Application of aquatic oligochaetes (Tubificidae) in ecogenotoxicological in situ and ex situ studies

Familija Tubificidae (Oligochaeta), po brojnosti jedinki i po biomasi, je jedna od najznačajnijih grupa u zajednicama akvatičnih makroinvertebrata. Ovi organizmi su prepoznati kao pouzdani bioindikatorski organizmi koji se koriste u proceni ekološkog potencijala akvatičnih ekosistema. U ovoj studiji akvatične oligohete su korišćene kao eksperimentalni organizmi u ekogenotoksikološkim istraživanjima. Uticaj različitih stresora in situ, kao i odabranih grupa zagađivača ex situ na integritet DNK molekula celomocita i hemocita, praćen je komet testom, a za procenu nivoa oštećenja korišćen je parametar TI (eng. Tail intensity). In situ istraživanje rađeno je tokom 2014. i 2015. godine na lokalitetu Duboko (Sava), i tokom 2014. godine na lokalitetu Višnjica (Dunav) na vrsti Branchiura sowerbyi, i mešovitom uzorku tubificidnih vrsta (Mix uzorak). Rezultati ukazuju na osetljivost celomocita i hemocita u detekciji sredinskog stresa. Nivo oštećenja DNK molekula kod različitih vrsta imao je sličan trend pri odgovoru na sredinske uslove i pokazao je značajne korelacije sa praćenim mikrobiološkim i fizičko-hemijskim parametrima kvaliteta vode. Vodostaj je izdvojen kao bitan stresor koji je tokom istraživanja znatno uticao na ostale praćene parametre. Brojnost jedinki i divezitet populacija akvatičnih oligoheta se nije menjala sa promenom stresora (poplave, ispuštanje otpadnih voda, spiranje zemljišta), s druge strane, uticaj stresora bio je evidentan na oštećenje DNK molekula. U ex situ istraživanu praćen je uticaj citostatika 5–fluorouracila i etopozida, kao i model mutagena kadmijum hlorida, na nivo oštećenja DNK molekula vrste Limnodrilus udekemianus. Sve supstance su značajno povećale nivo oštećenja DNK molekula.According to the number of individuals and their biomass, family Tubificidae (Oligochaeta) is one of the most important groups in the communities of aquatic macroinvertebrates. As bioindicators these organisms are used in the assessment of the ecological potential of aquatic ecosystems. In this study aquatic oligochaetes are used as experimental organisms in ecogenotoxicological assessment. For in situ studies impact of different stressors on the integrity of the DNA molecule of coelomocytes and haemocytes was evaluated, while the impact of a selected group of pollutants was studied ex situ. The comet assay was used for the evaluation of the genotoxic potential and parameter TI (Tail intensity) for the assessment of the level of DNA damage. In situ research was conducted on the species Branchiura sowerby and in the mixed sampled of tubificid species from the sampling sites Duboko (Sava) and Višnjica (Danube). Coelomocytes and haemocytes have shown to be sensitive to environmental stress. The level of DNA damage in different species showed a similar trend in response to environmental conditions and significantly correlated with microbial, physical and chemical water quality parameters. Water level was recognized as an important stressor that had significant impact on the other environmental parameters. Different stressors (floods, wastewater discharge, and runoffs) did not affect the number of individuals and the diversity of aquatic oligochaetes populations. On the other hand, the impact of stressors was evident on the level of DNA damage. The effects of cytostatic, 5-fluorouracil and etoposide and of model mutagen CdCl2, on the level of DNA damage of Limnodrilus udekemianus was examined ex situ. All tested substances significantly increased the level of DNA damage

