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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.

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.

Paper description: We compared a model for spectral sensitivity data designed in our laboratory with the widely used template. Our model was designed with fewer (four) parameters, which we believe brings us closer to understanding the true nature of the absorption curve. In the fitting of spectral sensitivity data it uses non-transformed wavelengths. As a result, the shape of the curve remains the same for a broad range of l max values. Abstract: A four-parameter model of spectral sensitivity curves was developed. Empirical equations were designed for A 1 - and A 2 -based visual pigments with the main a-band maximum absorptions (l max ) from 350 nm, near the ultraviolet, up to 635 nm in the far-red part of the spectrum. Subtraction of the a-band from the full absorbance spectrum left a “b-band” described by a l max -dependent Gaussian equation. Compatibility of our templates with A 1 -and A 2 -based spectra was tested on the electroretinographic (ERG-derived) scotopic action spectra recorded in dogfish shark, eel, Prussian carp and perch. To more precisely estimate the accuracy of our model, we compared it with widely used templates for visual pigments. There was almost no difference between the tested models in fitting the above-mentioned spectral data. One of the advantages of our model is that in the fitting of spectral sensitivity data it uses non-transformed wavelengths and the shape of the curve remains the same for a broad range of l max values. Compared to multiparameter templates of other authors, our model was designed with fewer (four) parameters, which we believe can bring us closer to un derstanding the true nature of the absorption curve. https://doi.org/10.2298/ABS180822052G Received: August 22, 2018; Revised: November 7, 2018; Accepted: November 14, 2018; Published online: November 20, 2018 How to cite this article: Gacic Z, Mickovic B, Gacic L, Damjanovic I. New spectral templates for rhodopsin and porphyropsin visual pigments. Arch Biol Sci. 2018;71(1):103-10.

In this study, the comparative results of the fan mussel Pinna nobilis larvae collection during 3 years (2016–2019) on the sites of the Montenegrin Adriatic coast and the Western Mediterranean, France are presented. In both countries, growth studies of collected fan mussel juveniles were carried out as well. After 1 year growth measurement of P. nobilis recruits in their natural habitat on the site Dobrota, Montenegro, mean shell length was 198.58 ± 17.77 mm for the recruits from Sv. Nedjelja and 206.73 ± 16.40 mm for the recruits collected from Ljuta. The growth study carried out in a laboratory tank in France indicated that the mean shell length of P. nobilis recruits after a 9 month period was 100.50 ± 7.59 mm for the recruits from Bomasse, 96.33 ± 11.06 mm from Basse Renette 1, and 95.75 ± 8.45 mm for recruits from Basse Renette 2, respectively. In spite of much larger mean shell lengths obtained in Montenegro, mean monthly growth rate of P. nobilis recruits bred in France was higher due to more stable conditions and access to food within the tank in contrast to variations of environmental parameters in their natural habitat. We have presented first data on P. nobilis recruitment and growth in Montenegro and showed higher growth rate in comparison with the other sites in the Adriatic Sea and Mediterranean. It was shown that the temperature is of high importance for the growth rate of P. nobilis juveniles in their natural habitat due to lower growth during winter. Anthropogenic pressure was the main obstacle for development of P. nobilis populations during the study period, while biological pollution as the main threat for P. nobilis survival will be the subject of further studies in the Montenegrin Adriatic coast.

Electrophysiological monitoring of saccadic eye movements in patients with hypoxic-ischemic encephalopathy (HIE) was carried out. Externally guided saccades (prosaccades) were recorded using a patented hardware-software complex for studying the subject's physical activity. Recordings were performed in two independent experimental procedures - for saccades separately and when they were in coordination with the motion of head and hand. In both cases statistically significant differences in latent periods and durations of saccadic eye movements were noticed in HIE patients in comparison with healthy subjects of the same age group (p

