Микробиолошки квалитет воде и детекција генотоксичног загађења различитих сектора реке Саве прокариотским и еукариотским тест системима

The Sava River Basin is under strong impact of stressors related to anthropogenic activity, yet the consequences that the stressors might have on aquatic biota are not properly addressed. This dissertation aims to provide data on genotoxic potential along the Sava River. Research was organized in two campaigns. In the first campaign, preliminary research was conducted to indicate optimal bioindicator, season, sample preservation and bioassays for performing the research on the scale of the whole Sava River. The second campaign consists of comprehensive survey along the 900 rkm of the Sava River. With combined in vivo and in vitro approach, 12 sites were analyzed in total. 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). Results indicated differential sensitivity of applied bioassays in detection of genotoxic pressure. Comet assay showed higher potential in differentiation of the sites based on genotoxic potential in comparison with other assays. Data presented in this dissertation are a snapshot of the current status of the river. Genotoxic potential along the river can be traced to the deterioration of water quality by communal and industrial wastewaters. The major highlight of the study is that the complex set of data was obtained from a single source (homogeneity of analyses for all samples).Слив реке Саве је под снажним утицајем стресора антропогеног порекла, међутим последице које стресори могу имати на водену биоту нису довољно испитане. Ова дисертација има за циљ да испита генотоксични потенцијал дуж реке Саве. Истраживање је организовано у две целине. У овиру прве целине испитивало се који биоиндикаторски организам је оптималан за истраживање на нивоу читаве реке. Такође испитивао се утицај сезоне на одговор биомаркера као и методологија очување узорка на резултате анализа. У оквиру друге целине рађено је комплексно истраживања дуж 900 ркм реке Саве. Коришћен је комбиновани in vivo и in vitro приступ, при чему је анализирано укупно 12 локација. Генотоксични потенцијал је праћен употребом сложене батерије биотестова на прокариотским и еукариотским организимима. Батерија је обухватала процену мутагеног потенцијала SOS/umuC тестом на бактерији Salmonella typhimurium TA1535/pSK1002. Ниво оштећења ДНК, као биомаркер излагања (кометни тест) и биомакер ефекта (микронуклеус тест) као и ниво оксидативног стреса (Fpg - модификовани кометни тест) праћен је у крвним ћелијама риба (Alburnus alburnus/Alburnoides bipunctatus). Резултати су показали различиту осетљивост примењених биолошких тестова у детекцији генотоксичног потенцијала. Комет тест је имао већи потенцијал у диференцирању локалитета по генотоксичном потенцијалу у поређењу са другим примењеним тестовима. Подаци представљени у овој дисертацији представљају слику тренутног стања реке. Генотоксични потенцијал забележен дуж реке се може везати за нарушен квалитет воде утицајем комуналних и индустријских отпадних вода. Подаци приказани у овој студији потичу из јединственог извора (хомогеност анализа за све узорке) упркос великој географској површини коју студија покрива што јој додатно даје на значају

Kako poplave modifikuju genotoksični odgovor slatkovodnih riba?

U ovom radu ispitivan je potencijal dve vrste roda Abramis (Abramis bjoerkna i Abramis sapa) kao bioindikatora genotoksičnog potencijala vode reke Save primenom komet testa. Izabrani lokalitet pripada oblasti intezivne poljoprivredne aktivnosti, izložen je otpadnim vodama grada Obrenovca i nalazi se u blizini najveće termoelektrane u Srbiji, “Nikola Tesla”. Uzorkovanje je vršeno od januara do avgusta 2014. godine, obuhvatajući mesec maj kada je došlo do intezivnih poplava u slivu reke Save. Dobijeni rezultati ukazuju da se ispitivane vrste mogu koristiti za procenu genotoksičnog potencijala

