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The appearance of COVID disease and the introduction of subsequent measures and specific medical treatments throughout the world in 2020 could have been expected to cause the drug content changes in the surface waters. We have studied the lower course of the Croatian Mrežnica River at three areas (rural, small town, and large town domains) in three periods (May 2020, April and September 2021). The applied method was liquid chromatography with tandem mass spectrometry, which enabled the screening of 256 compounds with 7-9% (19-24 drugs) being detected, depending on the sampling period. In the beginning of the pandemic, total quantity of drugs was much lower in the rural and small town domain (142-187 ng L- 1) compared to the large town (954 ng L-1), whereas by the autumn 2021 marked increase of drug levels in rural and small town areas was observed (443- 481 ng L-1). Increase was especially notable for analgesics: non-opioid (aminophenazon, acetaminophen, ibuprofen) and opioid (tramadol, oxycodone). In spring of 2020, nonopioids were detectable only in large town domain (94.3 ng L- 1), and their concentrations steadily increased throughout the pandemic (up to 172 ng L-1). In the vicinity of small town, sharp increase of nonopioids occurred already in spring 2021 (235 ng L-1) and in rural area in autumn 2021 (125 ng L- 1). The opioids appeared in the river water at all studied sites in the last sampling period (46-90 ng L-1). Contrary to analgesics, decrease in quantity was recorded for the stimmulants, i.e. coffeine and cotinine (nicotine metabolite), indicating less active socializing due to the COVID epidemiological measures.

Wastewater treatment plants are considered one of the main sources of organic micropollutants. Micropollutant continuous and unregulated release into the environment constitutes a threat to the water environment and human health. In this context, upgrading wastewater treatment seems a suitable option. In this paper, we study how to increase the removal efficiencies for 20 micropollutants by the addition of powdered activated carbon (PAC) to a full-scale membrane bioreactor mainly treating hospital wastewater. In addition, we study if PAC addition improves the overall quality of the final effluent and sludge flocs properties. A special focus is done on the influence of dissolved organic matter in the adsorption of the selected micropollutants on PAC.

To better understand the impact of conventional agricultural practices on soil and water resources, two main objectives were established in this study: to measure nitrate (NO3-) and sulphate (SO4 2-) concentrations in lysimeters and drainpipes leachates in response to different nitrogen fertilization levels and soil amendments and to identify organic contaminants that could have originated from the long-term use of agrochemicals in historically intensive agricultural area in Croatia. During the two-year study period leachate samples were collected from lysimeters and drainpipes. Research results indicate NO3- content in both lysimeters and drainpipes increased with rising doses of N fertilizer. The highest concentration of SO4 2- was recorded in the treatment with added phosphogypsum, while in all other treatments the concentrations were low. Over 40% of the 287 target substances were detected at least once during the two-year study period but only two substances (IPC/propham and carbosulfan) were found to have concentrations above 0.5 µg/L. Additionally, three priority substances were continuously present in the leachate samples: atrazine, simazine and isoproturon., Da bi se bolje razumio utjecaj konvencionalne poljoprivredne prakse na tlo i vodne resurse, u ovom radu uspostavljena su dva glavna cilja: mjerenje koncentracija nitrata (NO3-) i sulfata (SO4 2-) u lizimetarskim i drenskim procjednim vodama kao odgovor na gnojidbu različitim razinama dušika te poboljšivača tla te identificirati organske onečišćivače čije je moguće izvorište u dugotrajnoj uporabi pesticida na povijesno intenzivno poljoprivrednom području u Hrvatskoj. Tijekom dvogodišnjeg razdoblja uzorci procjednih voda prikupljani su iz lizimetara i drenskih cijevi. Rezultati istraživanja pokazuju da se sadržaj NO3- u lizimetrima i u drenskim cijevima povećavao s porastom doza N gnojiva. Najveća koncentracija SO4 2- zabilježena je u tretmanu s dodanim fosfogipsom, dok su u svim ostalim tretmanima koncentracije bile niske. Preko 40% od 287 ciljnih tvari otkriveno je barem jednom tijekom dvogodišnjeg razdoblja ispitivanja, ali utvrđeno je da su samo dvije tvari (IPC/propham i carbosulfan) imale koncentracije iznad 0,5 µg/L. Uz to, tri prioritetne tvari bile su kontinuirano prisutne u uzorcima procjednih voda: atrazin, simazin i izoproturon.

