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Iron plays vital roles in important biological processes in fish, but can be toxic in high concentrations. The information on metalloproteins that participate in maintenance of Fe homeostasis in an esocid fish, the northern pike, as an important freshwater bioindicator species, are rather scarce. The aim of this study was to identify main cytosolic constituents that sequester Fe in the northern pike liver. The method applied consisted of two-dimensional HPLC separation of Fe-binding biomolecules, based on anion-exchange followed by size-exclusion fractionation. Apparent molecular masses of two main Fe-metalloproteins isolated by this procedure were ~360 kDa and ~50 kDa, with the former having more acidic pI, and indicated presence of ferritin and hemoglobin, respectively. MALDI-TOF-MS provided confirmation of ferritin subunit with a m/z peak at 20.65 kDa, and hemoglobin with spectra containing main m/z peak at 16.1 kDa, and smaller peaks at 32.1, 48.2, and 7.95 kDa (single-charged Hb-monomer, dimer, and trimer, and double-charged monomer, respectively). LC-MS/MS with subsequent MASCOT database search confirmed the presence of Hb-β subunits and pointed to close relation between esocid and salmonid fishes. Further efforts should be directed towards optimization of the conditions for metalloprotein analysis by mass spectrometry, to extend the knowledge on intracellular metal-handling mechanisms., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Since fish metalloproteins are still not thoroughly characterized, the aim of this study was to investigate the acidic/basic nature of biomolecules involved in the sequestration of twelve selected metals in the soluble hepatic fraction of an important aquatic bioindicator organism, namely the fish species northern pike (Esox lucius). For this purpose, the hyphenated system HPLC-ICP-MS was applied, with chromatographic separation based on anion/cation-exchange principle at physiological pH (7.4). The results indicated predominant acidic nature of metal-binding peptides/proteins in the studied hepatic fraction. More than 90 % of Ag, Cd, Co, Cu, Fe, Mo, and Pb were eluted with negatively charged biomolecules, and >70 % of Bi, Mn, and Zn. Thallium was revealed to bind equally to negatively and positively charged biomolecules, and Cs predominantly to positively charged ones. The majority of acidic (negatively charged) metalloproteins/peptides were coeluted within the elution time range of applied standard proteins, having pIs clustered around 4-6. Furthermore, binding of several metals (Ag, Cd, Cu, Zn) to two MT-isoforms was assumed, with Cd and Zn preferentially bound to MT1 and Ag to MT2, and Cu evenly distributed between the two. The results presented here are the first of their kind for the important bioindicator species, the northern pike, as well as one of the rare comprehensive studies on the acidic/basic nature of metal-binding biomolecules in fish, which can contribute significantly to a better understanding of the behaviour and fate of metals in the fish organism, specifically in liver as main metabolic and detoxification organ., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The possible influence of historical contamination of water/sediments on the metal(loid) bioaccumulation in the digestive gland of mussel Unio crassus Philipsson, 1788, from two differently contaminated sites at the Mrežnica River was studied in three seasons. The first data for this species on total/cytosolic concentrations of 27 (non)essential elements were obtained by HR ICP-MS. Higher bioaccumulation was observed at the historically contaminated site, with several nonessential elements (Bi, Cs, Pb, Sb, Tl, U) found in 5-6 times higher concentrations compared to the reference site. Although both total and cytosolic levels revealed the influence of water/sediment contamination, the latter showed association between bioaccumulation and exposure for larger number of studied elements. At the reference site, several elements (Ba, Ca, Cd, Cr, Mn, Sr) were also found in 2-10 times higher concentrations compared to contaminated one, but it was attributed to background levels characteristic for karst rivers (for Ca and Cd), and to coaccumulation due to chemical similarity (for Ba, Cr, Mn, Sr). The seasonal variability was also observed, with generally highest metal(loid) concentrations in mussel digestive glands found in autumn which was associated to mussels reproductive period. Our results confirmed that sediment-dwelling mussels, specifically U. crassus, represent a good bioindicators for detection of historical pollution due to their direct contact/exposure to contaminants stored in sediments, with concurrent consideration of