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In this study, the possible ‘vector effect’ within the exposure of Mediterranean mussels (Mytilus galloprovincialis) to polystyrene microplastics with adsorbed fluoranthene was investigated by applying the multibiomarker approach. The major focus was placed on genotoxicological endpoints as to our knowledge there are no literature data on the genotoxicity of polystyrene microparticles alone or with adsorbed fluoranthene in the selected experimental organisms. DNA damage was assessed in haemocytes by comet assay and micronucleus test. For the assess- ment of neurotoxicity, acetylcholinesterase activity was measured in gills. Glutathione S-transferase was assessed in gills and hepatopancreas since these enzymes are induced for biotransformation and excretion of lipophilic compounds such as hydrocarbons. Finally, differences in physiological response within the exposure to polystyrene particles, fluoranthene, or particles with adsorbed fluoranthene were assessed by the variation of heart rate patterns studied by the noninvasive laser fibre-optic method. The uniform response of individual biomarkers within the exposure groups was not recorded. There was no clear pattern in variation of acetylcholinesterase or glutathione S-transferase activity which could be attributed to the treatment. Exposure to polystyrene increased DNA damage which was detected by the comet assay but was not confirmed by micronucleus formation. Data of genotoxicity assays indicated differential responses among the groups exposed to fluoranthene alone and fluoranthene adsorbed to polystyrene. Change in the heart rate patterns within the studied groups supports the concept of the Trojan horse effect within the exposure to polystyrene particles with adsorbed fluoranthene.

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.

More frequent application of biological parameters beside the conventional chemical parameters in national monitoring for marine ecosystem assessment is supported by UNEP/MAP. Accordingly, to evaluate the possible effects of pollution by determination of trace element content and genotoxicity assessment in marine organisms within the Boka Kotorska Bay, Montenegro (southern Adriatic), we carried a field study which included transplantation of the seabream (Sparus aurata) from aquaculture farm (Orahovac) to more impacted sites (Dobrota and port of Tivat), situated in vicinity of the main ports. On the sampling site Orahovac, the group of seabream specimens were placed in closed fish trap on 5 m depth (O1), another fish trap was placed in Dobrota, while the third fish trap with seabreams was placed at the site port of Tivat. The additional group was sampled from standard aquaculture cage on sampling site (O2) to reduce possible bias in genotoxicity data due to confined space within the fish trap. After two weeks of exposure, blood from fish heart was taken, afterwards muscle tissues were prepared for trace element determination. Genotoxicity was measured by DNA damage induction based on comet (single cell gel electrophoresis) assay parameter – Tail intensity (TI%), while trace element (As, Cd, Pb, Hg, Cr, Cu, Fe, Mn, Ni, Zn and V) content was determined by ICP. Mean values of TI% obtained from seabream specimens at the sampling site Orahovac were: 4.26 ± 0.17 – O1 and 2.56 ± 0.11 – O2, while TI% values for transplanted specimens on the sites Dobrota and port of Tivat were 6.38 ± 0.17 and 11.06 ± 0.23, respectively. Statistically significant differences (p<0,05) of TI% were observed between all specimens groups. The group of seabream from port of Tivat showed the highest TI% (higher level of DNA damage), most probably caused by marine pollution. Obtained trace element concentrations in seabream tissues were significantly lower in comparison to values recommended by FAO

Velika, Zapadna, Južna Morava, kao i Sava, Kolubara i Barička reka su pod značajnim uticajem komunalnih i industrijskih otpadnih voda. Ciljevi studije su procena genotoksičnog potencijala analiziranih lokaliteta i ispitivanje osetljivosti primenjenih biotestova i pristupa istraživanja. Studija slučaja Velika Morava obuhvatila je veliko ispitivano područje na osam lokaliteta na Velikoj, Zapadnoj i Južnoj Moravi, a u studiji Sava ispitivano je tri prostorno bliska lokaliteta koji su pod različitim pritiscima zagađenja. U in silico pristupu na osnovu literaturnih podataka analiziran je toksični/genotoksični potencijal jedinjenja detektovanih u vodi. Ex situ/in vitro pristup obuhvatio je ispitivanje genotoksičnog potencijala pomoću SOS/umuC testa, kao i primenom alkalnog komet testa na HepG2 ćelijskoj liniji. U in situ/in vivo istraživanju analizirano je DNK oštećenje ćelija krvi vrste Alburnus alburnus (uklija) pomoću alkalnog, Fpg-modifikovanog komet testa i mikronukleus testa. Rezultati su pokazali različitu osetljivost zavisno od upotrebljenih testova i pristupa, ali i različit odgovor kod uklija u zavisnosti od tipa zagađenja na lokalitetima. In silico i ex situ/in vitro pristupi su pokazali niži stepen osetljivosti u odnosu na in situ/in vivo testove, a alkalni komet test je pokazao najveći potencijal u diskriminaciji lokaliteta. Utvrđeno je da uklija može biti pouzdan bioindikator u ekogenotoksikološkim istraživanjima velikih ravničarskih reka.