The lower course of the Mrežnica River has long been exposed to different types of industrial loads. In Duga Resa, the cotton industry was active for more than a century, and a few kilometres downstream, industrial wastewaters of Karlovac entered the river until approximately a decade ago. Our aim was to determine the main inorganic and organic contaminants of river-water and sediments at two differently polluted sites compared to upstream reference site. Turbidity, chemical oxygen demand, and total nitrogen were somewhat increased in Karlovac, whereas total phosphorus was increased at the reference site. Although dissolved metal concentrations in the water were rather low, indicating a pristine river, an increasing trend towards Karlovac existed for several elements (e.g., Fe, Mn), where the highest metal concentrations in finer sediment fraction (

Quantitative chemical analyses of 428 organic contaminants (OCs) indicated the presence of 313 OCs in the sediment extracts from Sava River, Croatia. Pharmaceuticals were present in higher concentrations than pesticides thus confirming their increasing threat to freshwater ecosystems. Toxicity evaluation of the sediment extracts from four locations (Jesenice, Rugvica, Galdovo and Lukavec) using zebrafish embryotoxicity test (ZET) accompanied with semi-quantitative histopathological analyses exhibited correlation with cumulative number and concentrations of OCs at the investigated sites (10.05, 15.22, 1.25, and 9.13 μg/g respectively). Toxicity of sediment extracts and sediment was predicted using toxic unit (TU) approach and persistence, bioaccumulation and toxicity (PBT) ranking. Additionally, influential OCs and genes were identified by graph mining of the prior knowledge informed, site-specific chemical-gene interaction models. Predicted toxicity of sediment extracts (TUext) was similar to the results obtained by ZET and associated histopathology with Rugvica sediment being the most toxic, followed by Jesenice, Lukavec and Galdovo. Sediment TU (TUsed) favoured OCs with low octanolwater partition coefficients like herbicide glyphosate and antibiotics ciprofloxacin and sulfamethazine thus indicating locations containing higher concentrations of these OCs (Galdovo and Rugvica) as the most toxic. Results suggest that comprehensive in silico sediment toxicity predictions advocate providing equal attention to organic contaminants with either very low or very high log Kow.

Field sampling of water and sediments from Sava river in Zagreb, in a total length of 36, 73 km was collected in March 2019, with the goal to determine the geochemical characteristic of sediment in the vicinity of discharge at main outlet of a Zagreb central wastewater treatment plant. Samples were collected under the Jankomir bridge (a), Bridge of Youth (b), a zone of outlet (c) and in Rugvica (d) (Fig. 1). In all water sample pH and electrical conductivity (EC) were measured. Results are similar for 3 of 4 locations (pH around 8, and EC around 430 μs). The sample from location c shows an increase in electrical conductivity (844 μs) and decrease of pH value (7, 65). All sediment samples were dry to constant weight and divided into the two sub-samples. The first one was used for bulk analysis, while the second one was sieved and fraction < 0, 063 mm was collected for further analysis. The qualitative mineral phase omposition was performed, to determine the mineral phases by the Philips X’pert powder diffractometer with CuKα radiation from the tube operating at 40 kV and 45 mA. An X-ray diffraction data set was collected from 4° to 63° 2Ɵ. All analysed bulk samples have a similar composition, with the quartz as the main phase, calcite, dolomite, muscovite and kaolinite as phases presented in smaller amount. In the fraction < 0, 063 mm quartz was also determined as main mineral phase and all phases from bulk are present with adition phases, albite and magnesium calcite. The amount of carbonate component was also determined by the Austrian standard methods ÖNORM L1084 using Scheibler calcimeter. Amount of carbonate component decrease downstream, but all bulk samples have a high amount of a CaCO3 components, from 36% to 29, 5%. Qualitative and quantitative chemical analysis of metals in sediment and water as well as sequential extraction chemical analysis by BCR protocol was performed and results will be presented at the poster, due to the additional data processing which must be performed.