To shed light on thermoadaptive properties of Salmo trutta from lake Plav (Montenegro), we undertook kinetic studies of pyruvate reduction rates and thermal stability analyses of white muscle LDH. We compared these with the data obtained for trout of the same, confirmed by us, Danubian lineage living in rivers and streams of Serbia and Montenegro. We also tested the effect of acclimation in captivity at 4 and 14 °C. The lake trout was of a typical smoltified phenotype (the size, the elongated silver colored body). At physiological substrate concentration, the breaks in the Arrhenius plots (critical temperature - Tc) correlated with acclimation temperatures or habitat water temperatures. Q10 values for temperatures above Tc were close to one, in all cases except 4 °C acclimated trout. At temperatures below Tc Q10 was close to two, except in the case of 14 °C acclimated trout. Lake trout had a highest Q10 values at temperatures below Tc. It was conspicuous that within the entire range of tested temperatures the differences in Q10 resulted from the effect of environmental temperature. Higher Q10 values were obtained with LDH isolated from trout acclimated to 4 °C compared with LDH acclimated to 14 °C. Ea values were much lower at a temperature below Tc compared with temperatures above Tc. Thermal stability of muscle LDH was lower after acclimation to 14 compared to 4 °C, while extremely high thermostability was obtained with the lake trout enzyme. Our data support the concept that Tc values have distinct physiological significance.

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.

In this study a comprehensive genotoxicological survey throughout the upper and middle stretches of Adige river basin is presented. The study was carried out at 7 sites located along the Adige main course and one the most significant tributaries, the Noce creek, both presenting different levels of pollution pressure. To give an insight into the nature of the genotoxic activity we employed the battery of prokaryotic and eukaryotic assays. Mutagenicity in water samples was evaluated 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) were studied in blood cells of Salmo cenerinus Nardo, 1847 and Salmo marmoratus Cuvier, 1829. Within the applied bioassays, comet assay showed the highest potential for discriminating the sampling sites which are under lesser extent of pressure (sampling sites 1-Barnes at Bresimo and 4-Noce downstream S. Giustina) from the sites under high pressure (sampling sites 5-Noce at Mezzolombardo and 6/7-Adige upstream and downstream municipality of Trento). Significant correlation between the standard and Fpg - modified comet assay indicated that oxidative stress could be a major contributor to observed DNA damage in collected specimens. Science of The Total Environment (2016), 571: 1383-1391

The life‐history traits and population dynamics of the black bullhead Ameiurus melas (Rafinesque) were studied monthly from August 2009 to December 2012 in Lake Sava in Belgrade (Serbia). This period included a mass mortality event in May 2011 and subsequent population recovery during 2012. Available evidence suggests the mass mortality was caused by European catfish virus that only affected the black bullhead. Changes in key life‐history traits and the recovery potential (increase in catch‐per‐unit‐effort, doubling of young‐of‐the‐year to adult ratio, earlier maturation and increased fecundity) following the mass mortality event suggest population resilience typical of a recurring boom‐bust pattern and demonstrate the species’ capacity to recover rapidly and re‐establish following disturbance. Repeated systematic mass removals of both juveniles and adults (using fyke nets) will be required if future fishery management measures aimed at limiting the population size are to be effective.

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.

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 1000 μg/L) within 96 h 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.

The impact of etoposide (ETO), vincristine sulfate (VIN), and cisplatin (CP) on the DNA damage level was studied in vivo and in vitro on hemocytes of freshwater mussels Unio sp. (Unio pictorum/Unio tumidus) using the alkaline comet assay. For in vivo experiments, the mussels were exposed in static system for 72 h. For in vitro experiments, treatment was performed in primary cultures of hemocytes for 22 h. The DNA damage level was analyzed by Comet Assay IV software. Increase of damage was detected for ETO (40 and 100 μM) in vivo and in vitro and for VIN (0.04 and 0.1 μM) in vivo. CP did not induce an increase of DNA damage, but post-treatment with hydrogen peroxide indicated existence of DNA crosslinks in specimens treated with 4 μM CP. Effective concentrations of selected cytostatics are at least one (CP) or several orders of magnitude (ETO, VIN) higher comparing to the concentrations in hospital wastewater.

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.

