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The isoelectric point (pI) is a fundamental parameter intrinsic to any protein, representing the pH at which the protein has an overall neutral charge. This attribute is useful in characterizing the acidic or basic nature of complex proteins. Depending on the pH of the solution, proteins may carry a net positive or negative charge relative to their pI [1]. Consequently, ion exchange chromatography represents a powerful technique to achieve the separation of ionizable proteins based on their charge properties [2]. The aim of this study was to analyse the distribution of toxic elements Cd, Cs, Pb, and Tl among negatively and positively charged biomolecules in the digestive gland cytosol of the bivalve species Unio crassus using anion- and cation-exchange liquid chromatography (AEX-HPLC ; CEX-HPLC) coupled with inductively coupled plasma mass spectrometry (ICP- MS). Protein separation using both HPLC methods was performed with a liner salt gradient (0-500 mM ammonium acetate ; 20 minutes) in appropriate buffers at pH 7.4 (10 mM Tris buffer for AEX-ICP- MS ; 15 mM phosphate buffer for CEX-ICP-MS). In addition, AEX-ICP-MS profiles of six protein standards (alcohol dehydrogenase, carbonic anhydrase, metallothioneins 1 and 2, superoxide dismutase, and transferrin) and CEX-ICP-MS profiles of two protein standards (cytochrome-C and lysozyme) with known pI values were analysed. The results of AEX-ICP-MS showed that biomolecules that bind Cd, Pb and Tl were mainly eluted later than the measured protein standards. Only the first peak of the Pb-binding biomolecules had an elution time similar to that of transferrin (last eluted protein standard, 12.1 minutes, pI~6). Other Pb peaks, in addition to the peaks of Cd- binding biomolecules, were found in the last third of the salt gradient (375 mM – 500 mM ammonium acetate), indicating the more acidic nature of these metalloproteins. Moreover, Cd and Pb were eluted in overlapping peaks, revealing that they are bound to the biomolecules of the comparable negative charge in the digestive gland of U. crassus. This was confirmed by separation on CEX- column, where biomolecules that bind Cd and Pb, due to their appearant negative charge at neutral pH, were completely eluted in the void volume (in the first minute). Conversely, biomolecules that bind Cs were entirely eluted in the void volume (in the first minute) when AEX-column was used, but their elution was evident on the CEX-ICP-MS profile, with the main Cs peak appearing at 7.7 minutes and indicating that proteins that bind this metal are mainly positively charged at neutral pH. A peak of Tl-binding biomolecules was also observed on the CEX-ICP-MS profile (at ~15 minutes), indicating the presence of both acidic and basic cytosolic proteins responsible for Tl binding in U. crassus digestive gland. Protein standards (cytochrome-C with a pI value of 9.6 and lysozyme with a pI value of 11.1) had CEX-ICP-MS elution times at 19 and 24 minutes, respectively. This suggested that basic biomolecules that bind Cs and Tl have lower pI values than standards, probably closer to the pH of the buffer solution. Additional chromatographic separations and analyses by different techniques of mass spectrometry are needed to further identify and characterize these biomolecules.

Aspidogaster conchicola von Baer, 1826 is a common and widely distributed parasite of freshwater molluscs in the temperate zones of the Nearctic and Palearctic. Nevertheless, knowledge about its distribution and hosts in Croatia is still insufficient. Therefore, the aim of our research was to investigate the presence of A. conchicola in the freshwater mussel Unio pictorum (Linnaeus, 1758). This species from the family Unionidae is widely distributed in the rivers of Europe and Russia. The study was conducted in the Mrežnica River, where U. pictorum is a common species. Five specimens of U. pictorum were collected by scuba diving in October 2021. The dissection of mussels was carried out in the laboratory, and all tissues were inspected under a stereo microscope. Determination of the parasite was performed based on the number and arrangement of alveoli. The results showed that the prevalence of infection was 40 % (two out of five mussels were infected), and the intensity of infestation was 2 - 3 specimens/mollusc, which is higher than the values known so far. All parasites were detected in the mussels’ pericardial cavity. In conclusion, this is the first record of A. conchicola in U. pictorum in the Mrežnica River, and the first finding in this mussel species in Croatia. Further investigation is needed to elucidate its distribution among other hosts.

Cytosol is a metabolically active part of the cell that contains metalloproteins with isoelectric point (pI) values clustered between 4.0 and 6.0 [1]. Despite their responsibility for metabolic processes [2], metalloprotein characteristics (mass, charge, etc.) are poorly understood in many organisms, including mussel species. Therefore, the aim of this study was to analyse the distribution of 14 selected metals/metalloids among differently charged metal-binding biomolecules in the digestive gland cytosol of the mussel species Unio crassus (Philipson, 1788) using anion-exchange high-pressure liquid chromatography (AEX-HPLC) and mass spectrometric analysis (ICP-MS). Protein separation using AEX- HPLC was performed with a linear salt gradient (0- 500 mM NH4OAc ; 20 minutes) in 10 mM Tris buffer (pH 7.4). Metal-binding biomolecules with lower pI values tend to interact more strongly with AEX resins, requiring higher salt concentrations for elution [2]. In addition, the AEX- profiles of five protein standards with known pI values (alcohol dehydrogenase, carbonic anhydrase, metallothioneins 1 and 2, superoxide dismutase) were analysed. The results showed that the majority of biomolecules binding the elements Ag, As, Bi, Cd, Cs, Cu, Fe, Pb, Se, and Zn had elution times similar to those of the analysed protein standards (first 10 minutes ; pI higher or ≈ 4). Only a few elements (Ag, Co, Cd, Pb, and Mo) were bound to cytosolic biomolecules at the end of the salt gradient (15-20 min ; >375 mM NH4OAc), indicating the presence of more acidic proteins, i.e., proteins with very low pI values. Several elements (As, Co, Cs, Cu, Fe, Mn, Pb, Se, Zn) were eluted in the void volume indicating the presence of basic metal-binding proteins in the mussel digestive gland cytosol. Some elements (Ag, Co, and Mo ; Cd and Pb ; Mo and Zn ; Ag, Cu, and Cd) had overlapping peaks, suggesting that they are bound to the cytosolic biomolecules of the same charge in the digestive glands of this bivalve species. Further application of additional chromatographic separation and mass spectrometry techniques is needed to confirm the identity of these cytosolic biomolecules.

