Quantification of ß-lactamase producing bacteria in German surface waters with subsequent MALDI-TOF MS-based identification and ß-lactamase activity assay.

Environmental oligotrophic bacteria are suspected to be highly relevant carriers of antimicrobial resistance (AMR). However, there is a lack of validated methods for monitoring in the aquatic environment. Since extended-spectrum ß-lactamases (ESBLs) play a particularly important role in the clinical sector, a culturing method based on R2A-medium spiked with different combinations of ß-lactams was applied to quantify ß-lactamase-producing environmental bacteria from surface waters. In German surface water samples (n = 28), oligotrophic bacteria ranging from 4.0 × 103 to 1.7 × 104 CFU per 100 mL were detected on the nutrient-poor medium spiked with 3rd generation cephalosporins and carbapenems. These numbers were 3 log10 higher compared to ESBL-producing Enterobacteriales of clinical relevance from the same water samples. A MALDI-TOF MS identification of the isolates demonstrated, that the method leads to the isolation of environmentally relevant strains with Pseudomonas, Flavobacterium, and Janthinobacterium being predominant ß-lactam resistant genera. Subsequent micro-dilution antibiotic susceptibility tests (Micronaut-S test) confirmed the expression of ß-lactamases. The qPCR analysis of surface waters DNA extracts showed the presence of ß-lactamase genes (blaTEM, blaCMY-2, blaOXA-48, blaVIM-2, blaSHV, and blaNDM-1) at concentrations of 3.7 (±1.2) to 1.0 (±1.9) log10 gene copies per 100 mL. Overall, the results demonstrate a widespread distribution of cephalosporinase and carbapenemase enzymes in oligotrophic environmental bacteria that have to be considered as a reservoir of ARGs and contribute to the spread of antibiotic resistance.

Aerobic co-metabolic cis-Dichloroethene degradation with Trichloroethene as primary substrate and effects of concentration ratios.

Pollution with chloroethenes threaten groundwater resources worldwide. Cis-Dichloroethene (cDCE) and Trichloroethene (TCE) are widespread pollutants that often occur together at contaminated sites, either as primary discharges or as degradation products of anaerobic dechlorination. In this study, comprehensive microcosm experiments were conducted with groundwater samples of seven sites contaminated with chloroethenes. In total, twelve wells with different pollutant concentrations and chloroethene compositions were sampled, and aerobic microcosms including sterile controls were set up. The results revealed interactions as well as interferences between cDCE and TCE. First, co-metabolic cDCE degradation with TCE as growth substrate was detected for the first time in this work. Transformation yields Ty' (molar ratio of co-substrate degraded to primary substrate degraded) of the degradation process were determined and showed a linear relationship with the cDCE/TCE concentration ratio. At low cDCE/TCE ratio, aerobic metabolic TCE degradation can result in complete cDCE removal due to co-metabolic degradation. Secondly, interfering effects were detected at notable cDCE levels resulting in deceleration of TCE degradation and residual concentrations which were also correlating linearly with the cDCE/TCE concentration ratio. These findings are significant for investigating chloroethene contaminated sites and planning remediation strategies. In particular, the efficiency biological remediation methods in the presence of both pollutants can be evaluated more precisely through the knowledge of interactions and interferences. Our study emphasizes that co-contaminants and possible effects of contaminant mixtures on the degradation rates of individual substances should be considered in general.

Electrochemical biostimulation of aerobic metabolic TCE degradation in a bioaugmentation approach.

Chloroethenes are globally prevalent groundwater contaminants. Since 2014, TCE has been shown to be degradable in an aerobic metabolic process where it is used as sole energy source and growth substrate by a mixed bacteria culture (SF culture). In 2019, the SF culture was shown to be successfully used in bioaugmentation approaches under field-relevant conditions. In this study, a combined bio-/electro-approach to stimulate the TCE degradation by the SF culture was investigated in laboratory experiments. Column experiments were set up to compare a bioaugmentation approach with an electrochemical biostimulated bioaugmentation approach. Low strength direct current increased the amount of degraded TCE to about 150 % of the control. Through lowering the inflow concentration of oxygen, the effect of the electro-biostimulation in a low oxygen setting confirmed the potential of the bio-electro process for treatment of oxygen-deprived, TCE-contaminated sites.