Uticaj otpadnih voda na reku Dunav – ekogenotoksikološki aspekt ekspedicije JDS3

Narušavanje kvaliteta površinskih vodotokova otpadnim vodama se odražava na kvalitet akvatičnih ekosistema ali i na kvalitet života i zdravlja ljudi. Variranje u stepenu prerade otpadnih voda najčešće je posledica razlika u nacionalnim zakonskim regulativama što je veoma evidentno u slučaju reke Dunav, u nekim od podunavskih zemalja voda se prerađuje dok se u drugim nikakav tretman ne primenjuje pre ispuštanja. JDS3 (The Joint Danube Survey 3) je bila najveća rečna ekspedicija u 2013. godini koja je obuhvatila svih X sektora reke. Zbog obima uzoraka i različitih analiza, JDS je predstavljao idealnu priliku da se ispita prisustvo genotoksičnog zagađenja u ovoj reci. Komet test je rađen na hemocitama školjki Unio sp. i Sinanodanta woodiana. Najviši nivo genotoksičnog potencijala detektovan je u sektoru VI (Panonska ravan) gde veliki problem predstavljaju otpadne vode koje se bez prerade ispuštaju u Dunav. Ova studija je dala mapu zagađenja i kritičnih tačaka zagađenja duž reke Dunav i ukazala na bitnost prerade otpadnih voda za očuvanje i poboljšanje kvaliteta reke Dunav. Uvođenje molekularnih markera u monitoring kvaliteta voda znatno doprinosi razumevanju prirode i porekla zagađenja

Mapping of the microbiological water quality of surface waters in Serbia overlooked by the National monitoring programme.

Surface waters in Serbia are under high anthropogenic pressure. One of the major problems is untreated municipal and industrial wastewaters. Unfortunately, Serbia processes only 5 % of wastewaters before discharging. As a consequence, pollutants such as metals and metalloids from industrial wastewaters, pharmaceuticals, compounds from personal care products, etc. directly endure into surface water. Microbial faecal pollution, as an indicator of presence of human or/and animal pathogens, due to health hazard limits water usage for drinking, recreation, irrigation, etc. In regulations, faecal coliforms, with Escherichia coli as dominant representative, are widely used as faecal indicator bacteria. The aim of this study was to investigate the microbiological al water quality in the Republic of Serbia at the sites which are not routinely investigated within the national monitoring program. In this purpose 78 sites situated on canals, mountain springs, as well as lowland rivers were selected. Defined Substrate Technology was used for determination Most Probable Number (MPN) of E. coli using Colilert-18 System. The water classification system, developed for the Danube River (Kavka et al., 2006) was used. The results indicated that more than 47 % of selected sites are under critical (21.79 %) or strong (25.64 %) faecal pollution. On the other hand, about 32 % (32.05 %) of sites are little polluted and 20.51 % of sites showed moderate pollution. The increasing levels of faecal pollution detected on sites situated downstream of settlements indicated discharge of untreated municipal wastewaters directly into surface water. In further research, the focus will be placed on the determination of the origin of pollution by the employment of microbial source tracking technique. Obtained data will be used for modelling and predicting the effect of detected contamination on the water quality of the major water bodies in the Republic of Serbi

Ispitivanje osetljivosti testova i pristupa u ekogenotoksikološkim istraživanjima na velikim ravničarskim rekama–studije slučaja Velika Morava i Sava

Velika, Zapadna, Južna Morava, kao i Sava, Kolubara i Barička reka su pod značajnim uticajem komunalnih i industrijskih otpadnih voda. Ciljevi studije su procena genotoksičnog potencijala analiziranih lokaliteta i ispitivanje osetljivosti primenjenih biotestova i pristupa istraživanja. Studija slučaja Velika Morava obuhvatila je veliko ispitivano područje na osam lokaliteta na Velikoj, Zapadnoj i Južnoj Moravi, a u studiji Sava ispitivano je tri prostorno bliska lokaliteta koji su pod različitim pritiscima zagađenja. U in silico pristupu na osnovu literaturnih podataka analiziran je toksični/genotoksični potencijal jedinjenja detektovanih u vodi. Ex situ/in vitro pristup obuhvatio je ispitivanje genotoksičnog potencijala pomoću SOS/umuC testa, kao i primenom alkalnog komet testa na HepG2 ćelijskoj liniji. U in situ/in vivo istraživanju analizirano je DNK oštećenje ćelija krvi vrste Alburnus alburnus (uklija) pomoću alkalnog, Fpg-modifikovanog komet testa i mikronukleus testa. Rezultati su pokazali različitu osetljivost zavisno od upotrebljenih testova i pristupa, ali i različit odgovor kod uklija u zavisnosti od tipa zagađenja na lokalitetima. In silico i ex situ/in vitro pristupi su pokazali niži stepen osetljivosti u odnosu na in situ/in vivo testove, a alkalni komet test je pokazao najveći potencijal u diskriminaciji lokaliteta. Utvrđeno je da uklija može biti pouzdan bioindikator u ekogenotoksikološkim istraživanjima velikih ravničarskih reka