physiological/chemical factors. Historical contamination potentially can have serious impact on freshwater environment even long time after its cessation, and, therefore, a careful continuous monitoring is recommended., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Increased contamination of aquatic ecosystems with pharmaceuticals could have been expected due to the COVID-19 pandemic. Surface water from three domains (rural, semirural, urban) of the Mrežnica River (Croatia) was screened for 253 pharmaceuticals by SPE-UHPLC-MS/MS. At the beginning of the pandemic, the highest concentration of drugs (excluding veterinary) was detected at urban site (291.4 ng/L), followed by semirural (186.5 ng/L) and rural (141.6 ng/L). With the progression of pandemic, contamination increase was observed at all sites, but it was the most obvious at semirural (approximately 400-700 ng/L). The most pronounced concentration increases were observed for non-opioid analgesics, especially ibuprofen. In September 2021, the first notable occurrence of opioid analgesics was recorded. The most represented group of pharmaceuticals at the start of the pandemic (May 2020) was generally stimulants (caffeine, cotinine). In September 2021, the predominant group was analgesics at all sites (45-84%), whereas stimulants decreased to undetectable levels. The results of this study indicated that the epidemiological measures and medical treatments that were widely imposed/applied caused notable increase of the surface water contamination with drugs of a small river with limited dilution capacity, indirectly pointing to the changes that occurred in the behaviour and habits of the inhabitants of the affected areas., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In the present study, essential and nonessential metal content and biomarker responses were investigated in the intestine of fish collected from the areas polluted by mining. Our objective was to determine metal and biomarker levels in tissue responsible for dietary intake, which is rarely studied in water pollution research. The study was conducted in the Bregalnica River, reference location, and in the Zletovska and Kriva Rivers (the Republic of North Macedonia), which are directly influenced by the active mines Zletovo and Toranica, respectively. Biological responses were analyzed in Vardar chub (Squalius vardarensis; Karaman, 1928), using for the first time intestinal cytosol as a potentially toxic cell fraction, since metal sensitivity is mostly associated with cytosol. Cytosolic metal levels were higher in fish under the influence of mining (Tl, Li, Cs, Mo, Sr, Cd, Rb, and Cu in the Zletovska River and Cr, Pb, and Se in the Kriva River compared to the Bregalnica River in both seasons). The same trend was evident for total proteins, biomarkers of general stress, and metallothioneins, biomarkers of metal exposure, indicating cellular disturbances in the intestine, the primary site of dietary metal uptake. The association of cytosolic Cu and Cd at all locations pointed to similar pathways and homeostasis of these metallothionein-binding metals. Comparison with other indicator tissues showed that metal concentrations were higher in the intestine of fish from mining-affected areas than in the liver and gills. In general, these results indicated the importance of dietary metal pathways, and cytosolic metal fraction in assessing pollution impacts in freshwater ecosystems., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The remnants of historical industrial contamination can be detected in many aquatic ecosystems worldwide even at present time. Mrežnica is a river in Croatia that has been, for more than a hundred years, continually exposed to effluents of various industries, which have, in modern time, mostly ceased to operate. Our aim was to establish the level of current contamination and pollution of the Mrežnica river-water and sediments. The study of river contamination at three sites (reference site; site nearby former cotton industry facility in Duga Resa - DRF; industrial zone of Karlovac town - KIZ) in three sampling campaigns (May 2020, April and September 2021) encompassed analyses of physico-chemical water parameters, screening of 369 pesticides, measurement of metal (loid) concentrations in the sediments, and in the dissolved and particulate phases of the river-water. The sediment pollution was assessed through the analyses of total bacteria abundance (by targeting 16S rRNA genes), and their associated metal resistance genes (cnrA, pbrT and czcD) and class 1 integrons (intl1). At the DRF site, industrial organic contaminants that can be traced to textile production were detected (dye and nylon components), as well as increased levels of some metals bound to