Element concentrations in selected fish species from different trophic levels were analyzed. The following fish species were analyzed: common nase ( Chondrostoma nasus ), roach ( Rutilus rutilus ), freshwater bream ( Abramis brama ), barbel ( Barbus barbus ), Prussian carp ( Carassius gibelio ), chub ( Squalius cephalus ) , European perch ( Perca fluviatilis ) , wels catfish ( Silurus glanis ) and northern pike ( Esox lucius ). Fish were collected from the Međuvrsje Reservoir (West Morava River Basin, western Serbia) during 2012, and samples of liver, muscle and gills were analyzed for As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Li, Mn, Mo, Ni, Pb, Sr, and Zn using inductively coupled plasma optical emission spectrometry (ICP-OES). The liver and gills had the highest measured element concentrations, with Cu, Zn, Fe and Sr being the most prominent. The bioaccumulation of metals was species-specific, with the accumulation exhibiting the following trends: Prussian carp > northern pike > freshwater bream > European perch > chub > common nase > barbel > roach > wels catfish; Li>Pb>Zn>Cu>Fe>Ba>Mn>Sr>Cr>As>Mo>Ni>B, in all examined species; for Co, Cd and Hg, the bioaccumulation factor (BAF) was 0. Results of this study point to the tissue-specific differences in element concentrations, and to distinct differences between fish species regarding the accumulation patterns: common nase, with the highest accumulation observed in the liver, and Prussian carp, with the highest accumulation observed in the gills. DOI: 10.2298/ABS151104069D Key words : fish; metal bioaccumulation; trace element concentration; water reservoir; gills Received: November 4, 2015; Revised: January 15, 2016; Accepted: January 15, 2016; Published online: August 5, 2016 How to cite this article: Đikanovic V, Skoric S, Gacic Z. Concentrations of metals and trace elements in different tissues of nine fish species from the Međuvrsje reservoir (West Morava River Basin, Serbia). Arch Biol Sci. 2016;68(4):811-9.

Responses of direction selective (DS) units of retinal and tectal origin were recorded extracellularly from the tectum opticum (TO) of immobilized fish. The data were collected from three cyprinid species - goldfish, carp and roach. Responses of the retinal DS ganglion cells (GCs) were recorded from their axon terminals in the superficial layers of TO. According to their preferred directions DS GCs, characterized by small receptive fields (3-8°), can be divided in three distinct groups, each group containing ON and OFF subtypes approximately in equal quantity. Conversely, direction-selective tectal neurons (DS TNs), recorded at two different tectal levels deeper than the zone of retinal DS afferents, are characterized by large receptive fields (up to 60°) and are indifferent to any sign of contrast i.e. can be considered as ON-OFF type units. Fish DS TNs unlike the retinal DS GCs, select four preferred directions. Three types of tectal DS units prefer practically the same directions as those already selected on the retinal level - caudo-rostral, dorso-ventral and ventro-dorsal. The fact that three preferred directions of DS GCs and DS TNs coincide allows us to assume that three types of DS GCs are input neurons for corresponding types of DS TNs. The fourth group of DS TNs has the emergent rostro-caudal preference not explicitly present in any of the DS GC inputs. These units are recorded in deep TO layers exclusively. Receptive fields of these DS neurons could be entirely formed on the tectal level. Possible interrelations between retinal and tectal DS units are discussed.

Single-unit responses of retinal ganglion cells (GCs) were recorded extracellularly from their axonal terminals in the tectum opticum (TO) of the intact fish (goldfish, carp). The depths of retinal units consecutively recorded along the track of the microelectrode were measured. At the depth of around 50 μm, the responses of six types of direction-selective (DS) GCs were regularly recorded. Responses of two types of orientation-selective (OS) GCs and detectors of white and black spots occurred approximately 50 μm deeper. Responses of GCs with dark- and light-sustained activity were recorded deeper than all others, at about 200 μm. The receptive fields of consecutively recorded units overlap, so they analyze the same fragment of the visual scene, focused by eye optic on the photoreceptor raster. The responses of pairs of DS GCs (ON and OFF units that preferred same direction of stimulus movement) and OS GCs (detectors of vertical and horizontal lines) were often simultaneously recorded at one position of the microelectrode. (The paired recordings of certain units amounted about fourth part of all recordings.) This suggests that their axonal arborizations are located close to each other in the tectal retinorecipient layer. Electrophysiological method, thus, allows to indirectly clarify and make precise the morphology of the retino-tectal connections and to establish a morpho-physiological correspondence.

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 major objective of this study was to investigate the impact of wastewaters on the level of genotoxicity and the possible reflection of genotoxicity on the genetic variability in chub (Squalius cephalus) collected from the sites under different pollution pressures along the Sava River. To emphasize the impact of wastewaters, this study was carried out in a season with low water discharge. The level of genotoxic potential at the sites was studied by alkaline and Fpg- modified comet assay in fish blood, while RAPD technique was employed on the fish muscle tissue. The variations in the genotoxic potential were observed along the Sava River based on the level of DNA damage in blood of the specimens collected at the selected sites. Clustering of the impacted sites was also observed based on RAPD fingerprints. Comparison of the data obtained in RAPD and the data from the comet assay indicated the possible reflection of genotoxicity on the genetic variability in chub collected from the sites with different pollution pressure. This is a preliminary study which reports the differentiation of the population of chub along the Sava River based on RAPD analysis.