Taxonomical and functional composition of phytoplankton show clear seasonal patterns in the different lotic and lentic habitats. Compositional changes in phytoplanktic communities, with physical and chemical parameters of water were studied from April to September in2017 in two lentic habitats on Krka River, Visovac and Brljan lakes, for deepening the knowledge about the functioning and structural characteristics of these specific habitats. During sampling, temperature and oxygen were measured in depth profiles providing stratification curve and the position of deep chlorophyll a (Chla) maximum. Stratification was detected in the summer months in Lake Visovac, while there was no stratification in Lake Brljan. Deep Chl a maximum was not detected in these lakes, but clear anoxia was present in summer in Lake Visovac. Temperature and oxygen profiles indicated that Lake Brljan is a riverine system, while Lake Visovac is lake with much longer retention time. Taxonomic composition and Functional groups (FGs) clearly reflected to hydrological characteristics of the habitat. Centric diatoms belonging to FGs D and P as typical potamoplankton dominated in riverine Lake Brljan. Barrage Lake Visovac was characterized by the co-dominance and seasonal changes of centric diatoms, chrysophytes and dinoflagellates. Seasonal change in FGs composition (C-X2, U-L0) indicated the mesotrophic status of the stratified lake what was also confirmed by the results of physical and chemical parameters.

Toxic unit(TU)approach is currently currently prevailing in risk assessment of freshwater pollution (Ginebreda et al., 2014 ).It is defined as the the ratio between measured concentration of organic contaminants contaminants (OCs) in the water wand toxicological endpoints endpoints for aquatic organisms gathered from databases or by in - silico tools tools. To accentuate the importance of assessing both uptake paths in process of pollutant prioritization present in the the aquatic environment we propose an extension of the TU approach, by adding a factor containing the octanol-water partition coefficient as an estimator for lipid-water partitioning, sediment concentration and toxicity. It results in an estimative prioritization index, combined toxic unit (CTU). More effort must be invested in understanding diverse uptake paths of OCs into aquatic organisms. The CTU is an estimative index emphasizing both highly hydrophilic and lipophilic OCs no matter the uptake path.

Recent environmental monitoring approaches aim to identify chemicals of concern based on the prediction of biological targets they are likely to impact. One rapid screening approach for prioritization of environmental chemistry datasets is to estimate relative risk of chemicals based on the exposure-activity ratios (EARs). EARs integrate chemical occurence and potency data using high-throughput screening (HTS) data from the Toxicity Forecaster (ToxCast) database. The aim of this study was to: i) prioritize the sites along the middle course of the large transboundary river in the South Eastern Europe (Sava River) using EARs, and ii) highlight chemicals likely contributing to the observed biological activity by combining EARs and empirical in-vitro tests of estrogenic (ER), androgenic (AR) and glucocorticoid (GR) activity. Water samples were collected monthly in winter 2018 at 13 sites along the whole Croatian section of the Sava River. A total of 34 water samples were analysed using liquid chromatography/time-of- flight mass spectrometry (UHPLC-QTOF-MS). Out of 549 analysed organic contaminants, 184 were detected in Sava samples. To prioritize sites we calculated the cumulative EAR for each site (EARcumulative - sum of all individual EARs for each chemical - molecular target combination), as well as total EAR for each chemical (sum of EARs for all molecular targets ; EARtotal), and the EAR for each chemical-molecular target pair (EARtarget). The five sites with the highest EARcumulative were either in close proximity to major cities (two sites) or downstream of large tributaries and/or in the areas with intensive agriculture (three sites). The highest EARtarget values were observed for the nuclear receptors (androgen, estrogen and glucocorticoid in particular). Consequently, chemicals that exert estrogenic, androgenic and/or glucocorticoid activity were identified as chemicals of concern (e.g., 17-beta estradiol, mometasone fuorate and flumethasone had the highest EARtotal). EAR predictions were supported by the results of the empirical in vitro testing, two sites with the highest in vitro biological activity were also among the highest ranked sites by EAR. Future work will use mechanistic HTS data and EARs to idenify adverse outcome pathways for aquatic keystone species such as decapod crustaceans.

