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.

Raman microspectroscopy: toward a better distinction and profiling of different populations of dental stem cells.

AIM: To characterize stem cells originating from different dental tissues (apical papilla [SCAP], dental follicle [DFSC], and pulp [DPSC]) and test the capacity of Raman microspectroscopy to distinguish between the three dental stem cell types. METHODS: SCAP, DFSC, and DPSC cultures were generated from three immature wisdom teeth originating from three patients. Cell stemness was confirmed by inducing neuro-, osteo-, chondro-, and adipo-differentiaton and by mesenchymal marker expression analysis by flow-cytometry and real-time polymerase chain reaction. Cellular components were then evaluated by Raman microspectroscopy. RESULTS: We found differences between SCAP, DFSC, and DPSC Raman spectra. The ratio between proteins and nucleic acids (748/770), a parameter for discriminating more differentiated from less differentiated cells, showed significant differences between the three cell types. All cells also displayed a fingerprint region in the 600-700 cm-1 range, and characteristic lipid peaks at positions 1440 cm-1 and 1650 cm-1. CONCLUSION: Although different dental stem cells exhibited similar Raman spectra, the method enabled us to make subtle distinction between them.

Exercise improves growth, alters physiological performance and gene expression in common carp (Cyprinus carpio).

It has been suggested that induced swimming has the potential to improve the growth performance of fish. We tested this hypothesis by measuring growth, metabolic efficiency and physiological capacity of common carp (Cyprinus carpio). Fish were swum at different exercise regimes: 0.0 (control), 1.5 and 2.5 body lengths per second (BL/s) in 1600 L recirculating raceways for 4 weeks. The results showed a significant increase in weight gain, specific growth rate, improved feed conversion efficiency, and a higher hepatosomatic index for 2.5 BL/s exercised fish compared to control. Glycogen, protein and lipid energy stores in hepatic and muscular tissue showed limited differences among experimental groups. Likewise, plasma [Na+], [K+] and [Cl-] remained stable at all swimming regimes. Expression of genes controlling energy metabolism and growth (IGF-I axis, cytochrome oxidase) and stress response (cortisol receptor, heat shock protein 70) revealed clear regulatory roles as the mRNA transcript levels of IGF-I and growth hormone receptors in hepatic tissue were up-regulated in fish exercised for 3-4 weeks at 2.5 BL/s. Oxygen consumption rate and swimming performance (Ucrit) for each experimental group were evaluated in parallel in Blazka-type swim-tunnels (3.9 L) and showed no training effect while prolonged swimming at 1.5 and 2.5 BL/s facilitated ammonia excretion and prevented build-up of plasma ammonia. Overall, these data suggest that sustained exercise at 2.5 BL/s enhanced growth and physiological fitness without compromising energy metabolism or ion-regulation. Our study provides a prospective of implementing exercise as a tool to increase fish production efficiency in commercial aquaculture systems.

Effects of mine tailing and mixed contamination on metals, trace elements accumulation and histopathology of the chub (Squalius cephalus) tissues: Evidence from three differently contaminated sites in Serbia.

Chub (Squalius cephalus) specimens were collected in Korenita River seven months after spillover from the waste water of antimony mine tailing pond and compared with chub living in Kruscica reservoir (intended for water supply) and Meduvrsje reservoir (influenced by intense emission of industrial, urban and rural wastewater). Concentrations of 15 elements (Al, As, B, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Si, Sr, Zn) were determined in muscle, liver and gills of chub by inductively coupled plasma optical spectrometry (ICP-OES) and histopathological alterations in liver and gills were assessed. Chub specimens from Korenita River had higher concentrations of As, Ba and Pb in all three investigated tissues as well as higher total histopathological index values than chub from reservoirs. Specimens from Meduvrsje reservoir were characterized by higher values for concentrations of Cu and Si in muscle tissue and higher values for regressive histopathological alterations in gills. Individuals of chub from Kruscica reservoir had the highest concentrations of Fe in liver, Hg in muscle and Sr and Zn in muscle while gills had the lowest value of total histopathological index. The results from the present study showed higher level of histopathological alterations as a result of mine tailing accident. As a result of mixed contamination on the Meduvrsje site, histopathological index values of gills were in line with the index value from Korenita River. Increased values for Fe and Sr in chub tissue from Kruscica reservoir could be explained by geological structure of the site which is characterized by magmatic rock rich in Cu, Fe and Ni as well as dominant carbonate sediment complex of marine origin with increased level of Sr.

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.

Accumulation of metal trace elements in different body parts of terrestrial Roman snail Helix pomatia L., 1758 on three polluted sites in Serbia.