Sponges in the large rivers within the Danube River Basin (DRB) have not been adequately studied. Hence, the aim of this work was to undertake an investigation on the distribution of sponge species in the Danube and Sava rivers. Out of 88 localities covered by the study, sponges were found at 25 sites only (46 samples in total). By using morphological (light and scanning electron microscopy) and genetic (28S rDNA sequencing) analysis, four species were determined: Ephydatia fluviatilis (Linnaeus, 1759), Spongilla lacustris (Linnaeus, 1759), Eunapius fragilis (Leidy, 1851), Trochospongilla horrida Weltner, 1893. In the Danube, the predominant species was found to be E. fluviatilis making approximately 80% of collected samples, while in the Sava River S. lacustris dominated, representing 46% of the river sponges. Our work represents one of the few studies on freshwater sponges within the DRB from long stretches of the large lowland rivers (more than 2500 km of the Danube River and about 900 km of the Sava River). Moreover, molecular analysis for the identification of freshwater sponges was applied on the material collected from a wide area, thus contributing to the systematic studies on the distribution and abundance of the European freshwater invertebrate fauna in general.

Temperature dependence of electroretinogram (ERG) was investigated in 3 fish species occupying different habitats — dogfish shark ( Scyliorhinus canicula ), Prussian carp ( Carassius gibelio ) and European eel ( Anguilla anguilla ). Acute cooling of the shark isolated eyecup from 23 °C down to 6 °C induced suppression of the electroretinographic b-wave — a complete degradation of this component was observed at 6 °C. On the other hand, photoreceptor component of the ERG, the negative late receptor potential was not affected by cooling. The fact that the suppression of the dogfish shark b-wave at low temperatures was as a rule irreversible testifies about breakdown of neural retinal function at cold temperature extremes. Although in vivo experiments on immobilized Prussian carps have never resulted in complete deterioration of the b-wave at low temperatures, significant suppression of this ERG component by cooling was detected. Suppressing the effect of low temperatures on Prussian carp ERG might be due to the fact that C. gibelio , as well as other cyprinids, can be characterized as a warmwater species preferring temperatures well above cold extremes. The ERG of the eel, the third examined species, exhibited the strongest resistance to extremely low temperatures. During acute cooling of in situ eyecup preparations of migrating silver eels from 30 °C down to 2 °C the form of ERG became wider, but the amplitude of the b-wave only slightly decreased. High tolerance of eel b-wave to cold extremes shown in our study complies with ecological data confirming eurythermia in migrating silver eels remarkably adapted to cold-water environment as well.

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.

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. (C) 2014 Elsevier B.V. All rights reserved. Ministry of Science of Montenegro; OTKA (Hungarian Scientific - Research Foundation) {[}K112547] This is the peer reviewed version of the following article: Héberger K, Kolarević S, Kračun-Kolarević M, Sunjog K, Gačić Z, Kljajić Z, Mitrić M, Vuković-Gačić B. Evaluation of single-cell gel electrophoresis data: Combination of variance analysis with sum of ranking differences. Mutat Res Toxicol Environ Mutagen. 2014;771:15–22. [https://doi.org/10.1016/j.mrgentox.2014.04.028]

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.

We examined whether the freshwater bivalve Unio tumidus from the Sava River can serve as a bioindicator organism for long-term biomonitoring of river ecosystems for the presence of metal pollutants. To this end, we assessed in the digestive glands and gills of mussels, changes in activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), the phase II biotransformation enzyme glutathione-S-transferase (GST)), and changes in the concentrations of the non-enzymatic components of the antioxidant system (vitamin E and sulfhydryl groups (-SH), after exposure to metals in the environment. Mussels were collected at four sites where the concentrations of dissolved metals (Cu, Cd, Zn, Fe, Mn, Hg, Ni, As, Pb) were quantified. Cu, Ni and As exerted concentration-dependent inhibitory effects on CAT and GST activities. Increasing concentrations of Cd promoted increases in GSH-Px activity and -SH concentration. In response to increased Zn concentration GR activity increased whereas Fe promoted decreased enzymatic activity. Negative correlations between the concentrations of Cu and Cd and vitamin E, and a positive correlation between Mn and vitamin E concentrations were detected. The described correlations between components of the antioxidant system and metal levels in the environment reveal a high physiological sensitivity of freshwater mussels to pollution, supporting their use in biomonitoring of metal contamination in river ecosystems.

