Identification of hotspots of genotoxicological and faecal pollution along the Danube and Sava rivers – the whole river surveys

The level of genotoxic pollution was assessed along the Danube River and its most significant tributary Sava River by measuring the level of DNA damage in aquatic organisms collected from the selected sites. The Danube River survey was conducted within the Joint Danube Survey 3 project in 2013 on 34 sites along the 2285 rkm using mussels (Unio sp.) and fish (Alburnus alburnus) as bioindicators. The Sava River survey was conducted within the Globaqua project in 2015 at 12 sites along 900 rkm using fish (A. alburnus/Alburnoides bipunctatus) as bioindicators. The level of DNA damage was evaluated by the comet assay in haemocytes of mussels and blood cells of fish. The level of faecal pollution along the rivers was studied in parallel with genotoxicological surveys by using the standard indicators, total coliforms, Escherichia coli and Enterococci. Quantification was performed with Colilert/Enterolert Quanti-Tray 2000 and MPN approach. Quantitative PCR (qPCR)-based assays for analysis of human- or animal-associated genetic Bacteroidetes faecal markers have been used for tracking the source of pollution (microbial source tracking -MST). The human-associated BacHum and HF183II, the ruminantassociated BacR and the pig-associated Pig2Bac fecal markers were selected. The hotspots of faecal pollution were detected at both rivers. Presence of pollution was especially evident in the countries in which the legislation related to wastewater treatment and management is not fully implemented. In the case of the Danube River the most critical section of the river was the Pannonian plain (sector VI) while in the case of the Sava River the most affected section was the lower stretch of the river. The results of MST revealed the presence of human-associated fecal markers BacHum and HF183II in the majority of the analyzed samples. High correlation was observed between the standard fecal indicators and human associated faecal markers. Within the Danube survey, the highest levels of DNA damage were recorded in organisms from the section VI, which is under the impact of untreated wastewater discharges. In 2013 the Sava River was characterized with a lower level of both faecal and genotoxic pollution in comparison with the Danube. Similar observations were found within the Sava River survey in 2015 where the level of DNA damage in fish specimens from Sava was lower in comparison with the samples from the Danube. At both rivers detected genotoxic potential was traceable to the deterioration of quality by communal and industrial wastewaters. Acknowledgements: International Commission for the Protection of the Danube River, EU Seventh Framework Program GLOBAQUA (no. 603629-ENV-2013-6.2.1). Ministry of Education, Science and Technological Development of the Republic of Serbia projects no. 173045 and 173025, the bilateral project Serbia and Austria: SER Ev. No. 451-03-01039/2015-09/33

Assessment of the faecal contamination along the Sava River and identification of pollution sources

The contamination of water by faecal pollution leads to exposure to pathogens via drinking water production, recreation or irrigation. However, monitoring of microbiological quality of surface waters is quite neglected despite its importance for human health. In the case of Sava River Basin, many of the settlements situated on the river banks discharge high quantities of untreated or improperly treated wastewaters directly into surface waters. Due to usage of water for irrigation, the evaluation of microbiological quality of the Sava River becomes essential for further river management. Water samples were collected during September 2014 on 17 sites and during September 2015 on 15 sites situated along the Sava River. In 2015, additional samples were collected from 4 wastewater outlets detected onsite. Microbiological analyses comprised monitoring the standard indicators of faecal pollution within the surveys and long term monitoring data (obtained within 5 years of routine monitoring at 4 stations). For detection of total coliforms, Escherichia coli and enterococci, Defined Substrate Technology (DST) was used with quantification performed by Colilert Quanti-Tray 2000 system, which provides a Most Probable Number result. Detection of presumptive Clostridium perfringens was performed by membrane filtration method according to ISO 14189:2013. To identify the origin of pollution, microbial source tracking (MST) analyses were employed based on the human-associated BacHum and HF183II, the ruminant-associated BacR and the pigassociated Pig2Bac genetic Bacteroidetes faecal markers. Microbiological indicators showed the existence of hotsposts of faecal pollution in the Sava River. MST confirmed that the pollution is human associated. Long term data at selected sites indicated persistent faecal contamination which leads to conclusion that the sites are under the impact of continuous discharge of wastewaters

Have genetic targets for faecal pollution diagnostics and source tracking revolutionised water quality analysis yet?

The impacts on faecal pollution analysis using nucleic acid-based methods, such as PCR and sequencing, in health-related water quality research were assessed by rigorous literature analysis. A wide range of application areas and study designs has been identified since the first application more than 30 years ago (>1,100 publications). Given the consistency of methods and assessment types, we suggest defining this emerging part of science as a new discipline: genetic faecal pollution diagnostics (GFPD) in health-related microbial water quality analysis. Undoubtedly, GFPD has already revolutionised faecal pollution detection and microbial source tracking, the current core applications. GFPD is also expanding to many other research areas, including infection and health risk assessment, evaluation of microbial water treatment, and support of wastewater surveillance. In addition, storage of DNA extracts allows for biobanking, which opens up new perspectives. The tools of GFPD can be combined with cultivation-based standardised faecal indicator enumeration, pathogen detection, and various environmental data types, in an integrated data analysis approach. This comprehensive meta-analysis provides the scientific status quo of this field, including trend analyses and literature statistics, outlining identified application areas, and discussing the benefits and challenges of nucleic acid-based analysis in GFPD

Spring Water of an Alpine Karst Aquifer Is Dominated by a Taxonomically Stable but Discharge-Responsive Bacterial Community

