Linking antibiotic resistance gene patterns with advanced faecal pollution assessment and environmental key parameters along 2300 km of the Danube River.

The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, blaTEM with intermediate abundance and blaOXA-48like, blaCTX-M-1 group, blaCTX-M-9 group and blaKPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.

First report on the occurrence of Vibrio cholerae nonO1/nonO139 in natural and artificial lakes and ponds in Serbia: Evidence for a long-distance transfer of strains and the presence of Vibrio paracholerae.

Vibrio cholerae are natural inhabitants of specific aquatic environments. Strains not belonging to serogroups O1 and O139 are usually unable to produce cholera toxin and cause cholera. However, non-toxigenic V. cholerae (NTVC) are able to cause a variety of mild-to-severe human infections (via seafood consumption or recreational activities). The number of unreported cases is considered substantial, as NTVC infections are not notifiable and physicians are mostly unaware of this pathogen. In the northern hemisphere, NTVC infections have been reported to increase due to global warming. In Eastern Europe, climatic and geological conditions favour the existence of inland water-bodies harbouring NTVC. We thus investigated the occurrence of NTVC in nine Serbian natural and artificial lakes and ponds, many of them used for fishing and bathing. With the exception of one highly saline lake, all investigated water-bodies harboured NTVC, ranging from 5.4 × 101 to 1.86 × 104  CFU and 4.5 × 102 to 5.6 × 106 genomic units per 100 ml. The maximum values observed were in the range of bathing waters in other countries, where infections have been reported. Interestingly, 7 out of 39 fully sequenced presumptive V. cholerae isolates were assigned as V. paracholerae, a recently described sister species of V. cholerae. Some clones and sublineages of both V. cholerae and V. paracholerae were shared by different environments indicating an exchange of strains over long distances. Important pathogenicity factors such as hlyA, toxR, and ompU were present in both species. Seasonal monitoring of ponds/lakes used for recreation in Serbia is thus recommended to be prepared for potential occurrence of infections promoted by climate change-induced rise in water temperatures.

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.

Single and combined potential of polystyrene microparticles and fluoranthene in the induction of DNA damage in haemocytes of Mediterranean mussel (Mytilus galloprovincialis).

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 assessment 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.

Wastewater-based epidemiology in countries with poor wastewater treatment - Epidemiological indicator function of SARS-CoV-2 RNA in surface waters.

Wastewater-based epidemiology (WBE) surveillance of COVID-19 and other future outbreaks is a challenge for developing countries as most households are not connected to a sewerage system. In December 2019, SARS-CoV-2 RNA was detected in the Danube River at a site severely affected by wastewaters from Belgrade. Rivers are much more complex systems than wastewater systems, and efforts are needed to address all the factors influencing the adoption of WBE as an alternative to targeting raw wastewater. Our objective was to provide a more detailed insight into the potential of SARS-CoV-2 surveillance in Serbian surface waters for epidemiological purposes. Water samples were collected at 12 sites along the Sava and Danube rivers in Belgrade during the fourth COVID-19 wave in Serbia that started in late February 2021. RNA was concentrated using Amicon Ultra-15 centrifugal filters and quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. Microbiological (faecal indicator bacteria and human and animal genetic faecal source tracking markers), epidemiological, physicochemical and hydromorphological parameters were analysed in parallel. From 44 samples, SARS-CoV-2 RNA was detected in 31, but only at 4 concentrations above the level of quantification (ranging from 8.47 × 103 to 2.07 × 104 gc/L). The results indicated that surveillance of SARS-CoV-2 RNA in surface waters as ultimate recipients could be used as an epidemiological early-warning tool in countries lacking wastewater treatment and proper sewerage infrastructure. The performance of the applied approach, including advanced sampling site characterization to trace and identify sites with significant raw sewage influence from human populations, could be further improved by adaptation of the methodology for processing higher volumes of samples and enrichment factors, which should provide the quantitative instead of qualitative data needed for WBE.

A Study of Phytochemistry, Genoprotective Activity, and Antitumor Effects of Extracts of the Selected Lamiaceae Species.

