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

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

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.

Metal Accumulation in Muscle and Liver of the Common Nase (Chondrostoma nasus) and Vimba Bream (Vimbavimba) from the Danube River, Serbia: Bioindicative Aspects.

Accumulation of 17 elements in muscle and liver of common nase and vimba bream, caught between February and May 2016 in the Danube River (1173 river kilometer), were assessed by ICP-OES. The principal component analysis grouped muscle and liver samples based on element concentrations (muscle grouped by higher Ba and Sr values, and liver grouped by higher Al, B, Cd, Cr, Cu, Fe, Mn, and Zn values), but no grouping between the two species was observed. Concentrations of Ba, Cu, Fe, and Zn were significantly higher in muscle, and concentrations of Ba, Cd, Cu, and Mn in liver of common nase, while vimba bream had significantly higher concentrations of Cr and Fe in liver. Common nase has a higher affinity for bioaccumulation of Cu, Fe, and Zn in muscle, while vimba bream has a higher affinity for Al, Cd, and Cr in muscle.

Concentrations of Selected Elements in Liver Tissue of Grey Wolves (Canis lupus) from Serbia.

The grey wolf (Canis lupus) is a large carnivore species and a top predator in the ecosystems that it inhabits. Considering its role in food webs, wolves may be exposed to high concentrations of potentially harmful elements. Therefore liver samples from 28 legally hunted wolves were analyzed for concentrations of 16 elements using inductively coupled plasma optical emission spectrometry. The Mann-Whitney U test showed a significant difference between the genders only for Li, and there were no differences between individuals caught in different years. The majority of statistically significant correlations between element levels were positive, except for three cases. Compliance with several criteria for suitable bioindicator organisms imply that wolves may serve for monitoring environmental contamination.

Use of histopathology and elemental accumulation in different organs of two benthophagous fish species as indicators of river pollution.

The bottom feeding fish species have a good potential to be used for assessments of pollution, as they are under pressure from pollutants from both water and sediments. In this study, the level of similarity of histopathological responses to pollution in gills and liver between barbel (Barbus barbus) and sterlet (Acipenser ruthenus) from the Danube River was assessed, and compared with elemental concentrations in their gills, liver, and muscle. Results indicate that the detected metal concentrations were likely cause of different tissue responses in gills and liver of the two investigated fish species. Statistical analysis indicated a clear differentiation of the two species based on elemental concentrations and the level of histopathological changes in gills and liver. Metal concentrations exceeded maximum acceptable concentrations in a number of analyzed specimens, which indicates the importance of this type of monitoring. Results indicate that barbel is a better indicator for specific, rather narrow sites, whereas sterlet is a better indicator of larger (longer) water current segments. Obtained information could be of importance for both scientists and fishery and water management authorities working on the development of water monitoring programs.

Heavy metal and trace element bioaccumulation in target tissues of four edible fish species from the Danube River (Serbia).

Pikeperch (Sander lucioperca), European catfish (Silurus glanis), burbot (Lota lota), and common carp (Cyprinus carpio) were collected from the Danube River (Belgrade section, Serbia), and samples of liver, muscle, and gills were analyzed for Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Li, Mn, Mo, Ni, Pb, Se, Sr, and Zn using inductively coupled plasma optical emission spectrometry (ICP-OES) to highlight the importance of species and tissue selection in monitoring research, contaminant studies, and human health research. The Kruskal-Wallis test revealed significant differences between fish species in regard to metal levels in liver, muscle, and gills. The principal component analysis (PCA) indicated that the studied fish species could be grouped on the basis of the level of analyzed elements in liver and gills. The Mann-Whitney test showed two subsets (one comprising two piscivorous species, pikeperch and catfish, and the other, two polyphagous species, burbot and carp) in regard to Cr and Hg levels in liver (higher levels in piscivorous species), as well as B, Fe, and Hg in gills (B and Fe with higher levels in polyphagous and Hg in piscivorous species), and As in muscle (higher levels in polyphagous species). Carp had distinctly higher levels of Cd, Cu, and Zn in liver in comparison to other three species. None of the elements exceeded the maximum acceptable concentrations (MAC). However, since Hg levels are close to the prescribed MAC levels, the consumption of these fishes can be potentially hazardous for humans.

Assessment of macro-, trace- and toxic elements in Small Indian mongoose, Herpestes auropunctatus (Hodgson, 1836), from Montenegro: potential use for biomonitoring.

The aim of this study was to determine the concentrations of 28 elements in the liver, kidney, muscle, and heart of a small Indian mongoose from Montenegro. Element concentrations were determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Significant differences in elemental concentrations between analyzed tissues were observed for Ag, Al, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, P, S, Se, and Zn. The concentrations of Ag, Fe, Mo, and S were significantly different between all analyzed tissues. Muscle tissue is characterised by higher concentrations of some macroelements (K, Mg, S) and liver tissue by higher concentrations of some trace elements (Fe, Mn, Mo, and Zn). Cardiac tissue could be the target organ for bioaccumulation of Ag and Se. The molar ratio Se:Hg was higher than 1 in all studied tissues. There were few significant differences between element concentrations in mongooses from three analyzed (Airport, Saline, Inland) groups. Significantly higher concentrations of Ag and Se were found in liver tissue of Inland mongooses compared to animals from Airport and Saline. There were few significant differences (Zn in liver and muscle, Fe in muscle) in element concentrations between females and males.