Interlaboratory comparison using inactivated SARS-CoV-2 variants as a feasible tool for quality control in COVID-19 wastewater monitoring.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results. In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values. All PCR-laboratories reported SARS-CoV-2 genome copy equivalents (GCE) for all spiked samples with a mean intra- and inter-laboratory variability of 19 % and 104 %, respectively, largely reproducing the spike-in scheme. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/ml wastewater and had limited predictive value when analyzing at a genome coverage below 60 %. This interlaboratory test demonstrates that despite highly heterogeneous isolation and analysis procedures, overall SARS-CoV-2 GCE and mutations were determined accurately. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection.

Performance of bacterial and mitochondrial qPCR source tracking methods: A European multi-center study.

With the advent of molecular biology diagnostics, different quantitative PCR assays have been developed for use in Source Tracking (ST), with none of them showing 100% specificity and sensitivity. Most studies have been conducted at a regional level and mainly in fecal slurry rather than in animal wastewater. The use of a single molecular assay has most often proven to fall short in discriminating with precision the sources of fecal contamination. This work is a multicenter European ST study to compare bacterial and mitochondrial molecular assays and was set to evaluate the efficiency of nine previously described qPCR assays targeting human-, cow/ruminant-, pig-, and poultry-associated fecal contamination. The study was conducted in five European countries with seven fecal indicators and nine ST assays being evaluated in a total of 77 samples. Animal fecal slurry samples and human and non-human wastewater samples were analyzed. Fecal indicators measured by culture and qPCR were generally ubiquitous in the samples. The ST qPCR markers performed at high levels in terms of quantitative sensitivity and specificity demonstrating large geographical application. Sensitivity varied between 73% (PLBif) and 100% for the majority of the tested markers. On the other hand, specificity ranged from 53% (CWMit) and 97% (BacR). Animal-associated ST qPCR markers were generally detected in concentrations greater than those found for the respective human-associated qPCR markers, with mean concentration for the Bacteroides qPCR markers varying between 8.74 and 7.22 log10 GC/10 mL for the pig and human markers, respectively. Bacteroides spp. and mitochondrial DNA qPCR markers generally presented higher Spearman's rank coefficient in the pooled fecal samples tested, particularly the human fecal markers with a coefficient of 0.79. The evaluation of the performance of Bacteroides spp., mitochondrial DNA and Bifidobacterium spp. ST qPCR markers support advanced pollution monitoring of impaired aquatic environments, aiming to elaborate strategies for target-oriented water quality management.

Intrinsic and bioaugmented aerobic trichloroethene degradation at seven sites.

Trichloroethene (TCE) is one of the most prevalent contaminants in groundwater pollution worldwide. Aerobic-metabolic degradation of TCE has only recently been discovered at one field site. It has significant advantages over aerobic co-metabolism because no auxiliary substrates are required, and the oxygen demand is considerably lower. This study investigated the intrinsic degradation potential as well as the stimulation potential by bioaugmentation in microcosm experiments with groundwater from seven different sites contaminated with chloroethenes. An enrichment culture metabolizing TCE aerobically served as inoculum. The groundwater samples were inoculated with liquid culture in mineral salts medium as well as with immobilized culture on silica sand. Additionally, some samples were inoculated with groundwater from the site where the enrichment culture originated. The microcosms without inoculum proved the occurrence of aerobic TCE-metabolizing bacteria stimulated by the supply of oxygen in 54% of the groundwater samples. TCE degradation started in most cases after adaptation times of up to 92 d. The doubling time of 24 d indicated comparatively slow growth of the aerobic TCE degrading microorganisms. Bioaugmentation triggered or accelerated TCE-degradation in all microcosms with chlorothene concentrations below 100 mg L-1. All inoculation strategies (liquid and immobilized enrichment culture or addition of groundwater from the active field site) were successful. Our study demonstrates that aerobic-metabolic TCE degradation can occur and be stimulated across a broad hydrogeologic spectrum and should be considered as a viable option for groundwater remediation at TCE-contaminated sites.