Intensive agriculture is putting great pressure on the environment and therefore it is vital to rationalize its inputs, and to control and monitor the impact agricultural activities have on soil, water, and air, in order to achieve sustainability. For better understanding of the impact that conventional agricultural practices have not only on soil, but on water resources as well, two main objectives were established. The first one is to measure concentrations of nitrate (NO3-) and sulfate (SO42-) in lysimeter and drainpipe waters related to different nitrogen fertilization levels. The second one is to identify organic pollutants that potentially originate from used agrochemicals. The results of two-year investigating period (2014-2015) are presented in this study. The experimental field was established in Potok near Popovača on distric Stagnosols. The research contained ten treatments with four replications where different nitrogen fertilizer levels were applied: 1. Control (no fertilization), 2. N0 + P + K, 3. N100 + P + K, 4. N150 + P + K, 5. N200 + P + K, 6. N250 + P + K, 7. N250 + P + K + phosphogypsum, 8. N250 + P + K + zeolitic tuff + CaCO3, 9. N300 + P + K, and 10. Black fallow – tillage only. Phosphorous (P) and potassium (K) levels were constant, 120 and 180 kg/ha respectively. Each treatment area included two drainpipes (placed at the depth of 120 cm) and one lysimeter (at the depth of 80 cm) from which water samples were taken when discharge appeared. During the investigated period, 24 water samples for inorganic (13 from lysimeters and 11 from drainpipes) and 16 for organic substances analysis (six in 2014 and two in 2015 from lysimeters ; five in 2014 and three in 2015 from drainpipes) were taken. The concentrations of NO3- and SO42- were determined by suppressed ion chromatography (Dionex ICS-1000 system) using IonPac AS17-C separation column fitted with IonPac AG17 guard column. The gradient flow analysis was conducted using KOH as an eluent solution (10-30-10 mmol). Ultra-high performance liquid chromatography (UHPLC) coupled to time-of-flight mass spectrometry (Q- TOF/MS) was used for non-target screening and quantification of organic substances. This allowed the search of the entire spectrum of various organic compounds and their quantification at high resolution (> 10000 FWHM, full width half maximal), with accurate mass accuracy (< 1 ppm) and satisfactory sensitivity in fullacquisition mode. Research results indicated that NO3- content in both lysimeters and drainpipes increased with rising doses of N fertilizer. In the investigated period the daily concentration varied from 0.52–265 mg/L in both lysimeters and drainpipes, depending on the treatment, precipitation and agrotechnical measures. Higher average concentrations for each treatment were observed in water samples from drainpipes (up to 126 mg/L), which are placed deeper in the soil (120 cm) confirming that nitrates do not bond with soil particles and are leached into groundwater. Daily SO4 2- concentrations varied from 1-302 mg/L. As expected, the maximum average concentrations were recorded in treatment 7 with added phosphogypsum (184 mg/L in lysimeters and 105 mg/L in drainpipes), while the average concentrations on all other treatments were low (< 20 mg/L). Non-target screening revealed more than 400 different organic substances (agrochemicals, hormones, opioids, antibiotics and various other drugs) in water samples. The correlation with applied plant protection products was not observed. The focus of this study was three herbicides: atrazine, simazine, and isoproturon. Atrazine and simazine have been banned in Croatia since 2004, while isoproturon is still used. The average annual concentrations of atrazine, simazine and isoproturon in water from lysimeters in 2014 were 1.40, 0.20 and 0.21 μg/L, respectively. In drainpipes the concentrations were 0.44, 0.20 and 0.04 μg/L, respectively. The average annual concentration of atrazine, simazine and isoproturon in lysimeters in 2015 was 4.69, 8.70 and 0.78 μg/L, respectively. In drainpipes the concentrations were 0.82, 3.82 and 1.66 μg/L, respectively. Due to the fact that atrazine and simazine have not been used in Croatia since 2004 and there have been no records of isoproturon use in the investigated area since 1997, it can be assumed that these compounds persist in the environment for a considerable length of time.

