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Coupled measurements of nitrate (NO3−), nitrogen (N), and oxygen (O) isotopic composition (δ15NNO3 and δ18ONO3) were used to investigate the sources and processes of N cycling, while the microbial source tracking (MST) method was used to identify microbiological pollution in the surface water of the Sava River Basin (SRB) in autumn in 2014 and 2015 during high and low water discharge. Atmospheric nitrate deposition or nitrate-containing fertilizers were found not to be significant sources of riverine nitrate in the SRB. The ranges of isotope values suggest that NO3− in the SRB derives from soil nitrification, sewage, and/or manure, which were further supported by MST analysis. Microbiological indicators show the existence of hotspots of fecal pollution in the SRB, which are human associated. Long-term observations indicate persistent fecal contamination at selected locations caused by continuous discharge of untreated wastewaters into the SRB.

Biomarkers attract increasing attention in environmental studies, as a tool for detection of exposure and effects of pollution, from both natural and anthropogenic sources. This study aims to assess the impact of multiple stressors during distinctive seasons, covering also extreme hydrological events (extensive flooding in the mid May 2014), on different levels of biological organization in the liver and gills of three closely related freshwater breams. Our previous study on DNA damage in blood cells of these specimens showed increased DNA damage in June 2014, one month after the flooding event. As a continuation of that research, the present study was conducted. As a biomarker of exposure DNA damage was measured by applying the alkaline comet assay, while histopathological alterations were monitored as a biomarker of effect. Additionally, concentrations of metals and metalloids in gills, liver and muscle were assessed. Sampling of fish tissues was performed in 2014, during winter (January and February), spring (March and early June) and summer (late June, July and August). Significant seasonal difference in DNA damage was observed for both tissues. During spring and summer the level of DNA damage in gills was significantly higher when compared to the liver. Histopathological analyses showed higher frequency of alterations in gills during spring, and in liver during summer, but without a significant seasonal difference. Gills had the highest concentration of metals and metalloids during the spring and summer, and liver during winter. Muscle was the least affected tissue during all three seasons. This study highlighted the importance of the multiple biomarker approach and the use of different fish tissues in assessment of surface water pollution.

This study examines the use of freshwater bream (Abramis brama) as a sentinel organism for genotoxicity assessment of the Danube River using the comet assay. Sampling of bream was performed during February, April, August, and November in 2014 to assess seasonal variation of DNA damage level as a response to genotoxicity in annual cycle. Additionally, concentrations of fecal coliforms and enterococci were analyzed and they indicated a critical to strong level of fecal pollution on investigated locality during annual cycle. Comet assay was performed on blood, liver, and gill cells of bream. DNA damage level was expressed using tail intensity (TI %), Olive tail moment (OTM), and tail length (TL pix). According to TI and OTM, all three tissues had the highest level of DNA damage in August. The lowest level of DNA damage in liver was measured during February, in blood during November, and in gills during April. According to TL, gills had the highest level of DNA damage in February, and liver cells had the lowest level of damage during April. Multiple correspondence analysis (MCA) showed that DNA damage in blood cells is under the strong influence of variations in NO2, NO3-, NH4+ levels and also the variation in temperature and oxygen levels. DNA damage in liver cells is highly associated with the variations of Mn, Fe, Cu, Zn, and PO43- levels. DNA damage in gill cells is strongly affected by the variations of As, Cd, Pb, Cr, and COD (Mn) levels. Freshwater bream is shown to be a potentially good indicator organism in genotoxic potential field studies.

In this study we have performed a comprehensive genotoxicological survey along the 900 rkm of the Sava River. In total, 12 sites were chosen in compliance with the goals of GLOBAQUA project dealing with the effects of multiple stressors on biodiversity and functioning of aquatic ecosystems. The genotoxic potential was assessed using a complex battery of bioassays performed in prokaryotes and aquatic eukaryotes (freshwater fish). Battery comprised evaluation of mutagenicity by SOS/umuC test in Salmonella typhimurium TA1535/pSK1002. The level of DNA damage as a biomarker of exposure (comet assay) and biomarker of effect (micronucleus assay) and the level of oxidative stress as well (Fpg-modified comet assay) was studied in blood cells of bleak and spirlin (Alburnus alburnus/Alburnoides bipunctatus respectively). Result indicated differential sensitivity of applied bioassays in detection of genotoxic pressure. The standard and Fpg-modified comet assay showed higher potential in differentiation of the sites based on genotoxic potential in comparison with micronucleus assay and SOS/umuC test. Our data represent snapshot of the current status of the river which indicates the presence of genotoxic potential along the river which can be traced to the deterioration of quality of the Sava River by communal and industrial wastewaters. The major highlight of the study is that we have provided complex set of data obtained from a single source (homogeneity of analyses for all samples).