suspended particulate matter and sediments. At the most downstream KIZ site, occasional high level of industrial herbicide neburon was measured in the river-water, and metal contamination of suspended particulate matter and sediments was evident. Although, based on the comparison with legislation and literature data, the level of contamination was rather mild, the effects on microbial communities were unquestionable, confirmed by increased abundance of the czcD gene at DRF site and the intI1 gene at both industrially impacted sites. Obtained results indicated long-term sediment retention of some industrial contaminants at the places of historical freshwater contamination, and, thus, the necessity for their monitoring even after the termination of contamination sources., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Each of the bioindicator organisms studied, such as fish, crustaceans, or parasites, have certain advantages when used in the assessment of metal exposure. In this research, special attention was given to intestinal parasites, acanthocephalans, as effective metal bioaccumulators with the main aim of estimating and comparing metal accumulation in all organisms involved in their life cycle and testing their usage as sensitive bioindicators in metal exposure assessments., Methods: Efficiency of metal accumulation was evaluated in fish intestine (brown trout, Salmo trutta Linnaeus, 1758), gammarids (Gammarus balcanicus Schäferna, 1922) and acanthocephalans (Dentitruncus truttae Sinzar, 1955) in the Krka River at site threatened by industrial and municipal wastewaters (Town of Knin) and reference site (Krka River source) in two seasons (autumn and spring). All samples were digested in a drying oven at 85 °C for 3.5 h using nitric acid and hydrogen peroxide. Samples were diluted with ultrapure water and total concentrations of 15 trace and macroelements were measured by HR ICP-MS., Results: Higher concentrations of Co, Cu, Fe and Mn at the contaminated than at the reference site were recorded in all bioindicator organisms. Additionally, Se, Sr, Zn, Ca, K and Mg were elevated at the contaminated site in acanthocephalans, Cs, Pb, Zn and K in gammarids and Se, Sr and Ca in fish intestine. Opposite, concentrations of Cd, Cs and Rb were mostly higher in organisms from the reference than wastewater impacted site, which did not coincide with waterborne metal exposure but indicated importance of assessing dietary metal uptake., Conclusion: More effective metal accumulation was confirmed in acanthocephalans than fish and gammarids, especially of Cd and Pb in respect to gammarids, and of Cd, Cu, Pb, Mn and Sr in respect to fish intestine. Thus, both fish intestine and whole body of D. truttae were confirmed as sensitive indicators of low environmental metal exposure, reflecting site-specific differences, which proved their potential to be used in monitoring programmes and metal exposure assessment in real environmental situations., (Copyright © 2022. Published by Elsevier GmbH.)

Metal-handling strategies of various fish species are known to vary significantly in association with their intracellular metal behaviour. Thus, to better understand the possible consequences of increased metal exposure in fish it is important to perform comparative studies on metal-binding biomolecules in organs of different species. This study was the first of this kind on a liver of an esocid fish (northern pike, Esox lucius), and the gathered information were compared to fish belonging to three other families, Leuciscidae, Cyprinidae and Salmonidae. Distributions of ten elements among cytosolic biomolecules of different molecular masses were studied by size exclusion HPLC combined offline with high resolution ICP-MS. The results indicated predominant association of Co, Fe and Mo to high molecular mass biomolecules (>100 kDa), of Zn and Bi to both high and medium molecular mass biomolecules (>30 kDa), of Mn and Se to medium molecular mass biomolecules (30-100 kDa), and Ag, Cd and Cu to low molecular mass biomolecules (10-30 kDa), presumably metallothioneins. Evident binding to metallothioneins was also detected for Zn and Bi. For several metals, distinct differences were observed when cytosolic metal distributions of northern pike were compared to leuciscids, salmonids and cyprinids. More pronounced Zn binding to metallothioneins was recorded in leuciscids and cyprinids than both esocids and salmonids, whereas cytosolic Mn and Se distributions clearly differed between all studied fish families. Accordingly, in assessment of metal pollution it is vital to consider the exposed species, which requires prior comprehensive comparative research on numerous aquatic organisms., (Copyright © 2022 Elsevier Inc. All rights reserved.)