Cadmium accumulation above a certain threshold can adversely affect aquatic organisms. To evaluate the effects of Cd exposure on aquatic organisms, we monitored changes in the responses of the molecular biomarkers acetylcholinesterase (AChE), catalase (CAT), glutathione S-transferase (GST), malondialdehyde (MDA), metallothioneins (MT), total glutathione (tGSH), and total proteins (TP) in the digestive glands of Unio crassus mussels. In addition, the changes in concentrations of total and cytosolic Cd, Cu, and Zn in the digestive glands of the mussels were monitored. Mussels were exposed to 50 µg/L of Cd for 7 days. Metals were determined using a high-resolution inductively coupled plasma mass spectrometer, and biomarkers were measured spectrophotometrically. The results showed almost 30-fold higher total and cytosolic cadmium concentrations in the digestive glands of Cd-exposed mussels than in control mussels. Furthermore, the activities of biomarkers AChE and CAT were 50% and 70% lower, respectively, in exposed group than in control group. In addition, tGSH concentrations were 20% higher in exposed group than in control group. Other biomarkers showed similar activity/concentration trends in both studied mussel groups. The lack of MT induction in the presence of high Cd accumulation suggests possible toxic effects due to exceeding cellular inducible/defence capacity. Although our study examined only one concentration and exposure duration, the results suggest that Cd exposure can induce significant changes in metal accumulation and biochemical responses in mussels. Nevertheless, further studies are needed to determine the effects of Cd at different concentrations and exposure times and to clarify the mechanisms underlying toxic effects.

Acanthocephala or thorny headed worms is a group of obligatory endoparasites of Mandibulata and Gnathostomata which together with Rotifera form a monophyletic group referred to as Syndermata. Despite their close phylogenetic relationships, different lifestyles have led to different morphological and physiological characteristics of these two phyla. Although Acanthocephala is a relatively homogeneous group of parasites, considerable intraspecific variation within species can be observed. Here we present up-to-date status of phylogeny of acanthocephalans found in the freshwater fish in Croatia, including both the widespread species with broad range of hosts (e.g. Pomphorhynchus laevis) as well as the species confined to limited geographic areas with strong host specificity (e.g. Dentitruncus truttae). The sequences of three genes evolving at different rates (18S rDNA gene, COI gene and ITS region) were analysed in order to gain molecular insights into their phylogeny and genetic variability. 18S rDNA was useful for determination of the phylogenetic position of understudied (e.g. D. truttae) or not morphologically precisely identified acanthocephalans (Echinorhynchus sp.), but was non-informative for intrapopulation variability detection. For the most examined acanthocephalans the genetic variability of COI marker was low (≤1%) while the highest values were observed for the sister species of the genus Pomphorhynchus (0-10.3%). The similar results were obtained for the genetic variability of ITS region, with the highest values observed within Pomphorhynchus species (0-11.3%). Our results are important not just for further phylogeographic studies but also for precise identification of acanthocephalan genotypes which can serve as bioindicators in freshwater ecosystems contamination studies.

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.

Zbog rasta proizvodnje i primjene plastike te neprikladnog zbrinjavanja plastičnog otpada, plastika predstavlja jedan od najvećih onečišćivača današnjice. Plastika ima dugo vrijeme poluraspada, teško je razgradljiva, a pod utjecajem okolišnih faktora dolazi do njezina usitnjavanja. Mikroplastika predstavlja čestice manje od 5 mm koje zbog malih dimenzija lako dospijevaju u sve sastavnice okoliša, uključujući i organizme. Uz navedeno, dodatna okolišna prijetnja je sposobnost vezanja metala na mikroplastiku, poput kadmija (Cd), olova (Pb), nikla (Ni) jer njihovo međudjelovanje može imati višestruke štetne posljedice za organizme. Cilj eksperimenta je ispitati učinak Cd, čestica mikroplastike te vezanje Cd na mikroplastiku prateći eksperimentalni organizam vodenbuhu (Daphnia magna Straus, 1820). Provedeni su testovi toksičnosti tijekom tri dana pri različitim koncentracijama Cd (0, 25, 50, 100, 200, 400, 600, 800, 1000 mg L-1 ), zatim s različitim vrstama usitnjene mikroplastike (polistiren, polipropilen, polietilen niske, kao i visoke gustoće te kombinacije polietilen tereftalata i polietilena, polietilena niske gustoće i polipropilena te polietilena niske i visoke gustoće). Mikroplastika je bila usitnjena na veličinu koja nije dostupna unosu u vodenbuhu (0, 5-5 mm) te se kombinacijom s Cd ispitalo hoće li se vezanjem za čestice mikroplastike Cd ukloniti iz medija te biti manje raspoloživ vodenbuhama. Rezultati potvrđuju toksični učinak Cd na vodenbuhu, odnosno 100 % smrtnost za 100-1000 mg L1 Cd, dok je učinak mikroplastike manji, očito jer nije bila dostupna za unos u vodenbuhu. Kombinacijom Cd (50 mg L-1) i mikroplastike (5 mg L-1) potvrđena je manja smrtnost u odnosu na organizme izložene istoj koncentraciji Cd, što preliminarno može ukazati na vezanje Cd na čestice mikroplastike koje nisu dostupne organizmu i posljedično manju smrtnost (20-40 %). Sukladno literaturnim podacima, vezanje metala je bilo uspješnije na sitnije čestice mikroplastike te rezultati potvrđuju važnost praćenja učinaka mikroplastike, zbog sve veće prisutnosti u okolišu, ali i zbog svojstva vezanja drugih zagađivala te štetnog učinka na organizme.