Genotoxic response of freshwater mussels U. tumidus and U. pictorum to environmental stress was studied using comet assay on hemocytes and gill cells. The mussels were acclimated to controlled laboratory conditions for 10 days, and then exposed at 4 sites in the Sava and Danube rivers in the area of the city of Belgrade. Samples of each species were taken after 7, 14, and 30 days of exposure. The mussels sampled immediately after acclimation served as controls. Genotoxic response in both species was induced earlier at sites receiving untreated wastewaters from the city's main collectors (7 days), than at the site receiving only domestic wastewaters from small settlements located upstream from the city (30 days). There was a correlation between the comet tail intensity values in tissues of exposed mussels and the concentrations of zinc, copper, iron, and arsenic at the exposure sites. The genotoxic responses in both tissues of U. pictorum and in hemocytes of U. tumidus were similar, while U. tumidus gill cells failed to exhibit significant genotoxic response at two sites. These findings, together with higher mortality of U. tumidus at the most polluted sites, promote U. pictorum as a model for genotoxicity monitoring in freshwater environments. European Community {[}265264]

Anadromous fish could have intraspecific groups with different life histories; identification of these is very important for management and conservation. Geometric morphometrics represents a quick and cost-effective method to identify such intraspecific groups. In this study, geometric morphometrics of Pontic shad (Alosa immaculata) migrants from the Danube River were investigated, as was Fulton's condition factor (K), hepatosomatic index (HSI) and gonadosomatic index (GSI). Pontic shad specimens were collected during April and May of 2004 and 2006. The sample comprised four groups (65 males and 56 females in 2004; 110 males and 78 females in 2006). Geometric morphometrics revealed differences in shape that were statistically significant for both year (nested analysis of variance: F = 13.84; p < 0.001) and sex (nested analysis of variance: sex nested in year, F = 1.71; p < 0.01). The major differences between years were in head shape and position of fins. The most significant difference between females an...

The aim of this study was to use the data on the distribution of aquatic macroinvertebrates obtained from 14 sites within a 413 km long stretch of the Danube River in Serbia to show the relevance of the bordering zone between the Middle and Lower Danube. A total of 68 macroinvertebrate taxa were observed. Molluscs were the major component with regard to species richness and relative abundance. Lithoglyphus naticoides (C. Pfeiffer, 1828) was the most abundant species and Unio tumidus (Retzius, 1788) was the most frequent species. Product-moment correlation coefficients or Pearson r coefficient was used to analyse the relation between the sites based on macroinvertebrate distribution. The data obtained by product-moment correlation served as input for cluster analyses. According to a cluster analyses Danube River in Serbia could be separated in the free-flowing sector, the stretch with a backwater effect and the area of the Iron Gate.

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).

In this study we assessed the level of genotoxic pollution along the Danube River by measuring the level of DNA damage in the haemocytes of freshwater mussels of Unio sp. (Unio pictorum/Unio tumidus) and Sinanodonta woodiana. The comet assay was used for the assessment of DNA damage. The research was performed on 34 out of 68 sites analysed within the Joint Danube Survey 3 — the world's biggest river research expedition of its kind in 2013. During research, 2285 river kilometres were covered with an average distance of 68 km between the sites. The complex data set on concentrations of various substances present in water, suspended particulate matter and sediment on investigated sites gave the opportunity to identify the groups of xenobiotics which mostly affect the studied biomarker — DNA damage. The highest levels of DNA damage were recorded in the section VI (Panonnian Plain), which is under the impact of untreated wastewater discharges. Both positive and negative influences of the large tributaries on the level of genotoxicity in the Danube River were evident. Significant correlation in response was detected between the studied species of freshwater mussels. The level of DNA damage in mussels correlated with concentrations of compounds from the group of hazardous priority substances (polycyclic aromatic hydrocarbons), persistent organic pollutants (dioxins) and emerging pollutants (Oxazepam, Chloridazon-desphenyl).

