Long-term impact of basin-wide wastewater management on faecal pollution levels along the entire Danube River.

The Danube River is, at 2857 km, the second longest river in Europe and the most international river in the world with 19 countries in its catchment. Along the entire river, faecal pollution levels are mainly influenced by point-source emissions from treated and untreated sewage of municipal origin under base-flow conditions. In the past 2 decades, large investments in wastewater collection and treatment infrastructure were made in the European Union (EU) Member States located in the Danube River Basin (DRB). Overall, the share of population equivalents with appropriately biologically treated wastewater (without disinfection) has increased from 69% to more than 85%. The proportion of tertiary treatment has risen from 46 to 73%. In contrast, no comparable improvements of wastewater infrastructure took place in non-EU Member States in the middle and lower DRB, where a substantial amount of untreated wastewater is still directly discharged into the Danube River. Faecal pollution levels along the whole Danube River and the confluence sites of the most important tributaries were monitored during four Danube River expeditions, the Joint Danube Surveys (JDS). During all four surveys, the longitudinal patterns of faecal pollution were highly consistent, with generally lower levels in the upper section and elevated levels and major hotspots in the middle and lower sections of the Danube River. From 2001 to 2019, a significant decrease in faecal pollution levels could be observed in all three sections with average reduction rates between 72 and 86%. Despite this general improvement in microbiological water quality, no such decreases were observed for the highly polluted stretch in Central Serbia. Further improvements in microbiological water quality can be expected for the next decades on the basis of further investments in wastewater infrastructure in the EU Member States, in the middle and lower DRB. In the upper DRB, and due to the high compliance level as regards collection and treatment, improvements can further be achieved by upgrading sewage treatment plants with quaternary treatment steps as well as by preventing combined sewer overflows. The accession of the Western Balkan countries to the EU would also significantly boost investments in wastewater infrastructure and water quality improvements in the middle section of the Danube. Continuing whole-river expeditions such as the Joint Danube Surveys is highly recommended to monitor the developments in water quality in the future.

Wide-scope target screening characterization of legacy and emerging contaminants in the Danube River Basin by liquid and gas chromatography coupled with high-resolution mass spectrometry.

A state-of-the-art wide-scope target screening of 2,362 chemicals and their transformation products (TPs) was performed in samples collected within the Joint Danube Survey 4 (JDS4) performed in 2019. The analysed contaminants of emerging concern (CECs) included three major categories: plant protection products (PPPs), industrial chemicals and pharmaceuticals and personal care products (PPCPs). In total, 586 CECs were detected in the samples including 158 PPPs, 71 industrial chemicals, 348 PPCPs, and 9 other chemicals. A wide-variety of sample matrices were collected including influent and effluent wastewater, groundwater, river water, sediment and biota. Forty-five CECs (19 PPPs, 8 industrial chemicals, 18 PPCPs) were detected at levels above their ecotoxicological thresholds (lowest predicted no-effect concentration (PNEC) values) in one or more of the investigated environmental compartments, indicating potential adverse effects on the impacted ecosystems. Among them 12 are legacy substances; 33 are emerging and qualify as potential Danube River Basin Specific Pollutants (RBSPs). Moreover, the efficiency of the wastewater treatment plants (WWTPs) was evaluated using 20 selected performance indicator chemicals. WWTPs showed effective removal (removal rate ≥80%) and medium removal (removal rate 25-80%) for 6 and 8 of the indicator chemicals, respectively. However, numerous contaminants passed the WWTPs with a lower removal rate. Further investigation on performance of WWTPs is suggested at catchment level to improve their removal efficiency. WWTP effluents are proven to be one of the major sources of contaminants in the Danube River Basin (DRB). Other sources include sewage discharges, industrial and agricultural activities. Continuous monitoring of the detected CECs is suggested to ensure water quality of the studied area.

Target and suspect screening of 4777 per- and polyfluoroalkyl substances (PFAS) in river water, wastewater, groundwater and biota samples in the Danube River Basin.

Per- and polyfluoroalkyl substances (PFAS) are under regulatory scrutiny since some of them are persistent, bioaccumulative, and toxic. The occurrence of 4777 PFAS was investigated in the Danube River Basin (DRB; 11 countries) using target and suspect screening. Target screening involved investigation of PFAS with 56 commercially available reference standards. Suspect screening covered 4777 PFAS retrieved from the NORMAN Substance Database, including all individual PFAS lists submitted to the NORMAN Suspect List Exchange Database. Mass spectrometry fragmentation patterns and retention time index predictions of the studied PFAS were established for their screening by liquid chromatography - high resolution mass spectrometry using NORMAN Digital Sample Freezing Platform (DSFP). In total, 82 PFAS were detected in the studied 95 samples of river water, wastewater, groundwater, biota and sediments. Suspect screening detected 72 PFAS that were missed by target screening. Predicted no effect concentrations (PNECs) were derived for each PFAS via a quantitative structure-toxicity relationship (QSTR)-based approach and used for assessment of their environmental risk. Risk characterization revealed 18 PFAS of environmental concern in at least one matrix. The presence of PFAS in all studied environmental compartments across the DRB indicates a potentially large-scale migration of PFAS in Europe, which might require their further systematic regulatory monitoring.