The aquatic environment is exposed to an increasing amount of pollution from various sources. In order to protect the Earths diverse ecosystems, close monitoring and early detection of potentially dangerous substances is crucial. Polycyclic aromatic hydrocarbons (PAHs) present a group of hydrophobic organic pollutants which can have cargenogenic and mutagenic effects on the aquatic organisms. More hydrophobic PAHs usually adsorb and associate with organic particles in the aquatic environment, while less hydrophobic PAHs may be present in the dissolved form, making them more available for uptake by aquatic organisms. We are developing an electrochemical sensor which could be used for early detection of the dissolved fraction of PAHs present in waters. As it has been found, PAHs interact with phospholipid monolayers adsorbed on a mercury electrode surface, causing a disruption of the monolayers’ fluidity and structure. This interaction is monitored electrochemically by fast cyclic voltammetry using a semi-automated flow cell system which incorporates a chip based mercury film microelectrode. A mixed layer of a phospholipid palmitoyl-2-oleoyl-sn-glicero-3- phosphocholine and triolein, an oleic acid triglyceride, in 3:1 molar ratio, adsorbed onto a mercury film microelectrode, was used as a sensing element for PAH detection. The system proved sensitive to the presence of four different PAH molecules, anthracene, phenanthrene, pyrene and fluoranthene, in different matrices, with limits of detection down to 0.2 [micro]g/L [1]. The performance of the system was tested on a natural river sample, and its sensitivity compared to a conventional GC – MS method used for determination of PAHs. Reference: 1. Penezic, A., Gasparovic, B., Stipanicev, D., Nelson, A. In-situ electrochemical method for detecting freely dissolved polycyclic aromatic hydrocarbons in water, Environmental Chemistry 2014, 11(2), 173-180.

Acinetobacter baumannii is an emerging hospital pathogen causing outbreaks in Croatia since 2002 and is still present in Croatian hospitals. Clinical isolates of A. baumannii in Croatian hospitals are usually multi-drug resistant(MDR), with resistance to carbapenems dramatically increasing from 10% in 2008 to 82% in 2014. Although A. baumannii has been isolated from patients and hospital environment during outbreaks, crucial questions regarding its epidemiology remains incompletely understood. The aim of this study was to screen the hospital and municipal wastewater of the City of Zagreb for the presence of viable A. baumannii and carbapenems. Sampling of hospital wastewater was performed on 2 occasions in 2015 at the central manhole of one Zagreb’s hospital from which the clinical isolates of A. baumannii were recovered. Sampling of municipal wastewater was performed on 6 occasions in 2014/15 at the influent and effluent of the central Zagreb’s wastewater treatment plant. Concentration of carbapenems in wastewater was measured by UHPLC Q-TOF MS. The isolation of A. baumannii from wastewater was performed at 42C/48h on CHROMagar Acinetobacter without or with the addition of commercial supplement CR102 which allows the growth of carbapenem- resistant isolates. Presumptive A. baumannii colonies were characterized phenotypically, by using Vitek2 system, and MALDI-TOF MS. Antibiotic resistance profiles were determined by Vitek2 system and interpreted according to EUCAST criteria. Hospital and municipal wastewaters contained high concentrations of imipenem and meropenem which averaged: 894 and 129ng/L in hospital wastewater, 3060 and 380ng/L in raw and 497 and 311ng/L in treated municipal wastewater, respectively. From hospital wastewater, raw and treated municipal wastewater 8, 30 and 7 isolates of A. baumannii were recovered, respectively. All isolates from hospital wastewater and majority (33/37) of isolates from municipal wastewater were resistant to carbapenems and majority of tested antibiotics except colistin. The 7 MDR clinical isolates recovered in the same period showed comparable levels of antibiotic resistance to MDR isolates from hospital and municipal wastewater. These suggest that A. baumannii is able to survive in environment outside hospitals. However, 4 isolates from raw municipal wastewater were susceptible to carbapenems and other antibiotics. This finding opens the possibility that A. baumannii could have natural habitat in sewage system.