Atmospheric pollution remains one of the growing concerns in the twenty-first century, with particular focus on metal trace elements (MTE) from anthropogenic sources, due to their adverse effects on biota. The concentration and type of MTE in the atmosphere and in the soil are diverse, depending on the origin of pollutants, which can cause diverse detrimental effects on organisms living in the nearby environment. Three sites in Central Serbia with different origins of MTE pollution (urban contamination, smelting, and fly ash area) were assessed, using terrestrial Roman snails (Helix pomatia) as biomarker organisms. These snails are sentinel organisms and are known for their capacities for accumulation of MTE. Snails were sampled and their body was divided in three parts: viscera, foot, and shell and concentrations of MTE were determined in each of these body parts using inductively coupled plasma optical spectrometry. Results showed contrasting MTE accumulation patterns in body parts of the snails. Of three studied sites, snails sampled in the vicinity of fly ash containment had lower concentrations of MTE compared to other two polluted sites.

The European Chub (Squalius cephalus) as an indicator of reservoirs pollution and human health risk assessment associated with its consumption.

Five reservoirs (Vlasina, Medjuvrsje, Zaovine, Perucac, and Garasi) in Serbia were chosen as study sites, which differ by their position, purpose, stages of eutrophication, management policies, and levels of anthropogenic pressure. The objectives of this research were to: determine the concentrations of 26 elements in muscle, gills, and liver of the European chub by inductively-coupled plasma optical emission spectrometry (ICP-OES); determine the concentrations of 17 organochlorine pesticides in fish muscle by gas chromatography with mass spectrometric detection (GC-MS); compare these findings with condition factor (CF) and histopathological (HP) biomarkers; and assess the potential human health risks due to consumption of chub muscle tissue. The highest elemental accumulation was found in the gills. The European chub was not a good indicator of Pb pollution between reservoirs. Concentrations of Hg, As, and Cu were low and did not exceed the proscribed maximum allowed concentrations (MACs). 4,4'-DDE was detected only in individuals from Vlasina, 4,4'-DDD from Perucac and Zaovine, and heptachlor from Zaovine. Low to moderate levels of HP were observed for both gills and liver in all studied reservoirs. HP index for gills was significantly higher for Zaovine compared to Vlasina. Significantly lower HP index for liver and the total HP index value were observed for fish from Vlasina compared to Perucac. No significant human health risks due to the intake of examined pollutants in each reservoir were recorded; women were at higher risk compared to men. A reason for concern is a few muscle samples from Garasi, Vlasina, Perucac, and Medjuvrsje in which Cd exceeded the MAC. A reservoir for water supply (Garasi) is generally considered the safest for fish consumption.

Sex-specific elemental accumulation and histopathology of pikeperch (Sander lucioperca) from Garasi reservoir (Serbia) with human health risk assessment.

Accumulation of 26 elements (Ag, Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, S, Se, Si, Sr, and Zn) was analyzed in the gills, liver, and muscle of pikeperch males and females from Garasi reservoir using inductively coupled plasma optical emission spectrometry (ICP-OES). Histopathological (HP) changes in the gills and liver, and human health risk were also analyzed. The gills were most affected by metal pollution in both sexes. The concentrations of Hg in muscle tissue of four males, and Cd in two females and two males exceeded the maximum allowed concentrations. Statistical tests only revealed significant differences regarding the concentrations of Mg, K, and S in the muscle (higher in males) and Al, Ag, and Mn in the liver (higher in females) of individuals between sexes. Low to moderate levels of pathological changes were recorded for the gills and liver in both sexes. Significant differences between sexes were observed for inflammatory index of gills (IGI) and HP index of gills (IG), males had higher values compared to females, and for liver necrosis, where females had higher values compared to males. Gills were less affected by HP changes compared to the liver. There was no significant non-cancerogenic and cancerogenic health risk due to the consumption of pikeperch meat from the Garasi reservoir. However, women are under greater risk by consuming the meat of both male and female individuals, probably due to a longer lifetime and lesser body weight compared to the men.

Double whammy: Nitrate pollution heightens susceptibility to both hypoxia and heat in a freshwater salmonid.