The potential of large floodplains to remove nitrate in river basins - The Danube case.

Floodplains remove nitrate from rivers through denitrification and thus improve water quality. The Danube River Basin (DRB) has been affected by elevated nitrate concentrations and a massive loss of intact floodplains and the ecosystem services they provide. Restoration measures intend to secure and improve these valuable ecosystem services, including nitrate removal. Our study provides the first large-scale estimate of the function of large active floodplains in the DRB to remove riverine nitrate and assesses the contribution of reconnection measures. We applied a nutrient emission model in 6 river systems and coupled it with denitrification and flooding models which we adapted to floodplains. The floodplains have the capacity to eliminate about 33,200 t nitrate-N annually, which corresponds to 6.5 % of the total nitrogen emissions in the DRB. More nitrate is removed in-stream at regular flow conditions than in floodplain soils during floods. However, increasing frequently inundated floodplain areas reveals greater potential for improvement than increasing the channel network. In total, we estimate that 14.5 % more nitrate can be removed in reconnected floodplains. The largest share of nitrogen emissions is retained in the Yantra and Tisza floodplains, where reconnections are expected to have the greatest impact on water quality. In absolute numbers, the floodplains of the lower Danube convert the greatest quantities of nitrate, driven by the high input loads. These estimates are subject to uncertainties due to the heterogeneity of the available input data. Still, our results are within the range of similar studies. Reconnections of large floodplains in the DRB can, thus, make a distinct contribution to improving water quality. A better representation of the spatial configuration of water quality functions and the effect of floodplain reconnections may support the strategic planning of such to achieve multiple benefits and environmental targets.

An alien parasite affects local fauna-Confirmation of Sinergasilus major (Copepoda: Ergasilidae) switching hosts and infecting native Silurus glanis (Actinopterygii: Siluridae) in Hungary.

In 2016, an intense copepod infection was recorded from a reservoir in proximity to the Danube River in Hungary from visibly emaciated wels catfish, Silurus glanis. The parasite-induced pathology was described but parasite identity was not conclusive. Additional sample collections in 2017 and 2018 allowed for identification using both light and scanning electron microscopy, alongside genetic characterisation. The copepods were confirmed to be ergasilids, Sinergasilus major, distinctly different from any previous infection on silurids in Europe. This is the first record of this parasite from Hungary and the first host record from wels catfish.

A meta-analysis of the value of ecosystem services of floodplains for the Danube River Basin.

Floodplains provide ecosystem services (ES). Their evaluation is complex and integrative assessment remains challenging for sciences and practices. Studies have been published in the last two decades reporting ES monetary values of floodplains. Since ES are site-specific, we focus on those studies regarding the Europe's second largest river basin, namely the Danube River Basin (DRB). By analyzing these studies, we aim to answer the questions: "Do the significant predictor variables differ from previous meta-analyses?" and "Does the spatial database improve the meta-analysis?" In this context, we conducted a systematic review on Scopus and Web of Science combining the four themes "value", "ES", "floodplain", and "location". We conducted a meta-analysis of the Danube floodplains' ES values with different sub-groups based on the ES classes (provisioning, regulating, and cultural) and implemented model selection based on the corrected Akaike Information Criterion. We selected 251 entries from 25 studies to set up with a PostgreSQL spatial database, which provides limitless possibilities to enrich the information on the study areas. We observed that the most important variables to describe ES values of DRB floodplains depend on the ES class, but in general the area proportions of water bodies and riparian landscapes are important, together with the valuation method and the chemical or ecological status of the corresponding river section. Finally, we provided two versions of unconditional benefit-transfer functions to evaluate provisioning, regulating, and cultural ES. This paper complements previously conducted meta-analyses to recognize significant characteristics to value ES and it is a valid basis to help determine the ES value of Danube floodplains.

Occurrence and assessment of environmental risks of endocrine disrupting compounds in drinking, surface and wastewaters in Serbia.

