A Review of the Occurrence of Metals and Xenobiotics in European Hedgehogs (Erinaceus europaeus).

Monitoring data from several European countries indicate that European hedgehog (Erinaceus europaeus) populations are declining, and research exploring the causes of the decline, including exposure to potentially harmful xenobiotics and metals, may inform conservation initiatives to protect this species in the wild. Hedgehogs are ground-dwelling mammals, feeding on a range of insects, slugs, snails, and earthworms, as well as eggs, live vertebrates, and carrion, including carcasses of apex predator species representing higher levels of the food chain. Consequently, hedgehogs come into close contact with contaminants present in their habitats and prey. This review investigated the studies available on the subject of the occurrence of metals and organic xenobiotics in hedgehogs. This study found that a vast range of different pesticides; persistent organic pollutants (POPs), including organochlorine compounds and brominated flame retardants (BFRs); as well as toxic heavy metals could be detected. Some compounds occurred in lethal concentrations, and some were associated with a potential adverse effect on hedgehog health and survival. Due to their ecology, combined with the opportunity to apply non-invasive sampling techniques using spines as sampling material, we suggest that the European hedgehog is a relevant bioindicator species for monitoring the exposure of terrestrial wildlife to potential toxicants in urban and rural environments.

Ecotoxicity and rapid degradation of quaternary ammonium compounds (QACs) subjected to combined vacuum UV and UV-C treatment.

Quaternary ammonium compounds (QACs) are active ingredients in a palette of commercially available disinfectants, sanitizers, and biocides. QACs are widely used because of their broad-spectrum antimicrobial properties but the ubiquitous uses have resulted in frequent detection in aquatic and terrestrial matrices including domestic wastewater, surface waters, urban soils and sediments. An increased domestic QACs consumption has increased the environmental occurrence, and investigation of mitigation methods and effects on non-target organisms are in demand. In this study, we examined the potential ecotoxicity of six QACs and investigated the effect of combined vacuum UV (185 nm) and UV-C (254 nm) irradiation (VUV/UVC) on degradation and mitigation of ecotoxicity of QACs. The study showed that combined VUV/UVC irradiation facilitated rapid degradation of benzalkonium chloride, benzethonium chloride, didecyldimethylammonium chloride, dodecyltrimethylammonium chloride, and hexadecyltrimethylammonium chloride. The estimated half-lives varied between 2 and 7 min, and degradation was affected by the initial QAC concentrations, the UV fluence, and the water matrix. The potential ecotoxicity of QACs and VUV/UVC treated QACs was examined using a battery of test organisms that included the luminescent bacterium Aliivibrio fischeri, the gram-negative and gram-positive bacteria Escherichiacoli and Enterococcus faecalis, the freshwater microalga Raphidocelis subcapitata, and the crustacean Daphia magna. The potential for trophic transfer of QACs was investigated in a simplified aquatic food web. Test organisms from different trophic levels were included to assess adverse effects of bioactive compounds in VUV/UVC treated samples including transformation products. The study showed that several QACs were highly toxic to aquatic test organisms with EC50 and/or EC20 values < 1 µM. VUV/UVC treatment of QACs resulted in substantial photolysis of the parent compounds and comprehensive mitigation of the ecotoxicity potential. VUV/UVC represent an attractive oxidation technology for abatement QACs in contaminated water because the process does not require addition of catalysts or precursors.

Flocculating and dewatering of lake sediment: An in-situ pilot study comparing synthetic polymers and biopolymers for restoring lake water quality and reusing phosphorus.

Dredging of lake sediment is a method to remove accumulated phosphorus and nitrogen in lakes and thereby reducing the risk of eutrophication. After dredging, the sediment is dewatered to reduce the volume. It is important to get a high dry matter content and ensure that the filtrate does not contain harmful compounds so it can be returned to the lake. A pilot-scale belt filter and flexible intermediate bulk containers (FIBC) were used for dewatering lake sediment with the sediment treated with a synthetic polymer or three different biopolymers. The goal of the study was to retain the phosphorus in the filter cake while returning the filtrate to the lake with a minimal phosphorus content. Results showed dry matter content of up to 16 % in the dewatered sediment and the sediment retained 96-99 % of the phosphorus. Furthermore, nitrogen was reduced by 27-71 % in the filtrate water. Toxicity tests found low ecotoxicity for most biopolymer filtrates, whereas synthetic polymer showed the highest potential ecotoxicity. Consequently, biopolymers provided satisfactory results, proving more environmentally friendly despite requiring longer filtration time.