In the present study, we analysed spatial and temporal heterogeneity of the limnological characteristics to provide more detailed information about the processes taking place within Mantovo Reservoir (Republic of Macedonia). The relationship between principal macroinvertebrate species and environmental variables was analysed in order to explore factors that dominantly affect community distribution pattern. Unlike the most reservoirs, strong longitudinal gradient for suspended organic matter and nutrients (total phosphorous, nitrates and nitrites) along the reservoir doesn't exist. However, the process of thermal stratification has a strong influence on the metabolism and structure of the Mantovo ecosystem, which can be demonstrated by the vertical and longitudinal distribution of dissolved oxygen (DO), CO2, pH and metals concentrations. Canonical Correspondence Analysis (CCA) indicated that the main factors controlling spatial distribution of Limnodrilus hoffmeisteri and Chironomus plumosus group were temperature, dissolved oxygen and manganese, including sulphates for C. plumous group. Chaoborus crystallinus showed opposite distribution pattern. Cladotanytarsus mancus group was strongly associated with shallower part (littoral and sublittoral) of Mantovo Reservoir characterized by favourable oxygen condition. None of the environmental variables included in CCA showed any relationship with density of Procladius sp. null

In this study, we investigated the level of sanitary pollution and organic contamination of the Velika Morava River, the largest river in central Serbia. Samples of water for microbiological analysis were collected at 5 sites, monthly, from April 2010 to February 2011. Sanitary analysis, i.e. enumeration of total and fecal coliforms and intestinal enterococci, indicated moderate to critical fecal contamination, while organic load assessment (oligotroph to heterotroph ratio, index of phosphatase activity) revealed the category of moderately polluted water. We also investigated seasonal variations of these groups of bacteria and the factors that could contribute to these differences. Our results showed that the micro- biological quality of the water in the Velika Morava River during different seasons is affected by numerous factors such as unequal loading of wastewaters, solar irradiation, and relations of flow/dilution and rainfall/runoff. [Acknowledgments. This study was financially supported by the Ministry of Education and Science of the Republic of Serbia: projects No 173025, No 177045 and No 043002.]

Summary This paper presents the status and trends in management of sturgeon species and the development of sturgeon aquaculture in the Lower Danube countries: Romania, Bulgaria, Serbia, Ukraine and Moldova. Sturgeon fishery moratoria and aquaculture development represent first steps in the Lower Danube countries to combat extirpation. Supportive stocking was used as a compensation for the impact of sturgeon fishery and dam construction, but these efforts unfortunately lacked adequate cooperation and coordination between and among region countries. Unsolved problems remain, such as the presence of illegal sturgeon fishery, water pollution, habitat destruction and fragmentation. Construction of fish passes and habitat restoration project developments are two key issues that have yet to be tackled in the Lower Danube region.

The accumulation of heavy metals in fish has been extensively studied and well documented. However, the research has been mainly focused on the muscle tissue, while the distribution patterns among other tissues, such as liver and gills, have been mostly neglected. Within the present study, the concentrations of Ag, Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Se, Sr, Zn and Li in the muscle, gills, liver and intestine of the sterlet (Acipenser ruthenus) from the Danube River have been assessed by using the inductively-coupled plasma optical spectrometry. The analysis has revealed a high degree of differential accumulation of the studied heavy metals and trace elements in the tissues of the Danube sterlet. Liver appeared to be the main heavy metal storage tissue, while the lowest levels of analyzed metals were found in muscles. Heavy metal concentrations in the muscle were at acceptable levels for human consumption, except partly for cadmium. We conclude that, in order to enable a more efficient control of contaminants in fish products, there is an apparent need to resolve the problem of lack of legal limits for many metals in European legislation.

The aim of this work was to examine the impact of urban wastewaters on the water quality of the Danube River in Serbia. Samples of water and sediments for microbiological analysis and genotoxicity monitoring were collected from 6 sites during spring and/or autumn 2010. Sanitary analysis, i.e. enumeration of total and fecal coliforms and intestinal enterococci, indicated moderate to critical fecal contamination, while organic load assessment (oligotroph to heterotroph ratio, index of phosphatase activity) revealed the category of moderately polluted water. Mercury-resistant bacteria were detected in all water samples, with high numbers at locations positioned downstream of Belgrade. There was no correlation of the microbiological parameters of the sediment and water samples. Genotoxicity monitoring, performed by the comet assay on hemocytes of mussels Sinanodonta woodiana, indicated a significant increase of DNA damage in mussels collected from the studied sites compared with the control group.

Three localities, representing three distinct coastal areas, were investigated: 1. the mouth of the Sutorina River; 2. the mouth of the Jaska River; 3. the mouth of the Bojana River (right branch). Regarding the composition of young mullet assemblages, Lisa saliens, Lisa ramada and Lisa aurata were dominant at the first locality; L. aurata and Chelon labrosus at the second; and at the third L. ramada was clearly dominant, Mugil cephalus occupied the subdominant position, while L. aurata was absent. The seasons of the first appearance of migratory fry were October, April, November, October and July, for M. cephalus, C. labrosus, L. ramada, L. aurata and L. saliens, respectively. Migratory periods varied from 2 (C. labrosus) to 7 (L. ramada) months.