The present study is the first comprehensive monitoring of 13 selected endocrine disrupting compounds (EDCs) in untreated urban and industrial wastewater in Serbia to assess their impact on the Danube River basin and associated freshwaters used as sources for drinking water production in the area. Results showed that natural and synthetic estrogens were present in surface and wastewater at concentrations ranging from 0.1 to 64.8 ng L-1. Nevertheless, they were not detected in drinking water. For alkylphenols concentrations ranged from 1.1 to 78.3 ng L-1 in wastewater and from 0.1 to 37.2 ng L-1 in surface water, while in drinking water concentrations varied from 0.4 to 7.9 ng L-1. Bisphenol A (BPA) was the most abundant compound in all water types, with frequencies of detection ranging from 57% in drinking water, to 70% in surface and 84% in wastewater. Potential environmental risks were characterized by calculating the risk quotients (RQs) and the estrogenic activity of EDCs in waste, surface and drinking water samples, as an indicator of their potential detrimental effects. RQ values of estrone (E1) and estradiol (E2) were the highest, exceeding the threshold value of 1 in 60% of wastewater samples, while in surface water E1 displayed potential risks in only two samples. Total estrogenic activity (EEQt) surpassed the threshold of 1 ng E2 L-1 in about 67% of wastewater samples, and in 3 surface water samples. In drinking water, EEQt was below 1 ng L-1 in all samples.

Characterization of wastewater effluents in the Danube River Basin with chemical screening, in vitro bioassays and antibiotic resistant genes analysis.

Averaged 7-day composite effluent wastewater samples from twelve wastewater treatment plants (WWTPs) in nine countries (Romania, Serbia, Hungary, Slovenia, Croatia, Slovakia, Czechia, Austria, Germany) in the Danube River Basin were collected. WWTPs' selection was based on countries' dominant technology and a number of served population with the aim to get a representative holistic view of the pollution status. Samples were analyzed for 2248 chemicals of emerging concern (CECs) by wide-scope target screening employing LC-ESI-QTOF-MS. 280 compounds were detected at least in one sample and quantified. Spatial differences in the concentrations and distribution of the compounds classes were discussed. Additionally, samples were analyzed for the possible agonistic/antagonistic potencies using a panel of in vitro transactivation reporter gene CALUX® bioassays including ERα (estrogenics), anti-AR (anti-androgens), GR (glucocorticoids), anti-PR (anti-progestins), PPARα and PPARγ (peroxisome proliferators) and PAH assays. The potency of the wastewater samples to cause oxidative stress and induce xenobiotic metabolism was determined using the Nrf2 and PXR CALUX® bioassays, respectively. The signals from each of the bioassays were compared with the recently developed effect-based trigger values (EBTs) and thus allowed for allocating the wastewater effluents into four categories based on their measured toxicity, proposing a putative action plan for wastewater operators. Moreover, samples were analyzed for antibiotics and 13 antibiotic-resistant genes (ARGs) and one mobile genetic element (intl1) with the aim to assess the potential for antibiotic resistance. All data collected from these various types of analysis were stored in an on-line database and can be viewed via interactive map at https://norman-data.eu/EWW_DANUBE.

Assessing the radiological load on the environment in the middle Danube river basin on the basis of a study of the Kopacki Rit Nature Park, Croatia.

A study of the environmental radioactivity in the Kopacki Rit Nature Park, Croatia, is presented. This wildlife reserve is part of the Middle Danube River Basin, and it is exposed to various pollutants due to a number of human activities in the surroundings, where there is a nuclear power plant and also urban centres and areas of intense agricultural production. Results for the activity concentrations of soil and surface water samples do not indicate any elevated radioactivity level, which is confirmed by on-site measurements of ambient dose rate equivalent. An assessment of the radiological load on the local biota, carried out using the ERICA tool, implies an overall low radiological risk even if conservative values of the risk quotient are used. Therefore, human activities do not have a significant effect on the radiological load on the Kopacki Rit area. A similar conclusion might be made with regard to numerous similar environments in the Middle Danube River Basin.

Common barbel (Barbus barbus) as a bioindicator of surface river sediment pollution with Cu and Zn in three rivers of the Danube River Basin in Serbia.

River sediments are a major source of metal contamination in aquatic food webs. Due to the ability of metals to move up the food chain, fishes, occupying higher trophic levels, are considered to be good environmental indicators of metal pollution. The aim of this study was to analyze the metal content in tissues of the common barbel (Barbus barbus), a rheophilous cyprinid fish widely distributed in the Danube Basin, in order to find out if it can be used as a bioindicator of the metal content in the river sediment. We analyzed bioavailable concentrations of 15 elements (Al, As, B, Ba, Co, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Se, Sr, and Zn) in sediments of the Danube (D), the Zapadna Morava (ZM), and the Juzna Morava (JM) using the inductively coupled plasma spectroscopy (ICP-OES). The barbel specimens were collected in the proximity of sediment sampling sites for the analysis of metals in four tissues, gills, muscle, intestine, and liver. The sediment analysis indicated that the ZM is the most polluted with Cu, Ni, and Zn compared to other two rivers. The JM had the lowest concentrations of almost all observed elements, while the Danube sediments were mainly characterized by higher concentrations of Pb. The fish from the ZM had the highest concentration of Cu and Ni in the liver and intestine, and of Zn in the muscle tissue, which was in accordance with the concentrations of these metals in the sediment. Scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM-EDS) was used for further analyses of metal interactions with fish tissues. The results suggest that the barbel can potentially be used as a bioindicator of sediment quality with respect to metal contamination.