Seasonal variations of microbiological parameters of water quality of the Velika Morava river Serbia

In this study, we investigated the level of sanitary pollution and organic contamination of the Velika Morava River, the largest river in central Serbia. Samples of water for microbiological analysis were collected at 5 sites, monthly, from April 2010 to February 2011. Sanitary analysis, i.e. enumeration of total and fecal coliforms and intestinal enterococci, indicated moderate to critical fecal contamination, while organic load assessment (oligotroph to heterotroph ratio, index of phosphatase activity) revealed the category of moderately polluted water. We also investigated seasonal variations of these groups of bacteria and the factors that could contribute to these differences. Our results showed that the micro- biological quality of the water in the Velika Morava River during different seasons is affected by numerous factors such as unequal loading of wastewaters, solar irradiation, and relations of flow/dilution and rainfall/runoff. [Acknowledgments. This study was financially supported by the Ministry of Education and Science of the Republic of Serbia: projects No 173025, No 177045 and No 043002.

Cytostatics as emerging pollutants – is there a threat for aquatic invertebrates?

Seeking for a better quality of life, consumption of pharmaceuticals is constantly increasing. Pharmaceuticals are designed to be more potent, degradation resistant, to affect protein targets at relatively low doses. Many of them are not selective and became threat to non-target organisms, especially to one living in aquatic ecosystems. Because of the inability of wastewater treatment plants to eliminate these compounds in the end they reach aquatic environments through sewage system. Aquatic invertebrates spend at least a part of their life cycle in the aquatic environment. Mobility is not one of the traits that characterize many species of aquatic invertebrates, especially freshwater mussels and aquatic worms which are almost sedentary organisms. Due to their way of life, this species are under the influence of variety of pollutants via sediment and via water column. Genotoxic effects of cytostatics with different mode of action – alkylation agent (cisplatin - CP), antimetabolite agent (5-fluorouracil – 5-FU), plant alkaloids (etoposide - ETO, vincristine sulphate - VIN) and other neoplastic agent (imatinib mesylate - IM) were studied in vivo and in vitro on haemocytes of two freshwater mussels species Unio sp. (U. pictorum/U. tumidus), and in vivo on haemocytes and coelomocytes of tubificid species Limnodrilus udekemianus. Experiments were organized as short-term treatments (72h for mussels/96h for worms) in static system. Level of DNA damage was evaluated by alkaline single-cell gel electrophoresis (comet assay). Based on our results ranking of cytostatics by their effects on mussels was VIN>5-FU>ETO>CP>IM and on worms was 5-FU>ET. Worms have shown higher sensitivity for the negative effects of 5-FU and ET on the integrity of DNA molecule comparing with mussels. The lowest observed effect concentration (LOEC) for 5-FU was 52 μg/L for mussels and 0.52μg/L for worms. In the case of ET LOEC was 24 mg/L for mussels and 0.024 mg/L for worms. For VIN was detected difference in the response in U. pictorum and U. tumidus – LOEC for U. pictorum was 3.7 μg/L, while for U. tumidus was 36.9 μg/L. Significant damage of DNA wasn’t detected for CP and IM. Although the PEC values for tested cytostatics are lower than the ones used in our study, it must be emphasized that in the environment, organisms are under constant influence of these pollutants and organisms are struggling with the effects of mixture of pharmaceuticals and mixture of different pollutants. Impacts of these mixtures on the aquatic organisms are still unknown, and therefore, further research should consider this fact and the studies should be organized in this direction. Acknowledgements: EU Seventh Framework Programme – Cytothreat (no. 265264). Ministry of Education, Science and Technological Development of the Republic of Serbia project no. 037009

Determination of the level and source of microbial pollution in the Sava River Basin