This study was designed to evaluate the genoprotective, antigenotoxic, as well as antitumor potential of methanolic, ethanolic, and aqueous extracts of Melissa officinalis, Mentha × piperita, Ocimum basilicum, Rosmarinus officinalis, Salvia officinalis, and Satureja montana (Lamiaceae), in different model systems. The polyphenols in these extracts were quantified both spectrophotometrically and using HPLC-DAD technique, while DPPH assay was used to assess the antioxidant activity. The genoprotective potential was tested on pUC19 Escherichia coli XL1-blue, and the antigenotoxicity on Salmonella typhimurium TA1535/pSK1002 and human lung fibroblasts, while the antitumor activity was assessed on colorectal cancer cells. Rosmarinic acid, quercetin, rutin, and luteolin-7-O-glucoside were among the identified compounds. Methanolic extracts had the best DPPH-scavenging and SOS-inducing activities, while ethanolic extracts exhibited the highest antigenotoxicity. Additionally, all extracts exhibited genoprotective potential on plasmid DNA. The antitumor effect was mediated by modulation of reactive oxygen species (ROS), nitric oxide (NO) production, and exhibition of genotoxic effects on tumor cells, especially with O. basilicum ethanolic extract. Generally, the investigated extracts were able to provide antioxidant protection for the acellular, prokaryotic, and normal human DNA, while also modulating the production of ROS and NO in tumor cells, leading to genotoxicity toward these cells and their decrease in proliferation.

Temporal variation of biomarkers in common bream Abramis brama (L., 1758) exposed to untreated municipal wastewater in the Danube River in Belgrade, Serbia.

This study was conducted on the Danube River locality Visnjica, 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.

A comprehensive assessment of the chemical composition, antioxidant, genoprotective and antigenotoxic activities of Lamiaceae species using different experimental models in vitro.

This research was aimed to assess the potential of Glechoma hederacea, Hyssopus officinalis, Lavandula angustifolia, Leonurus cardiaca, Marrubium vulgare and Sideritis scardica (Lamiaceae) methanolic, ethanolic and aqueous extracts against the damaging effects of oxidative stress using different experimental models. The chemical characterization was done spectrophotometrically by quantifying total phenolics, phenolic acids, flavonoids and flavonols in the extracts, as well as by employing HPLC-DAD technique. Moreover, DPPH assay was used to assess the extracts' radical scavenging potential. Genoprotective properties of the extracts were evaluated using plasmid pUC19 Escherichia coli XL1-Blue, whereas their antigenotoxic potential was determined using Salmonella typhimurium TA1535/pSK1002 and normal human lung fibroblasts. All of the extracts showed antioxidant activity in DPPH assay. Furthermore, the results have shown that aqueous extracts provided the best protection for plasmid DNA, while alcoholic extracts most effectively contributed to the preservation of prokaryotic DNA. Additionally, each of the tested samples significantly protected the eukaryotic cells against genomic damages. Finally, despite not showing exceptional results in DPPH assay, S. scardica extracts are regarded as the most favorable in maintaining the integrity of DNA, which might be due to high quantities of phenolics such as quercetin (up to 17.95 mg g-1), naringin (up to 5.07 mg g-1) and luteolin-7-O-glucoside (up to 3.54 mg g-1). Overall, this comprehensive concept highlights the ability of these Lamiaceae species to safeguard the DNA from reactive oxygen species, to curtail the inflicted damage and also improve the efficiency of the DNA repair mechanisms, while emphasizing the importance of polyphenols as their active principles.

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.

The impact of different extracts of six Lamiaceae species on deleterious effects of oxidative stress assessed in acellular, prokaryotic and eukaryotic models in vitro.