Background Wastewater-based epidemiology surveillance of COVID-19 and other outbreaks in the future is a challenge for developing countries, as the majority of households are not connected to sewerage systems. In December 2019, we have detected SARS-CoV-2 RNA in the Danube River at a site that is severely affected by wastewater of Belgrade. Considering that rivers are much more complex systems in comparison to wastewater, additional efforts are needed to address all the factors which might influence the adoption of WBE as an alternative of targeting raw wastewater. Objectives The major objective of this study was to provide a more detailed insight in the potential of SARS-CoV-2 surveillance in Serbian surface waters under consideration of epidemiological, microbiological, physico-chemical and hydro-morphological parameters for epidemiological purposes. Methodology Water samples were collected at 12 sites at Sava and Danube Rivers in the Belgrade city area during the fourth COVID-19 wave in Serbia that started in late February 2021. SARS-CoV-2 RNA was quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. Microbiological (standard fecal indicator bacteria and microbial faecal source tracking markers), epidemiological, physico-chemical and hydro-morphological parameters were analysed in parallel. Results Out of 44 samples analyzed, 31 were positive for at least one of the target regions of SARS-CoV-2. The results indicated that surveillance of SARS-CoV-2 RNA in surface waters in context with the large amount of epidemiological and environmental metadata can be used as epidemiological early-warning tool in countries with poor wastewater treatment.

Within the past decade, our research group placed great effort in exploration of the impact of treated/untreated wastewaters on aquatic ecosystems as less than 13 % of collected municipal wastewaters are processed before their release to receiving waters in Serbia. Having in mind that more than 90% of the rivers within the territory of Serbia belong to the Danube River Basin, our major focus is naturally on the large transnational waters such as the Danube and Sava rivers. Depended on the concept of the study and hydro-morphological characteristics of the sites, various approaches have been employed for in situ assessment of eco/geno-toxicity which use different organisms in passive and active biomonitoring. Aquatic organisms of different trophic levels have diverse life strategies, metabolism pathways, and consequently, they have a different response to pollutant pressure. In eco/geno-toxicological studies we have successfully employed several species of freshwater mussels (Unio tumidus, Unio pictorum and Sinanodonta woodiana) as well as few economically important fish species (common carp (Cyprinus carpio), freshwater bream (Abramis brama), bleak (Alburnus alburnus, chub (Squalius cephalus)).Comet assay is our primary choice for the assessment of DNA damage considering its sensitivity and cost effectiveness. Selection of certain types of tissues for the assessment (such as blood or haemolymph), which do not require sacrificing of the animals or additional manipulation in preparation of cell suspension coupled with mini-gel format of the slides enables high-throughput screening of the genotoxic potential in situ. Additionally, cryopreservation of the blood samples directly onsite expands the research area as it overcomes issues related to safe transportation of the samples to the laboratory. Data obtained so far indicated that the genotoxic response in the studied indicator organisms significantly differ which is understandable considering the difference in uptake

The middle section of the Danube River basin in the previous three Joint Danube Surveys (JDS) was found to be under intensive pollution, mainly affected by wastewaters. As a consequence of the lack of wastewater treatment plants, complex xenobiotics mixtures may get into surface water and decrease water quality and organisms’ health. Some xenobiotics may induce different types of DNA damage. Hence, DNA damage could be a useful biomarker in the detection of “early warning signals” of organisms’ exposure to genotoxic compounds, while the usage of bioassays batteries can provide better insight into a genotoxic potential. This study was conducted in 2019, during the JDS4 expedition, to assess the genotoxic potential of nine sites on the section of the Danube River which goes through Serbia and its major tributaries: the Sava, Tisza and Velika Morava. Blood and muscle of Alburnus alburnus (bleak) were used for genotoxicity assessment by comet, micronucleus and RAPD assays. In comet assay, the highest level of DNA damage was recorded at the Velika Morava mouth (JDS 39) and on the Danube River, downstream Radujevac (JDS 41). The highest frequency of micronucleus was recorded at the Sava mouth (JDS 36) and on the Danube River, downstream Pančevo (JDS 37). The lowest level of DNA damage in both tests was recorded on the Sava, site Jamena (JDS 35). The RAPD analysis distinct three major groups: Tisza mouth (JDS 33) and sites on the Sava (JDS 35, JDS 36), then two sites on the Danube River (JDS 37, JDS 41) and one site on the Danube River, Ram (JDS 40) with the Velika Morava mouth (JDS 39). Analyzed bioassays showed different sensitivity where the comet assay had the highest potential in discrimination of sites based on genotoxicity. Bleak was proved to be a reliable bioindicator in eco/ genotoxicological studies.