Cascade Filtration With PCR Detection and Field-Flow-Fractionation Online With ICP-MS for the Characterization of DNA Interaction With Suspended Particulate Matter.

The variety of applied antibiotics in animal and human medicine results in the release, development, and spread of relevant numbers of antibiotic resistance genes (ARGs) in the environment. The majority of ARGs are present in intracellular forms (in bacteria). Neglected aspects are extracellular variants of ARGs (eARGs) and their fragments, which have been detected in surface-water samples and sediments. The stability of eARGs is expected to be low; however, binding to particulate matter is likely to improve their stability and also affect their transport and dissemination behavior. Few studies have investigated DNA particle interactions, mostly via indirect characterization of adduct formation in model systems but not in real environmental matrices. Therefore, our study aims at a novel approach for direct characterization of desoxyribonucleic acid (DNA) particle interactions using both cascade filtration and field-flow fractionation. Cascade filtration with quantitative polymerase chain reaction (qPCR) detection indicated retention of ARGs on filters with much larger pore sizes supporting the hypothesis of ARG-particle interactions. However, artifacts from membrane clogging or DNA-membrane interaction cannot be excluded. Consequently, asymmetric flow field-flow fractionation was investigated as an alternative separation technique with the advantage of particle separation in a thin channel, reducing the risk of artifacts. The key method parameters, membrane composition, molecular weight cut off, and carrier composition, were systematically investigated using a calf-thymus DNA-spiked surface-water sample as a model. The results clearly showed a shift in the elution time of clay particles suggesting the presence of DNA-clay adducts. Multi-element detection by inductively coupled plasma mass spectrometry (ICP-MS) enabled monitoring of clay via the Al, Fe, and Si signals and DNA via the P signal. Matching peak profiles for the new fraction in the fractograms of the ARG and DNA-spiked water sample support adduct formation. Further evidence was provided by a novel post-channel filtration approach for the separation of free DNA from DNA-clay adducts.

Transport-based source tracking of contaminants in a karst aquifer: Model implementation, proof of concept, and application to event-based field data.

Identification and location of contamination sources is crucial for water resource protection - especially in karst aquifers which provide 25% of the world´s population with water but are highly vulnerable to contamination. Transport-based source tracking is proposed and verified here as a complementary approach to microbial and chemical source tracking in karst aquifers for identifying and locating such sources of contamination and for avoiding ambiguities that might arise from using one method alone. The transport distance is inversely modelled from contaminant breakthrough curves (BTC), based on analytical solutions of the 1D two-region non-equilibrium advection dispersion equation using GNU Octave. Besides the BTC, the model requires reliable estimates of transport velocity and input time. The model is shown to be robust, allows scripted based, automated 2D sensitivity analyses (interplay of two parameters), and can be favourable when distributed numerical models are inappropriate due to insufficient data. Sensitivity analyses illustrate that the model is highly sensitive to the input time, the flow velocity, and the fraction of the mobile fluid region. A conclusive verification approach was performed by applying the method to synthetic data, tracer tests, and event-based field data. Transport distances were correctly modelled for a set of artificial tracer tests using a discharge-velocity relationship that could be established for the respective karst catchment. For the first time such an approach was shown to be applicable to estimate the maximum distance to the contamination source for coliform bacteria in karst spring water combined with microbial source tracking. However, prediction intervals for the transport distance can be large even in well-studied karst catchments mainly related to uncertainties in the flow velocity and the input time. Using a maximum transport distance is proposed to account for less permeable, "slower" pathways. In general, transport-based source tracking might be used wherever transport can be described by the 1D two-region non-equilibrium model, e.g. rivers and fractured or porous aquifers.