Accumulation of 20 elements in great cormorant (Phalacrocorax carbo) and its main prey, common carp (Cyprinus carpio) and Prussian carp (Carassius gibelio).

In this study, 20 heavy metals and trace elements (Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Ni, Pb, Si, Se, Sr and Zn) were analyzed in different tissues (muscle, liver, intestine, feather and bone) of the great cormorant (Phalacrocorax carbo) and in different tissues (muscle, liver, gills, kidney and gonad) of their main prey (common carp-Cyprinus carpio and Prussian carp-Carassius gibelio) during the nesting season. Cormorant and fish specimens were collected at the Ecka Fishing Farm (Serbia) and from the nearby river. Principal components analysis (PCA) showed that cormorant livers were differentiated from other four tissues by higher concentrations of Hg, Fe, Cu and Mo, feathers by higher concentrations of Al and Si, and bones by higher concentrations of Sr and Mg. Differentiation among the three age classes of cormorants was observed only with regard to elemental concentrations in their feathers: subadult cormorants were differentiated by higher Hg and Zn concentrations, while the adults were separated by higher concentrations of Fe. In comparison with their prey (common carp and Prussian carp) cormorants were differentiated by higher concentrations of Fe, Hg and Cu in the muscle. Toxic mercury concentrations above 4µgg(-1)dw were determined in the liver of juvenile cormorants, as well as in the liver and feathers of subadults. Hg concentrations were significantly correlated among all studied tissues. Cormorant had significantly higher concentrations of Fe and Hg in muscle and liver than two studied fish species.

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

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

Seasonal variations of macro-, micro-, and toxic elements in tissues of vimba bream (Vimba vimba) from the Danube River near Belgrade, Serbia.

Vimba bream (Vimba vimba) individuals were collected from the Danube River near Belgrade, from May 2016 to May 2017. Samples of muscle and liver tissue were analyzed for concentrations of 25 elements (macro-, micro-, and toxic elements) using inductively coupled plasma optical emission spectrometry (ICP-OES). For both tissues, the highest concentrations of most elements were observed in late spring/early summer (months of May and June). Majority of statistically significant correlations between elements were positive in both tissues. Contributions of Ca, K, and Mg to human diet were in a similar range. The lowest contribution of all macroelements was observed for Na, while the contribution of Se was well above the recommended dietary allowance for this element. Levels of detected elements were well below MAC values prescribed by national and international regulations. The highest value of Hg liver/muscle index (>1) was observed in August, which could indicate a higher contamination of the sampling location in that month. Se:Hg molar ratio never drops below 1, which could indicate that Hg toxicity is prevented. THQ values showed little variation thought the year and were well below the threshold of 1, with the exception of THQ for Al in May (0.92). This element also strongly affects the hazard index.

Temporal analysis of electroretinographic responses in fishes with rod-dominated and mixed rod-cone retina.

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.

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.

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.

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

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

Mapping differential elemental accumulation in fish tissues: assessment of metal and trace element concentrations in wels catfish (Silurus glanis) from the Danube River by ICP-MS.

Studies of metal accumulation in fish are mainly focused on the muscle tissue, while the metal accumulation patterns in other tissues have been largely neglected. Muscle is not always a good indicator of the whole fish body contamination. Elemental accumulation in many fish tissues and organs and their potential use in monitoring programs have not received proper attention. In the present study, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, and Zn concentrations were assessed by inductively coupled plasma mass spectrometry (ICP-MS) in the following 14 tissues of the wels catfish (Silurus glanis) from the Danube River: muscle, gills, spleen, liver, kidneys, intestine, gizzard, heart, brain, gallbladder, swim bladder, vertebra, operculum, and gonads. A high level of differential elemental accumulation among the studied tissues was observed. The maximum overall metal accumulation was observed in the vertebra, followed by the kidneys and liver, with the metal pollution index (MPI) values of 0.26, 0.25, and 0.24, respectively. The minimum values were observed in the gallbladder, muscle, brain, and swim bladder, with MPI values of 0.03, 0.06, 0.07, and 0.09, respectively. Average metal concentrations in the fish muscle were below the maximum allowed concentrations for human consumption. The mean As, Cd, Pb, Cu, Fe, and Zn concentrations in the muscle were 0.028, 0.001, 0.001, 0.192, 3.966, and 3.969 µg/g wet weight, respectively. We believe that the presented findings could be of interest for the scientific community and freshwater ecosystem managers. There is a need for further research that would assess less studied tissues in different fish species.