Attenuation of toxicity and occurrence of degradation products of the fungicide tebuconazole after combined vacuum UV and UVC treatment of drinking water.

Antifungal azoles are the most frequently used fungicides worldwide and occur as active ingredients in many antifungal pharmaceuticals, biocides, and pesticides. Azole fungicides are frequent environmental contaminants and can affect the quality of surface waters, groundwater, and drinking water. This study examined the potential of combined vacuum UV (185 nm) and UVC (254 nm) irradiation (VUV/UVC) of the azole fungicide tebuconazole and the transformation product 1,2,4-trizole on degradation and changes in ecotoxicity. In vivo ecotoxicity was examined before and after UV treatment using bioassays with test organisms from different trophic levels to integrate changes in biological effect of the parent compound and the degradation products. The test battery included the luminescent bacterium Aliivibrio fischeri, the Gram-positive bacterium Bacillus subtilis, the fungus Fusarium graminearum, the green microalga Raphidocelis subcapitata, and the crustacean Daphnia magna. The combined VUV/UVC treatment of tebuconazole in drinking water efficiently degraded the parent compound at the µg/L-mg/L level and resulted in transformation products with lower toxicity than the parent compound. A direct positive correlation was observed between the applied UV dose (fluence, J/cm2), the disappearance of tebuconazole, and the decrease in ecotoxicity. The combined VUV/UVC process does not require addition of supplementary oxidants or catalysts and our study suggests that VUV/UVC-mediated photolysis of azole fungicides in water can decrease the overall toxicity and represent a potentially environmentally friendly treatment method.

Effect of UV-A, UV-B and UV-C irradiation of glyphosate on photolysis and mitigation of aquatic toxicity.

The active herbicide ingredient glyphosate [N-(phosphonomethyl)glycine] is frequently detected as a contaminant in groundwater and surface waters. This study investigated effects of UV-A (365 nm), UV-B (302 nm) and UV-C (254 nm) irradiation of glyphosate in water on photolysis and toxicity to aquatic organisms from different trophic levels. A test battery with bacteria (Bacillus subtilis, Aliivibrio fischeri), a green microalga (Raphidocelis subcapitata), and a crustacean (Daphnia magna) was used to assess biological effect of glyphosate and bioactive transformation products before and after UV irradiation (4.7-70 J/cm2). UV-C irradiation at 20 J/cm2 resulted in a 2-23-fold decrease in toxicity of glyphosate to aquatic test organisms. UV-B irradiation at 70 J/cm2 caused a twofold decrease whereas UV-A did not affect glyphosate toxicity at doses ≤ 70 J/cm2. UV-C irradiation of glyphosate in drinking water and groundwater with naturally occurring organic and inorganic constituents showed comparable or greater reduction in toxicity compared to irradiation in deionized water. High-resolution mass spectrometry analyses of samples after UV-C irradiation showed > 90% decreases in glyphosate concentrations and the presence of multiple transformation products. The study suggests that UV mediated indirect photolysis can decrease concentrations of glyphosate and generate less toxic products with decreased overall toxicity to aquatic organisms.

Methods for the identification of farm escapees in feral mink (Neovison vison) populations.