Specimens of Pontic shad (Alosa pontica Eichwald, 1838) were collected during April and May of 2006 in the Danube River (rkm 863) just downstream of the Iron Gate (Djerdap) II dam. Twenty-eight scales and 30 vertebrae were prepared and given to five interpreters with fish aging experience of from 3 to 30 years and with shad aging experience of from 0 to 12 years. Interpreters with experience in shad age determination showed lower values for the index of average percent error (IAPE) and an index of coefficient of variation (ICV). In regard to within-interpreter reproducibility, no significant differences were found between scale and vertebrae as structure for age determination. The most experienced reader showed the lowest value for IAPE, while the least experienced reader showed the highest value for IAPE. The results of this work indicate that experience in age determination by particular structure had more impact on age determination precision than specificity of structure, scale or vertebra. More work is needed on adopting standard protocols that must include some sort of common interaction between responsible age readers.

Photoreceptor content of fish retinas could be accessed by comparative electroretinographic (ERG) studies using flickering light stimuli that could separate rod-mediated vision where critical flicker frequency (CFF, frequency when the eye loses its ability to resolve individual light pulses) is usually less than 15 Hz from cone-mediated vision. Four fish species inhabiting different photic environments (small-spotted dogfish shark--Scyliorhinus canicula, eel--Anguilla anguilla, painted comber--Serranus scriba, Prussian carp--Carassius gibelio) were investigated. Dogfish shark b-wave amplitudes significantly decreased at low frequency of stimulation and CFF was reached at 3.2 Hz. A similar effect on the b-wave amplitude was observed in the eel, but CFF occurred at around 20 Hz. Conversely, b-waves of painted comber and Prussian carp remained unaltered under intermittent low-frequency stimulation, and CFFs were around 25 and 30 Hz, respectively. Additional support in accessing the receptor content of fish retinas was given by the characterization of the OFF-response (d-wave) after light adaptation. Monotonous time course of the b-wave dark adaptation indicated a rod dominated retina of the dogfish shark. Observed results indicate that the dogfish shark possesses preponderantly rod retina, that of the eel is rod-dominated, while Prussian carp and painted comber have cone-rich retinae.

The fish assemblage from reservoir Medjuvrsje was monitored over the 1955–2000 period. The Medjuvrsje Reservoir is one of the oldest Serbian reservoirs formed in 1953. Ichthyofauna was sampled in 1955, 1984, 1991 and 2000. Modified index of biotic integrity (IBI) metrics were used to assess changes in biotic integrity. The relative abundance of omnivorous, phytophilic and tolerant species increased, that of lithophilic, intolerant and rheophilic species decreasing during the 45 years. The total IBI decreased from 44 in 1955 to 24 in 2000, while the sediment deposition rate increased from 26.8% in 1963 to 70.4% in 2005. There was a significant negative correlation between the IBI and the sediment deposition rate. This study showed that the IBI could be used to characterize the status of the reservoir.

In spectral sensitivity studies, electroretinograms (ERG) were recorded from the in situ eyecup of immobilized perch (Perca fluviatilis) electrofished in the floodplain zone of the Danube River. It is shown that the ERG b-wave is a good indicator of spectral sensitivity, although it reflects the activity of photoreceptors indirectly. With a maximum around 542 mm, the scotopic spectral sensitivity of perch obtained using fitted amplitude-log intensity functions for threshold calculation and two models developed in our laboratory for computer-assisted fitting of spectral sensitivity curves did not differ from the sensitivity determined using the microspectrophotometric and isolated pigment methods (maximum around 541 nm).

Genotoxicity of 5-fluorouracil (5-FU), etoposide (ET) and cadmium chloride (CdCl2) was evaluated in Limnodrilus udekemianus, cosmopolitan tubificid species, by alkaline single-cell gel electrophoresis (comet assay). Groups of 50 individuals were exposed in vivo in water-only short-term (96 h) tests to 5-FU (0.004, 0.04, 0.4, 4 and 40 μM), ET (0.004. 0.04, 0.4 and 4 μM) and CdCl2 (0.004, 0.04, 0.4, 4 and 40 μM). Mortality of worms was observed only for CdCl2 (4 and 40 μM). Cell viability lower than 70 % was detected for 5-FU (0.4, 4 and 40 μM), ET (4 μM) and CdCl2 (0.4 and 4 μM). All tested substances induced significant increase of DNA damage except 0.004 μM of ET. L. udekemianus being sensitive to all tested substances indicates that it can be used in ecogenotoxicology studies. Concern should be raised to cytostatics, especially to 5-FU, since concentration of 0.004 μM induced DNA damage is similar to ones detected in wastewaters.