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.

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.

Surface waters in Serbia are under high anthropogenic pressure. One of the major problems is untreated municipal and industrial wastewaters. Unfortunately, Serbia processes only 5 % of wastewaters before discharging. As a consequence, pollutants such as metals and metalloids from industrial wastewaters, pharmaceuticals, compounds from personal care products, etc. directly endure into surface water. Microbial faecal pollution, as an indicator of presence of human or/and animal pathogens, due to health hazard limits water usage for drinking, recreation, irrigation, etc. In regulations, faecal coliforms, with Escherichia coli as dominant representative, are widely used as faecal indicator bacteria. The aim of this study was to investigate the microbiological al water quality in the Republic of Serbia at the sites which are not routinely investigated within the national monitoring program. In this purpose 78 sites situated on canals, mountain springs, as well as lowland rivers were selected. Defined Substrate Technology was used for determination Most Probable Number (MPN) of E. coli using Colilert-18 System. The water classification system, developed for the Danube River (Kavka et al., 2006) was used. The results indicated that more than 47 % of selected sites are under critical (21.79 %) or strong (25.64 %) faecal pollution. On the other hand, about 32 % (32.05 %) of sites are little polluted and 20.51 % of sites showed moderate pollution. The increasing levels of faecal pollution detected on sites situated downstream of settlements indicated discharge of untreated municipal wastewaters directly into surface water. In further research, the focus will be placed on the determination of the origin of pollution by the employment of microbial source tracking technique. Obtained data will be used for modelling and predicting the effect of detected contamination on the water quality of the major water bodies in the Republic of Serbia

Recently, cancer research has focused on searching for new and more effective antitumor agents of natural origin that can activate multiple defence mechanisms and selectively damage transformed cells. The goal of this research was to assess different antitumor mechanisms of ethanolic extracts of 18 Lamiaceae species traditionally used in Serbian folk medicine and cuisine, as well as their genotoxic potential towards HCT-116 (colorectal cancer) cells. The viability of treated HCT-116 cells was assessed by MTT assay; the production of reactive oxygen species (ROS) by treated HCT- 116 cells was determined using NBT assay, while their production of nitric oxide (NO) was evaluated using Griess assay. The genotoxic activity of the extracts on HCT-116 cells was tested in Comet assay, using etoposide as a positive control. The results indicated that lavender, basil, and rosemary inhibited the proliferation of these cells, significantly lowering their viability. Moreover, lavender and thyme extracts displayed a significant increase in ROS production, whereas ground- ivy, hyssop, lemon balm, peppermint, basil, rosemary, sage, and winter savory have significantly lowered their production. The results of the Griess assay suggested that lavender, motherwort, peppermint, basil, rosemary, sage, winter savory, ironwort, and thyme have significantly increased the production of NO. Furthermore, Comet assay results pointed out that motherwort, peppermint, basil, oregano, marjoram, winter savory, ironwort, wild thyme, thyme, and mountain germander possess genotoxic potential towards HCT-116 cells, while only basil had genotoxic activity statistically similar to etoposide. The obtained results are in accordance with our previous findings, which indicated that these extracts have antigenotoxic and genoprotective activities towards normal cells. Finally, it can be concluded that these traditionally valued plants might act as potent antitumor agents by modulating the proliferation and prod