In Denmark, American mink (Neovison vison) have been bred for their fur since the mid-1920s. Mink escaping from farms may supply the feral population. Often, it is of biological and management interest to separate the population of feral mink (i.e. mink caught in the wild) in two groups: 1) mink born on farms i.e., escapees, and 2) mink born in the wild. In this study, two methods were used for separating feral mink into the two groups: a) Comparison of body length of farmed mink and feral mink, and b) Presence of a biomarker (tetracycline: an oral antibiotic used on mink farms). A total of 367 wild caught mink (from the mainland of Denmark and the island of Bornholm), and 147 mink from farms, collected during the period 2014-2018, were used for the analysis of body length. For the testing of tetracycline (TC) as a biomarker, 78 mink from farms where there was knowledge about TC treatment (with or without) were examined for fluorescent markings in the canine teeth. Results from both univariate analyses and Gaussian mixture model analysis demonstrated clear divisions between the mean body length (mean ± S.E., range) of farmed males (52.1 cm ± 0.4, 48-68) and farmed females (mean 44.0 ± 0.2, 40-50), and between farmed mink and wild caught mink. Mixture analysis identified two groups within each sex of the wild caught mink, one assigned to farmed mink (born in captivity) and another group of smaller mink suspected of being born in the wild. On Bornholm, the mean (±SD, range) length of males born in the wild was 43.7cm (± 0.3, 36-57) and for females 37.5cm (± 0.3, 32-45). The mean length (±SD, range) of males born in the wild in the mainland of Denmark was 42.5cm (± 2.3, 36-46) and for females 36.1cm (± 1.0, 34-37). Among the feral mink from mainland Denmark, 28.4% of males and 21.6% of females were identified as escapees, while 0% of the males and 1% of the females were identified as escapees among the wild caught mink on Bornholm. Eight percent of mink from farms using tetracycline were false negatives, while no false positives were found among mink from farms not using TC. TC fluorescence was found in five of 217 mink caught in the wild equivalent to 22% escapees in mainland Denmark. No TC markings were found in mink caught in the wild on Bornholm. In conclusion, both methods a) the body length of mink, and b) fluorescent biomarkers in canine teeth are considered as useful tools to identifing mink that have escaped from farms.

Behavioral responses and starvation survival of Daphnia magna exposed to fluoxetine and propranolol.

Fluoxetine and propranolol are neuroactive human pharmaceuticals that occur frequently as pollutants in surface waters. The potential effects of these pharmaceuticals on aquatic organisms have raised concern but many adverse effects are not well characterized for a broad range of concentrations and endpoints. In this study, 6 biological parameters in the freshwater Cladoceran Daphnia magna were compared for their responses to fluoxetine or propranolol exposure: mobility (dichotomous response), active swimming time, swimming distance, swimming velocity, swimming acceleration speed, and survival in the absence of food (starvation-survival). Changes in swimming behavior was quantified by video tracking followed by image analyses at six exposure concentrations between 100 ng/L and 10 mg/L. Active swimming time and swimming distance were the most responsive parameters among the behavioral traits. Sublethal exposure concentrations resulted in nonmonotonic responses and behavior profiling suggested that fluoxetine and propranolol stimulated swimming activity at 1-10 µg/L whereas lower (0.1-1 µg/L) and higher exposure concentrations (>100 µg/L) inhibited swimming activity. The ability to survive in the absence of food when exposed to fluoxetine or propranolol resulted in EC50 and EC10 values that were lower than for swimming behavior (EC50 of 0.79-0.99 mg/L; EC10 of 1.4-2.9 µg/L). Starvation-survival appeared to be a potentially simple and sensitive endpoint for adverse effects in D. magna at intermediate exposure concentrations. Nonmonotonic behavioral responses at low exposure concentrations and decreased ability to survive starvation should be considered in assessment of adverse effects of pharmaceuticals to freshwater invertebrates.

Ingestion and Egestion of Microplastics by the Cladoceran Daphnia magna: Effects of Regular and Irregular Shaped Plastic and Sorbed Phenanthrene.