The effects of ex situ exposure of Mytilus galloprovincialis to Superdispersant-25 (S-25), diesel oil and dispersed diesel oil mixtures were studied by the impact on level of DNA damage in haemocytes (comet assay) and the cardiac activity patterns of mussels. Specimens were exposed for 72 h in a static system to diesel oil (100 mu L/L and 1 mL/L), S-25 (5 and 50 mu L/L), and dispersed diesel oil mixtures M1 (diesel oil 100 g mu L/L + S-25 5 mu L/L) and M2 (diesel oil 1 mL/L + S-25 50 mu L/L). For positive control 40 mu M CdCl2 was used. The comet assay results indicated genotoxic potential of S-25 while the effects of diesel oil alone were not observed. The highest response was detected for M1 while the effects of M2 were not detected. The heart rate disorders were recorded for the diesel oil (1 mL/L), S-25 (50 mu L/L) and both dispersed diesel oil mixtures. (C) 2015 Elsevier Ltd. All rights reserved.

Genotoxicity monitoring of the lower stretch of the Sava River was performed by the combined approach of in situ assessment of genotoxicity and active biomonitoring of two species of mussels from the Unionidae family, Unio pictorum and Unio tumidus. Genotoxic response was studied using comet assay on hemocytes. For active biomonitoring, the mussels were acclimated to controlled laboratory conditions for 10 days and then exposed at two sites in the Sava River in the area of the city of Belgrade. Hemolymph of exposed specimens of each species was taken after 7, 14, and 30 days of exposure. For in situ assessment, the mussels were collected from five sites in the lower flow of the Sava River. The mussels were sampled immediately after the acclimation served as controls in both types of monitoring procedures. The results of our studies indicated the presence of genotoxic pollution at all studied sites at the Sava River. The level of DNA damage varied at different sites depending on the source and level of pollution. The response to genotoxic pollution was evident at the site in the urban area of Belgrade city, as well as at the sites far from the large urban settlements, suggesting that the lower flow of the Sava River is under pollution pressure. Milačić R, Ščančar J, Paunović M, editors. The Sava River. Berlin, Heidelberg: Springer-Verlag; 2015. p. 437-52.

We applied an innovative technology, based on infrared light sensors, to point out the possible harmful effects of diesel oil and the oil slick dispersant Superdispersant-25 (SD-25) on the cardiac system of the Mediterranean mussels (Mytilus galloprovincialis L.). Heart rate (Hr) measurements were conducted under laboratory conditions by noninvasive procedure for the registration and analysis of cardiac activity of molluscs and crustaceans. No response was observed of the Hr within a group of mussels in 2 h of exposure to 1 ml/l of diesel oil. However, a decrease of Hr in application of 10 µl/l of diesel oil dispersed by the same amount of SD-25 was recorded. Moreover, the very low concentration of SD-25, 2 µl/l and a hundred times higher one, caused a significant bradycardia which could be the evidence for high toxicity of this chemical. The resulting changes of the mussels Hr were reversible. The main advantages of the technique applied are data accuracy and real time information about changes of the environmental conditions.

The action spectrum of the electroretinographic (ERG) b-wave and the unmasked late receptor potential (LRP) were measured under a variety of conditions (isolated eyecup, detached retina, b-wave recording in fresh preparations, LRP measurements after low-temperature aging, dark and light adaptation). It was shown that in the dogfish, Scyliorhinus canicula (L.), eyecup spectral sensitivity matched closely the pigment 502 spectral curve like other rhodopsin-possessing marine species. The ERG b-wave is as good an indicator of spectral sensitivity as the unmasked LRP that directly reflects the responsiveness of photoreceptors. Differences in spectral sensitivity were not revealed between dogfish preparations studied under a variety of conditions (isolated eyecup vs. detached retina; b-wave recording in fresh preparations vs. LRP measurements after low-temperature aging; dark vs. light adaptation). We developed a new model for spectral sensitivity data.