Pharmaceuticals are complex molecules with different physicochemical and biological properties and functionalities. Although they are present in the aquatic environment in low (ng/L) concentrations, they are continually being released into the environment what can lead to unwanted effects on the living organisms. Many studies showed that the main points of collection and subsequent release of pharmaceuticals into the environment are wastewater treatment plants, suggesting that their upgrade and implementation of advanced treatment technologies are required. One of the possible solutions for degradation and/or removal of pharmaceuticals from the wastewaters is the use of advanced oxidation processes (AOP) as an additional treatment step. In this study the degradation of albendazole (ALB) was investigated using three UV based processes (with 185/254 and 365 nm radiation sources): (i) UV photolysis, (ii) UV photocatalysis (UV light + TiO2 nano film) and (iii) UV+O3 process. Screening of albendazole and related degradates was performed using liquid chromatography tandem mass spectrometry (HPLC- MS/MS) and ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) with direct injection. The fastest degradation of ALB, for both UV-C and UV-A radiation, was obtained by UV+O3 process with removal efficiency higher than 90%, achieved in 10 minutes. Although the slowest degradation of ALB was obtained using UV-A processes, they have a potential for practical use since they could use natural solar radiation as a source of UV-A radiation and therewith significantly reduce the price of the treatment step.

Increasing amounts of pollutants in the aquatic environment urge the development of simple and low cost methods for early detection of these dangerous substances. Such pollutants include polycyclic aromatic hydrocarbons (PAHs), hydrophobic molecules with potentially cargenogenic and mutagenic effects on the aquatic organisms. PAHs which are more hydrophobic usually adsorb and associate with organic particles in the aquatic environment, while PAHs which are less hydrophobic can be present in the dissolved form, which makes them more available for uptake by aquatic organisms. We are thus developing an electrochemical sensor which would be used for early detection of the dissolved fraction of PAHs present in waters. It has been found that PAHs can interact with phospholipid monolayers on a mercury electrode surface, causing a disruption of the monolayer fluidity and structure. This interaction can be monitored electrochemically by means of rapid cyclic voltammetry using a semi-automated flow cell system which incorporates a chip – based mercury microelectrode[1]. A mixed layer of palmitoyl-2-oleoyl-sn-glicero-3-phosphocholine and triolein, an oleic acid triglyceride, in 3:1 molar ratio, adsorbed onto a mercury microelectrode, was used as a sensing element for PAH detection. The system proved sensitive to the presence of four different PAH molecules, namely anthracene, phenanthrene, pyrene and fluoranthene, in different matrices, with a limit of detection of 0.3 ug L-1[2]. The performance of the system was tested on a natural river sample, in order to compare the sensitivity of the electrochemical sensor with a conventional GC – MS method used for determination of PAHs in natural samples. Further optimisation of the system is being carried out in order to improve sensitivity and enable subsequent use of the system in-situ. References: 1. Z. Coldrick, A. Penezic, B. Gasparovic, P. Steenson, J. Merrifield, A. Nelson, High throughput systems for screening biomembrane interactions on fabricated mercury film electrodes. J. Appl. Electrochem. 2011, 41. 939-949 2. A. Penezić, B. Gašparović, D. Stipaničev, A. Nelson. In-situ electrochemical method for detecting freely dissolved polycyclic aromatic hydrocarbons in water, Environmental Chemistry 2014, 11. 173-180

