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

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

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

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

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

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

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

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

Significance of genotoxicity and toxicity evaluation of freshwater bodies

Fish communities are excellent indicators of biological and ecological integrity due to their continuous exposure to water conditions. Freshwater biodiversity constitute a valu able natural resource but extensive pollution of freshwater can result in biodiversity de cline. For that reason it is important to employ bioassays for purpose of detecting these conditions. In this work we monitored chub and bream species in 2 reservoirs, Uvac and Garasi, and 4 rivers: Dunav, Sava, Pestan and Beljanica. For assessment of metal and metalloid in fish tissues (liver, gills, gonads and muscle) we have used ICP-OES. The comet assay or single cell gel electrophoresis (SCGE) was selected as an in vivo geno toxicity assay, for measuring DNA damage in blood, liver and gills. Histopathological alterations were monitored in liver and gills.The high quality of Uvac water was con firmed by low values of DNA damage in all tissues compared to other sites. An analysis metals in tissues showed a high degree of their differentiation, as well as significant dif ferences in the distribution of tissue elements between the sites tested. The highest con centrations of most of the analyzed metals were found in gills, liver and gonads, and the lowest in muscles at all sites. In chub, blood showed the lowest DNA damage compared to liver and gills, while in all breams DNA damage was the highest in blood cells, follow ing gills and liver. Histopathological analysis performed on bream specimens on the Sava and Danube River revealed a higher degree of alterations in liver compared to the gills