Karst rivers are characterized by complex and unpredictable variations in water quality due to the strong interaction between surface and groundwater, as well as high sensitivity to pollution. The present study aimed to evaluate the seasonal and spatial variations in physicochemical, organic (COD, TOC, DOC, mineral oils) and inorganic (total and dissolved Al, As, Ba, Ca, Cd, Co, Fe, Hg, K, Li, Mg, Mn, Mo, Na, Rb, Sb, Sr, U, V, Zn) parameters in the karst Krka River, Croatia. Although most of the Krka River watercourse is protected as a national park (NP), it is affected by improperly treated upstream wastewater discharges. The sampling was performed in four seasons of 2021 at eight sites: Krka River source (KRS, reference site), industrial wastewater from the screw factory (IWW), municipal sewage (MWW), tributaries Krčić (TKR, directly influences KRS), Orašnica (TOR, impacted by IWW), Kosovčica (TKO, situated nearby gypsum factory), Butišnica (TBU, influenced by agricultural runoff) and Brljan Lake (KBL, located in the Krka NP). Water quality was more site than season dependent and, in all seasons, showed poor ecological status for IWW and moderate for MWW and TOR according to Directive on water quality status, especially for nutrients, TOC, DOC, and mineral oils. Compared to KRS, poorer values of some physicochemical water parameters were also recorded at TBU and TKO, as a result of agricultural and industrial activity, respectively. Dissolved and particulate metal(loid) forms showed comparable variations, with the highest levels at IWW, especially those used in industry (Sr, Fe, Zn, Mn, Ba, Al), but increase of many elements was also evident at MWW and TOR compared to KRS in all seasons. Seasonal variations of physicochemical water parameters were not obvious, especially at wastewater impacted sites which reflected discharge peaks. Concentrations of TOC, DOC, and mineral oils were higher during the wet season when transport from the catchment area and exchange between surface and groundwater were increased by the rainfall. However, phenol and metal(loid) levels were higher during the dry period, due to lower water levels and less effective purification processes in the river. Presented results pointed to deteriorated water quality near wastewater outlets, which also affect natural seasonal variations of the main ecological parameters. Proper purification of the wastewaters and monitoring plans are mandatory to protect this sensitive karst ecosystem.

One of the main challenges of the modern world is the degradation of water quality due to human activities. The continuous interaction between sediment and water regulates the adsorption- desorption behaviour and thus the bioavailability of pollutants in rivers. The aim of this study was to compare the granulometric and geochemical characteristics of river sediments at five sites of the karst Krka River, which are under different anthropogenic impact: Krka River source (KRS, reference site), Krka River influenced by municipal wastewater (KRK-MWW), tributaries impacted by industrial effluents (Orašnica (TOR) and Kosovčica (TKO)) and by agricultural runoff (Butišnica (TBU)). After sampling the river sediments in July of 2021, carbonate content, particle size distribution, mineral oils, total organic carbon (TOC) and concentrations of Al, As, Ba, Cd, Cr, Cu, Fe, Mn, Ni, Pb, V and Zn were determined. Carbonate content ranged 22.8 (TOR) - 92.8% (KRK), while the predominant particle size fractions were sand and silt at all locations. The particle size distribution indicated multiple material sources for the studied sediments. The obtained characteristics of the sediments were typical for karst rivers, but some parameters indicated anthropogenic impact. Compared to the other sites, levels of mineral oils and common industrial metals were high (Al≥Fe>Zn>Mn>Ba) at TOR and TKO, while TOC was elevated at TKO and KRK. Geochemical data of sediment from the Krka River watercourse indicated more evidence for industrial impact than urban and agricultural. Since pollutants in the sediment from karst rivers can easily become bioavailable, strict protection plans and continuous monitoring of the Krka River are recommended.

Rijeka Krka poznata je po krškom krajobrazu, sedrenim slapovima te brojnosti endemičnih i ugroženih vrsta. Zbog iznimne prirodne vrijednosti, veći dio toka je 1985. proglašen nacionalnim parkom, ali potencijalna prijetnja čitavom ekosustavu su neadekvatno pročišćene industrijske i komunalne otpadne vode koje se ulijevaju u Krku 2 km uzvodno od granice parka. Cilj ovog istraživanja bio je procijeniti kakvoću vode uzvodnog dijela rijeke Krke mjerenjem fizikalno-kemijskih čimbenika te procijeniti toksični učinak testom akutne toksičnosti na vodenbuhama (Daphnia magna Straus, 1820). Uzorkovanje je provedeno u listopadu 2021. na pet lokacija: izvoru rijeke Krke (KRS) kao referentnoj postaji, kod komunalnog ispusta grada Knina (KRK), u bazenima s industrijskom otpadnom vodom (IWW) te pritokama Orašnici (TOR), koja teče uz IWW, i Butišnici (TBU), na koju utječu poljoprivredne aktivnosti. Ovisno o očekivanom stupnju onečišćenja, pripremljene su odgovarajuće serije razrjeđenja uzoraka vode medijem za uzgoj vodenbuha te je za svaku određen postotak nepokretnih jedinki nakon 24 h i 48 h. Kao referentni toksikant korišten je K2Cr2O7. Osnovni fizikalno-kemijski čimbenici ukazali su na dobro stanje vode na lokacijama KRS i TBU, a umjereno stanje na lokacijama TOR i KRK. Iako su na TOR i, osobito, KRK izmjerene povišene koncentracije nutrijenata, uzorci vode s tih lokacija nisu imali toksični učinak. Postotak pokretnih vodenbuha nakon oba razdoblja izlaganja bio je > 90 % u svim uzorcima, osim u IWW, u kojoj već nakon 24 h nije bilo preživjelih vodenbuha. Dobiveni rezultati u skladu su s izmjerenim razinama nitrata, nitrita i ukupnog dušika, koje su u IWW bile višestruko veće od propisanih graničnih vrijednosti. Rezultati istraživanja potvrdili su toksični učinak industrijskih otpadnih voda te narušenu kakvoću riječne vode kod komunalnog ispusta, koji je potencijalna opasnost za rijeku Krku. Stoga su sanacija zagađenih bazena, pročišćavanje otpadnih voda i redoviti monitoring neophodni za zaštitu ovog osjetljivog krškog sustava i Nacionalnog parka Krka.