Species persistence in a changing world will depend on how they cope with co-occurring stressors. Stressors can interact in unanticipated ways, where exposure to one stressor may heighten or reduce resilience to another stressor. We examined how a leading threat to aquatic species, nitrate pollution, affects susceptibility to hypoxia and heat stress in a salmonid, the European grayling (Thymallus thymallus). Fish were exposed to nitrate pollution (0, 50 or 200 mg NO3- L-1) at two acclimation temperatures (18 °C or 22 °C) for eight weeks. Hypoxia- and heat-tolerance were subsequently assessed, and the gills of a subset of fish were sampled for histological analyses. Nitrate-exposed fish were significantly more susceptible to acute hypoxia at both acclimation temperatures. Similarly, in 18 °C- acclimated fish, exposure to 200 mg NO3- L- 1 caused a 1 °C decrease in heat tolerance (critical thermal maxima, CTMax). However, the opposite effect was observed in 22 °C-acclimated fish, where nitrate exposure increased heat tolerance by ~1 °C. Further, nitrate exposure induced some histopathological changes to the gills, which limit oxygen uptake. Our findings show that nitrate pollution can heighten the susceptibility of fish to additional threats in their habitat, but interactions are temperature dependent.

Liver, gills, and skin histopathology and heavy metal content of the Danube sterlet (Acipenser ruthenus Linnaeus, 1758).

The sterlet (Acipenser ruthenus L.) is a bottom-feeding fish species with a direct exposure to contaminants from water and sediments. Although heavy metal pollution is believed to be one of the main threats to the sterlet population in the Danube River basin, there is a lack of knowledge of the exact impact of heavy metals on their survival. In the present study, effects of heavy metal pollution on sterlet in the Danube basin were assessed as well as the utility of different sterlet organs and tissues as indicators of heavy metal contamination. The sterlet were sampled at three different sites in the Danube basin, in Hungary and Serbia, isolated from each other by dams. Heavy metal analysis included measurement of Cd, As, Pb, Cr, Hg, Cu, Ni, Fe, Mn, and Zn concentrations in sterlet gills, muscle, liver, and intestine, and histopathological analyses comprised assessment and scoring of the extent and intensity of alterations in skin, gills, and liver tissue. Analysis revealed a significant presence of sublethal histopathological changes that were most pronounced in the liver and skin and increased accumulation of heavy metals, with the highest concentrations in the liver. Canonical discriminant analysis showed significant differentiation among the three studied localities, suggesting that the heavy metal concentrations in sterlet populations were site specific. The present study concludes that the accumulation of heavy metals is a response to the presence of these pollutants in the environment, and, together with other pollutants, it affects the vital organs of natural sterlet populations.

Toxicity and bioaccumulation of Cadmium, Copper and Zinc in a direct comparison at equitoxic concentrations in common carp (Cyprinus carpio) juveniles.

The individual toxicity and bioaccumulation of cadmium, copper and zinc for common carp juveniles was evaluated in a direct comparison in two experimental setups. First, fish were exposed for 10 days to different metal concentrations in order to link metal bioaccumulation to LC50 values (concentration lethal to 50% of the animals) and incipient lethal levels (ILL, concentration where 50% survives indefinitely). Accumulated metals showed a positive dose dependent uptake for cadmium and copper, but not for zinc. Toxicity was in the order cadmium>copper>zinc with 96h LC50 values for cadmium at 0.20±0.16 µM, for copper at 0.77±0.03 µM, and for zinc at 29.89±9.03 µM respectively. For copper, the 96h exposure was sufficient to calculate the incipient lethal level and therefore 96h LC50 and ILL levels were the same, while for cadmium and zinc 5 to 6 days were needed to reach ILL resulting in slightly lower values at 0.16 µM and 28.33 µM respectively. Subsequently, a subacute exposure experiment was conducted, where carp juveniles were exposed to 2 equitoxic concentrations (10% and 50% of LC50 96 h) of the three metals for 1, 3 and 7 days. Again a significant dose-dependent increase in gill cadmium and copper, but not in zinc, was observed during the 7-day exposure. Copper clearly affected sodium levels in gill tissue, while zinc and cadmium did not significantly alter any of the gill electrolytes. The overall histopathological effects (e.g. hyperemia and hypertrophy) of the metal exposures were mild for most of the alterations. Our study showed that copper an cadmium (but not zinc) showed dose dependent metal accumulation, however this bioaccumulation was only correlated with mortality for cadmium. Metal specific alterations were reduced gill sodium levels in copper exposed fish and oedema of the primary epithelium which typically occurred in both levels of zinc exposure.

Impact of reservoir properties on elemental accumulation and histopathology of European perch (Perca fluviatilis).