The presence of microplastics in aquatic ecosystems is of increasing global concern. This study investigated ingestion, egestion and acute effects of polyethylene microplastics in Daphnia magna. Fate of regular shaped microplastic beads (10-106 µm) were compared with irregular shaped microplastic fragments (10-75 µm). Daphnia magna ingested regular and irregular microplastic with uptake between 0.7 and 50 plastic particles/animal/day when exposed to microplastic concentrations of 0.0001-10 g/L. Egestion of irregular fragments was slower than that of microplastic beads. The EC50 for irregular microplastic was 0.065 g/L whereas microplastic beads were less inhibitory. The potential of microplastic to act as vector for hydrophobic pollutants was examined using [14C]phenanthrene as tracer. Polyethylene microplastic sorbed less [14C]phenanthrene compared to natural plankton organisms (bacteria, algae, yeast). As microplastics are much less abundant in most aquatic ecosystems compared to plankton organisms this suggests a limited role as vector for hydrophobic pollutants under current environmental conditions.

Effects of Ammonia and Density on Filtering of Commensal and Pathogenic Escherichia coli by the Cladoceran Daphnia magna.

Grazing by cladocerans can reduce the survival of enteric bacteria associated with fecal pollution. This study examined the potential of Daphnia magna to filter commensal and pathogenic Escherichia coli of human origin. Grazing on commensal and pathogenic bacteria was comparable, but slightly greater at 20 compared to 15 and 25°C. Filtering activity was strongly dependent on D. magna and E. coli densities at environmentally relevant bacterial concentrations. Maximum feeding rates were >107 cells h-1 daphnid-1, clearance rates were 1-6 mL h-1 daphnid-1, and filtering was independent of bacterial cell sizes between 0.7 and 1.8 µm. Filtering and ingestion of E. coli by D. magna was susceptible to acute inhibition by unionized ammonia with a 24 h EC50 of 0.18 mg L-1 NH3-N, and a LOEC of 0.09 mg L-1 NH3-N. The study indicated that biological and chemical constraints should be considered when applying Daphnia for attenuation of fecal pollution.

Behavioral responses of juvenile Daphnia magna after exposure to glyphosate and glyphosate-copper complexes.

Glyphosate (N-(phosphonomethyl)glycine) is the active ingredient in a range of popular broad-spectrum herbicide formulations. Glyphosate is a chelating agent that can form stable complexes with divalent metal ions including Cu(II). Little is known about the bioavailability and ecotoxicity of glyphosate-Cu(II) complexes to aquatic organisms. In this study, we used video tracking and behavior analysis to investigate sublethal effects of binary mixtures of glyphosate and Cu(II) to juvenile D. magna. Behavioral responses were quantified for individual D. magna after 24h and 48h exposure to glyphosate and glyhosate-Cu(II) mixtures. Sublethal concentrations resulted in decreases in swimming velocity, acceleration speed, and distance moved whereas inactive time of D. magna increased. Distance moved and inactive time were the most responsive parameters to glyphosate and glyphosate-Cu(II) exposure. On a molar basis, glyphosate-Cu(II) complexes appeared more toxic to D. magna than glyphosate alone. The 48h EC50 for glyphosate and glyphosate-Cu(II) determined from swimming distance were 75.2µM and 8.4µM, respectively. In comparison, traditional visual observation of mobility resulted in 48h EC50 values of 52.8µM and 25.5µM for glyphosate and glyphosate-Cu(II), respectively. The behavioral responses indicated that exposure of D. magna to mixtures of glyphosate and Cu(II) attenuated acute metal toxicity but increased apparent glyphosate toxicity due to complexation with Cu(II). The study suggests that glyphosate is a likely mediator of aquatic metal toxicity, and that video tracking provides an opportunity for quantitative studies of sublethal effects of pesticide complexes.

Automated swimming activity monitor for examining temporal patterns of toxicant effects on individual Daphnia magna.