Wastewater-Based Epidemiology for SARS-CoV-2 Biomarkers: Evaluation of Normalization Methods in Small and Large Communities in Southern Germany.

In the context of the COVID-19 pandemic, wastewater-based epidemiology (WBE) emerged as a useful tool to account for the prevalence of SARS-CoV-2 infections on a population scale. In this study, we analyzed wastewater samples from three large (>300,000 people served) and four small (<25,000 people served) communities throughout southern Germany from August to December 2021, capturing the fourth infection wave in Germany dominated by the Delta variant (B.1.617.2). As dilution can skew the SARS-CoV-2 biomarker concentrations in wastewater, normalization to wastewater parameters can improve the relationship between SARS-CoV-2 biomarker data and clinical prevalence data. In this study, we investigated the suitability and performance of various normalization parameters. Influent flow data showed strong relationships to precipitation data; accordingly, flow-normalization reacted distinctly to precipitation events. Normalization by surrogate viruses CrAssphage and pepper mild mottle virus showed varying performance for different sampling sites. The best normalization performance was achieved with a mixed fecal indicator calculated from both surrogate viruses. Analyzing the temporal and spatial variation of normalization parameters proved to be useful to explain normalization performance. Overall, our findings indicate that the performance of surrogate viruses, flow, and hydro-chemical data is site-specific. We recommend testing the suitability of normalization parameters individually for specific sewage systems.

SARS-CoV-2 wastewater surveillance in Germany: Long-term RT-digital droplet PCR monitoring, suitability of primer/probe combinations and biomarker stability.

In recent months, wastewater-based epidemiology (WBE) has been shown to be an important tool for early detection of SARS-CoV-2 circulation in the population. In this study, a detection methodology for SARS-CoV-2 RNA (wildtype and variants of concern) in wastewater was developed based on the detection of different target genes (E and ORF1ab) by polyethylene glycol (PEG) precipitation and digital droplet PCR. This methodology was used to determine the SARS-CoV-2 concentration and the proportion of N501Y mutation in raw sewage of the wastewater treatment plant of the city of Karlsruhe in south-western Germany over a period of 1 year (June 2020 to July 2021). Comparison of SARS-CoV-2 concentrations with reported COVID-19 cases in the catchment area showed a significant correlation. As the clinical SARS-CoV-2 official case report chain takes time, viral RNA titre trends appeared more than 12 days earlier than clinical data, demonstrating the potential of wastewater-based epidemiology as an early warning system. Parallel PCR analysis using seven primer and probe systems revealed similar gene copy numbers with E, ORF, RdRP2 and NSP9 assays. RdPP1 and NSP3 generally resulted in lower copy numbers, and in particular for N1 there was low correlation with the other assays. The occurrence of the N501Y mutation in the wastewater of Karlsruhe was consistent with the occurrence of the alpha-variant (B.1.1.7) in the corresponding individual clinical tests. In batch experiments SARS-CoV-2 RNA was stable for several days under anaerobic conditions, but the copy numbers decreased rapidly in the presence of dissolved oxygen. Overall, this study shows that wastewater-based epidemiology is a sensitive and robust approach to detect trends in the spread of SARS-CoV-2 at an early stage, contributing to successful pandemic management.

[Innovative SARS-CoV-2 crisis management in the public health sector: Corona dashboard and wastewater surveillance using the example of Berchtesgadener Land, Germany]. / Innovatives SARS-CoV-2-Krisenmanagement im öffentlichen Gesundheitswesen: Corona-Dashboard und Abwasserfrühwarnsystem am Beispiel Berchtesgadener Land.

