessment process because of their sensitivity and apparent relationships to higher levels. By identifying and establishing relationships between levels of biological organization we should be better able to understand the mechanisms of stress responses in ecological systems that could ultimately result in improved predictive capability of ecological risk assessment and also allow for more informed decisions regarding remedial actions.     

Rapid screening for soil ecotoxicity with a battery of luminescent bacteria tests

A bacterial test battery, involving i) Microtox, an aquatic test, ii) the Flash assay, a soil-suspension test (with Vibrio fischeri as the test organism), and iii) the Metal Detector assay, a semi-specific aquatic test for heavy metals (with recombinant luminescent Escherichia coli), was used in a combined toxicological and chemical hazard assessment of Estonian soils sampled from a former Soviet military airfield (13 samples) and from traffic-influenced roadsides (5 samples). The soils showed slightly elevated levels of total petroleum hydrocarbons (TPH), but not of heavy metals. In most of the samples, the levels of TPH did not exceed the Estonian permitted limit values set for residential areas. Toxicity testing was performed on both fresh and dried soils, after aqueous extraction for 1 hour and 24 hours. The toxicity results obtained with the Microtox test did not significantly differ in all of the sample treatment schemes; however, it appeared that the drying and sieving of the soils increased the bioavailability of toxicants, probably due to an enlarged reactive soil surface area. According to chemical analysis of the soils and the data from the Microtox test and the Metal Detector assay (performed on aqueous elutriates of the soils), these soils would not be considered to be hazardous. In contrast, the Flash assay performed on soil-water suspensions of dried soils, showed that most of the soils were toxic and thus probably contained undetermined particle-bound bioavailable toxicants. The photobacterial toxicity test (the Flash assay) can be recommended for the rapid screening of soils, as it is sensitive, cheap and inexpensive, and provides valuable information on particle-bound bioavailable toxicants, useful for complementing a chemical analysis and for assessing the risks originating from polluted soils.     

Changes in Prokaryote and Eukaryote Assemblages Along a Gradient of Hydrocarbon Contamination in Groundwater

Groundwater biota are particularly sensitive to environmental perturbations such as groundwater contamination. The diversity of prokaryotic and eukaryotic biota has been examined along a gradient of chlorinated hydrocarbon (CHC) contamination in the Botany Sands, an urban coastal sand-bed aquifer (Sydney, Australia). Molecular techniques were used to analyze the richness and composition of prokaryote and eukaryote assemblages using 16S and 18S rDNA, respectively. Taxon richness did not change significantly along the gradient for either prokaryotes or eukaryotes; however, significant shifts in assemblage composition were evident for both groups. Assemblage changes were most strongly correlated with concentrations of the major CHC, cis-1,2-dichloroethene, but the concentrations of a number of the contaminants were also correlated, making it difficult to infer if effects were due to any particular contaminant. The presence of cis-1,2-dichloroethene and other secondary ethenes suggests in situ breakdown of the primary CHCs via natural attenuation. The current focus of management of the Botany aquifer is to stop the contaminant plume reaching the adjoining estuary. This approach is clearly justified given the changes evident in the microbial assemblages in the groundwater, which are a likely consequence of the contamination.     

Direct addition of cultures of tester bacteria into semi-permeable membrane devices (SPMDs) as a modified procedure for preliminary detection of mutagenic pollution of the marine environment by use of microbiological mutagenicity assays

Mutagenic pollution of the natural environment, including marine waters, is a very serious ecological problem. However, since chemical mutagens usually occur and act at low concentrations, their detection and identification is technically difficult, laborious and time-consuming. Therefore, preliminary detection of mutagenic pollution is commonly based on biological mutagenicity assays. On the other hand, triolein-containing semi-permeable membrane devices (SPMDs) provide a method for concentration of hydrophobic organic contaminants, including a large fraction of the mutagens. Combinations of SPMDs with microbiological toxicity and mutagenicity assays have already been described, but only SPMD-derived extracts, prepared with various organic solvents, were tested in such a way to date. We found that the presence of these solvents could interfere with the Vibrio harveyi bioluminescence-based mutagenicity assay. Moreover, preparation of the extracts from SPMD takes usually at least 48 h. Here, we propose a modified procedure, based on direct addition of tester bacteria cultures into SPMD. We found that this procedure is significantly (at least two times) more rapid and several times more sensitive than that based on testing the extracts. This optimization is presented in this report. Moreover, we have performed preliminary studies on samples of marine waters. Positive results (i.e. detection of mutagenic activity) were obtained when test samples came from a region known to be highly contaminated by industrial pollution, while negative results were observed in the case of samples from a region supposed to be of low risk for mutagenic pollution.