This study was conducted on individuals of European perch from six reservoirs in Serbia with different properties, used for different purposes, and situated at different altitudes. Samples of muscle, gills, and liver, as well as water samples, were used for analysis of elemental accumulation. Histopathological (HP) analysis of gills and liver, and Fulton's condition factor (CF) were used as biomarkers. Analysis of Ag, Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, S, Se, Si, Sr, and Zn was conducted by inductively-coupled plasma optical emission spectrometry (ICP-OES), and accordingly, metal pollution index and bioaccumulation factor (BAF) were calculated. Elemental accumulation varied considerably both among tissues and reservoirs. Reservoirs located on large low-altitude rivers were the most heavily polluted with metals (Meduvrsje, on the Zapadna Morava River, and Lake Sava, on the Sava River). Of three tissues analyzed, the gills were most affected by pollution. The levels of Hg in muscle decreased with an increase in reservoir altitude. Specimens sampled from the highest altitude reservoir were the least affected by HP alterations; however, HP differences between the reservoirs were not statistically significant, and the severity of HP changes was generally low. No significant correlations between the elemental accumulation or HP indices with CF were observed.

Estimating volumes from common carp hepatocytes using design-based stereology and examining correlations with profile areas: Revisiting a nutritional assay and unveiling guidelines to microscopists.

Assessing fish liver status is common in aquaculture nutrition assays. This often implies determining hepatocytes profile areas in routine thin (5-7 µm) histological sections. However, there are theoretical problems using planar morphometry in thin sections: inherent sampling cells biases, too small numbers of sampled cells, under/overestimation of size, measuring size as areas when cells are three-dimensional (3D) entities. The gold standard for assessing/validate cell size is stereology using thick sections (20-40 µm). Here, we estimated the volume of hepatocytes and their nuclei by the nucleator and optical disector stereological probes (in thick sections), and, innovatively, in thin sections too (using single-section disectors). The liver of common carp eating feed containing either low or high level of lipids was targeted. Results were compared with prior profile areas from planar morphometry using thin sections, and with profile areas estimated here with the two-dimensional (2D) nucleator. Ratios between nucleus and cell/cytoplasm (N/C) areas and volumes were calculated and compared. There was high positive correlation between volumes in thin and thick sections (r = .85 to .89; p < .001), empirically validating the single-section disector. Strong correlations existed between profile-derived versus 2D-nucleator areas (r = .74 to .83; p < .001). There was systematic underestimation of cells and nucleus size using planar morphometry. The N/C ratios derived from the 2D-nucleator data were higher than those from planar morphometry. Despite theoretical premises for using simple planar morphometry in thin sections are flawed, our results support that such morphometry on carp/fish hepatocytes may offer some valid biological conclusions. Anyway, we advanced guidelines for implementing proper methods.

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.

The Antibacterial Activity of Coriolus versicolor Methanol Extract and Its Effect on Ultrastructural Changes of Staphylococcus aureus and Salmonella Enteritidis.

The antibacterial activity of methanol extract obtained from fruiting body of industrially grown basidiomycete Coriolus versicolor was examined. The Minimum Inhibitory Concentration (MIC) values against various bacteria ranged from 0.625 to 20 mg mL(-1). C. versicolor expressed bactericidal activity against both Gram-positive and Gram-negative bacteria. The growth curves of Staphylococcus aureus and Salmonella enterica serovar Enteritidis, measured at 630 nm, and confirmed with macrodilution method showed that the obtained extract could inhibit the growth of tested bacteria. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the loss of 260-nm-absorbing material were used to examine the ultrastructural changes in bacteria induced by the extract. When S. aureus was exposed to the MIC of C. versicolor, elongated and malformed cells were observed by SEM, while S. Enteritidis treated cells appeared shorter and aggregated with ruptured cell walls. TEM revealed the formation of non-membrane-enclosed bodies and depleted inner content of S. aureus. Larger and irregular periplasmic space and deformed and scattered components of the cell envelope were observed in treated S. Enteritidis. The loss of 260-nm-absorbing material indicated that the disruptive action of the extract on cytoplasmic membrane was more pronounced in S. aureus than in S. Enteritidis treated cells. The UV and FTIR spectrophotometric analyses revealed diverse composition of C. versicolor extract and high content of total phenolics. Altogether, mushroom extracts could be used to develop nutraceuticals or drugs effective against pathogenic microorganisms.

Subacute and subchronic toxicity of Avalon(®) mixture (bentazone+dicamba) to rats.

Subacute and subchronic toxicity of the herbicide Avalon(®), a mixture of bentazone and dicamba, were tested on rats. Avalon(®) was administered at dose levels of 250, 500 and 1000mg/kg body weight/day for 28 and 90 days. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities were monitored together with biochemistry parameters. The results showed that the mixture caused increases in the activities of ALT, AST and ALP, elevated concentrations of sodium, albumin and albumin/globulin ratio in males. In females, ALT activity, cholesterol and phosphate levels were increased. The changes generally were dose related and, in most cases, females exhibited lower susceptibility than males. The effects of a mixture are, in the most cases, different from the effects of the individual substances. The effects of bentazone were not prevalent which would be expected taking the composition of the mixture into account.