Aquatic pollutants are often biologically active at low concentrations and impact on biota in combination with other abiotic stressors. Traditional toxicity tests may not detect these effects, and there is a need for sensitive high-throughput methods for detecting sublethal effects. We have evaluated an automated infra-red (IR) light-based monitor for recording the swimming activity of Daphnia magna to establish temporal patterns of toxicant effects on an individual level. Activity was recorded for 48 h and the sensitivity of the monitor was evaluated by exposing D. magna to the reference chemicals K2 Cr2 O7 at 15, 20 and 25 °C and 2,4-dichlorophenol at 20 °C. Significant effects (P < 0.001) of toxicant concentrations, exposure time and incubation temperatures were observed. At 15 °C, the swimming activity remained unchanged for 48 h at sublethal concentrations of K2 Cr2 O7 whereas activity at 20 and 25 °C was more biphasic with decreases in activity occurring after 12-18 h. A similar biphasic pattern was observed after 2,4-dichlorophenol exposure at 20 °C. EC50 values for 2,4-dichlorophenol and K2 Cr2 O7 determined from automated recording of swimming activity showed increasing toxicity with time corresponding to decreases in EC50 of 0.03-0.07 mg l(-1) h(-1) . EC50 values determined after 48 h were comparable or lower than EC50 values based on visual inspection according to ISO 6341. The results demonstrated that the swimming activity monitor is capable of detecting sublethal behavioural effects that are toxicant and temperature dependent. The method allows EC values to be established at different time points and can serve as a high-throughput screening tool in toxicity testing. Copyright © 2015 John Wiley & Sons, Ltd.

A fluorescence-based hydrolytic enzyme activity assay for quantifying toxic effects of Roundup® to Daphnia magna.

Daphnia magna is a widely used model organism for aquatic toxicity testing. In the present study, the authors investigated the hydrolytic enzyme activity of D. magna after exposure to toxicant stress. In vivo enzyme activity was quantified using 15 fluorogenic enzyme probes based on 4-methylumbelliferyl or 7-amino-4-methylcoumarin. Probing D. magna enzyme activity was evaluated using short-term exposure (24-48 h) to the reference chemical K2 Cr2 O7 or the herbicide formulation Roundup®. Toxicant-induced changes in hydrolytic enzyme activity were compared with changes in mobility (International Organization for Standardization standard 6341). The results showed that hydrolytic enzyme activity was quantifiable as a combination of whole body fluorescence of D. magna and the fluorescence of the surrounding water. Exposure of D. magna to lethal and sublethal concentrations of Roundup resulted in loss of whole body enzyme activity and release of cell constituents, including enzymes and DNA. Roundup caused comparable inhibition of mobility and alkaline phosphatase activity with median effective concentration values at 20 °C of 8.7 mg active ingredient (a.i.)/L to 11.7 mg a.i./L. Inhibition of alkaline phosphatase activity by Roundup was lowest at 14 °C and greater at 20 °C and 26 °C. The results suggest that the fluorescence-based hydrolytic enzyme activity assay (FLEA assay) can be used as an index of D. magna stress. Combining enzyme activity with fluorescence measurements may be applied as a simple and quantitative supplement for toxicity testing with D. magna.

Microbial toxicity of methyl tert-butyl ether (MTBE) determined with fluorescent and luminescent bioassays.

The inhibitory effects of the fuel additive methyl tert-butyl ether (MTBE) and potential degradation products tert-butanol (TBA) and formaldehyde was examined using mixed microbial biomass, and six strains of bioluminescent bacteria and yeast. The purpose was to assess microbial toxicity with quantitative bioluminescent and fluorescent endpoints, and to identify sensitive proxies suitable for monitoring MTBE contamination. Bioluminescent Aliivibrio fischeri DSM 7151 (formerly Vibrio fischeri) appeared highly sensitive to MTBE exposure, and was a superior test organisms compared to lux-tagged Escherichia coli DH5α, Pseudomonas fluorescens DF57-40E7 and Saccharomyces cerevisiae BLYR. EC10 and EC50 for acute MTBE toxicity in A. fischeri were 1.1 and 10.9 mg L(-1), respectively. Long term (24h) MTBE exposure resulted in EC10 values of 0.01 mg L(-1). TBA was significantly less toxic with EC10 and EC50 for acute and chronic toxicity >1000 mg L(-1). Inhibition of bioluminescence was generally a more sensitive endpoint for MTBE toxicity than measuring intracellular ATP levels and heterotrophic CO2 assimilation. A weak estrogenic response was detected for MTBE at concentrations ⩾ 3.7 g L(-1) using an estrogen inducible bioluminescent yeast strain (S. cerevisiae BLYES). Microbial hydrolytic enzyme activity in groundwater was affected by MTBE with EC10 values of 0.5-787 mg L(-1), and EC50 values of 59-3073 for alkaline phosphatase, arylsulfatase, beta-1,4-glucanase, N-acetyl-beta-d-glucosaminidase, and leucine-aminopeptidase. Microbial alkaline phosphatase and beta-1,4-glucanase activity were most sensitive to MTBE exposure with EC50 ⩽ 64.8 mg L(-1). The study suggests that bioassays with luminescent A. fischeri, and fluorescent assays targeting hydrolytic enzyme activity are good candidates for monitoring microbial MTBE toxicity in contaminated water.

