Combined use of biomarkers to assess the impact of untreated wastewater from the Danube River, Serbia.

In this study a battery of bioassays, both in vivo (metals and metalloids concentrations, erythrocyte morphometry, comet assay, micronucleus assay, and histopathological analyses) on vimba bream Vimba vimba (L., 1758) and white bream Blicca bjoerkna (L., 1758), and in vitro (treatment of HepG2 cells with native water samples) was applied to assess the harmful potential of untreated wastewater. Faecal indicator bacteria were quantified to assess the microbiological water quality. Vimba bream had significantly higher Fe concentrations in both liver and muscle, while white bream had higher Ca and Cu concentrations in liver. Vimba bream had a significantly higher level of DNA damage in both liver and blood cells, in comparison to white bream. Low levels of micronucleus and nuclear abnormalities were observed in both species. Erythrocytes morphometry did not show significant interspecific differences. Histopathological analyses revealed a similar response of the studied species, with a significantly higher presence of ceroid pigments in the liver of vimba bream. Treatment of HepG2 cells revealed the high genotoxic potential of water downstream of the discharge point. The results of this study clearly demonstrate the importance of effect-based monitoring, in order to enforce more efficient management of natural resources and implementation of wastewater treatment systems.

In situ detection of the genotoxic potential as one of the lines of evidence in the weight-of-evidence approach-the Joint Danube Survey 4 Case Study.

Environmental studies which aim to assess the ecological impact of chemical and other types of pollution should employ a complex weight-of-evidence approach with multiple lines of evidence (LoEs). This study focused on in situ genotoxicological methods such as the comet and micronucleus assays and randomly amplified polymorphic DNA analysis as one of the multiple LoEs (LoE3) on the fish species Alburnus alburnus (bleak) as a bioindicator. The study was carried out within the Joint Danube Survey 4 (JDS4) at nine sites in the Danube River Basin in the Republic of Serbia. Out of nine sampling sites, two were situated at the Tisa, Sava, and Velika Morava rivers, and three sites were at the Danube River. The three additionally employed LoEs were: SumTUwater calculated based on the monitoring data in the database of the Serbian Environmental Protection Agency (SEPA) (LoE1); in vitro analyses of JDS4 water extracts employing genotoxicological methods (LoE2); assessment of the ecological status/potential by SEPA and indication of the ecological status for the sites performed within the JDS4 (LoE4). The analyzed biomarker responses in the bleak were integrated into the unique integrated biomarker response index which was used to rank the sites. The highest pollution pressure was recorded at JDS4 39 and JDS4 36, while the lowest was at JDS4 35. The impact of pollution was confirmed at three sites, JDS4 33, 40, and 41, by all four LoEs. At other sampling sites, a difference was observed regarding the pollution depending on the employed LoEs. This indicates the importance of implementing a comprehensive weight-of-evidence approach to ensure the impact of pollution is not overlooked when using only one LoE as is often the case in environmental studies.

Detection of SARS-CoV-2 RNA in the Danube River in Serbia associated with the discharge of untreated wastewaters.

In Serbia less than 13% of collected municipal wastewaters is being treated before their release in the environment. This includes all municipal wastewater discharges from Belgrade (capital city of Serbia; population 1,700,000). Previous research has identified the impacts of raw wastewater discharges from Belgrade on the Danube River, and this study investigated if such discharges also provided a pathway for SARS-CoV-2 RNA material. Samples were collected during the most critical circumstances that occurred so far within the COVID-19 pandemics in Serbia. Grab and composite samples were collected in December 2020, during the peak of the third wave (in terms of reported cases) at the site which receives the wastewater loads in Belgrade. Grab samples collected upstream and downstream of Belgrade were also analyzed. RNA was quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. SARS-CoV-2 RNA (5.97 × 103 to 1.32 × 104 copies/L) was detected only in samples collected at the site strongly impacted by the wastewaters where all three applied primer sets gave positive signals. Determined concentrations correspond to those reported in wastewater influents sampled at treatment plants in other countries indicating an epidemiological indicator function of used approach for rivers with high pollution loads in countries with poor wastewater treatment.

Genotoxicity of fluoride subacute exposure in rats and selenium intervention.