Macroparasites and their communities of the European eel Anguilla anguilla (Linnaeus) in the Czech Republic.

This paper summarises the results of parasitological examinations of the European eel Anguilla anguilla (Linnaeus) in the Czech Republic, carried out at the Institute of Parasitology, Czech Academy of Sciences (previously the Czechoslovak Academy of Sciences) within the period of 50 years (1958-2008). Even though this survey is limited to the Czech Republic, it provides extensive data probably incomparable with any other study anywhere regarding the number of eels examined and parasites found. A total of 723 eels was examined from 42 localities that belong to all of the three main river drainage systems in the country, i.e. the Elbe, Danube and Oder river basins. Of the 31 species of adult and larval macroparasites including Monogenea (4 species), Trematoda (3), Cestoda (3), Nematoda (11), Acanthocephala (5), Hirudinea (1), Bivalvia (1), Copepoda (1), Branchiura (1) and Acariformes (1), most of them (30) were recorded from the Elbe River basin. These parasites can be divided into three main groups regarding their host specificity: parasites specific for eels (26%), non-specific adult parasites occurring also in other fishes (61%) and non-specific larvae (13%). The highest number (19) of parasite species was recorded in the Mácha Lake fishpond system in northern Bohemia. The parasite communities in eels from the individual localities exhibited large differences in their species composition and diversity depending on local ecological conditions. The parasite fauna of A. anguilla in the Czech Republic is compared with that in other European countries. The nematode Cucullanus egyptae Abdel-Ghaffar, Bashtar, Abdel-Gaber, Morsy, Mehlhorn, Al Quraishy et Mohammed, 2014 is designated as a species inquirenda.

Genetic and morphological variability of the European mudminnow Umbra krameri (Teleostei, Umbridae) in Serbia and in Bosnia and Herzegovina, a basis for future conservation activities.

As a basis for future conservation activities, the genetic and external body morphology variability of the European mudminnow Umbra krameri, a highly endangered fish species in Serbia and in Bosnia and Herzegovina, was determined for existing populations with the use of molecular markers (mitochondrial and microsatellite DNA) and geometric morphometric methods. Mitochondrial DNA cytochrome b gene analysis revealed two previously undescribed haplotypes: Da1 (the Lugomir population from the Danube River basin) and Sa1 (the Bakreni Batar and the Gromizelj populations from the Sava River system), with a corresponding genetic distance of 0·7%. Paired values of FST and DAS distances for microsatellite marker data show that the difference between the Danube and the Sava populations is seven to nine times higher than the difference between the populations within the Sava River system. Geometric morphometric analyses also support a clear separation of the Lugomir population from the Bakreni Batar and the Gromizelj populations. The analysis of the body shape variation, however, indicates a significant difference between the two genetically indistinguishable Sava populations. The observed genetic and phenetic relationships of the analysed mudminnow populations most probably represent a consequence of historical, geographical and ecological factors. These results will offer guidelines for future protection, conservation and sustainable management of this species in the region.

Characterization of the Danube River sediments using the PMF multivariate approach.

Chemical composition data for the Danube River and its tributaries sediments were analyzed using positive matrix factorization (PMF). The objective was to identify both natural and anthropogenic sources affecting Danube Basin. During the Joint Danube Survey 2 (JDS2) campaign 148 bottom sediments samples were collected. The following elements were analyzed using the X-ray fluorescence technique: Al, As, Ca, Cd, Cl, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, S, Si, Ti, V and Zn. Mercury was determined by cold vapour atomic absorption spectrometry. Three factors were obtained considering the whole dataset (Danube and tributaries), identified as: (i) carbonate component characterized by Ca and Mg; (ii) alumino-silicate component dominated by Si and Al content and the presence of some metals attributed to natural processes; (iii) anthropogenic source identified by Hg, S, P and some heavy metals load. To better characterize the role of tributaries, the Danube and tributaries datasets, were also analyzed separately. The same three factor structures were identified in the Danube dataset. For the tributaries, a four-factor source model gave one further factor dominated by S and P, which could be attributed to the use of fertilizers in agriculture.