Introduction: Dentitruncus truttae is an acanthocephalan species endemic to limited areas in Croatia, Italy, and Bosnia and Herzegovina. Since these parasites are characterised as effective metal accumulators, we estimated for the first time application of D. truttae as bioindicator of metal exposure. Therefore, metal distribution, seasonal variation and accumulation were compared in D. truttae and its host Salmo trutta from the Krka River in order to evaluate their sensitivity and indications of metal exposure in the moderately contaminated watercourse. Methods: Sampling was conducted in autumn 2015, spring 2016 and autumn and spring 2021 at Krka River source (KRS) as reference site, and location near the Town of Knin (KRK) impacted by industrial and municipal wastewaters. Molecular analysis confirmed species D. truttae and its taxonomic classification in the family Illiosentidae. Metals were measured by HR ICP-MS in aciddigested acanthocephalans and fish intestine, and their ratio was used to calculate bioconcentration factors (BCFs = C[parasite]/C[host intestine]), as possible indication of exposure duration. Results: Parasite prevalence ranged 92-100% at KRS and 80-97% at KRK. Fish and acanthocephalans showed higher levels of most elements at KRK compared to KRS, while metal accumulation was more efficient in acanthocephalans than fish. That is especially valid for Cd, Cu, Mn, Pb or Sr, resulting in higher BCFs and therefore, reflecting their higher input into Krka River watercourse in recent times. Conclusions: D. truttae showed more effective metal accumulation than fish and even significant differences in metal accumulation between two sites, confirming this parasite as a promising bioindicator species sensitive even to low environmental metal concentrations

Procesi sekvestracije metala pomoću citosolskih proteina i/ili netopljivih staničnih depozita (granule) ovise o vrsti školjkaša i prisutnom elementu. Cilj ovog istraživanja bio je utvrditi postoje li razlike u unutarstaničnoj raspodjeli (raspodjela između topljive citosolske i netopljive frakcije tkiva, raspodjela među citosolskim biomolekulama različitih molekulskih masa) toksičnih metala Ag i Cd u probavnim žlijezdama triju slatkovodnih vrsta školjkaša (Anodonta anatina, Unio crassus, Unio pictorum) iz porodice Unionidae. Dodatni cilj bio je utvrditi važnost toplinski stabilnih citosolskih biomolekula u vezivanju dvaju navedenih elemenata. Školjkaši su uzorkovani na lokaciji niskog antropogenog utjecaja na rijeci Mrežnici. Utvrđivanje raspodjele navedenih elemenata među citosolskim biomolekulama različitih molekulskih masa ostvareno je kromatografskim razdvajanjem uz isključenje po veličini, a mjerenja metala provedena su pomoću spektrometrije masa visoke rezolucije s induktivno spregnutom plazmom. Koncentracije Ag bile su više u netopljivoj frakciji, dok je Cd bio gotovo u potpunosti zastupljen u citosolskoj frakciji tkiva probavnih žlijezda svih triju vrsta školjkaša. Citosolsko Ag bilo je primarno vezano za biomolekule niskih molekulskih masa (NMM: 10-40 kDa) u svim školjkašima u području koje odgovara metalotioneinima. U vrstama A. anatina i U. pictorum, Ag je dodatno eluiralo u području visokih molekulskih masa (> 300 kDa). Kod svih školjkaša, eluiranje Cd zabilježeno je u NMM području te se podudaralo s eluiranjem Ag. Toplinskom obradom citosola probavnih žlijezda te usporedbom absorbanci kromatografskih profila pri valnim duljinama 254 i 280 nm dobiveni su rezultati koji ukazuju na prisutnost toplinsko stabilnih metalotioneina s malim udjelom aromatskih aminokiselina. Zaključno, rezultati ove studije ukazuju na postojanje značajnih razlika u unutarstaničnoj raspodjeli Ag i Cd, kao i na različito sudjelovanje metalotioneina u detoksikaciji ova dva toksična elementa. Potrebna su daljnja istraživanja s višim koncentracijama metala kako bi se bolje razumjele razlike u toleranciji i mehanizmima detoksikacije toksičnih metala u različitim vrstama školjkaša.

Once present in aquatic environment, metals may remain dissolved in the water as free ions, complexes and colloids, or adsorbed by suspended particulate matter, both of which serve as pathways of uptake into benthic animals. In many freshwaters, bivalves have a key role as filter- feeding, sediment-dwelling animals. The lower course of the Mrežnica River has been long exposed to various types of industrial loads which may adversely affect them. To assess the metal contamination of the Mrežnica River affected by anthropogenic influences, the concentrations of five non-essential elements (Ag, Cd, Cs, Pb and Tl) were measured in dissolved and particulate phases in the water and in the digestive gland of Unio crassus mussels at two sampling sites (the industrial area of Karlovac as a polluted site and a location upstream from the Town of Duga Resa as the reference site) in spring 2021. Water samples were filtered through a 0.45 μm pore diameter filter. The water filtrates containing dissolved metals were acidified with nitric acid, while the suspended material remaining on the filter was dried and then digested with nitric acid. Tissue samples were digested with mixture of nitric acid and hydrogen peroxide. The metal concentrations were measured using a high resolution inductively coupled plasma mass spectrometer. In dissolved and/or particulate phase in water, elevated Cs and Tl concentrations were detected at Karlovac, while Cd was higher at the reference site. Increased concentrations of these metals coincided with increased concentrations in U. crassus specimens. Although higher concentrations of Ag and Pb in the particulate phase were also detected at Karlovac, expected changes in the bioaccumulation of those metals were not observed in the digestive glands of U. crassus. Trend of higher concentrations of highly toxic metals at the Karlovac industrial zone indicated a potential risk for the Mrežnica River ecosystem. Thus, further monitoring of both water and biota is recommendable.