BACKGROUND: The rise of an infectious disease crisis such as the SARS-CoV­2 pandemic posed significant challenges for the administrative structures of the public health service, which resulted in varying levels of efficiency in outbreak management as a function of staffing and digital resources. This substantially impeded the integration of innovative pandemic outbreak management tools. Innovative crisis management, such as cluster tracking, risk group testing, georeferencing, or the integration of wastewater surveillance recommended by the EU Commission, was made significantly more difficult. AIM: In this case study in Berchtesgadener Land, we present the integration of an area-wide georeferenced wastewater surveillance system that captured 95% of the entire population since November 2020. METHODOLOGY: Sampling occurred twice a week at nine municipal wastewater treatment plants and directly from the main sewer at three locations. Samples were pre-treated by centrifugation and subsequently analyzed by digital droplet polymerase chain reaction (PCR) targeting four specific genes of SARS-CoV­2. RESULTS: The integration of an area-wide georeferenced wastewater surveillance system was successful. Wastewater occurrences are plotted for each municipality against cumulative infections over seven days per 100,000 inhabitants. Changes in the infection pattern in individual communities are noticeable ten days ahead of the official case numbers with a sensitivity of approximately 20 in 100,000 inhabitants. DISCUSSION: The integration of this innovative approach to provide a comprehensive overview of the situation by employing a digital dashboard and the use of an early warning system via quantitative wastewater surveillance resulted in very efficient, proactive management, which might serve as a blueprint for other municipalities in Germany.

Beneficial effects of dynamic groundwater flow and redox conditions on Natural Attenuation of mono-, poly-, and NSO-heterocyclic hydrocarbons.

Natural Attenuation (NA) processes have been demonstrated to reduce pollutant loads at different contaminated groundwater sites world-wide and are increasingly considered in contaminated site management concepts. However, data are mainly available for steady state groundwater flow and stable redox conditions as well as pollutants listed in standard regulatory schemes. In this study, the influence of transient groundwater flow and redox conditions on NA was examined at a former gas works site near the river Rhine in Germany. The investigated 78 pollutants included 40 mono- and polyaromatic hydrocarbons (MAHs, PAHs) and 38 NSO-heterocyclic aromatic hydrocarbons (NSO-HET). In the highly polluted areas, the MAHs benzene, indene and indane, the PAHs naphthalene, acenaphthene, 1- and 2-methylnaphthalene and the NSO-HET 2-methylquinoline, carbazole, benzothiophene, dibenzofuran and benzofuran were predominant. Pollutant concentrations decreased with increasing distance from the sources of contamination. At the plume fringes, the MAHs benzene and indane, the PAH acenaphthene, the NSO-HET carbazole, 5-methylbenzothiophene, 2- and 3-methylbenzofuran and 2-methyldibenzofuran were predominant, indicating low retention and slow intrinsic biodegradation of these compounds. The influence of surface water on groundwater level, pollutant concentrations, and redox conditions in the monitoring wells was observed with a permanently installed groundwater sensor. The temporary availability of oxygen was observed at the plume fringes, resulting in aerobic and ferric iron reducing biodegradation processes. Field and laboratory data were used to set-up a groundwater flow and reactive transport model used for quantification of the field mass transfer rates. In conclusion, the study demonstrates that NA is effective under transient flow and redox conditions. A conceptual model and reactive transport simulation can facilitate the interpretation of pronounced fluctuations of pollutant concentration in monitoring wells. Based on the analysis of 78 pollutants, indane, indene and several NSO-HET like carbazole, benzothiophene and 2-methyldibenzofuran are recommended for monitoring at tar oil polluted sites, besides EPA-PAHs and BTEX.

Bacteriophages Are Good Estimators of Human Viruses Present in Water.

The detection of fecal viral pathogens in water is hampered by their great variety and complex analysis. As traditional bacterial indicators are poor viral indicators, there is a need for alternative methods, such as the use of somatic coliphages, which have been included in water safety regulations in recent years. Some researchers have also recommended the use of reference viral pathogens such as noroviruses or other enteric viruses to improve the prediction of fecal viral pollution of human origin. In this work, phages previously tested in microbial source tracking studies were compared with norovirus and adenovirus for their suitability as indicators of human fecal viruses. The phages, namely those infecting human-associated Bacteroides thetaiotaomicron strain GA17 (GA17PH) and porcine-associated Bacteroides strain PG76 (PGPH), and the human-associated crAssphage marker (crAssPH), were evaluated in sewage samples and fecal mixtures obtained from different animals in five European countries, along with norovirus GI + GII (NoV) and human adenovirus (HAdV). GA17PH had an overall sensitivity of ≥83% and the highest specificity (>88%) for human pollution source detection. crAssPH showed the highest sensitivity (100%) and specificity (100%) in northern European countries but a much lower specificity in Spain and Portugal (10 and 30%, respectively), being detected in animal wastewater samples with a high concentration of fecal indicators. The correlations between GA17PH, crAssPH, or the sum of both (BACPH) and HAdV or NoV were higher than between the two human viruses, indicating that bacteriophages are feasible indicators of human viral pathogens of fecal origin and constitute a promising, easy to use and affordable alternative to human viruses for routine water safety monitoring.