Effect of floods on DNA damage of two cyprinid fish in the Sava River

Freshwater ecosystems are constantly deteriorated due to the multitude of human activities, intensification of the industrial processes, wastewater discharge and agricultural chemicals. Besides, climate change provokes extreme weather and hydrological conditions leading to prolonged periods of droughts and floods. Floods can cause movements of polutants already present in the environment and introduce pathogenic bacteria in the flooded area. Many of the substances that reach environment could exhibit genotoxic potential on the genetic material of aquatic organisms, either alone or in complex mixtures of chemicals. Unfavorable hydrological conditions during 2014 resulted in extensive flooding in May in the Sava River basin. Obrenovac city was one of the most affected settlements on the river with esstimation that nearly 90% of town area was flooded. This led to evacuation of majority of inhabitants from the affected area. Our research was conducted from January to December 2014 on the Sava River, site Duboko, situated downstream of Obrenovac city. This is the area of intensive agricultural activity. Few km upstream the sampling site is the mouth of Kolubara river which is receiving untreated municipal wastewater of the town Obrenovac (50.000 inhabitants) and thermal power plant Nikola Tesla A with belonging ash fild. On monthly basis we have performed the measurements of basic phisico-chemical parameters, sampling water for microbiology analysis and fish tissue for the comet assay. Single Cell Gel Electrophoresis (SGCE) or Comet assay is widley accepted tool in ecogenotoxicology studies, in which DNA damage is measured on the level of a single eukaryotic cell. For the detection and quantification of E. coli and Enterococci we have used enzymathic methods and MPN approach. For comet assay analysis blood, liver and gill tissues were sampled from two cyprinid fishes. The highest water level was observed in May 2014, when flooding occured. Evacuation of inhabitants had led to exclusion of urban wastewater discharge which was refelcted through the lowest concentration of faecal pollution indicator bacteria, E. coli and Enterococci. With return of inhabitants in June their concentration in water started to increase gradually. In contrast, with withdrawl of water from the flooded agricultural land and ash disposal field high amounts of potentialy genotoxic substances were introduced into the Sava river, which was observed in sudden increase of DNA damage level in all three fish tissues during June. This study showed that flood occurence had major impact on the microbiological quality and genotoxic potential of the Sava River. Faecal pollution is mainly under the impact of urban wastewater discharge. On the other hand, genotoxic substances are most likely of industrial and agricultural origin. Acknowledgements This research was funded by project OI173045 „Fishes as water quality indicators in open waters of Serbia” (Ministry of Education, Science and Technological Development of the Republic of Serbia). This research was conducted in the Laboratory at the Chair of Microbiology, Center for genotoxicology and ecogenotoxicology, Faculty of Biology, University of Belgrade, and Institute for Multidisciplinary research, University of Belgrade

Primena IBR metode (Integrated Biomarker Response) u mikrobiološkoj analizi vodenih ekosistema

UVOD: Mikrobiološki indikatori sanitarnog i organskog zagađenja čine neophodan segment svake procene kvaliteta vode. Međutim, kako se velike količine neprerađenih ili nepropisno prerađenih otpadnih voda ispuštaju u vodotokove teško je razdvojiti sanitarno od organskog zagađenja. Vizuelni prikaz rezultata IBR metodom pokazao se veoma korisnim u analizi međusobnih odnosa različitih biomarkera i ima široku primenu u biomonitoringu. Osim primene na biomarkerima, metoda može da pokaže i uzajamne odnose različitih hemijskih parametara. Metoda IBR do sada nije korišćena za analizu uzajamnih odnosa mikrobioloških parametara. CILJEVI: Primena IBR metode u proceni kvaliteta vode, pri čemu su mikrobiološki parametri fekalnog i organskog zagađenja korišćeni kao “biomarkeri“. METODE: Za potrebe ove studije odabrana su dva seta podataka. Prvi set je dobijen tokom višemesečnog monitoringa na reci Savi tokom 2014. godine, uključujući i period sa poplavom, tj. maj mesec. Od indikatora fekalnog zagađenja praćeni su totalni koliformi, Escherichia coli, Enterococcus faecalis i Clostridium perfringens. Nivo organskog zagađenja određen je na osnovu odnosa heterotrofnih i oligotrofnih bakterija, i indeksa fosfatazne aktivnosti. Drugi set podataka dobijen je tokom višemesečnog monitoringa sezonskog variranja mikrobioloških indikatora na reci Peštan, tokom 2012. godine. Od indikatora fekalnog zagađenja praćeni su totalni koliformi, E. coli i E. faecalis. Nivo organskog zagađenja određen je na osnovu odnosa heterotrofnih i oligotrofnih bakterija. REZULTATI: Vizuelni prikaz rezultata IBR metodom dao je jasan uvid u minimalne i maksimalne vrednosti svakog od indikatora fekalnog i organskog zagađenja tokom različitih meseci. Za prvi set podataka, na osnovu grafika i na osnovu dobijenih IBR vrednosti, jasno se izdvaja mesec u kome je došlo do poplava. Za drugi set podataka očigledno je variranje mikrobioloških parametara tokom različitih sezona. ZAKLJUČAK: Metoda IBR je pogodna za prikaz i analizu kompleksnog seta mikrobioloških podataka