The lower part of the Mrežnica River has long been exposed to industrial pollution from the cotton industry near the Town of Duga Resa. To assess the level of metal pollution of the Mrežnica River affected by anthropogenic activities, total metal concentrations were measured in liver and muscle of northern pike (Esox lucius), as a representative fish species. The liver was selected as the main metabolic site in organism, while the muscle, as the edible part, could pose a health risk to humans in case of elevated metal levels. Fish were caught in autumn 2021 at two sites: upstream of the known pollution source as the reference site (REF) and downstream of the Town of Duga Resa (DRT) as the contaminated site. Tissue samples were digested with mixture of HNO3 and H2O2, and metal concentrations (As, Bi, Cd, Co, Cs, Cu, Fe, K, Mg, Mn, Na, Pb, Rb, Se, Tl and Zn) were measured using HR ICP-MS. The concentrations of Pb were below detection limits in both organs, and of Cd only in the muscle. Obtained results confirmed liver as metabolically more active organ than muscle, as accumulation of most elements was 2-250 times higher in liver. The most prominent difference was observed for Fe, Cu, Co and Zn at both investigated sites, with concentrations in the liver higher 10-250 times compared to the muscle. However, Cs, K, Mg and Rb showed higher bioaccumulation in the muscle. Despite different roles and structures of the two organs, they reflected environmental conditions in a similar manner. Significant differences between REF and DRT sites were evident for Bi, Rb and Se in both organs, and for Cd, Mn and Zn only in liver. Bismuth and Zn showed higher levels at DRT, whereas enhanced accumulation of Cd, Mn, Se and Rb was observed at REF. These results can be partially explained by differences of metal exposure from water and sediments. Namely, patterns of Bi, Cd, Mn, and Zn are consistent with their exposure levels in sediments or water [1], while the causes of increased Se and Rb accumulation at REF should be further investigated. Some spatial variability in both organs were also observed for As, Co, Fe, K, Mg and Na, but were not shown significant. Comparison to literature revealed that metal(loid) concentrations in analyzed organs of northern pike indicate certain increase of bioaccumulation due to anthropogenic pressure. The concentrations in the fish muscle, however, were acceptable at both sites based on the national and European legislations [2, 3], which provide permissible levels for As, Cd and Pb. Therefore, moderate impact of the historical contamination was observed in the Mrežnica River, and further monitoring of the environment and biota is still recommended

The Croatian karst river Mrežnica has been in some parts exposed to pollution from various types of anthropogenic activities, especially from industry. Although most industrial plants now provide proper treatment and discharge of wastewater, pollutants previously accumulated in sediments may still pose a threat to aquatic organisms. Molecular biomarkers are considered the most sensitive and earliest response to pollutants. Therefore, it is useful for pollution assessment to obtain information about the harmful effects of pollutants on biota by analysing different biomarkers. As benthic inhabitants and filter- feeding animals, mussels are capable of accumulating large amounts of pollutants and are therefore considered good bioindicator organisms for assessing water pollution. In order to assess water pollution and evaluate possible effects of pollutants on aquatic biota, a set of molecular biomarkers was analysed in the digestive glands of Unio crassus mussels from the Mrežnica River. The set included following biomarkers: metallothioneins (MT) for metal exposure, total glutathione (tGSH) and catalase (CAT) for oxidative capacity and malondialdehyde (MDA) for oxidative damage. In addition, cytosolic concentrations of several essential and non- essential (Ag, Cd, Cu, Pb and Zn) elements were determined. Biomarkers were measured spectrophotometrically, and element concentrations were quantified using a high-resolution inductively coupled plasma mass spectrometer. Mussel samples were collected from two sites at the Mrežnica River: in the industrial zone of the town of Karlovac (contaminated site) and upstream from the town of Duga Resa (reference site). The MDA levels and the activity of CAT were significantly increased in the mussels from the contaminated site, indicating possible overproduction of reactive oxygen species and oxidative stress in these mussels. The elevated levels of these two biomarkers correlated with the increased concentrations of cytosolic Ag, Cu and Pb. Higher tGSH and MTs concentrations at the reference site coincided with higher cytosolic concentrations of Cd, a known inducer of MTs. Cytosolic Zn concentration was similar in mussels from both sites of the Mrežnica River. The presented results suggest differences in biochemical responses of mussels from two sites with different pollutant loads. Therefore, the application of molecular biomarkers is a very useful tool for diagnosing the effects of environmental conditions on organisms.

Toxicity assessment is performed by acute tests that indicate potential toxic effect on aquatic organisms. In our research, toxicity testing was performed in the upstream flow of the Krka River, directly impacted by inappropriately treated industrial and municipal wastewaters, using green algae (Pseudokirchneriella subcapitata (Korshikov) F. Hindák, 1990). Toxicity of water in karst area was tested at five sites, Krka River source (KRS, reference site), near municipal (MWW) and industrial wastewater outlets (IWW) and in tributaries Orašnica (TOR) and Butišnica (TBU). The average algal specific growth and the percentage of inhibition was calculated according to ISO 8692:2012 after 24, 48 and 72 h incubation by measuring the optical density on a spectrophotometer at 670 nm. The final result of the test is presented by the highest concentration at which no inhibition was observed (

The common approach of measuring only the total concentrations of macro- and trace elements in biological materials does not provide complete information about their bioavailability and toxicity, which is mostly associated with their binding to essential molecules in the cytosol [1]. The aim of this study was to apply and validate multielement analysis in the cytosolic fractions of northern pike (Esox lucius) liver and Unio crassus digestive gland, based on the procedure previously described for the analysis of several metals in the hepatic cytosol of European chub (Squalius cephalus) [2]. After tissue homogenization, taking in consideration the specificities of each tissue, the homogenates were centrifuged at 50.000×g for 2 hours at 4 °C and the supernatants (cytosolic fractions containing metabolically active proteins and microsomes [3]) were obtained. An oxidation mixture containing concentrated HNO3 and 30% H2O2 (v/v 3:1) was added to the cytosolic fractions (v/v 1:1). Digestion was performed in a laboratory drying oven at 85 °C for 3.5 h. Thereafter, the samples were diluted with Mili-Q water, five times for Ca/trace element analysis and 20 times for Na/K/Mg analysis on HR ICP-MS. Indium was added to all samples as an internal standard. Appropriate dilutions of the multi-element stock standard solution in 2% HNO3 were used for external calibration. The method was validated by determining the limits of detection (LOD) and quantification (LOQ) for 30 elements (26 trace and four macro), the repeatability of measurement in duplicates, and measurement accuracy based on analysis of quality control samples. Out of 30 analyzed elements, the method enabled the reliable measurement of 19 elements in the E. lucius liver and of 27 elements in the U. crassus digestive gland. The study demonstrated that a high number of elements, both essential and highly toxic nonessential elements, can be accurately measured in the digested cytosols of fish liver and mussels digestive glands using ICP- MS. Metal quantification in the cytosolic fraction containing metalloproteins could provide more information about the biological effects of metals than a study limited to metal concentrations in whole tissues.