Performance of Layer-by-Layer-Modified Multibore® Ultrafiltration Capillary Membranes for Salt Retention and Removal of Antibiotic Resistance Genes.

Polyether sulfone Multibore® ultrafiltration membranes were modified using polyelectrolyte multilayers via the layer-by-layer (LbL) technique in order to increase their rejection capabilities towards salts and antibiotic resistance genes. The modified capillary membranes were characterized to exhibit a molecular weight cut-off (at 90% rejection) of 384 Da. The zeta-potential at pH 7 was -40 mV. Laboratory tests using single-fiber modified membrane modules were performed to evaluate the removal of antibiotic resistance genes; the LbL-coated membranes were able to completely retain DNA fragments from 90 to 1500 nt in length. Furthermore, the pure water permeability and the retention of single inorganic salts, MgSO4, CaCl2 and NaCl, were measured using a mini-plant testing unit. The modified membranes had a retention of 80% toward MgSO4 and CaCl2 salts, and 23% in case of NaCl. The modified membranes were also found to be stable against mechanical backwashing (up to 80 LMH) and chemical regeneration (in acidic conditions and basic/oxidizing conditions).

Bioactivation of Quinolines in a Recombinant Estrogen Receptor Transactivation Assay Is Catalyzed by N-Methyltransferases.

Hydroxylation of polyaromatic compounds through cytochromes P450 (CYPs) is known to result in potentially estrogenic transformation products. Recently, there has been an increasing awareness of the importance of alternative pathways such as aldehyde oxidases (AOX) or N-methyltransferases (NMT) in bioactivation of small molecules, particularly N-heterocycles. Therefore, this study investigated the biotransformation and activity of methylated quinolines, a class of environmentally relevant N-heterocycles that are no native ligands of the estrogen receptor (ER), in the estrogen-responsive cell line ERα CALUX. We found that this widely used cell line overexpresses AOXs and NMTs while having low expression of CYP enzymes. Exposure of ERα CALUX cells to quinolines resulted in estrogenic effects, which could be mitigated using an inhibitor of AOX/NMTs. No such mitigation occurred after coexposure to a CYP1A inhibitor. A number of N-methylated but no hydroxylated transformation products were detected using liquid chromatography-mass spectrometry, which indicated that biotransformations to estrogenic metabolites were likely catalyzed by NMTs. Compared to the natural ER ligand 17ß-estradiol, the products formed during the metabolization of quinolines were weak to moderate agonists of the human ERα. Our findings have potential implications for the risk assessment of these compounds and indicate that care must be taken when using in vitro estrogenicity assays, for example, ERα CALUX, for the characterization of N-heterocycles or environmental samples that may contain them.

Ecotoxicity of Nitrogen, Sulfur, or Oxygen Heterocycles and Short-Chained Alkyl Phenols Commonly Detected in Contaminated Groundwater.

