A robust, scalable, and cost-efficient approach to whole genome sequencing of RSV directly from clinical samples.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections causing significant morbidity and mortality among children and the elderly; two RSV vaccines and a monoclonal antibody have recently been approved. Thus, there is an increasing need for a detailed and continuous genomic surveillance of RSV circulating in resource-rich and resource-limited settings worldwide. However, robust, cost-effective methods for whole genome sequencing of RSV from clinical samples that are amenable to high-throughput are still scarce. We developed Next-RSV-SEQ, an experimental and computational pipeline to generate whole genome sequences of historic and current RSV genotypes by in-solution hybridization capture-based next generation sequencing. We optimized this workflow by automating library preparation and pooling libraries prior to enrichment in order to reduce hands-on time and cost, thereby augmenting scalability. Next-RSV-SEQ yielded near-complete to complete genome sequences for 98% of specimens with Cp values ≤31, at median on-target reads >93%, and mean coverage depths between ~1,000 and >5,000, depending on viral load. Whole genomes were successfully recovered from samples with viral loads as low as 230 copies per microliter RNA. We demonstrate that the method can be expanded to other respiratory viruses like parainfluenza virus and human metapneumovirus. Next-RSV-SEQ produces high-quality RSV genomes directly from culture isolates and, more importantly, clinical specimens of all genotypes in circulation. It is cost-efficient, scalable, and can be extended to other respiratory viruses, thereby opening new perspectives for a future effective and broad genomic surveillance of respiratory viruses. IMPORTANCE: Respiratory syncytial virus (RSV) is a leading cause of severe acute respiratory tract infections in children and the elderly, and its prevention has become an increasing priority. Recently, vaccines and a long-acting monoclonal antibody to protect effectively against severe disease have been approved for the first time. Hence, there is an urgent need for genomic surveillance of RSV at the global scale to monitor virus evolution, especially with an eye toward immune evasion. However, robust, cost-effective methods for RSV whole genome sequencing that are suitable for high-throughput of clinical samples are currently scarce. Therefore, we have developed Next-RSV-SEQ, an experimental and computational pipeline that produces reliably high-quality RSV genomes directly from clinical specimens and isolates.

Small proteins in bacteria - Big challenges in prediction and identification.

Proteins with up to 100 amino acids have been largely overlooked due to the challenges associated with predicting and identifying them using traditional methods. Recent advances in bioinformatics and machine learning, DNA sequencing, RNA and Ribo-seq technologies, and mass spectrometry (MS) have greatly facilitated the detection and characterisation of these elusive proteins in recent years. This has revealed their crucial role in various cellular processes including regulation, signalling and transport, as toxins and as folding helpers for protein complexes. Consequently, the systematic identification and characterisation of these proteins in bacteria have emerged as a prominent field of interest within the microbial research community. This review provides an overview of different strategies for predicting and identifying these proteins on a large scale, leveraging the power of these advanced technologies. Furthermore, the review offers insights into the future developments that may be expected in this field.

Neighbourhood watch: genomic epidemiology of SARS-CoV-2 variants circulating in a German federal state, Mecklenburg-Western Pomerania, in 2020-2022.

ABSTRACTGlobal and even national genome surveillance approaches do not provide the resolution necessary for rapid and accurate direct response by local public health authorities. Hence, a regional network of microbiological laboratories in collaboration with the health departments of all districts of the German federal state of Mecklenburg-Western Pomerania (M-V) was formed to investigate the regional molecular epidemiology of circulating SARS-CoV-2 lineages between 11/2020 and 03/2022. More than 4750 samples from all M-V counties were sequenced using Illumina and Nanopore technologies. Overall, 3493 (73.5%) sequences fulfilled quality criteria for time-resolved and/or spatially-resolved maximum likelihood phylogenic analyses and k-mean/ median clustering (KMC). We identified 116 different Pangolin virus lineages that can be assigned to 16 Nextstrain clades. The ten most frequently detected virus lineages belonged to B.1.1.7, AY.122, AY.43, BA.1, B.1.617.2, BA.1.1, AY.9.2, AY.4, P.1 and AY.126. Time-resolved phylogenetic analyses showed the occurrence of virus clades as determined worldwide, but with a substantial delay of one to two months. Further spatio-temporal phylogenetic analyses revealed a regional outbreak of a Gamma variant limited to western M-V counties. Finally, KMC elucidated a successive introduction of the various virus lineages into M-V, possibly triggered by vacation periods with increased (inter-) national travel activities. The COVID-19 pandemic in M-V was shaped by a combination of several SARS-CoV-2 introductions, lockdown measures, restrictive quarantine of patients and the lineage specific replication rate. Complementing global and national surveillance, regional surveillance adds value by providing a higher level of surveillance resolution tailored to local health authorities.