Mycobacterium avium complex in day care hot water systems, and persistence of live cells and DNA in hot water pipes.

The Mycobacterium avium complex (MAC) is a group of opportunistic human pathogens that may thrive in engineered water systems. MAC has been shown to occur in drinking water supplies based on surface water, but less is known about the occurrence and persistence of live cells and DNA in public hot water systems based on groundwater. In this study, we examined the occurrence of MAC in hot water systems of public day care centers and determined the persistence of live and dead M. avium cells and naked DNA in model systems with the modern plumbing material cross-linked polyethylene (PEX). The occurrence of MAC and co-occurrence of Legionella spp. and Legionella pneumophila were determined using cultivation and qPCR. Co-occurrences of MAC and Legionella were detected in water and/or biofilms in all hot water systems at temperatures between 40 and 54 °C. Moderate correlations were observed between abundance of culturable MAC and that of MAC genome copies, and between MAC and total eubacterial genome copies. No quantitative relationship was observed between occurrence of Legionella and that of MAC. Persistence in hot water of live and dead M. avium cells and naked DNA was studied using PEX laboratory model systems at 44 °C. Naked DNA and DNA in dead M. avium cells persisted for weeks. Live M. avium increased tenfold in water and biofilms on PEX. The results suggest that water and biofilms in groundwater-based hot water systems can constitute reservoirs of MAC, and that amplifiable naked DNA is relatively short-lived, whereas PEX plumbing material supports persistence and proliferation of M. avium.

State of the art molecular markers for fecal pollution source tracking in water.

Most environmental waters are susceptible to fecal contamination from animal and/or human pollution sources. To attenuate or eliminate such contamination, it is often critical that the pollution sources are rapidly and correctly identified. Fecal pollution source tracking (FST) is a promising research area that aims to identify the origin(s) of fecal pollution in water. This mini-review focuses on the potentials and limitations of library independent molecular markers that are exclusively or strongly associated with fecal pollution from humans and different animals. Fecal-source-associated molecular markers include nucleic acid sequences from prokaryotes and viruses associated with specific biological hosts, but also sequences such as mitochondrial DNA retrieved directly from humans and animals. However, some fecal-source-associated markers may not be absolutely specific for a given source type, and apparent specificity and frequency established in early studies are sometimes compromised by new studies suggesting variation in specificity and abundance on a regional, global and/or temporal scale. It is therefore recommended that FST studies are based on carefully selected arrays of markers, and that identification of human and animal contributions are based on a multi-marker toolkit with several markers for each source category. Furthermore, future FST studies should benefit from increased knowledge regarding sampling strategies and temporal and spatial variability of marker ratios. It will also be important to obtain a better understanding of marker persistence and the quantitative relationship between marker abundance and the relative contribution from individual fecal pollution source types. A combination of enhanced pathogen screening methods, and validated quantitative source tracking techniques could then contribute significantly to future management of environmental water quality including improved microbial risk assessment.

Characterization and validation of a chemiluminescent assay based on 1,2-dioxetanes for rapid detection of viable Escherichia coli.