Among various pollutants in the aquatic ecosystems, metals pose a serious threat due to their toxicity and potential for bioaccumulation. To assess the overall impact of metal exposure in aquatic environments, bioindicator organisms are commonly used. An early warning biological sign of metal exposure is the induction of metallothioneins (MTs), cytosolic proteins responsible for metal homeostasis and detoxification [1]. In this study, MT levels were used to evaluate metal exposure in fish intestinal parasite Dentitruncus truttae Sinzar, 1955 and its host brown trout (Salmo trutta Linnaeus, 1758) from the karst Krka River. Although the lower part of the Krka River course was proclaimed a national park, its upper course is impacted by inadequately treated wastewaters, which are the main anthropogenic source of metals in the river. MT concentrations were compared among fish liver, as a site of metal detoxification, fish intestine, as a site of metal uptake, and D. truttae, which absorb nutrients from the intestinal tract of fish but also efficiently accumulate metals. Fish were caught at the Krka River source (KRS), the reference site, downstream of the wastewater outlets (KRK) and at hydropower plant Miljacka, situated in the national park (KNP). MTs were quantified by differential pulse voltammetry (DPV) using modified Brdička procedure, a commonly used electrochemical method for the determination of proteins containing thiol groups. DPV was confirmed as a reliable and sensitive method for measuring MTs in biological samples. MT concentrations were significantly higher in D. truttae than in fish gut and liver, and represent the first data for these parasites [2]. D. truttae also showed differences among locations, with the highest MT levels in KRK (2, 2 mg g-1 wet tissue), indicating metal-induced stress near the wastewater outlets.

Industrial activities are nowadays one of the most important sources of metal contamination in the aquatic environments. Such example in Croatia is the Krka River, which lower part is protected as the national park, but upstream part is impacted by improperly purified industrial effluents from the screw factory. Industrial impact was assessed in July 2021 by comparison of element concentrations (As, Ba, Co, Cu, Fe, Hg, Mo, Mn, Na, Ni, Sr, U, Zn) in water and sediments from three sites: industrial wastewaters (IWW), the Orašnica tributary (TOR) directly affected by IWW and Krka River source (KRS) as the reference site. In addition, physicochemical parameters in water were considered. Chemical oxygen demand, ammonia and total nitrogen indicated degraded water quality at IWW and TOR, which was related to significant increases of element concentrations in sediments and water compared to KRS. The highest concentrations of all elements were evident at IWW, except higher Zn in water and Hg, Mn in sediments at TOR. The highest increase was observed for Fe, Mo, Co, Ni and Cu, elements commonly used in industry. Compared to KRS, IWW had up to three orders of magnitude higher element levels in water (Mn>Co>Fe) and even more in sediments (Zn>Co>Mo). TOR, which pose the direct threat to the Krka River, as IWW basins sometimes overflow into TOR, also had much higher concentrations of all elements than KRS. Thus, presented research indicates a deterioration of the Krka River by industrial effluents and highlights the need of adequate purification and monitoring strategies to protect the living world and tufa barriers of this sensitive karst ecosystem.

U ovom osvrtu predstavljen je rad na HrZZ projektu METABIOM.

High metal contamination of aquatic ecosystems can have a huge impact on water quality and lead to series of health issues in aquatic organisms. The Mrežnica River, a Croatian karst river, has long been exposed to various types of industrial loads in its lower course. Freshwater mussels from family Unionidae, are benthic, filter-feeding organisms which are very susceptible to any changes of water chemistry. Since they reflect a quality of water and sediment, these species are considered as the best indicators of ecological conditions. In addition, these bivalves live relatively long and are therefore suitable for monitoring long-term metal exposure. For this investigation we select U. crassus because this species has very abundant populations in the river Mrežnica. Aim of this study was to compare concentrations of twenty eight metals in the digestive gland of U. crassus from two different locations of Mrežnica River, the industrial zone of Karlovac (KAR) and upstream from the Town of Duga Resa as the reference site (REF), and establish if river contamination with metals in industrial zone of Karlovac city has influenced metal bioaccumulation in bivalves. An additional aim was to determine whether total or cytosolic metal concentrations in digestive gland of U. crassus are better indicators of metal exposure in freshwater ecosystems. Measurements of metal concentrations in homogenates (total metal) and water-soluble fraction of digestive gland tissue (cytosolic metal) were performed using a high-resolution inductively coupled plasma mass spectrometer (HR ICP-MS) after sample digestion with mixture of nitric acid and hydrogen peroxide. Higher total concentrations of several elements (Bi, Cs, Cu, Mo, Pb, Sb and U) were found in specimens at the KAR site, whereas Cd and Se were, unexpectedly, increased at the REF site. Among the elements significantly increased at the KAR site, the largest differences compared with the REF site were recorded for Cs and Cu, which were approximately twice as high. Only total concentrations of Cs correlated with concentration in water, while seven elements (Ca, Cu, Cs, Mo, Sb and U) followed the trend of concentrations in sediments. Metal concentrations in cytosolic fractions were increased for almost half of analyzed elements in digestive gland of U. crassus from the KAR site. Significantly higher cytosolic concentrations at KAR site were additionally proven for Al, Co, Fe and Ni, which correlated with their higher concentrations in water and sediment. Results suggest that U. crassus is a good indicator of water quality, which provides information of different bioavailable metal species and forms in aquatic environments, especially by analysing cytosolic (potentially toxic) metal concentrations in their digestive glands.

