Procena genotoksičnosti korišćenjem kometa testa u različitim tkivima klena iz rečne vode

Cilj ovog rada je bila analiza oštećenja molekula DNK primenom komet testa (engl. Single Cell Gel Electrophoresis, SCGE) na klenu (Squalius cephalus L.) kao potencijalnom model organizmu za procenu genotoksičnosti vode Kolubarskog basena. Kolubarski basen je bogat nalazištima lignita pa kao takav trpi veliki pritisak rudarskog postrojenja, “Kolubara”, pored već postojećeg uticaja komunalnih voda kao i spiranja sa obradivih površina. Uzorkovanja su vršena mesečno tokom sezone 2011/2012 god. Jedinke klena sa Zlatarskog jezera, Uvac, su korišćene kao prirodna kontrolna populacija. Komet test je osetljiva i relativno jednostavna metoda primenljiva na velikom broju različitih organizama i tkiva. Za detekciju oštećenja DNK molekula u ovom radu korišćena su tri tkiva: krv, jetra i škrge

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

Analiza kvaliteta vode reke Jablanice na osnovu zajednice vodenih makroinvertebrata

Cilj rada je da se prikažu rezultati ocene kvaliteta vode reke Jablanice (sliv Kolubare) zasnovane na prisutnoj zajednici vodenih makroinvertebrata. Jablanica je relativno kratka (25 km) desna sastavnica, koja u blizini Valjeva sa Obnicom (leva sastavnica) formira Kolubaru. Kolubara, kao poslednja veća pritoka Save, protiče kroz područje sa izraženim i različitim antropogenim uticajima. Sama Jablanica nastaje spajanjem većeg broja potoka sa obronaka planina Jablanik i Medvednik. U skladu sa zahtevima EU prilikom ocene stanja rečnih sistema neophodno je utvrđivanje referentnih uslova. Da bi se utvrdilo recentno stanje, i usaglašenost sa zahtevima za dostizanje referentnog statusa, izvršeno je ispitivanje ove reke u aprilu 2013. godine na tri lokaliteta gornjeg toka. Kao parametri za ocenu kvaliteta vode, korišćeni su sledeći pokazatelji: ukupan broj taksona, saprobni indeks (SI; Zelinka & Marvan), BMWP i ASPT skorovi, broj taksona grupa Ephemeroptera, Plecoptera and Trichoptera (EPT) i indeks diverziteta (SWI; Shannon-Wiener’s indeks). Zabeležena su ukupno 72 taksona makroinvertebrata. Kao najraznovrsnije grupe izdvajaju se Trichoptera (16 taksona) i Ephemeroptera (15 taksona). U odnosu na saprobnu vrednost, utvrđeno je da je većina prisutnih taksona osetljiva na organsko zagađenje (ksenosaprobni i oligosaprobni organizmi). Dobijene vrednosti praktično svih korišćenih pokazatelja kvaliteta vode, upućuju na veoma dobar status (klasa I), čime su ispunjeni uslovi da se ispitivani deo toka Jablanice može smatrati referentnim stanjem, za potrebe istraživanja sliva Kolubare. Puštanje u rad akumulacije u Rovnima (srednji tok Jablanice) može izmeniti zatečeno stanje, zbog čega je neophodan redovni monitoring

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

Procena genotoksičnog potencijala voda u Srbiji komet testom

Sistemi površinskih voda često su pod pritiskom otpadnih voda iz industrijskih, domaćih i poljoprivrednih izvora. Različiti sastojci prisutni u zagađenim vodama mogu da izazovu biološke promene, koje mogu uticati na populacije i čitave ekosisteme. Mnogi akvatični organizmi, među kojima bitno mesto zauzimaju ribe, imaju sposobnost bioakumulacije genotoksičnih agenasa u tkivima, te se mogu koristiti kao bioindikatori u ekogenotoksikološkim studijama.1,2 Procena genotoksičnosti površinskih voda vršena je na osnovu detekcije oštećenja DNK molekula alkalnim komet testom, testom elektroforeze pojedinačnih ćelija, (engl. SCGE), u krvi, jetri i škrgama klena (Squalius cephalus L.). Ekološki status površinskih voda određivao se na dve akumulacije, Garaši (vodozahvat za pijaću vodu) i latar (Uvac, zaštićeno prirodno dobro), kao i četiri reke u okviru sliva reke Kolubare (područje intenzivnih kopova uglja): Kolubara, Peštan, Beljanica i Bubanj Potok, tokom 2011./2012. godine. Na osnovu analize fizičko-hemijskih i mikrobioloških parametara, izdvojile su se akumulacije, Zlatar i Garaši, u I klasu kvaliteta voda. Sa druge strane, analize vode sa Peštana i Beljanice odgovarale su III i IV klasi kvaliteta voda. Više vrednosti oštećenja DNK molekula su zabeležena na rekama u okviru sliva Kolubare, dok su oštećenja na tkivima klena sa Garaša i latara bile značajno niže. 1. Sunjog, K., Kolarević, S., Kračun-Kolarević, M., et al., 2014, Environ. Toxicol. Pharmacol. 37:967-974. 2. Sunjog, K., Kolarević, S., Kračun-Kolarević, M., et al., 2016, Environ. Pollut. 213:600-607. Zahvalnica: Ovaj rad je finansiran od strane Ministarstva prosvete, nauke i tehnološkog razvoja Republike Srbije, projekat OI173045