The role of stormwater infrastructure in regional methane emissions.

Stormwater infrastructure has been recently indicated as a potential hotspot for methane (CH4) emissions. Although local assessments based on direct CH4 measurements are increasingly available, there is currently no standardized approach for evaluating CH4 emissions from different types of stormwater infrastructure, including permanently impounded or fast-draining structures in Urban Drainage Systems (UDS). Therefore, a comparative analysis with wastewater infrastructure systems, such as wastewater treatment plants (WWTPs), is not yet possible. Here, we present a conceptual framework for the first-order quantification and upscaling of CH4 emissions from stormwater infrastructure at local and national scales. We combined in-situ and ex-situ measurements of CH4 emissions with purposely acquired data from selected stormwater facilities to provide initial estimates of CH4 emissions and emission factors for stormwater infrastructure in Germany. The results show that while stormwater infrastructure might emit comparable amounts of CH4 per area as natural and anthropogenically impacted inland waters, it may exhibit higher mean emission factors (up to 7 times) than conventional WWTPs, indicating less efficiency in limiting CH4 emissions than WWTPs. This is particularly true for permanently impounded facilities, which showed substantially higher mean surface CH4 emissions (up to 632 mg m-2 d-1) than fast-draining infrastructure (0.5-1.28 mg m-2 d-1). Permanently impounded sedimentation basins for stormwater management alone may reach up to 60% of the total CH4 emissions originating from WWTPs in Germany. These results are in conflict with the ongoing trend towards increasing implementation of impounded stormwater infrastructure systems, highlighting the urgent need for more extensive assessments of their impact on CH4 dynamics.

MpoxRadar: a worldwide MPXV genomic surveillance dashboard.

The mpox virus (MPXV) is mutating at an exceptional rate for a DNA virus and its global spread is concerning, making genomic surveillance a necessity. With MpoxRadar, we provide an interactive dashboard to track virus variants on mutation level worldwide. MpoxRadar allows users to select among different genomes as reference for comparison. The occurrence of mutation profiles based on the selected reference is indicated on an interactive world map that shows the respective geographic sampling site in customizable time ranges to easily follow the frequency or trend of defined mutations. Furthermore, the user can filter for specific mutations, genes, countries, genome types, and sequencing protocols and download the filtered data directly from MpoxRadar. On the server, we automatically download all MPXV genomes and metadata from the National Center for Biotechnology Information (NCBI) on a daily basis, align them to the different reference genomes, generate mutation profiles, which are stored and linked to the available metainformation in a database. This makes MpoxRadar a practical tool for the genomic survaillance of MPXV, supporting users with limited computational resources. MpoxRadar is open-source and freely accessible at https://MpoxRadar.net.

Lessons learned: overcoming common challenges in reconstructing the SARS-CoV-2 genome from short-read sequencing data via CoVpipe2.

Background: Accurate genome sequences form the basis for genomic surveillance programs, the added value of which was impressively demonstrated during the COVID-19 pandemic by tracing transmission chains, discovering new viral lineages and mutations, and assessing them for infectiousness and resistance to available treatments. Amplicon strategies employing Illumina sequencing have become widely established for variant detection and reference-based reconstruction of SARS-CoV-2 genomes, and are routine bioinformatics tasks. Yet, specific challenges arise when analyzing amplicon data, for example, when crucial and even lineage-determining mutations occur near primer sites. Methods: We present CoVpipe2, a bioinformatics workflow developed at the Public Health Institute of Germany to reconstruct SARS-CoV-2 genomes based on short-read sequencing data accurately. The decisive factor here is the reliable, accurate, and rapid reconstruction of genomes, considering the specifics of the used sequencing protocol. Besides fundamental tasks like quality control, mapping, variant calling, and consensus generation, we also implemented additional features to ease the detection of mixed samples and recombinants. Results: We highlight common pitfalls in primer clipping, detecting heterozygote variants, and dealing with low-coverage regions and deletions. We introduce CoVpipe2 to address the above challenges and have compared and successfully validated the pipeline against selected publicly available benchmark datasets. CoVpipe2 features high usability, reproducibility, and a modular design that specifically addresses the characteristics of short-read amplicon protocols but can also be used for whole-genome short-read sequencing data. Conclusions: CoVpipe2 has seen multiple improvement cycles and is continuously maintained alongside frequently updated primer schemes and new developments in the scientific community. Our pipeline is easy to set up and use and can serve as a blueprint for other pathogens in the future due to its flexibility and modularity, providing a long-term perspective for continuous support. CoVpipe2 is written in Nextflow and is freely accessible from \href{https://github.com/rki-mf1/CoVpipe2}{github.com/rki-mf1/CoVpipe2} under the GPL3 license.