Evaluation of the Sava and the Danube river genotoxicity by Comet assay on different cyprinid fish

A number of biological consequences of DNA damage can be initiated at the cellular, organ, whole animal, population and community levels. The comet assay, single cell gel electrophoresis (SCGE), is a sensitive and rapid technique for detection of DNA damage in individual cells based on the migration of denatured DNA during electrophoresis, in which damaged nuclei form comet-like shapes. Since, the large rivers are constantly exposed to variety of chemicals we tested genotoxic potential of rivers Sava and Danube in the Belgrade region, on cyprinid fish. DNA damage was monitored in blood, liver and gill cells of Blicca bjoerkna, Ballerus sapa and Abramis brama by comet assay. The specimens of B. bjoerkna and B. sapa were sampled at the site Duboko (Sava river), which is exposed to untreated municipal wastewater, effluents of the Thermal Power Plant and intensive agricultural activity. The specimens of A. brama were sampled at the site Višnjica (Danube river), which is exposed to untreated municipal wastewater from surrounding Belgrade municipalities. Sampling was performed during February and August 2014. Comet IV Computer Software (Perceptive Instruments, UK) was used for the quantification of DNA damage. The increased level of DNA damage obtained in all tissues of B. bjoerkna and B. sapa (Sava river), and A. brama (Danube river) is in correlation with high chemical pollution detected at Duboko and Višnjica sites. Moreover, this study showed the influence of seasonal variations on the level of DNA damage

Uticaj poplava na mikrobiološki kvalitet vode reke Save na teritoriji grada Obrenovca

U ovom istraživanju praćen je mikrobiološki kvalitet vode reke Save, na lokalitetu nizvodno od grada Obrenovca, pre i nakon velikih poplava koje su se dogodile u maju 2014. godine. Praćeni su indikatori fekalnog zagađenja (ukupni koliformi, Escherichia coli, Enterococcus faecalis) kao i pokazatelji organskog opterećenja (heterotrofi). Dobijeni rezultati pokazuju da je tokom meseca sa najvišim vodostajem (maj) došlo do velikog pada u koncentraciji glavnih pokazatelja fekalnog zagađenja, u prvom redu E. coli. Sa porastom vodostaja tokom perioda poplava uočen je i pad koncentracije heterotrofnih bakterija. S druge strane, detektovan je porast koncentracije ukupnih koliforma tokom poplava. U ovom ispitivanju jasno je pokazano da poplave dovode do promena u trendu variranja koncentracija mikrobioloških parametara kvaliteta vode

The impact of the Sava river pollution on biomarkers response in the liver and gills of three cyprinid species