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

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

Ocena ekološkog potencijala lokaliteta Duboko (Sava) i Višnjica (Dunav) na osnovu zajednice akvatičnih Oligochaeta

Na Savi (Duboko) i Dunavu (Višnjica) vršena je procena ekološkog potencijala na osnovu zajednice akvatičnih oligoheta. Na Savi istraživanje je rađeno tokom 2014. i 2015. godine, a na Dunavu tokom 2014. godine. Na lokalitetu Duboko veća raznovrsnost oligohetne faune bila je zabeležena 2015. godine (11 vrsta), u odnosu na 2014. godine (9 vrsta), a na lokalitetu Višnjica zabeleženo je prisustvo 17 vrsta oligoheta. Na oba lokaliteta familija Tubificidae je bila dominantno zastupljena (>90 %) sa vrstama Limnodrilus hoffmeisteri i L. claparedeanus. Visoka zastupljenost familije Tubificidae u uzorcima ukazuje na veliko organsko zagađenje koje je prisutno na ovim lokalitetima

Freshwater mussels in the ecogenotoxicological studies-application of comet assay

Presence of pollutants in aquatic ecosystems can be detected by a range of physiological, histological and molecular responses, including abnormal morphology, alterations of antioxidative status and DNA integrity. The integrity of cellular DNA is continuously attacked by various agents resulting in DNA lesions. Unrepaired DNA lesions may block replication and transcription, potentially leading to cell death or generating mutations. The main objectives of ecogenotoxicology studies are detection of the origin of genotoxic pollution and their effect on individuals and populations. Mussels are commonly employed in the ecogenotoxicological studies. They have several characteristics, such as wide distribution, filter feeding, a sessile life form and an ability to accumulate pollutants, which makes them favorable organisms for estimating the environmental pollution level and the bioavailability of various types of pollutants. Different approaches are used for assessing the conditions of ecosystems, the passive (collection of mussels from selected locations) and the active biomonitoring (subsequent exposure of mussels from unstressed populations at polluted sites). Moreover, in ex situ assessment, the genotoxic effect of different compounds can be detected in laboratory conditions. 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 (damaged nuclei form comet-like shapes). It has been accepted as one of the major tools for assessing pollution related genotoxicity in aquatic organisms. In our experiments we performed alkaline version of comet assay described by Singh et al. (1988). Images of 250 nuclei per each concentration of test substance were analyzed with a fluorescence microscope and scored using analysis software (Comet Assay IV Image analysis system, PI, UK). Tail intensity and Olive tail moment are chosen as relevant measure of DNA damage. Active biomonitoring implies translocation of specimens from unstressed populations to selected sites of interest. In active approach issues related to availability of specimens and effects of age, gender and physiological condition are avoided. This approach enables research on highly polluted sites. Specimens of Unio sp. were exposed at selected sites at the Sava and Danube rivers which are under the impact of untreated wastewaters of Belgrade city. Haemolymph was sampled after 7, 14 and 30 days of exposure. Passive biomonitoring implies collection of specimens which inhabit selected sites of interest. Easily performed, suitable when research area is situated far from the laboratory. Study was performed on the Velika Morava River, significant Danubes tributary. Specimens of Sinanodonta woodiana collected monthly on two sites with different pollution pressure. Significant correlation was detected between the level of DNA damage in haemocytes and concentration of haevy metals in water. Seasonal variation in the level of DNA damage was observed. Studies performed ex situ are significant research tool for prediction of the effect of single compounds in environment. Obtained data are useful for risk assessment. Among emerging pollutants, cytostatics represent hot topic as serious threat to non-target organisms due to their cytotoxicity, genotoxicity and mutagenicity. As part of research for Cytotreat project (EU Commision, FP7) we are investigating impact of short term exposure to mostly used drugs: 5-Fluorouracil, Cisplatin, Etoposide Vincristine and Imatinib mesylate with Cadmium chloride as positive control. Data obtained so far indicate that cytistatics can induce increase in DNA damage in concentrations similar to ones measured in wastewater. Our studies indicate that freshwater mussels can be used as reliable bioindicators. Research performed in situ showed that selected species can detect genotoxic pollution present in environment. Results of the research performed ex situ showed harmful effects of environmentally relevant concentrations of cytostatics. Studies performed ex situ on mussels coul be significant research tool for prediction of the effect of single compounds in environment

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