The aims of this study were to: (i) examine the toxic effects of sodium fluoride (NaF) in blood, liver, spleen, and brain cells of Wistar rats after the subacute exposure; (ii) explore the potential protective properties of selenium (Se) against fluoride toxicity after the simultaneous administration. Twenty male Wistar rats, eight weeks old, weighing approximately 140-190 g, were divided into four experimental groups (n = 5) as follows: I control-tap water; II NaF 150 ppm; III NaF 150 ppm and Se 1.5 mg/L; IV Se 1.5 mg/L, and had available water with solutions ad libitum for 28 days. DNA damage detected by comet assay was confirmed in the liver, spleen, and brain cells, but not in blood. Selenium supplementation together with NaF decreased DNA damage in liver and spleen cells. According to the histological findings, no changes were observed in spleen and brain tissues after NaF administration. Unlike the observed Se protective effect on the DNA level, no significant reduction of liver tissue injury was observed after the NaF and Se treatment, resulting in mild inflammation. Data of this study suggest that DNA damage after NaF subacute exposure at moderately high concentration was reduced in liver and spleen cells due to Se supplementation, but a similar change was not seen in the brain.

Sensitivity of Bleak (Alburnus alburnus) in Detection of the Wastewater Related Pressure in Large Lowland Rivers.

This study deals with bleak (Alburnus alburnus) sensitivity in detecting of the wastewater related pressure in large lowland rivers. The major objective was to investigate if the response measured in bleak should be linked to a certain stretch of the river and characterised as "stretch specific", or it should be linked to the sampling site and characterised as "site specific". The response was evaluated via condition index, metal pollution index, DNA damage and cell viability using integrated biomarker response approach. The study was conducted at 3 sub-sites characterized by different pollution levels in a relatively short stretch (2 km) of the Sava River (Serbia). Results indicated that the response of the biomarkers in bleak can be interpreted as "site specific". Among the studied biomarkers, DNA damage assessed by comet assay and micronucleus test has shown high sensitivity in differentiation of the sites.

Selection of assay, organism, and approach in biomonitoring significantly affects the evaluation of genotoxic potential in aquatic environments.

In this study, few different evaluation concepts were used for the assessment of genotoxic potential at the stretch of the Danube River identified as a significant hotspot of pollution originated through the untreated wastewaters. Three sites were chosen: one site upstream of the wastewater outlet in Novi Sad (Serbia), one at the outlet of wastewaters, and one site few kilometer downstream. Ex situ approach comprised prokaryotic SOS/umuC test on Salmonella typhimurium TA1535/pSK1005 and comet assay on human hepatoma cell line (HepG2). In situ approach was based on the active monitoring (cage approach) using freshwater mussels Sinanodonta woodiana and fish Cyprinus carpio. The comet and micronucleus assays were selected for evaluation of DNA damage in mussel haemocytes and fish blood cells. Within the ex situ part of the study, our results indicated that the eukaryotic model system is more sensitive compared to the prokaryotic one. In situ bioassays are recommended for obtaining a better insight into ecosystem status and in the case of our study the complete insight of genotoxic pressure. However, the choice of animals as bioindicators also has a significant impact on the quality of the obtained information. Differential response between fish and mussels was observed at the highly polluted site suggesting possible involvement of additional protective mechanism such as valve closure in mussels.

Seasonal variation in metal concentration in various tissues of the European chub (Squalius cephalus L.).

Due to the increasing industrialization, metals are discharged into all spheres of the environment, in particular, in river courses, which leads to the need for constant monitoring. Metals do not degrade into harmless end products; they are very persistent and have high potential for accumulation in biota. Metals in the fish body are accumulated in different amounts in the tissue specific matter. In relation to the biotic conditions and fish metabolism, the load of food, or the stage of the reproductive cycle, the seasonal variation of metal levels can be expected. Because of that, the objective of our present study was to analyze 15 metals and metalloids in liver, gills, muscle, and gonads of European chub (Squalius cephalus) throughout the 4 seasons, autumn, winter, spring, and summer. The specimens were collected from two rivers, Pestan and Beljanica at the Kolubara basin, and their concentrations were determined with inductively coupled plasma optical emission spectrometry (ICP-OES). Specimens from both rivers have shown similarities in metal accumulation like the highest accumulation of majority of elements in gills, lowest accumulation of majority of elements in muscle (except for Hg), and higher accumulation of some elements in summer (Cu, Fe, Zn). In addition, Cu and Fe showed affinity for liver, while Ba, Cr, Sr, and Zn were specific for gills. Also, Al, B, Fe, Ni, and Pb did not show significant differences in concentrations among different seasons in all investigated tissues.