Nitrogen, sulfur, or oxygen heterocyclic aromatic hydrocarbons (NSO-HETs) and short-chained alkyl phenols (SCAPs) are commonly detected in groundwater at contaminated sites and in the surrounding environment. It is now scientific consensus that these chemicals pose a risk to human and ecosystem health. However, toxicity data are comparably fragmentary, and only few studies have addressed the ecotoxicity of NSO-HETs and SCAPs in a systematic and comparative fashion. To overcome this shortcoming, we tested 18 SCAPs, 16 NSO-HETs, as well as the homocyclic hydrocarbons indane and indene in the Microtox® assay with Aliivibrio fischeri, the growth inhibition test with Desmodesmus subspicatus, the acute immobilization assay with Daphnia magna, as well as the fish embryo toxicity test with embryos of the zebrafish (Danio rerio). Because of the physicochemical properties of the tested chemicals (limited water solubility, volatility, and sorption to test vessels), actual exposure concentrations in test media and their dissipation over time were analytically quantified by means of gas chromatography with mass spectrometry. Analytically corrected effect levels (median effect and lethal concentrations) ranged from 0.017 to 180 mg L-1 , underlining the environmental relevance of some NSO-HETs and SCAPs. Para-substituted phenols showed the overall greatest toxicities in all 4 toxicity tests. We provide, for the first time, a complete high-quality data set in support of better environmental risk assessments of these chemicals. Environ Toxicol Chem 2019;38:1343-1355. © 2019 SETAC.

Evaluation and application of molecular denitrification monitoring methods in the northern Lake Tai, China.

Worldwide, excessive reactive nitrogen in groundwater and surface waters is a growing problem, especially in areas that face rapid urbanization and industrialization. One example for environmental nitrogen pollution is the Lake Tai, China's third largest freshwater Lake, located in the Yangtze River basin. Due to the rapid development of the surrounding area, nitrogen compounds like nitrate are discharged into the Lake. Consequently, eutrophication and harmful algae blooms increased and led to the production of toxins directly affecting water consumers through the water supply chain. Denitrification is the main process that attenuates nitrate by converting it into atmospheric nitrogen and represents an intrinsic natural process to compensate the excess reactive nitrogen. In this study, the methodology to detect nitrate reducing bacteria on a functional gene and transcriptional level was optimized and verified in laboratory experiments with a pure culture of Pseudomonas veronii, isolated from Lake Tai. We demonstrated that transcripts analysis (mRNA) did correspond with nitrate reduction activity. Subsequently, the abundance and the activity of nitrate reducing bacteria in Lake Tai were assessed using the developed methods. We demonstrated that nitrate reducing bacteria can be found throughout all sediment and water samples taken from the northern Lake Tai in September 2017. Measurements of narG transcripts also indicated the activity of the membrane-bound nitrate reductase in the water samples. However, the bioinformatic analysis of narG sequences showed varying binding efficiency of primer and gene sites in dependence of phylogenetic groups, which may lead to an underestimation in the qPCR method. Thus, it is important to point out the precautions and limitations of primer systems to monitor nitrogen transformation by qPCR in the environment. Based on this study, mRNA detection methods are suitable for improved microbiological monitoring of denitrification, as an intrinsic process in Lake Tai to mitigate the inflowing reactive nitrogen compounds.

Aerobic metabolic trichloroethene biodegradation under field-relevant conditions.

Chloroethenes belong to the most widely distributed groundwater contaminants. Since 2014, it has been known that trichloroethene (TCE) can be degraded aerobically and metabolically as growth substrate by a mixed bacterial enrichment culture (named SF culture). In this study, the degradation capabilities under a range of field-relevant conditions were investigated in fixed-bed reactors as well as in batch experiments. Aerobic metabolic TCE degradation was stable over the long term, with degradation optima at 22 °C and pH 7. Degradation of up to 400 µM TCE was observed. The longest starvation period after which degradation of TCE was regained was 112 days. The possible co-contaminants perchloroethene, trans-1,2-dichloroethene, and cis-1,2-dichloroethene did not inhibit TCE degradation, even though they were not degraded themselves. The presence of equimolar amounts of 1,1-dichloroethene and vinyl chloride inhibited TCE degradation. Experiments with groundwater from different chloroethene-contaminated field sites proved the potential of the SF culture for bioaugmentation. Thus, aerobic metabolic TCE degradation should be considered as a promising method for the bioremediation of field sites with TCE as the main contaminant.