The main objective of this research was to evaluate the impact of methanolic, ethanolic and aqueous extracts of Origanum majorana L., Origanum vulgare L., Teucrium chamaedrys L., Teucrium montanum L., Thymus serpyllum L. and Thymus vulgaris L. (Lamiaceae) on the effects of free radicals using different model systems. The extracts were characterized on the basis of the contents of total phenolics, phenolic acids, flavonoids and flavonols, and also using high-performance liquid chromatography with diode-array detection. Antioxidant activity in vitro was assessed using DPPH assay. The genoprotective properties were tested using plasmid relaxation assay on pUC19 E. coli XL1-Blue, while SOS/umuC assay on Salmonella typhimurium TA1535/pSK1002 and Comet assay on human lung fibroblasts were used to assess the antigenotoxicity of the extracts. Ethanolic extracts had the most phenolics (up to 236.20 mg GAE/g at 0.5 mg/mL), flavonoids (up to 42.47 mg QE/g at 0.5 mg/mL) and flavonols (up to 16.56 mg QE/g at 0.5 mg/mL), and they exhibited the highest DPPH activity (up to 92.16% at 0.25 mg/mL). Interestingly enough, aqueous extracts provided the best protection of plasmid DNA (the lowest IC50 value was 0.17 mg/mL). Methanolic extracts, on the other hand, most efficiently protected the prokaryotic DNA, while all the extracts had a significant impact against genomic damages inflicted on human fibroblasts. O. vulgare extracts are considered to be the most promising in preserving the overall DNA integrity against oxidative genomic damages. Moreover, HPLC-DAD analysis highlighted rosmarinic acid as the most abundant in the investigated samples (551.45 mg/mL in total in all the extracts), followed by luteolin-7-O-glucoside (150.19 mg/mL in total), while their presence correlates with most of the displayed activities. The novelty of this study is reflected in the application of a prokaryotic model for testing the antigenotoxic effects of Lamiaceae species, as no previous reports have yet been published on the genoprotective potential of these species.

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.

The impact of multiple stressors on the biomarkers response in gills and liver of freshwater breams during different seasons.

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.

Characterization of sections of the Sava River based on fish community structure.

Sampling was undertaken, with the same fishing gear and along the Sava River, from its source to its confluence, in September 2014 and September 2015. In total, 44 fish species were identified, of which 37 were native species and 7 were alien. Fish samples revealed independence in terms of both species composition and their abundance under different hydrological conditions. During flooding and high water levels in 2014, pelagic fish species were sampled in greater proportion than at lower water levels in 2015 when benthic fish species were more abundant. The flood wave in 2014 was accompanied by catch of common carp, Cyprinus carpio, a typical lower rhithron fish species in the upper course, and of tench, Tinca tinca, a typical potamon fish species of backwaters, in the main channel of the lower Sava River. One specimen of bighead goby, Ponticola kessleri, which is common in the potamon fish community, was caught during the 2015 sampling close to the boundary between the upper and middle sections of the Sava. This is the first record of Ponto-Caspian gobies in the inland waters of Slovenia. Its finding far upstream indicates a strong effect of an as yet unidentified stress along the Sava River up to the spot where the bighead goby was sampled. Finally, these results indicate that pelagic fish species are more resistant to the stressful effect of flooding than benthic species, and that the structure of fish communities is influenced/affected by flooding as a short-term stressor. The progressively increasing number of alien fish species downstream in the Sava River point to the effects of long-term human-induced stressors in the area.

Comparative assessment of cardiac activity and DNA damage in haemocytes of the Mediterranean mussel Mytilus galloprovincialis in exposure to tributyltin chloride.

This study gives an insight in sensitivity of heart rate (Hr) of Mytilus galloprovincialis as a physiological biomarker. Impact of tributyltin chloride (TBT-Cl) on Hr was studied in parallel with evaluation of mutagenic, genotoxic and cytotoxic potential of TBT-Cl (10, 100 and 1000µg/L) within 96h treatment in static conditions. Mutagenic potential was assessed by SOS/umuC assay while genotoxicity was assessed in haemocytes of M. galloprovincialis by using the comet assay and the micronucleus test. Benzo(a)pyrene (B(a)P) was used as a positive control. Hr variations detected in TBT-Cl treatments can be linked to data obtained in the genotoxicological assays indicating that Hr can be considered and used as a reliable physiological biomarker for detecting the presence of organotin compounds. However despite the observed genotoxic potential of B(a)P, a noteworthy Hr response was not observed which further questions the potential of Hr in the detection of different types of pollutants.