Marine ecosystems are threatened by variety of anthropogenic activities and pollutants, of which metals present serious threat due to their persistence, toxicity and potential to bioaccumulate. Metal concentrations in water may not reflect the chronic pollution load so bioindicator organisms such as native fish species are used to get an overall measure of contaminant exposure in the aquatic environments (Kraemer et al., 2006). Our goal was to estimate metal exposure in the area impacted by harbor activities, agriculture and tourism near the Port of Ploče and Neretva River mouth in the Southern Adriatic Sea in Croatia, using 30 individuals of three fish species (Mullus barbatus Linnaeus, 1758, Merluccius merluccius Linnaeus, 1758, Pagellus erythrinus Linnaus, 1758) as bioindicators. Biometric characteristics revealed the highest total length and masses of M. merluccius compared to other two species. Fulton condition index, as a measure of fish feeding and health, pointed to better condition of M. merluccius and M. barbatus compared to P. erythrinus. Metal exposure was assessed by determination of metal concentrations in water and fish muscle, as well as biomarker responses of metal exposure (metallothioneins), oxidative (malondialdehyde) and general stress (total proteins) in fish muscle. Accumulation of the most elements showed considerable species-specific differences except for Al, Fe, K and Mg, which concentrations were comparable in all species. The most elements showed significantly higher metal accumulation in M. barbatus (As, Co, Cu, Mn, Pb, Rb) and the lowest in M. merluccius (Co, Cu, Pb, Sr and Zn) when compared with other two species. Species-specific differences were also observed in biomarker responses, possibly as an adaptive response to the present ecological disturbances. Due to the complexity of anthropogenic releases of contaminants and their potential toxic effects, it is crucial to analyze larger set of relevant chemical and toxicological variables including metals and biomarkers, which can be preferably assessed in combination to provide information on marine ecosystem health. Such comprehensive research can provide adequate monitoring strategies in order to preserve the nature and living organisms, but also to provide marine ecosystems goods and services to human population

Due to its travertine waterfalls, many cascades and lakes, very rich and diverse flora and fauna, the Krka River represents exceptional natural value in the Republic of Croatia and worldwide. In accordance, middle and lower course of the river were proclaimed Krka National Park (KNP). Only 2 km upstream of the park border municipal and industrial wastewaters, which are released without proper purification, may threaten living world and tufa barriers of this karst phenomenon [1]. The aim of the present study was to evaluate water quality and metal exposure by means of physico- chemical water parameters (turbidity, conductivity, total dissolved salts, chemical oxygen demand, ammonia, nitrates, nitrites, total nitrogen and total phosphorus) and dissolved metal levels (Al, As, Cd, Co, Fe, Hg, Li, Mn, Mo, Ni and Zn). Sampling was performed at eight locations: Krka River source (KRS) as reference site ; tributary Krčić (TKR) ; industrial wastewater (IWW) ; municipal sewage of the Town of Knin (KRK) ; tributary Orašnica (TOR), impacted by industrial wastewater ; tributary Kosovčica (TKO), nearby Knauf factory ; tributary Butišnica (TBU), influenced by agricultural runoff and Brljan Lake (KBL), located in the KNP. Sampling campaigns were conducted in April and July 2021, as two seasons of higher and lower water level, respectively. Physico-chemical parameters were measured in situ and by Standard Methods for the Examination of Water & Wastewater, while metals were measured by high resolution inductively coupled plasma-mass spectrometry (HR ICP-MS). Water quality was estimated following the Directive on water quality status [2]. Chemical water status was the lowest in industrial effluents (IWW) in both sampling campaigns, which were therefore confirmed as a serious threat to the river water. Physico- chemical water parameters (especially chemical oxygen demand, ammonia, total nitrogen and phosphorus) also pointed to deteriorated water quality at anthropogenically impacted locations (TKO, KRK, TOR, TBU) but also at KBL, which is situated within KNP. Dissolved metal levels, especially of Mn, Fe, Co and Ni, showed the highest increase at IWW, TOR, KRK and TKO compared to KRS in both seasons. Those metals are known by their use in industry and confirmed potential impact of IWW to the Krka River and its tributaries. Only Hg levels were low at all sites, even below limit of detection. Concentrations of most metals were higher in July than April at all sampling sites, probably due to lower water level in the summer period. Presented results indicated the influence of direct pollution sources and emphasized its potential threat to the preservation of the natural beauties of the Krka River. Therefore, proper purification of the wastewaters and implementation of water quality monitoring strategies of the Krka River are required. Acknowledgements The financial support of the Croatian Science Foundation for the project: “Integrated evaluation of aquatic organism responses to metal exposure: gene expression, bioavailability, toxicity and biomarker responses (BIOTOXMET)” (IP-2020-02-8502) is highly acknowledged.

Rijeka Krka poznata je kao krški fenomen zahvaljujući velikom bogatstvu sedrenih slapova i jezera te raznolikosti vrsta pa je veći dio toka proglašen nacionalnim parkom (NP) 1985. godine. Međutim, samo 2 km uzvodno od granice NP Krka u rijeku se ispuštaju neadekvatno pročišćene industrijske (tvornica vijaka) i komunalne otpadne vode (grad Knin) koje ugrožavaju i kakvoću vode zaštićenog dijela vodotoka [1]. Stoga je cilj istraživanja bio procijeniti antropogeni utjecaj na kakvoću vode rijeke Krke mjerenjem fizikalno- kemijskih čimbenika te koncentracija otopljenih metala u vodi na šest lokacija tijekom tri uzorkovanja između 2015.-2021. godine. Izvor rijeke Krke (KRS) odabran je kao referentna lokacija, dok su ostale pod različitim antropogenim utjecajima: otpadna voda tvornice vijaka (IWW), pritoka Orašnica (TOR) koja prolazi uz bazene s otpadnom vodom, Krka nizvodno od ispusta komunalnih voda grada Knina (KRK), pritoka Butišnica (TBU) koja je pod utjecajem poljoprivredne djelatnosti i jezero Brljan (KBL) kao nizvodna lokacija unutar NP Krka. Tijekom sva 3 uzorkovanja potvrđena je najlošija kakvoća vode u otpadnoj vodi tvornice (IWW). Značajno više koncentracije mnogih elemenata i lošije vrijednosti pojedinih fizikalno-kemijskih čimbenika u odnosu na izvor zabilježene su i na lokacijama TOR, TBU i KRK, dok je KBL, vezano uz izraženu sedimentaciju u jezeru, imao relativno niske koncentracije metala. Značajno je da su i koncentracije Hg na svim postajama niske, čak i ispod razine detekcije. Najviši porast na postajama pod antropogenim utjecajem zabilježen je za Co, Fe, Mn, Ni i Zn, metale koji se koriste u industriji, kao i višestruko više razine K, Mo, Se na lokacijama IWW, TOR i TBU u odnosu na izvor. Višegodišnje istraživanje ukazuje na slične trendove, iako se tokom godina uočava blagi pad koncentracija metala u rijeci Krki i njezinim pritokama, vjerojatno kao rezultat djelomičnog ulaganja u efikasnije pročišćavanje otpadnih voda. Međutim, zabilježen porast razina metala u blizini ispusta otpadnih voda ukazuje na potencijalnu opasnost za ovaj osjetljivi krški ekosustav i važnost monitoringa i zaštite NP Krka.