Listeria monocytogenes genes supporting growth under standard laboratory cultivation conditions and during macrophage infection.

The Gram-positive bacterium Listeria monocytogenes occurs widespread in the environment and infects humans when ingested along with contaminated food. Such infections are particularly dangerous for risk group patients, for whom they represent a life-threatening disease. To invent novel strategies to control contamination and disease, it is important to identify those cellular processes that maintain pathogen growth inside and outside the host. Here, we have applied transposon insertion sequencing (Tn-Seq) to L. monocytogenes for the identification of such processes on a genome-wide scale. Our approach identified 394 open reading frames that are required for growth under standard laboratory conditions and 42 further genes, which become necessary during intracellular growth in macrophages. Most of these genes encode components of the translation machinery and act in chromosome-related processes, cell division, and biosynthesis of the cellular envelope. Several cofactor biosynthesis pathways and 29 genes with unknown functions are also required for growth, suggesting novel options for the development of antilisterial drugs. Among the genes specifically required during intracellular growth are known virulence factors, genes compensating intracellular auxotrophies, and several cell division genes. Our experiments also highlight the importance of PASTA kinase signaling for general viability and of glycine metabolism and chromosome segregation for efficient intracellular growth of L. monocytogenes.

A decade of monitoring micropollutants in urban wet-weather flows: What did we learn?

Urban wet-weather discharges from combined sewer overflows (CSO) and stormwater outlets (SWO) are a potential pathway for micropollutants (trace contaminants) to surface waters, posing a threat to the environment and possible water reuse applications. Despite large efforts to monitor micropollutants in the last decade, the gained information is still limited and scattered. In a metastudy we performed a data-driven analysis of measurements collected at 77 sites (683 events, 297 detected micropollutants) over the last decade to investigate which micropollutants are most relevant in terms of 1) occurrence and 2) potential risk for the aquatic environment, 3) estimate the minimum number of data to be collected in monitoring studies to reliably obtain concentration estimates, and 4) provide recommendations for future monitoring campaigns. We highlight micropollutants to be prioritized due to their high occurrence and critical concentration levels compared to environmental quality standards. These top-listed micropollutants include contaminants from all chemical classes (pesticides, heavy metals, polycyclic aromatic hydrocarbons, personal care products, pharmaceuticals, and industrial and household chemicals). Analysis of over 30,000 event mean concentrations shows a large fraction of measurements (> 50%) were below the limit of quantification, stressing the need for reliable, standard monitoring procedures. High variability was observed among events and sites, with differences between micropollutant classes. The number of events required for a reliable estimate of site mean concentrations (error bandwidth of 1 around the "true" value) depends on the individual micropollutant. The median minimum number of events is 7 for CSO (2 to 31, 80%-interquantile) and 6 for SWO (1 to 25 events, 80%-interquantile). Our analysis indicates the minimum number of sites needed to assess global pollution levels and our data collection and analysis can be used to estimate the required number of sites for an urban catchment. Our data-driven analysis demonstrates how future wet-weather monitoring programs will be more effective if the consequences of high variability inherent in urban wet-weather discharges are considered.

Costs and benefits of combined sewer overflow management strategies at the European scale.

Combined sewer overflows (CSOs) may represent a significant source of pollution, but they are difficult to quantify at a large scale (e.g. regional or national), due to a lack of accessible data. In the present study, we use a large scale, 6-parameter, lumped hydrological model to perform a screening level assessment of different CSO management scenarios for the European Union and United Kingdom, considering prevention and treatment strategies. For each scenario we quantify the potential reduction of CSO volumes and duration, and estimate costs and benefits. A comparison of scenarios shows that treating CSOs before discharge in the receiving water body (e.g. by constructed wetlands) is more cost-effective than preventing CSOs. Among prevention strategies, urban greening has a benefit/cost ratio one order of magnitude higher than grey solutions, due to the several additional benefits it entails. We also estimate that real time control may bring on average a CSO volume reduction of just above 20%. In general, the design of appropriate CSO management strategies requires consideration of context-specific conditions, and is best made in the context of an integrated urban water management plan taking into account factors such as other ongoing initiatives in urban greening, the possibility to disconnect impervious surfaces from combined drainage systems, and the availability of space for grey or nature-based solutions.