Evaluation of genotoxic potential of avarol, avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models.

In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3'-methoxyavarone, 4'-(methylamino)avarone and 3'-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3'-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3'-methoxyavarone and 3'-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.

Cyanobacterial effects in Lake Ludos, Serbia - Is preservation of a degraded aquatic ecosystem justified?

Cyanobacteria are present in many aquatic ecosystems in Serbia. Lake Ludos, a wetland area of international significance and an important habitat for waterbirds, has become the subject of intense research interest because of practically continuous blooming of cyanobacteria. Analyses of water samples indicated a deterioration of ecological condition and water quality, and the presence of toxin-producing cyanobacteria (the most abundant Limnothrix redekei, Pseudanabaena limnetica, Planktothrix agardhii and Microcystis spp.). Furthermore, microcystins were detected in plants and animals from the lake: in macrophyte rhizomes (Phragmites communis, Typha latifolia and Nymphaea elegans), and in the muscle, intestines, kidneys, gonads and gills of fish (Carassius gibelio). Moreover, histopathological deleterious effects (liver, kidney, gills and intestines) and DNA damage (liver and gills) were observed in fish. A potential treatment for the reduction of cyanobacterial populations employing hydrogen peroxide was tested during this study. The treatment was not effective in laboratory tests although further in-lake trials are needed to make final conclusions about the applicability of the method. Based on our observations of the cyanobacterial populations and cyanotoxins in the water, as well as other aquatic organisms and, a survey of historical data on Lake Ludos, it can be concluded that the lake is continuously in a poor ecological state. Conservation of the lake in order to protect the waterbirds (without urgent control of eutrophication) actually endangers them and the rest of the biota in this wetland habitat, and possibly other ecosystems. Thus, urgent measures for restoration are required, so that the preservation of this Ramsar site would be meaningful.

Evaluation of genotoxic potential in the Velika Morava River Basin in vitro and in situ.

The Velika Morava River is the greatest national Serbian river and the significant tributary of the Danube River. The major problems in the Velika Morava River Basin (VMRB) represent untreated industrial and municipal wastewaters. In this study, the level of genotoxic potential at the sites along the VMRB was evaluated by parallel in vitro and in situ approach. Within in vitro testing, genotoxicity of native water samples collected from the sites in VMRB was evaluated by SOS/umuC test on Salmonella typhimurium TA1535/pSK1002 and by the comet assay on HepG2 cells. DNA damage in situ was assessed in bleak (Alburnus alburnus) erythrocytes by the comet (alkaline and Fpg-modified comet) and micronucleus assays. Additionally, the concentration of heavy metals in fish tissue was measured and this data, compiled with the data of the physico-chemical parameters measured in water, was used as a measure of the pollution pressure at the sites. Results showed that applied in vitro tests with native water samples are less sensitive in comparison with in situ tests and should be taken with precaution when making predictions on the status of the ecosystem. Within applied battery of in situ assays differential sensitivity of assays was observed where alkaline comet assay showed the highest potential in differentiation of the sites based on genotoxic potential. Integrated biomarker response showed that usage of the battery of bioassays provides better insight in a genotoxic effects in animals, and consequently, that the holistic approach is more suitable for this type of study.

Assessment of status of three water bodies in Serbia based on tissue metal and metalloid concentration (ICP-OES) and genotoxicity (comet assay).

