Karolina Sunjog

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.

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.

Genotoxicological Studies of Lower Stretch of the Sava River

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.

Evaluation of genotoxic potential of avarol, avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models.

Abstract In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3′-methoxyavarone, 4′-(methylamino)avarone and 3′-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3′-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3′-methoxyavarone and 3′-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.

Evaluation of genotoxic potential in the Velika Morava River Basin in vitro and in situ

The Velika Morava River is the greatest national Serbian river and the significant tributary of the Danube River. The major problems in the Velika Morava River Basin (VMRB) represent untreated industrial and municipal wastewaters. In this study, the level of genotoxic potential at the sites along the VMRB was evaluated by parallel in vitro and in situ approach. Within in vitro testing, genotoxicity of native water samples collected from the sites in VMRB was evaluated by SOS/umuC test on Salmonella typhimurium TA1535/pSK1002 and by the comet assay on HepG2 cells. DNA damage in situ was assessed in bleak (Alburnus alburnus) erythrocytes by the comet (alkaline and Fpg-modified comet) and micronucleus assays. Additionally, the concentration of heavy metals in fish tissue was measured and this data, compiled with the data of the physico-chemical parameters measured in water, was used as a measure of the pollution pressure at the sites. Results showed that applied in vitro tests with native water samples are less sensitive in comparison with in situ tests and should be taken with precaution when making predictions on the status of the ecosystem. Within applied battery of in situ assays differential sensitivity of assays was observed where alkaline comet assay showed the highest potential in differentiation of the sites based on genotoxic potential. Integrated biomarker response showed that usage of the battery of bioassays provides better insight in a genotoxic effects in animals, and consequently, that the holistic approach is more suitable for this type of study.

Microbial Characterisation of the Sava River

Data on the microbiological quality of the freshwater systems under the anthropogenic influence, such as the Sava River, are of the major importance for the water resource management. Furthermore, analyses of the microbial quality of fish meat provide information of the fish as a valuable food resource from the investigated river basin. The health status of the fish, including dynamics of infection and biodiversity of endoparasites, is important bioindicator of changes in the ecosystem structure and function. For the ecosystem-based approach to the Sava River management, investigations of microbiological quality of the Sava River water and the meat of the European chub as the bioindicator organism, as well as dynamics of infection/biodiversity of intestinal parasites Acanthocephala, were performed. The survey comprised the data collected in periods 2005, 2006 and 2012. Microbiological investigation of water was performed in 2006 and 2012, while microbiological analyses of fish meat and ichthyo-parasitological investigation took place during 2005–2006. A high number of heterotrophic bacteria were recorded during 2006 survey, confirmed by the distinctly higher values of the three faecal indicators (total coliform, E. coli and enterococci), and indicated poor water quality downstream of the cities Zagreb and Velika Gorica, as a result of the municipal sewage outlets. The results from 2012 survey indicated the existence of moderate to critical faecal and organic pollution in all samples. Accumulation of the bacteria in the European chub meat was mainly uniform along the watercourse within standards and limitations for the human consumption. Sampling sites downstream cities of Zagreb and Velika Gorica were characterised with the lower prevalence and abundance of two common species of the chub intestinal acanthocephalan parasites, Pomphorhynchus laevis and Acanthocephalus anguillae. Poor microbiological quality of the water and lower distribution of chub intestinal parasites were related to the anthropogenic influence, downstream of the urban areas.

Cyanobacterial effects in Lake Ludoš, Serbia - Is preservation of a degraded aquatic ecosystem justified?

Cyanobacteria are present in many aquatic ecosystems in Serbia. Lake Ludoš, a wetland area of international significance and an important habitat for waterbirds, has become the subject of intense research interest because of practically continuous blooming of cyanobacteria. Analyses of water samples indicated a deterioration of ecological condition and water quality, and the presence of toxin-producing cyanobacteria (the most abundant Limnothrix redekei, Pseudanabaena limnetica, Planktothrix agardhii and Microcystis spp.). Furthermore, microcystins were detected in plants and animals from the lake: in macrophyte rhizomes (Phragmites communis, Typha latifolia and Nymphaea elegans), and in the muscle, intestines, kidneys, gonads and gills of fish (Carassius gibelio). Moreover, histopathological deleterious effects (liver, kidney, gills and intestines) and DNA damage (liver and gills) were observed in fish. A potential treatment for the reduction of cyanobacterial populations employing hydrogen peroxide was tested during this study. The treatment was not effective in laboratory tests although further in-lake trials are needed to make final conclusions about the applicability of the method. Based on our observations of the cyanobacterial populations and cyanotoxins in the water, as well as other aquatic organisms and, a survey of historical data on Lake Ludoš, it can be concluded that the lake is continuously in a poor ecological state. Conservation of the lake in order to protect the waterbirds (without urgent control of eutrophication) actually endangers them and the rest of the biota in this wetland habitat, and possibly other ecosystems. Thus, urgent measures for restoration are required, so that the preservation of this Ramsar site would be meaningful.

The Genetic Variability (RAPD) and Genotoxicity In Vivo (Alkaline and Fpg-Modified Comet Assay) in Chub (Squalius cephalus): The Sava River Case 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.Graphical abstract