Water pollution by toxic micropollutants, which is predominantly the consequence of human activities (industry, agriculture and urbanisation) is one of the most critical problems concerning drinking water resources and environmental protection of water bodies. Usage of plant test species has proven essential in investigation, detection and quantification of toxic activity in the natural environment. Toxic effects were investigated in several surface waters (Sava River basin, Croatia) collected monthly over a 3 month-monitoring period. Duckweed (Lemna minor L.) is often used as a plant model because it is a widely spread monocot which multiplies rapidly. Heavy metals were determinated by atomic absorption spectrometry, while nutrients determination was conducted using spectrometry and ionic chromatography. The fitotoxic indicators (relative frond number, relative fresh weight, chlorophyll and carotenoid contents) were monitored after seven days of exposure. All samples of tested waters caused growth inhibition and decrease of chlorophyll and carotenoid contents. The biological effects of water samples appeared related to the physicochemical characteristics. Therefore, bioassays should be included, along with conventional chemical analysis, in water quality monitoring programs. The results also suggest that duckweed should be used in the biomonitoring of water quality because of its simplicity, sensitivity and cost-effectiveness.

Industrijske otpadne vode, kanalizacijske vode kao i ispirne vode s poljoprivrednih površina glavni su izvori onečišćenja vode i tla. Izuzetno važnim u ispitivanju, detekciji i kvantifikaciji fito-, cito- i genotoksičnih djelovanja u okolišu pokazala se primjena biljnih testnih organizama kao biomarkera. Nasađivanjem vodene leće i običnog luka na površinske vode (Sava Županja, Sava Jesenice, Sutla Prišlin, Krapinica Krapina, Toplica nizvodno od Daruvara, Kutinica prije utoka u Ilovu, Glogovnica Mostari, jezero Kozjak) uzorkovane tijekom 3 mjeseca te na otpadne vode ZGOS (uzorkovane kroz 24 sata), Jakuševac (jednokratno uzorkovanje) i Gorjak (uzorkovan tijekom četiri mjeseca) istraživani su fitotoksični i genotoksični učinci voda na dvije modelne biljke vodenoj leći (Lemna minor L.) i običnom luku (Allium cepa L.). Vodena leća se često koristi zbog svoje osjetljivosti na različite toksične tvari te zbog jednostavnog i brzog uzgoja. Običan luk je vrlo pogodan za istraživanje genotoksičnosti zbog malog broja relativno velikih kromosoma (2n = 16). Biljni materijal je uziman nakon tjedan dana (vodena leća) ili 24h (luk) inkubacije. Metodom atomske apsorpcijske spektrometrije u uzorkovanim vodama su određeni esencijalni i neesencijalni teški metali a ionskom kromatografijom i spektrofotometrijom hranjive tvari. Kao pokazatelji fitotoksičnosti praćeni su rast vodene leće, aktivnost gvajakol peroksidaze, sadržaj klorofila i karotenoida te malondialdehida koji je ujedino i pokazatelj opsega peroksidacije lipida. Kao pokazatelji genotoksičnosti praćene su morfološke promjene na korijenu luka koje nastaju kao posljedica djelovanja genotoksičnih tvari, mitotski indeks kao i učestalost kromosomskih aberacija. Komet-testom jezgara vodene leće uzgajanih na testnim vodama tjedan dana detektirana je prisutnost lomova DNA. Svi uzorci voda su izazvali inhibiciju rasta, smanjenje sadržaja klorofila, karotenoida i aktivnosti gvajakol peroksidaze. Pokazatelj opsega peroksidacije lipida bio je značajno povećan kod svih uzoraka otpadnih voda te kod površinskih voda Sutla Prišlin, Krapinica Krapina i jezera Kozjak. Zajednički učinak teških metala i povišenih koncentracija hranjivih tvari uzrokovao je inhibiciju rasta korjenčića i morfološke promjene kao što su savijanje vrškova u obliku kuke, smanjenje mitotskog indeksa i broja stanica u pojedinim fazama staničnog ciklusa. Citogenetička analiza pokazala je najveći broj kromosomskih aberacija u otpadnim vodama te površinskoj vodi Sutla Prišlin, Krapinica i jezero Kozjak. Najčešće kromosomske aberacije prisutne u uzorcima (sljepljivanje, c-mitoza i zaostali kromosomi) su vjerojatno posljedica povećanih količina olova, kroma i žive u uzorcima vode. Upotreba primarnih producenata za ispitivanje površinskih i otpadnih voda u kombinaciji s fizikalno kemijskim analizama može biti dobar indikator stanja okoliša, a rezultati dobiveni u ovom radu na dvjema biljnim vrstama, vodenoj leći i luku, ukazuju na prisutnost fito-, cito- i genotoksičnih tvari u uzorkovanim vodama i indiciraju potencijalan rizik za sve žive organizme.