Introduction. The contamination of water bodies by sewage or manure is generally determined by fecal indicator bacteria. Specific differentiation between sources of contamination is of particular importance, since the health risk to humans is usually considered higher from human, than from animal fecal contamination. Quantitative PCR (qPCR)-based assays for analysis of human- or animal-associated genetic Bacteroidetes fecal markers have gained increased popularity in the field of microbial source tracking (MST). Aim. The aim of the present study was to assess the level and source of microbial pollution in the Sava River Basin (SRB). Materials and methods. The sampling at the SRB was performed in September 2015, at 15 locations. Additional samples were collected from 4 wastewater outlets. Quantification of total coliforms, E. coli and enterococci was performed with Colilert Quanti-Tray 2000 and MPN approach. The human-associated BacHum and HF183II, the ruminant-associated BacR and the pig-associated Pig2Bac fecal markers were enumerated by quantitative PCR (qPCR). Results. According to the concentrations of fecal indicator bacteria, the river samples were scattered within the classes I and II (slight to moderate pollution). According to all monitored indicators, samples collected at 3 wastewater outlets were excessively contaminated. The results of MST revealed the presence of human-associated fecal markers BacHum and HF183II at 13 locations. The ruminant-associated BacR and the pig-associated Pig-2-Bac markers were not detected. High correlation was observed between the standard fecal indicators and human associated fecal markers. Conclusions. The results reveal human origin of fecal pollution in the SRB and indicates the urgent need for effective wastewater treatment plants in water management

Effects of 5-Fluorouracil, Etoposide and CdCl2 in Aquatic Oligochaeta Limnodrilus udekemianus Claparede (Tubificidae) Measured by Comet Assay

Genotoxicity of 5-fluorouracil (5-FU), etoposide (ET) and cadmium chloride (CdCl2) was evaluated in Limnodrilus udekemianus, cosmopolitan tubificid species, by alkaline single-cell gel electrophoresis (comet assay). Groups of 50 individuals were exposed in vivo in water-only short-term (96 h) tests to 5-FU (0.004, 0.04, 0.4, 4 and 40 mu M), ET (0.004. 0.04, 0.4 and 4 mu M) and CdCl2 (0.004, 0.04, 0.4, 4 and 40 mu M). Mortality of worms was observed only for CdCl2 (4 and 40 mu M). Cell viability lower than 70 \% was detected for 5-FU (0.4, 4 and 40 mu M), ET (4 mu M) and CdCl2 (0.4 and 4 mu M). All tested substances induced significant increase of DNA damage except 0.004 mu M of ET. L. udekemianus being sensitive to all tested substances indicates that it can be used in ecogenotoxicology studies. Concern should be raised to cytostatics, especially to 5-FU, since concentration of 0.004 mu M induced DNA damage is similar to ones detected in wastewaters.Water Air and Soil Pollution (2015), 226(242

Microbial quality and pollution origin of montenegrin surface waters, the first comprehensive study

Background: Montenegro is characterized by numerous mountain springs and larger streams. Many of them are used as sources of drinking water, irrigation, but also for tourists’ recreation: kayaking, rafting, etc. Unfortunately, there is a lack of wastewater treatment plants as well as information about microbial water quality of many surface and ground waters. Objectives: Taking into consideration issue of scarce data about microbial water quality, the aim of this study was to locate hotspots of faecal pollution and investigate the source of pollution. Within the study, 25 sites were selected while three of them were not included in National monitoring program. Methods: To quantify the most probable number of Escherichia coli we used Colilert-18 System. The water quality based on this parameter was assessed according to Kirshner et al. (2009). For determination of the pollution origin, microbial source tracking method was employed. Indicators of human faecal pollution, markers BacHum and HF183II, and animal faecal pollution, markers BacR and Pig2Bac, were quantified with qPCR. Results: According to most probable number of E. coli, 40% sites were little polluted, 36% moderately, 16% critically and per 4% heavily and excessively polluted. Analysis of genetic faecal markers indicated the prevalence of human-as- sociated faecal pollution at all sites. Considering the results of the study, four sites were classified as hotspots of faecal pollution: downstream Pljevlja (Ćehotina River), downstream Vezišnica (Ćehotina River), wastewater outlet at Podgori- ca (Morača River), and downstream Bijelo Polje (Lim River)