CovRadar: continuously tracking and filtering SARS-CoV-2 mutations for genomic surveillance.

SUMMARY: The ongoing pandemic caused by SARS-CoV-2 emphasizes the importance of genomic surveillance to understand the evolution of the virus, to monitor the viral population, and plan epidemiological responses. Detailed analysis, easy visualization and intuitive filtering of the latest viral sequences are powerful for this purpose. We present CovRadar, a tool for genomic surveillance of the SARS-CoV-2 Spike protein. CovRadar consists of an analytical pipeline and a web application that enable the analysis and visualization of hundreds of thousand sequences. First, CovRadar extracts the regions of interest using local alignment, then builds a multiple sequence alignment, infers variants and consensus and finally presents the results in an interactive app, making accessing and reporting simple, flexible and fast. AVAILABILITY AND IMPLEMENTATION: CovRadar is freely accessible at https://covradar.net, its open-source code is available at https://gitlab.com/dacs-hpi/covradar. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SARS-CoV-2 Omicron variants BA.1 and BA.2 both show similarly reduced disease severity of COVID-19 compared to Delta, Germany, 2021 to 2022.

German national surveillance data analysis shows that hospitalisation odds associated with Omicron lineage BA.1 or BA.2 infections are up to 80% lower than with Delta infection, primarily in ≥ 35-year-olds. Hospitalised vaccinated Omicron cases' proportions (2.3% for both lineages) seemed lower than those of the unvaccinated (4.4% for both lineages). Independent of vaccination status, the hospitalisation frequency among cases with Delta seemed nearly threefold higher (8.3%) than with Omicron (3.0% for both lineages), suggesting that Omicron inherently causes less severe disease.

Advancing Precision Vaccinology by Molecular and Genomic Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 in Germany, 2021.

BACKGROUND: Comprehensive pathogen genomic surveillance represents a powerful tool to complement and advance precision vaccinology. The emergence of the Alpha variant in December 2020 and the resulting efforts to track the spread of this and other severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern led to an expansion of genomic sequencing activities in Germany. METHODS: At Robert Koch Institute (RKI), the German National Institute of Public Health, we established the Integrated Molecular Surveillance for SARS-CoV-2 (IMS-SC2) network to perform SARS-CoV-2 genomic surveillance at the national scale, SARS-CoV-2-positive samples from laboratories distributed across Germany regularly undergo whole-genome sequencing at RKI. RESULTS: We report analyses of 3623 SARS-CoV-2 genomes collected between December 2020 and December 2021, of which 3282 were randomly sampled. All variants of concern were identified in the sequenced sample set, at ratios equivalent to those in the 100-fold larger German GISAID sequence dataset from the same time period. Phylogenetic analysis confirmed variant assignments. Multiple mutations of concern emerged during the observation period. To model vaccine effectiveness in vitro, we employed authentic-virus neutralization assays, confirming that both the Beta and Zeta variants are capable of immune evasion. The IMS-SC2 sequence dataset facilitated an estimate of the SARS-CoV-2 incidence based on genetic evolution rates. Together with modeled vaccine efficacies, Delta-specific incidence estimation indicated that the German vaccination campaign contributed substantially to a deceleration of the nascent German Delta wave. CONCLUSIONS: SARS-CoV-2 molecular and genomic surveillance may inform public health policies including vaccination strategies and enable a proactive approach to controlling coronavirus disease 2019 spread as the virus evolves.

Prevalence and predictive factors for depressive symptoms among medical students in Germany - a cross-sectional study.