Alpine karst aquifers are important groundwater resources for the provision of drinking water all around the world. Yet, due to difficult accessibility and long-standing methodological limitations, the microbiology of these systems has long been understudied. The aim of the present study was to investigate the structure and dynamics of bacterial communities in spring water of an alpine limestone karst aquifer (LKAS2) under different hydrological conditions (base vs. event flow). The study was based on high-throughput 16S rRNA gene amplicon sequencing, study design and sample selection were guided by hydrology and pollution microbiology data. Spanning more than 27 months, our analyses revealed a taxonomically highly stable bacterial community, comprising high proportions of yet uncultivated bacteria in the suspended bacterial community fraction. Only the three candidate phyla Parcubacteria (OD1), Gracilibacteria (GN02), Doudnabacteria (SM2F11) together with Proteobacteria and Bacteroidetes contributed between 70.0 and 88.4% of total reads throughout the investigation period. A core-community of 300 OTUs consistently contributed between 37.6 and 56.3% of total reads, further supporting the hypothesis of a high temporal stability in the bacterial community in the spring water. Nonetheless, a detectable response in the bacterial community structure of the spring water was discernible during a high-discharge event. Sequence reads affiliated to the class Flavobacteriia clearly increased from a mean proportion of 2.3% during baseflow to a maximum of 12.7% during the early phase of the studied high-discharge event, suggesting direct impacts from changing hydrological conditions on the bacterial community structure in the spring water. This was further supported by an increase in species richness (Chao1) at higher discharge. The combination of these observations allowed the identification and characterization of three different discharge classes (Q1–Q3). In conclusion, we found a taxonomically stable bacterial community prevailing in spring waters from an alpine karst aquifer over the entire study period of more than 2 years. Clear response to changing discharge conditions could be detected for particular bacterial groups, whereas the most responsive group – bacteria affiliated to the class of Flavobacteriia – might harbor potential as a valuable natural indicator of “system disturbances” in karst aquifers

Global Distribution of Human-Associated Fecal Genetic Markers in Reference Samples from Six Continents

Numerous bacterial genetic markers are available for the molecular detection of human sources of fecal pollution in environmental waters. However, widespread application is hindered by a lack of knowledge regarding geographical stability, limiting implementation to a small number of well-characterized regions. This study investigates the geographic distribution of five human-associated genetic markers (HF183/BFDrev, HF183/BacR287, BacHum-UCD, BacH, and Lachno2) in municipal wastewaters (raw and treated) from 29 urban and rural wastewater treatment plants (750-4»400»000 population equivalents) from 13 countries spanning six continents. In addition, genetic markers were tested against 280 human and nonhuman fecal samples from domesticated, agricultural and wild animal sources. Findings revealed that all genetic markers are present in consistently high concentrations in raw (median log10 7.2-8.0 marker equivalents (ME) 100 mL-1) and biologically treated wastewater samples (median log10 4.6-6.0 ME 100 mL-1) regardless of location and population. The false positive rates of the various markers in nonhuman fecal samples ranged from 5% to 47%. Results suggest that several genetic markers have considerable potential for measuring human-associated contamination in polluted environmental waters. This will be helpful in water quality monitoring, pollution modeling and health risk assessment (as demonstrated by QMRAcatch) to guide target-oriented water safety management across the globe.Fil: Mayer, René E.. Vienna University of Technology; Austria. Interuniversity Cooperation Centre for Water and Health; AustriaFil: Reischer, Georg. Vienna University of Technology; AustriaFil: Ixenmaier, Simone K.. Vienna University of Technology; Austria. Interuniversity Cooperation Centre for Water and Health; AustriaFil: Derx, Julia. Vienna University of Technology; AustriaFil: Blaschke, Alfred Paul. Vienna University of Technology; AustriaFil: Ebdon, James E.. University of Brighton; Reino UnidoFil: Linke, Rita. Vienna University of Technology; Austria. Interuniversity Cooperation Centre Water And Health; AustriaFil: Egle, Lukas. Vienna University of Technology; AustriaFil: Ahmed, Warish. Csiro Land And Water; AustraliaFil: Blanch, Anicet R.. Universidad de Barcelona; EspañaFil: Byamukama, Denis. Makerere University; UgandaFil: Savill, Marion. Affordable Water Limited;Fil: Mushi, Douglas. Sokoine University Of Agriculture; TanzaniaFil: Cristobal, Hector Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; ArgentinaFil: Edge, Thomas A.. Canada Centre for Inland Waters. Environment and Climate Change Canada; CanadáFil: Schade, Margit A.. Bavarian Environment Agency; AlemaniaFil: Aslan, Asli. Georgia Southern University; Estados UnidosFil: Brooks, Yolanda M.. Michigan State University; Estados UnidosFil: Sommer, Regina. Interuniversity Cooperation Centre Water And Health; Austria. Medizinische Universitat Wien; AustriaFil: Masago, Yoshifumi. Tohoku University; JapónFil: Sato, Maria I.. Cia. Ambiental do Estado de Sao Paulo. Departamento de Análises Ambientais; BrasilFil: Taylor, Huw D.. University of Brighton; Reino UnidoFil: Rose, Joan B.. Michigan State University; Estados UnidosFil: Wuertz, Stefan. Nanyang Technological University. Singapore Centre for Environmental Life Sciences Engineering and School of Civil and Environmental Engineering; SingapurFil: Shanks, Orin. U.S. Environmental Protection Agency; Estados UnidosFil: Piringer, Harald. Vrvis Research Center; AustriaFil: Mach, Robert L.. Vienna University of Technology; AustriaFil: Savio, Domenico. Karl Landsteiner University of Health Sciences; AustriaFil: Zessner, Matthias. Vienna University of Technology; AustriaFil: Farnleitner, Andreas. Vienna University of Technology; Austria. Interuniversity Cooperation Centre Water And Health; Austria. Karl Landsteiner University of Health Sciences; Austri

Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis

Niederösterreichische Forschungs- und Bildungsgesellschaft (NFB), Life Science Call 2013Austrian Science Fund (FWF