[(4-methoxy-4(3-beta-D-galactose-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.0(2,7)]tridec-2,7-ene] ("sbeta-Gal 102") and sodium [4-methoxy-4(3-beta-D-glucuronic acid-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.0(2,7)]tridec-2,7-ene] ("sbeta-Glucor 102") are carbohydrate-containing 1,2-dioxetane compounds that produce chemiluminescence upon enzymatic hydrolysis by beta-D-galactosidase, and beta-D-glucuronidase, respectively. In this study, we have characterized and validated a sensitive detection principle for viable Escherichia coli based on enzymatic cleavage of sbeta-Gal 102 and sbeta-Glucor 102 ("ColiLight II"). The proposed chemiluminescent assay was optimized with respect to analytical requirements including incubation time, temperature, pH, enzyme induction, and cell permeabilization. The sensitivity and specificity rates of the assay were tested on ten different bacterial genera. The assay was found to be representative based on low coefficients of variations for both accuracy and precision. The analysis time was less than 1 h and the analytical detection limit was 10(2) to 10(3) E. coli cells. In combination with membrane filtration and a brief resuscitation step of 4 h, the proposed assay was capable of detecting low concentrations of stressed E. coli in potable water (<30 CFU 100 ml(-1)). The proposed chemiluminescent enzyme assay may be used for assessing the metabolic activity of E. coli in oligotrophic environments and for early warning detection of low concentrations of E. coli in water for human consumption.

Isotope array analysis of Rhodocyclales uncovers functional redundancy and versatility in an activated sludge.

Extensive physiological analyses of different microbial community members in many samples are difficult because of the restricted number of target populations that can be investigated in reasonable time by standard substrate-mediated isotope-labeling techniques. The diversity and ecophysiology of Rhodocyclales in activated sludge from a full-scale wastewater treatment plant were analyzed following a holistic strategy based on the isotope array approach, which allows for a parallel functional probing of different phylogenetic groups. Initial diagnostic microarray, comparative 16S rRNA gene sequence, and quantitative fluorescence in situ hybridization surveys indicated the presence of a diverse community, consisting of an estimated number of 27 operational taxonomic units that grouped in at least seven main Rhodocyclales lineages. Substrate utilization profiles of probe-defined populations were determined by radioactive isotope array analysis and microautoradiography-fluorescence in situ hybridization of activated sludge samples that were briefly exposed to different substrates under oxic and anoxic, nitrate-reducing conditions. Most detected Rhodocyclales groups were actively involved in nitrogen transformation, but varied in their consumption of propionate, butyrate, or toluene, and thus in their ability to use different carbon sources in activated sludge. This indicates that the functional redundancy of nitrate reduction and the functional versatility of substrate usage are important factors governing niche overlap and differentiation of diverse Rhodocyclales members in this activated sludge.

Escherichia coli phylogenetic groups are associated with site of infection and level of antibiotic resistance in community-acquired bacteraemia: a 10 year population-based study in Denmark.

OBJECTIVES: The aim of this study was to assess whether Escherichia coli phylogenetic groups were associated with the site of infection and the level of antibiotic resistance in community-acquired bacteraemia (CAB). METHODS: The population-based cohort study included 1533 unique isolates of E. coli from Danish patients with CAB during a 10 year period. Triplex PCR was used to classify the phylogenetic groups, and susceptibility testing was performed by disc diffusion. Data were analysed using contingency tables and logistic regression. RESULTS: Overall, 65.9% of the 1533 E. coli isolates belonged to phylogroup B2, 16.6% to D, 13.1% to A and 4.4% to B1. B2 was the most prevalent group for all sites of infection, ranging from 69.9% in cases with a urinary tract site of infection to 54.8% in cases with a hepatobiliary tract site of infection. Antibiotic resistance to one and more than three antibiotics, respectively, was most frequent in group D (11.4%/33.9%), followed by A (5.5%/26.9%), B1 (5.9%/19.1%) and B2 (6.7%/7.5%). Regression analysis, with group B2 as reference, confirmed that groups A and B1 were associated with a site of infection other than the urinary tract and that groups A and D were associated with resistance to antibiotics including ampicillin, sulphonamide, trimethoprim, gentamicin and quinolones. CONCLUSIONS: Phylogenetic group B2 was predominant in E. coli CAB. This was the least resistant of the four groups. Phylogroups A and B1 were associated with sites of infection other than the urinary tract, and resistance to multiple antibiotics was most prevalent for groups A and D.