Background: Elevated levels of depressive symptoms among medical students have been the subject of international and national research before, yet associated risk factors and protective factors are to be determined. This study aims to show the burdens of depression at different stages of academic medical education with a special emphasis on correlated risk factors and protective factors. Methods: A total number of n=1103 medical students of a middle-sized German university were sampled and surveyed regarding depressive symptoms and correlating factors. The assessment of potential depressive symptoms was based on the BDI-II. Correlating factors were surveyed through a self-designed questionnaire consisting of possible cofactors for depressive symptoms based on established scientific literature. Results: Survey response rate was 90.2% (1103/1223). The prevalence of depressive symptoms was 11% for mild, 5.6% for moderate and 2.4% for severe symptoms. The sample's most prevalent risk factors were feeling unable to confide one's own worries to someone else (88%); and experiencing a lack of time for partner, friends and family (77%) or hobbies (76%). Significant predictors for depressive symptoms were neuroticism above all, insufficient emotional support, eating irregular meals, use of medication or drugs to calm down, and mental overload. Factors associated with less depressive symptoms could be identified as: spending time with partner, friends, family, hobbies and exercise; and confiding worries to classmates. Conclusions: Every fifth medical student surveyed reported at least mild depressive symptoms. The majority of the surveyed medical students felt unable to confide their worries to someone else and lamented not having enough time for social interaction with peers, family and hobbies. Certain personality traits - such as neuroticism - and insufficient emotional support showed to play important roles in making medical students more prone to developing depressive symptoms. Based on this research, control of the surveyed cofactors associated with depressive symptoms and possible intervention programs targeted to these are proposed to be a key subject of further research.

The association of sleep patterns and depressive symptoms in medical students: a cross-sectional study.

OBJECTIVE: Mental health is a fundamental aspect in ensuring the stable and successful professional life of future physicians. Depressive symptoms can negatively affect the work-life-balance and efficiency at work of medical students. To date, there have been very few studies involving medical students that examine the association between single sleep characteristics and the outcome of the Beck Depression Inventory-II score. Therefore, the aim of the present study is to investigate this possible association. A classroom survey using socio-demographic characteristics, the Beck Depression Inventory-II, and the Pittsburgh Sleep Quality Index was conducted amongst students at a German medical school from December 2017 to September 2018. Data analysis was performed with descriptive statistics and binary logistic regression. RESULTS: Of the students surveyed, 19% showed depressive symptoms with a Beck Depression Inventory-II score over 13 and 42% of these cases were moderate or severe. The occurrence of relevant depressive symptoms was associated with lower sleep quality, higher sleep latency, and the consumption of sleeping pills. In general, female students and students from abroad had a higher risk of depressive symptoms. Addressing these relevant findings in medical school can increase awareness of mental health.

Critical Assessment of MetaProteome Investigation (CAMPI): a multi-laboratory comparison of established workflows.

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

Protein expression profiling of Staphylococcus aureus in response to the bacteriocin bovicin HC5.

Alternative strategies to antibiotic treatment are required to inhibit pathogens, including Staphylococcus aureus. Bacteriocins, such as the lantibiotic bovicin HC5, have shown potential to control pathogens. This study aims to evaluate the stress response of S. aureus to bovicin HC5 using a proteomic approach. Sublethal concentrations of the bacteriocin repressed the synthesis of 62 cytoplasmic proteins, whereas 42 proteins were induced in S. aureus COL. Specifically, synthesis of several proteins involved in amino acid biosynthesis, mainly products of ilv-leu operon, and DNA metabolism, such as DNA polymerase I, decreased following bovicin treatment while proteins involved in catabolism, mainly tricarboxylic acid cycle metabolism, and chaperones were over-expressed. The levels of CodY and CcpA, important regulators involved in the stationary phase adaptation and catabolite repression, respectively, also increased in the presence of the bacteriocin. These results indicate that stress caused by the sublethal concentration of bovicin HC5 in the cell membrane results in growth reduction, reduced protein synthesis, and, at the same time, enhanced the levels of chaperones and enzymes involved in energy-efficient catabolism in an attempt to restore energy and cell homeostasis. These results bring relevant information to amplify the knowledge concerning the bacterial physiological changes in response to the stress caused by the cell exposition to bovicin HC5. New potential targets for controlling this pathogen can also be determined from the new protein expression pattern presented. KEY POINTS: • Bovicin HC5 changed the synthesis of cytoplasmic proteins of S. aureus. • Bovicin HC5 interfered in the synthesis of proteins of amino acids biosynthesis. • Synthesis of chaperones enhanced in the presence of sublethal dosage of bovicin HC5.

Mutations in the gdpP gene are a clinically relevant mechanism for ß-lactam resistance in meticillin-resistant Staphylococcus aureus lacking mec determinants.