Metals and metalloids are natural components of the biosphere, which are not produced per se by human beings, but whose form and distribution can be affected by human activities. Like all substances, they are a contaminant if present in excess compared to background levels and/or in a form that would not normally occur in the environment. Samples of liver, gills, gonads and muscle from European chub, Squalius cephalus, were analyzed for Al, As, B, Ba, Cr, Cu, Fe, Hg, Mn, Mo, Sr and Zn using inductively coupled plasma optical emission spectrometry (ICP-OES) to highlight the importance of tissue selection in monitoring research. The comet assay or single cell gel electrophoresis (SCGE) was selected as an in vivo genotoxicity assay, a rapid and sensitive method for measuring genotoxic effects in blood, liver and gills of the European chub. Microscopic images of comets were scored using Comet IV Computer Software (Perceptive Instruments, UK). The objective of our study was to investigate two reservoirs, Zlatar and Garasi, and one river, Pestan by: (i) determining and comparing metal and metalloid concentrations in sediment, water and tissues of European chub: liver, gills, muscle and gonads (ii) comparing these findings with genotoxicity of water expressed through DNA damage of fish tissues. A clear link between the level of metals in water, sediment and tissues and between metal and genotoxicity levels at examined sites was not found. This suggests that other xenobiotics (possibly the organic compounds), contribute to DNA damage.

Evaluation of single-cell gel electrophoresis data: combination of variance analysis with sum of ranking differences.

Specimens of the mussel Mytilus galloprovincialis were collected from five sites in the Boka Kotorska Bay (Adriatic Sea, Montenegro) during the period summer 2011-autumn 2012. Three types of tissue, haemolymph, digestive gland were used for assessment of DNA damage. Images of randomly selected cells were analyzed with a fluorescence microscope and image analysis by the Comet Assay IV Image-analysis system. Three parameters, viz. tail length, tail intensity and Olive tail moment were analyzed on 4200 nuclei per cell type. We observed variations in the level of DNA damage in mussels collected at different sites, as well as seasonal variations in response. Sum of ranking differences (SRD) was implemented to compare use of different types of cell and different measure of comet tail per nucleus. Numerical scales were transferred into ranks, range scaling between 0 and 1; standardization and normalization were carried out. SRD selected the best (and worst) combinations: tail moment is the best for all data treatment and for all organs; second best is tail length, and intensity ranks third (except for digestive gland). The differences were significant at the 5% level. Whereas gills and haemolymph cells do not differ significantly, cells of the digestive gland are much more suitable to estimate genotoxicity. Variance analysis decomposed the effect of different factors on the SRD values. This unique combination has provided not only the relative importance of factors, but also an overall evaluation: the best evaluation method, the best data pre-treatment, etc., were chosen even for partially contradictory data. The rank transformation is superior to any other way of scaling, which is proven by ordering the SRD values by SRD again, and by cross validation.

The impact of in vivo and in vitro exposure to base analogue 5-FU on the level of DNA damage in haemocytes of freshwater mussels Unio pictorum and Unio tumidus.

The impact of in vivo and in vitro exposure to anticancer drug 5-Fluorouracil (5-FU) on the level of DNA damage was evaluated using comet assay on haemocytes of freshwater mussels Unio pictorum and Unio tumidus. For the in vivo experiment, the groups of 5 mussels per concentration were exposed for 72 h to 5-FU (0.04, 0.4, 4, 40 and 100 µM) with 0.4 µM being the lowest concentration to induce significant DNA damage. For the in vitro experiment, the primary cultures of haemocytes were treated with 0.04, 0.4, 4 and 40 µM 5-FU for 22 h and the treatment with CdCl2 was used as a positive control. In contrast to in vivo exposure, 5-FU did not induce significant increase of DNA damage in vitro, possibly because of the absence of haemocytes proliferation in primary cultures.

Heavy metal accumulation and the genotoxicity in barbel (Barbus barbus) as indicators of the Danube river pollution.

The aim of this study was to analyze 16 trace elements (Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Li, Mn, Mo, Ni, Pb, Sr, and Zn) in different barbel (Barbus barbus) tissues and to detect the presence of genotoxic effects in erythrocytes with the alkaline comet assay. Barbel specimens were collected in the Danube river near Belgrade, Serbia, where the discharge of untreated communal and industrial wastewaters is likely to produce negative effects on fish residing in this area. The highest concentrations of Sr, Mn, Fe, Ba, B, and Al were found in gills, Mo and Cu in liver, and As and Zn in gonads. Concentrations of Zn and Fe were above maximum acceptable concentrations (MACs) in a number of gonad, gill, and liver samples. Three-year-old barbel specimens had higher tail moment and Zn concentrations in gills (1.71 and 51.20 µg/g dw, resp.) than 5-year-old specimens (0.85 and 42.51 µg/g dw, resp.). Results indicate that the younger barbel specimens might be more suitable for the monitoring of environmental pollution.