Zoran Gačić

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.

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.

Monitoring of DNA damage in haemocytes of freshwater mussel Sinanodonta woodiana sampled from the Velika Morava River in Serbia with the comet assay

This study was undertaken to investigate the potential of the freshwater mussel Sinanodonta woodiana for detection of genotoxic pollution of the environment. Study was performed at two sites in the Velika Morava River, from May 2010 to February 2011. The alkaline comet assay on haemocytes was used, and the olive tail moment (OTM) was chosen as a measure of DNA damage. The specimens held on acclimation under controlled laboratory conditions for 10d were used as a control. Chemical analysis revealed the presence of phosphates and increased concentrations of zinc, copper and nickel at both sites during the entire sampling period. The values of OTM in mussels collected from the environment, significantly correlated with the concentration of zinc (r=0.6248), temperature (r=0.7006) and dissolved oxygen (r=0.7738). Seasonal variations in genotoxic response were observed, with the highest OTM values obtained during summer months. Preliminary results of the in vitro study indicated the effect of water temperature on genotoxic response to zinc and cadmium in S. woodiana suggesting that the presence of genotoxic pollutants during months with lower temperature could be under-estimated. Obtained results indicate that S. woodiana could be a valuable tool for active biomonitoring of aquatic environments and emphasizes the importance of seasonal genotoxic monitoring with this organism.

Changes in the phytoplankton community and dominance of Cylindrospermopsis raciborskii (Wolosz.) Subba Raju in a temperate lowland river (Ponjavica, Serbia)

Species composition and seasonal succession of phytoplankton were analyzed in a lowland river located in South Banat (Ponjavica, Serbia). As a result of human activity, a network of irrigation channels was built on the Ponjavica River’s upper and lower courses. These channels reduce the water level in the river and help to create favorable conditions for accelerated eutrophication processes. Eight phytoplankton divisions with 444 taxa were present. Physical and chemical testing of water, phytoplankton abundance and biomass, as well as chlorophyll a concentration showed a high level of eutrophication. Cyanobacterial biomass developed in 2002 with dominance of Microcystis aeruginosa and Aphanizomenon flos-aquae. Changes in phytoplankton composition were observed during 2008, characterized by the appearance and dominance of Cylindrospermopsis raciborskii when this species accounted for more than 85% of the total phytoplankton biovolume. The change in dominant phytoplankton functional groups was observed: codons H1 and M characterizing summer in 2002 were replaced by codons SN and S1 in 2008. This study compares the presence of C. raciborskii with other phytoplankton species in relation to water quality parameters. High abundance of this species correlated positively with biochemical oxygen demand and turbidity, and negatively with NO3-N concentrations during the study period.

Assessment of the genotoxic potential along the Danube River by application of the comet assay on haemocytes of freshwater mussels: The Joint Danube Survey 3

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 worlds 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).

Intestinal helminths infection of rats (Ratus norvegicus) in the Belgrade area (Serbia): the effect of sex, age and habitat

Gastrointestinal helminths of Norway rat (Rattus norvegicus) from the Belgrade area were studied as a part of a wider ecological research of rats in Serbia (data on the distribution, population ecology, economic and epizoothiological-epidemiological importance, and density control). Rats were captured from May 2005 to July 2009 at both urban and suburban-rural sites. Of a total of 302 trapped rats 48% were males and 52% females, with 36.5% and 38.8% of juvenile-subadult individuals, per sex respectively. Intestinal helminth infection was noted in 68.5% of rats, with a higher prevalence in male hosts and in adult individuals. Higher numbers of infected juveniles-subadults were noted in suburban-rural habitats, while an opposite tendency was noted in adult rats. Seven helminth species were recovered, of which five were nematode (Heterakis spumosa, Nippostrongylus brasiliensis, Capillaria sp., Trichuris muris and Syphacia muris) and two cestode species (Hymenolepis diminuta and Rodentolepis fraterna). The most prevalent parasites were Heterakis spumosa (36.7%) and Hymenolepis diminuta (30.5%). Sex and habitat-related differences were noted in the prevalence of infection with Capillaria sp. and Trichuris muris, while there were no age-related differences in the prevalence of infection with any individual helminth species. Significantly higher prevalence of infection was noted in summer as compared to spring or winter, with a tendency to be higher in autumn as compared to spring. The only significant difference in the prevalence of infection between habitat-related was noted during spring. H. spumosa was most prevalent in summer, while H. diminuta and N. brasiliensis in autumn. The mean intensity of infection with H. spumosa, R. fraterna, S. muris and T. muris was higher in autumn than in the other seasons, while N. brasiliensis and Capillaria sp. occured in winter. No more than four helminth species were found in one host.

Evaluation of single-cell gel electrophoresis data: Combination of variance analysis with sum of ranking differences

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.

Flooding modifies the genotoxic effects of pollution on a worm, a mussel and two fish species from the Sava River

Extreme hydrological events, such as water scarcity and flooding, can modify the effect of other stressors present in aquatic environment, which could result in the significant changes in the ecosystem functioning. Presence and interaction of various stressors (genotoxic pollutants) in the environment can influence the integrity of DNA molecules in aquatic organisms which can be negatively reflected on the individual, population and community levels. Therefore, in this study we have investigated the impact of flooding, in terms of genotoxicity, on organisms belonging to different trophic levels. The study was carried out on the site situated in the lower stretch of the Sava River which faced devastating effects of severe flooding in May 2014. The flooding occurred during our field experiment and this event provided a unique opportunity to assess its influence to the environment. The in situ effects of this specific situation were monitored by measuring physical, chemical and microbiological parameters of water, and by comparing the level of DNA damage in coelomocytes and haemocytes of freshwater worms Branchiura sowerbyi, haemocytes of freshwater mussels Unio tumidus and blood cells of freshwater fish Abramis bjoerkna/Abramis sapa, by means of the comet assay. Our study indicated that the flooding had a significant impact on water quality by decreasing the amount and discharge rate of urban wastewaters but simultaneously introducing contaminants from the nearby fly ash disposal field into river by runoff, which had diverse effects on the level of DNA damage in the studied organisms. This indicates that the assessment of genotoxic pollution in situ is strongly affected by the choice of the bioindicator organism.

Spectral sensitivity of the dogfish shark (Scyliorhinus canicula)

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.

Electroretinographic evaluation of spectral sensitivity in yellow and silver eels (Anguilla anguilla)

Although differences in visual pigments between developmental stages of the European eel are well known, the expected differences in spectral sensitivity have not been demonstrated at the electrophysiological level. In fact, one past electroretinographic study led to the conclusion that in eels there is no change in scotopic sensitivity, with increasing sexual maturity. In the present experiments, electroretinograms (ERGs) were recorded from in situ eyecups of immobilized eels Anguilla anguilla (L.) caught in coastal running waters. It was shown that the ERG b-wave is as good an indicator of spectral sensitivity as the unmasked late receptor potential (LRP) which directly reflects the responsiveness of photoreceptors. Complete spectral-sensitivity curves, based on b-wave thresholds and on thresholds of LRP subsequently isolated by means of sodium iodate, have been obtained in the same eel. Using fitted amplitude-log intensity functions for threshold calculation, and two models for computer-assisted fitting of spectral-sensitivity curves, significant differences in lambda max were found between yellow and silver developmental stages of the eel, identified by ocular index measurements.