In Staphylococcus aureus, resistance to ß-lactamase stable ß-lactam antibiotics is mediated by the penicillinbinding protein 2a, encoded by mecA or by its homologues mecB or mecC. However, a substantial number of meticillin-resistant isolates lack known mec genes and, thus, are called meticillin resistant lacking mec (MRLM). This study aims to identify the genetic mechanisms underlying the MRLM phenotype. A total of 141 MRLM isolates and 142 meticillin-susceptible controls were included in this study. Oxacillin and cefoxitin minimum inhibitory concentrations were determined by broth microdilution and the presence of mec genes was excluded by PCR. Comparative genomics and a genome-wide association study (GWAS) approach were applied to identify genetic polymorphisms associated with the MRLM phenotype. The potential impact of such mutations on the expression of PBP4, as well as on cell morphology and biofilm formation, was investigated. GWAS revealed that mutations in gdpP were significantly associated with the MRLM phenotype. GdpP is a phosphodiesterase enzyme involved in the degradation of the second messenger cyclic-di-AMP in S. aureus. A total of 131 MRLM isolates carried truncations, insertions or deletions as well as amino acid substitutions, mainly located in the functional DHH-domain of GdpP. We experimentally verified the contribution of these gdpP mutations to the MRLM phenotype by heterologous complementation experiments. The mutations in gdpP had no effect on transcription levels of pbp4; however, cell sizes of MRLM strains were reduced. The impact on biofilm formation was highly strain dependent. We report mutations in gdpP as a clinically relevant mechanism for ß-lactam resistance in MRLM isolates. This observation is of particular clinical relevance, since MRLM are easily misclassified as MSSA (meticillin-susceptible S. aureus), which may lead to unnoticed spread of ß-lactam-resistant isolates and subsequent treatment failure.

Up-to-date monitoring data of wastewater and stormwater quality in Germany.

A comprehensive dataset of pollutant concentrations in German urban wastewater systems is available from recently completed monitoring projects. It contains up to 1000 concentration values for each of 79 substances in wastewater treatment plant (WWTP) effluents from 49 sites, and up to 157 values for each of 95 substances in combined sewer overflows (CSOs) from 12 sites. WWTP influents and stormwater outfalls were sampled to a lesser extent. All sampling methods were harmonised and aimed at collecting event or multi-day composite samples over periods of ≥1 year. Among the substances analysed were biocides and pesticides, polycyclic aromatic hydrocarbons, perfluorinated alkyl substances, metals, pharmaceuticals, benzotriazoles, phenols, acesulfame, di-(2-ethylhexyl)phthalate, and hexabromocyclododecanes. Occurrence, concentration ranges, and removal rates of selected WWTPs are presented. CSOs can be confirmed as an important pathway of metals and PAH to receiving waters when compared to WWTPs on the basis of annual per capita loads. The derived volume-weighted site mean concentrations are qualified to be used as representative input data for estimation of average substance emissions in large areas, e.g. on river basin scale, if no site-specific data are available. As such, they will contribute to the development of strategies to reduce substance emissions, taking into account not only WWTPs but also stormwater-related discharges.

Modelling copper emissions from antifouling paints applied on leisure boats into German water bodies.

Copper-containing antifouling paints (AFP) are widely used for leisure boat maintenance. Cu emitted from AFP into German surface water bodies has been suggested to be a significant source of heavy metal pollution, threatening water quality. We developed two scenarios to model Cu emissions from AFP applied on leisure boats on national scale, which allow identifying regional hotspots. The top-down approach (scenario A) was based on a previous study on national AFP consumption, while in the bottom-up approach (scenario B), median and interquartile range of Cu release rates depending on salinity conditions were considered for emission estimation. Both scenarios clearly highlighted the locally high emission pressure on inland waters in popular watersport regions, identifying these as requiring intense protection. Scenario B generally predicted lower Cu emissions (sea: 11.05-25.53 t a-1, inland: 14.15-34.59 t a-1) than scenario A (sea: 22.53 t a-1, inland: 47.97 t a-1). To evaluate their relevance, scenario results were compared to emissions modelled with MoRE (Modelling of Regionalized Emissions), which is used as reporting tool on substance emissions by Germany. According to scenarios A and B, the emission from AFP accounted for 13 % and 4-9 % of the total Cu emissions into inland waters in 2016, respectively. Scenario results were similar or higher than other emission pathways such as industrial direct dischargers. Thus, we consider Cu emissions from AFP as a significant pathway to be included in the MoRE emission inventory. We recommend scenario B for implementation as it allows a more flexible adaptation for future modelling.