Mrežnica River is karst Croatian river included in ecological network of European Union NATURA 2000 which protects important areas for preservation of species and types of habitats. However, its lower course has been exposed to the impact of cotton industry for more than one hundred years, including release of metal polluted ash and slag material from coal burning [1]. To evaluate the current pollution status of the Mrežnica River and possible early toxic effects in organisms which occur due to exposure to industrial wastes, we have applied multibiomarker approach in the liver of the northern pike (Esox lucius), as a representative fish species. Analyzed set of biomarkers included the activity of enzymes catalase (CAT) and acetylcholinesterase (AChE), and concentrations of malondialdehyde (MDA), glutathione (GSH), total cytosolic proteins (TP) and metallothioneins (MTs). Additionally, we have measured cytosolic concentrations of several metals. Fish sampling was performed at the reference site, upstream of the known pollution source, and near the former cotton industry facility as the presumed site of historical contamination. Some significant differences in biological responses between the fish from two sites, when we analyzed all sampled fish, were observed. However, strong impact of historical contamination was not confirmed when only fish with similar body masses were compared, which indicated the influence of body size. Still, trend of higher accumulation of some metals (Bi, Cs, Cu, Fe) at the contaminated site was evident, but not followed by biomarker responses. Higher levels of GSH and MT, as well as Mn and Tl, and lower AChE activity, were recorded at the reference site, possibly due to agricultural practices. Two liver colorations, probably due to different feeding habits, were observed at both sites ; „green“ in most of smaller and probably more invertebrate- feeding individuals, and „yellow“ in bigger fish which are almost completely piscivorus [2]. Green livers in fish can be caused by lower fish meal diet due to the defficiency of specific proteins involved in excretion of bile pigments [3]. Differences betwen green and yellow livers within each site were small for biomarkers. For metals, higher levels in fish with yellow liver were observed for Bi, Cs and Fe, and in fish with green liver for Mn, Mo and Co, suggesting different metal availability depending on the diet. Differences between sites for metals were more obvious in fish with yellow liver, with significantly higher Bi, Cu and Fe accumulation at the former factory site, and Tl at reference site. Among biomarkers, significant difference between sites was observed only for AChE activity in fish with yellow liver. Beside water and sediments as contamination sources, our research showed the importance of feeding habits for fish biological responses. Diet of fish with yellow liver is probably more diverse, which contributes to variations in biomarker and metal levels between two groups of northern pike. Our results point to moderate impact of former factory and contribute to evaluation of the influence of long-term historical contamination, but also of feeding behavior on condition of fish in the Mrežnica River.

Metals are common pollutants in aquatic systems, but they are also crucial for normal functioning of organisms. Freshwater bivalves, as filter- feeders, are suitable bioindicators of metal contamination but their accumulation capacity and intracellular distribution may vary by species. Therefore, the aim of this study was to compare the bioaccumulation and intracellular distribution of nine selected metals/metalloids (essential: Cu, Zn, Fe, Co, Mo and Se ; non-essential: Cd, Ag and As) in the digestive gland of two freshwater bivalve species, Anodonta anatina and Unio pictorum, collected from a reference site on the Mrežnica River (Croatia). Higher total metal concentrations were found in U. pictorum for all of the elements studied, except for Ag where concentrations were comparable in both species. The largest differences were found for Cd being 13 times higher, and for Co and Fe with about 3.5 fold increased values in U. pictorum. In both species, Cd and Se were primarily accumulated in the cytosol, while Fe was found mainly in the insoluble tissue fraction. The distribution of Cd and Cu did not differ significantly between species, while for all other elements except As, a significantly higher presence in the cytosolic fraction was found in A. anatina. Analysis of metal distribution among cytosolic biomolecules by size exclusion HPLC allowed the definition of molecular masses of biomolecules involved in binding of particular metals. The obtained metal profiles were comparable in both of the studied species for all of the studied elements. Our results will contribute to a better understanding of accumulation and metal metabolism in the studied freshwater bivalve species.

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 (

Modern life results in continuous impact of different pollutants to aquatic ecosystems, of which marine plastic is considered as global environmental problem. According to United Nations, 30% of marine fish contained microplastics in their tissues, which consequently can lead to gut blockages, reduced energy budgets or starvation and toxic effects (Rochman et al., 2019). Our research was conducted in Croatia, in the Southern Adriatic Sea near the Port of Ploče and Neretva River mouth, known by the pollution impact of harbor activities, agricultural runoff and tourism. Three fish species, Mullus barbatus Linnaeus, 1758, Merluccius merluccius Linnaeus, 1758, Pagellus erythrinus Linnaeus, 1758, were used as bioindicators of contaminant exposure by measurement of oxidative stress biomarkers in muscle tissue, comparing fish from nearshore and offshore area. Oxidative stress is a commonly observed response to microplastic impact, but also to metal and organic exposure (Qiao et al., 2019). Our goal was to compare different biomarkers in native fish as indication of oxidative stress (malondialdehyde, MDA and glutathione, GSH), organic and metal exposure (acetylcholinesterase, AChE) and general stress (total proteins, TP). Results showed that 10% of the total trawl catch contained waste, mostly plastic. Isolation of microplastics from the fish gut content confirmed its presence in almost all selected individuals. Although Adriatic Sea was reported as the third location in Europe by plastic waste abundance (Zeri et al., 2018), our results pointed that fish species still don’t show significant differences in biological responses to oxidative stress or metal and organic exposure between nearshore and offshore area. But presence of microplastics in the gut content of sampled fish highlights the importance of continuous biomonitoring and assessment of plastic accumulation in marine environment and organisms.