Due to the enormous number of potentially polluting substances contained in surface water, a chemical-specific approach is insufficient to provide the information about water quality. Therefore it is essential to use biological test systems with living cells or organisms that give a global response to the pool of micropollutants present in the sample. The aim of this study was to evaluate and compare the suitability of certain endpoints: growth parameters, pigment content, peroxidase activity, lipid peroxidation and alkaline comet assay as biomarkers for environmental monitoring of surface waters using duckweed plants as a test system according to the standardized protocol ISO 20079. Duckweeds (Lemnaceae) possess physiological properties (small size, high multiplication rates, and vegetative propagation), which make them an ideal test system. Moreover, duckweed can be used in a wide range of pH-values (pH 3.5-10). In the proposed test protocol, plants are exposed to a toxicant over a period of seven days, when the consequent potential growth inhibition based on frond number and biomass is estimated. The surface waters were collected monthly over a three-month period at three sampling sites along the river Sava and its confluents (Croatia). The pH value of all samples was in the range 7.8– 8.0. All samples caused inhibition of growth rates derived from frond (leaves) number, decrease of chlorophylls content and of peroxidase activity. Contrary to that, dry to fresh weight ratio markedly increased in duckweed exposed to tested water samples. Tail extent moment (measure of DNA strand breaks) and lipid peroxidation (measure of biomembrane injury) showed increase only in more polluted water samples. In the present study frond number and final biomass proved to be almost equally sensitive parameters, followed by dry to fresh weight ratio, peroxidase activity and chlorophyll a and carotenoid contents. Simplified Comet procedure modified for plants tissues allows rapid yet sensitive determination of DNA damage. The results obtained suggest that phyto- and genotoxicity tests with Lemna minor should be used in the biomonitoring of municipal, agricultural and industrial effluents because of their simplicity, sensitivity and cost-effectiveness.

Analysis of genotoxicity is of a great importance due to the pollution of the environment by genotoxicants including risk assessment evaluation. In this study the response of the Allium cepa and Lemna minor L. genetic material to the presence of potential cytotoxic and genotoxic substances in surface and wasterwater samples was monitored. Also the suitability of the Allium test and alkaline comet assay as systems for environmental monitoring of surface and wastewaters were investigated. The surface waters were collected at three sampling sites along the river Sava and its confluents while wastewater samples were collected from sewage and industrial effluents near Zagreb, Croatia. Morphological modifications in the A. cepa roots, inhibition of root growth and cell division, aberrant cells in metaphase and anaphase as parameters of Allium test were observed. Tail extent moment as a measure of DNA strand breaks was evaluated by using comet assay on Lemna minor plants. The most polluted water samples caused the inhibition of root growth over 50% (even up to 65%), decrease of mitotic index over 40%, increase of aberrant cells for more than 10 times and increase of tail extent moment for more than 35 times in comparison to control. Obtained data demonstrate the mutagenic activity in some samples of tested waters and suitability of both tests for complex assessment of surface as well as waste waters.