Due to the presence of a large number of different pollutants, monitoring of the surface water quality based solely on the analysis of a limited number of xenobiotics, cannot be considered as reliable. Beside toxic, these agents can exert genotoxic effects, inducing damage in the DNA molecule, which, if not repaired, could lead to mutations and alterations in cells, tissues, organism, whole population and the ecosystem. The surface waters are under the pressure of both anthropogenic and natural sources of pollution. Additionally, extreme hydrological events, such as water scarcity and flooding, may further impair the state of freshwater bodies. Fish may be exposed to harmful substances through water, sediment and food. In ecogenotoxicity studies, gills are used as they represent the first organ in direct contact with water and waterborne pollutants, while the liver, as a key organ that controls many life functions is used as a major organ for metabolic breakdown of xenobiotics. Common bream (Abramis brama), white bream (Blicca bjoerkna) and white-eye bream (Ballerus sapa) are three closely related, benthivorous cyprinids, native for the Sava River. The sampling site Duboko (23 rkm), on the Sava River, is chosen as it is exposed to the untreated wastewater from the town of Obrenovac (more than 70,000 inhabitants), intensive agricultural activity and close proximity to the largest thermal power plant in Serbia (TENTA) and belonging ash field. This study was conducted to assess the impact of multiple stressors during different seasons on different levels of biological organization, subcellular (genotoxic effect) and cellular/tissue level (histopathological effects), in the liver and gills of three bream species. 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. In parallel, concentration of metals and metalloids were assessed in gills, liver and muscle. Basic physical (pH, temperature, oxygen concentration, electrical conductivity) and chemical (NO2, NO3-, NH4+, PO43-) parameters were measured on site. Microbiological indicators of faecal pollution, total coliforms (TC), Escherichia coli (EC) and Enterococci (EF) were assessed by using a most probable number approach (MPN). Presumptive Clostridium perfringens (CP) numbers were determined by using membrane filtration and incubation on TSC (Tryptose Sulphite Cycloserine) media. Sampling of fish tissue for comet assay and histopathological analyses was performed in 2014, during winter (January and February), spring (March and early June), and summer (late June, July, and August), once per month, for a total of 52 specimens. Analysis of metals and metalloids was performed only on fish sampled in February, early June and in August, in total 15 specimens. In the mid May extreme hydrological events led to extensive flooding in the studied area. Comet assay was conducted on the liver and gill cells according to Kostić et al. Tail intensity, % of DNA in the comet tail (TI) was used to express the DNA damage level. Histopathological analyses included the use of conventional histological methods, staining of tissues sections using hematoxylin eosine differential staining, and examined under the microscope. The type and the extent of histological alterations were described by using a method developed by Bernet et al. Analysis of metals and metalloids was performed by inductively-coupled plasma optical emission spectrometry (ICP-OES), and included assessment of concentrations of 16 elements (Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Li, Mn, Mo, Ni, Pb, Sr and Zn). To compare the total metal content in different tissues and through different seasons metal pollution index (MPI) was calculated according to equation MPI = (cf1 x cf2 x cf3 x…cfn) 1/n, where cfn = concentration of the metal n in the sample. Statistical analysis of data from the individual months showed the highest DNA damage in gill cells during early June (spring). Gill histopathological index (IG) did not show significant seasonal variations, however it was the lowest during winter, the highest in spring, and slightly decreased in summer. A possible cause of this incidence could be a withdrawal of water which took place in June, after the flooding event that occurred in the middle of May. In liver the highest DNA damage was observed during August. Histopathological index of liver showed significantly higher values in summer in comparison to spring. This could be prescribed to a higher metabolic rate of fish liver during warm seasons and also could be a consequence of processing a large quantities of xenobiotics introduced into the water column due to withdrawal of water after floods and sediment disturbance. According to the MPI, gills were under the highest pressure of metal pollution during spring and summer. Liver was under the highest pressure of metal pollution during winter, while the muscle was the least affected tissue during all three seasons. Overall, gills as the first organ in direct contact with water showed a higher response in terms of DNA damage (molecular level), while the liver as the major organ for processing of xenobiotics both from water and food showed a higher degree of histopathological alterations in comparison to gills (tissue/organ level). Increased response of both biomarkers during spring and summer indicates a joint effect of the flooding event and seasonal changes of climate and hydrological parameters

DNA damage and histological changes in gills and liver of cyprinid fish as biomarkers of the Sava River pollution