Degradation of phthalate esters in an activated sludge wastewater treatment plant.

Efficient removal of phthalate esters (PE) in wastewater treatment plants (WWTP) is becoming an increasing priority in many countries. In this study, we examined the fate of dimethyl phthalate (DMP), dibutyl phthalate (DBP), butylbenzyl phthalate (BBP), and di-(2-ethylhexyl) phthalate (DEHP) in a full scale activated sludge WWTP with biological removal of nitrogen and phosphorus. The mean concentrations of DMP, DBP, BBP, and DEHP at the WWTP inlet were 1.9, 20.5, 37.9, and 71.9 microg/L, respectively. Less than 0.1%, 42%, 35%, and 96% of DMP, DBP, BBP, and DEHP was associated with suspended solids, respectively. The overall microbial degradation of DMP, DBP, BBP, and DEHP in the WWTP was estimated to be 93%, 91%, 90%, and 81%, respectively. Seven to nine percent of the incoming PE were recovered in the WWTP effluent. Factors affecting microbial degradation of DEHP in activated sludge were studied using [U-(14)C-ring] DEHP as tracer. First order rate coefficients for aerobic DEHP degradation were 1.0 x 10(-2), 1.4 x 10(-2), and 1.3 x 10(-3) at 20, 32, and 43 degrees C, respectively. Aerobic degradation rates decreased dramatically under aerobic thermophilic conditions (<0.1 x 10(-2)h(-1) at 60 degrees C). The degradation rate under anoxic denitrifying conditions was 0.3 x 10(-2)h(-1), whereas the rate under alternating conditions (aerobic-anoxic) was 0.8 x 10(-2)h(-1). Aerobic DEHP degradation in activated sludge samples was stimulated 5-9 times by addition of a phthalate degrading bacterium. The phthalate degrading bacterium was isolated from activated sludge, and maintained a capacity for DEHP degradation while growing on vegetable oil. Collectively, the results of the study identified several controls of microbial PE degradation in activated sludge. These controls may be considered to enhance PE degradation in activated sludge WWTP with biological removal of nitrogen and phosphorus.

Degradation of organic pollutants by methane grown microbial consortia.

Microbial consortia were enriched from various environmental samples with methane as the sole carbon and energy source. Selected consortia that showed a capacity for co-oxidation of naphthalene were screened for their ability to degrade methyl-tert-butyl-ether (MTBE), phthalic acid esters (PAE), benzene, xylene and toluene (BTX). MTBE was not removed within 24 h by any of the consortia examined. One consortium enriched from activated sludge ("AAE-A2"), degraded PAE, including (butyl-benzyl)phthalate (BBP), and di-(butyl)phthalate (DBP). PAE have not previously been described as substrates for methanotrophic consortia. The apparent Km and Vmax for DBP degradation by AAE-A2 at 20 degrees C was 3.1 +/- 1.2 mg l(-1) and 8.7 +/- 1.1 mg DBP (g protein x h)(-1), respectively. AAE-A2 also showed fast degradation of BTX (230 +/- 30 nmol benzene (mg protein x h)(-1) at 20 degrees C). Additionally, AAE-A2 degraded benzene continuously for 2 weeks. In contrast, a pure culture of the methanotroph Methylosinus trichosporium OB3b ceased benzene degradation after only 2 days. Experiments with methane mono-oxygenase inhibitors or competitive substrates suggested that BTX degradation was carried out by methane-oxidizing bacteria in the consortium, whereas the degradation of PAE was carried out by non-methanotrophic bacteria co-existing with methanotrophs. The composition of the consortium (AAE-A2) based on polar lipid fatty acid (PLFA) profiles showed dominance of type II methanotrophs (83-92% of biomass). Phylogeny based on a 16S-rRNA gene clone library revealed that the dominating methanotrophs belonged to Methylosinus/Methylocystis spp. and that members of at least 4 different non-methanotrophic genera were present (Pseudomonas, Flavobacterium, Janthinobacterium and Rubivivax).