This study was conducted on the Danube River locality Višnjica, exposed to the discharge of the largest wastewater collector in Serbia’s capital, Belgrade. Concentrations of metals and metalloids (Al, Fe, Cr, As, Sr, Mn, Cd, Zn, Mo, Cu, Li, Ni, B, Co, Pb, and Ba) and histopathological alterations were investigated in different tissues of common bream during one representative month of each season in 2014. This is the first study in which these two biomarkers were assessed parallelly in common bream. The highest concentrations of examined elements were noticed in gills and the lowest in muscle. Statistically significant differences in element concentrations between different seasons were noticed only in gills for Al, Cu, and Fe. Concentrations of As and Pb in fish muscle were below the maximum acceptable concentrations (MAC). The histopathological index (HI) of the liver showed higher values in comparison to the HI of the gills. Histopathological index of the gills had a significantly higher score in November in comparison to August. The liver HI had the highest score in April, and the lowest in August, while the total histopathological index had the highest score in November, and the lowest in August, both without significant differences between the months. This study endorses gills and liver as reliable organs for studying accumulation and histopathology as biomarkers of environmental changes. A faster reaction of the gills was confirmed since seasonal variations of both biomarkers were observed in this organ. Common bream proved as a good indicator of the state of organisms in polluted environments.

Neprerađene komunalne i industrijske otpadne vode su jedan od glavnih zagađivača površinskih voda u Srbiji. Sa komunalim otpadnim vodama u površinske vode dospevaju hemijski polutanti i fekalno zagađenje koje može sadržati za čoveka patogene mikroorganizme. U okviru istraživanja vršena je analiza mikrobiološkog kvaliteta vode sa 40 lokaliteta na teritoriji Republike Srbije koji nisu obuhvaćeni programom rutinskog monitoringa. Prisustvo i broj Escherichia coli je odabrano kao parametar fekalnog zagađenja. Rezultati su pokazali da voda sa 57,5% lokaliteta pripada III i IV klasi. Najveći broj zagađenih lokaliteta se nalazi nizvodno od naseljenih mesta, što potvrđuje značaj izgradnje postrojenja za preradu otpadnih voda.

Background: Montenegro is characterized by numerous mountain springs and larger streams. Many of them are used as sources of drinking water, irrigation, but also for tourists’ recreation: kayaking, rafting, etc. Unfortunately, there is a lack of wastewater treatment plants as well as information about microbial water quality of many surface and ground waters. Objectives: Taking into consideration issue of scarce data about microbial water quality, the aim of this study was to locate hotspots of faecal pollution and investigate the source of pollution. Within the study, 25 sites were selected while three of them were not included in National monitoring program. Methods: To quantify the most probable number of Escherichia coli we used Colilert-18 System. The water quality based on this parameter was assessed according to Kirshner et al. (2009). For determination of the pollution origin, microbial source tracking method was employed. Indicators of human faecal pollution, markers BacHum and HF183II, and animal faecal pollution, markers BacR and Pig2Bac, were quantified with qPCR. Results: According to most probable number of E. coli, 40% sites were little polluted, 36% moderately, 16% critically and per 4% heavily and excessively polluted. Analysis of genetic faecal markers indicated the prevalence of human-as- sociated faecal pollution at all sites. Considering the results of the study, four sites were classified as hotspots of faecal pollution: downstream Pljevlja (Ćehotina River), downstream Vezišnica (Ćehotina River), wastewater outlet at Podgori- ca (Morača River), and downstream Bijelo Polje (Lim River).

Untreated municipal wastewaters are one of the major negative contributors on freshwater quality, and consequently on ecosystem balance. Some compounds such as pharmaceuticals, cosmetic products, etc. could be genotoxic and lead to somatic and/or germinative mutations. This mutation should be reflected on organisms’ health and reproductive potential. Comet assay is a widely used test in ecogenotoxicology for detection of primary DNA damage. In that way, this sensitive test could provide information about early warning signs of potential stressors effects before those have an impact on the population or ecosystem level. The scope of our study was to assess the level of DNA damage of Alburnus alburnus specimens’ gill cells and erythrocytes. Three sites on the Sava river were chosen: reference site Zabran suited upstream the municipal discharging; second at the confluence of the Kolubara river and the Sava river; and the third at the confluence of the Barička river and the Sava river. The Kolubara river and the Barička river are chosen as important recipients of untreated wastewaters. From each site, 5 bleak specimens were collected and blood and gills were taken. Tail Intensity (TI%) was chosen as a parameter for evaluating the level of DNA damage. Besides that, cell viability and extremely damaged cells - hedgehogs (HH) were determined. The significant difference in TI% values was recorded in erythrocytes and gills cells in comparison between the Zabran (TI%=14.01±0.61 in erythrocytes, TI%=15.25±1.03 in gills cells) and the second site (TI%=18.53±0.65 in erythrocytes, TI%=22.78±1.89 in gills cells). Correlation between cell viability and HH frequency was not observed. According to the results, we could conclude that the second site is the most affected by pollutants. Also, usage of comet assay on freshwater organisms could be appropriate for preliminary screening of ecosystem state.