Fish species living in freshwater ecosystems are exposed to a various range of xenobiotics. Two of the most important pathways of xenobiotics intake by fish are either via the digestive or respiratory routes or by absorption of waterborne chemicals through the gills. As a biomarker of exposure to genotoxic agents, the single cell gel electrophoresis (SCGE), the comet assay, is widely used for detecting DNA damage in different tissues of aquatic organisms. As biomarkers of effect, histopathological analyses are considered efficient and sensitive for monitoring the fish health and pollution of aquatic ecosystems. Examination of multiple biomarkers in exposed organisms may give important information on organisms’ exposure to xenobiotics and related stress response. The main objective of this study was to evaluate the level of the Sava River pollution in the Belgrade region by measuring genotoxic and histopathological biomarker responses in liver and gills of cyprinid fish. Specimens of Abramis brama, Blicca bjoerkna and Ballerus sapa, were collected in winter, spring, summer and autumn of 2014 from the site Duboko. This site is exposed to untreated urban wastewaters, agricultural and industrial activity. DNA damage in liver and gills is expressed by Tail Intensity (TI), while histological changes were expressed as organ index which represents the extent of organ injuries. The highest level of DNA damage in liver and gills was detected in summer. The lowest level of DNA damage in liver was observed during spring and in gills during autumn. HP index of liver (IL) and gills (IG) showed no significant difference between seasons, but IL showed gradual increase from winter to autumn and IG showed a gradual increase from winter to summer. In conclusion, summer is marked as a season in which fish are under higher pressure of pollution according to both DNA damage and histopathological changes in tissues

How flooding modifies genotoxic response in freshwater fish?

U ovom radu ispitivan je potencijal dve vrste roda Abramis (Abramis bjoerkna i Abramis sapa) kao bioindikatora genotoksičnog potencijala vode reke Save primenom komet testa. Izabrani lokalitet pripada oblasti intezivne poljoprivredne aktivnosti, izložen je otpadnim vodama grada Obrenovca i nalazi se u blizini najveće termoelektrane u Srbiji, “Nikola Tesla”. Uzorkovanje je vršeno od januara do avgusta 2014. godine, obuhvatajući mesec maj kada je došlo do intezivnih poplava u slivu reke Save. Dobijeni rezultati ukazuju da se ispitivane vrste mogu koristiti za procenu genotoksičnog potencijala

Assessment of the faecal contamination along the Sava River and identification of pollution sources

The contamination of water by faecal pollution leads to exposure to pathogens via drinking water production, recreation or irrigation. However, monitoring of microbiological quality of surface waters is quite neglected despite its importance for human health. In the case of Sava River Basin, many of the settlements situated on the river banks discharge high quantities of untreated or improperly treated wastewaters directly into surface waters. Due to usage of water for irrigation, the evaluation of microbiological quality of the Sava River becomes essential for further river management. Water samples were collected during September 2014 on 17 sites and during September 2015 on 15 sites situated along the Sava River. In 2015, additional samples were collected from 4 wastewater outlets detected onsite. Microbiological analyses comprised monitoring the standard indicators of faecal pollution within the surveys and long term monitoring data (obtained within 5 years of routine monitoring at 4 stations). For detection of total coliforms, Escherichia coli and enterococci, Defined Substrate Technology (DST) was used with quantification performed by Colilert Quanti-Tray 2000 system, which provides a Most Probable Number result. Detection of presumptive Clostridium perfringens was performed by membrane filtration method according to ISO 14189:2013. To identify the origin of pollution, microbial source tracking (MST) analyses were employed based on the human-associated BacHum and HF183II, the ruminant-associated BacR and the pigassociated Pig2Bac genetic Bacteroidetes faecal markers. Microbiological indicators showed the existence of hotsposts of faecal pollution in the Sava River. MST confirmed that the pollution is human associated. Long term data at selected sites indicated persistent faecal contamination which leads to conclusion that the sites are under the impact of continuous discharge of wastewaters