As the most successful crayfish invader and possible vector for infectious agents, signal crayfish Pacifastacus leniusculus is among the major drivers of the native crayfish species decline in Europe. We describe histopathological manifestation and frequency of newly detected idiopathic necrotizing hepatopancreatitis along the invasion range of the signal crayfish in the Korana River in Croatia. Our results show extremely high prevalence of necrotizing hepatopancreatitis (97.3%), with 58.9% of individuals displaying mild and 31.5% moderate histopathological changes in the hepatopancreas, also reflected in the lower hepatosomatic index of analysed animals. Recorded histopathological changes were more frequent in the invasion core where population density is higher. Our preliminary screening of co-occurring native narrow-clawed crayfish Pontastacus leptodactylus showed lower incidence (33.3%) and only mild hepatopancreatic lesions, but potentially highlighted the susceptibility of native crayfish populations to this disease. Pilot analyses of dissolved trace and macro elements in water, sediment fractions and crayfish hepatopancreas do not highlight alarming or unusually high concentrations of analysed elements. Hepatopancreas microbiome analysis, using 16S rRNA gene amplicon sequencing, identified taxonomic groups that should be further investigated, along with impacts of the disease on health and viability of both invasive and native crayfish populations., (© 2021 John Wiley & Sons Ltd.)

Cytosolic distribution of six essential elements and nonessential Cd among biomolecules of different molecular masses was investigated in the intestine of brown trout (Salmo trutta) from the karst Krka River and Prussian carp (Carassius gibelio) from the lowland Ilova River. Fish were sampled at two locations (reference and contaminated) and in two seasons (autumn and spring). Analyses were conducted by size exclusion high performance liquid chromatography and high resolution inductively coupled plasma mass spectrometry. Although studied salmonid and cyprinid fish have different biological characteristics, obtained profiles often showed mostly similar patterns in both species. Specifically, Cd and Cu were dominantly bound to metallothioneins in both species, but the same association was not observed for Zn, whereas Mo distribution was similar in the intestine of both fish species with two well shaped and clear peaks in HMM (100-400 kDa) and VLMM (2-8 kDa) range. In brown trout, Se was mostly associated with biomolecules of very low molecular masses (VLMM, <10 kDa), whereas significant additional elution in HMM region (30-303 kDa) was observed only in Prussian carp. Iron binding to VLMM biomolecules (1.8-14 kDa) was observed only in brown trouts, and of Zn in Prussian carps. Cobalt was mostly bound to HMM biomolecules (85-235 kDa) in brown trout and to VLMM biomolecules (0.7-18 kDa) in Prussian carp. Comparison of intestinal profiles with previously published data on liver and gills revealed some similarities in distribution, but also organ-specific differences due to the different function and composition of each organ. As so far there is no published data on intestinal trace metal distribution, the obtained results represent the novel findings, and the key point for the exact identification of specific metal-binding biomolecules which could eventually be used as biomarkers of metal exposure or effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

To address and to compare the respective impact of gold and silver nanoparticles (Au and Ag NPs) in soil invertebrate, the earthworm Eisenia fetida was exposed to soil containing 2, 10, and 50 mg/kg of Au and Ag in both nanoparticulate and ionic forms for 10 days. Both metal NPs were 2-15 times less bioavailable than their ionic forms, and displayed similar transfer coefficients from soil to earthworm tissues. Both metal NPs triggered the onset of an oxidative stress as illustrated by increased glutathione S-transferase levels, decreased catalase levels, and increased malondialdehyde concentrations. Protein carbonylation distinguished the nanoparticular from the ionic forms as its increase was observed only after exposure to the highest concentration of both metal NPs. Au and Ag NPs triggered DNA modifications even at the lowest concentration, and both repressed the expression of genes involved in the general defense and stress response at high concentrations as did their ionic counterparts. Despite the fact that both metal NPs were less bioavailable than their ionic forms, at equivalent concentrations accumulated within earthworms tissues they exerted equal or higher toxic potential than their ionic counterparts. Capsule: At equivalent concentrations accumulated within earthworm tissues Au and Ag NPs exert equal or higher toxic potential than their ionic forms.

Pigmented macrophage aggregates (MAs) are known to change under influence of various factors, such as aging, season, starvation, and/or pollution. In this study, changes in the pigment content of the MAs in the spleen of Vardar chub (Squalius vardarensis, Karaman) (n = 129) collected in spring and autumn, from three rivers with different pollution impact was examined: Zletovska River (metals), Kriva River (metals and municipal wastewater), and Bregalnica River (municipal wastewater). Collected data revealed increased relative volume and number of MAs containing hemosiderin under the influence of metals, significant in autumn (p < .05). In chub exposed to metals combined with municipal wastewater, significant increase of lipochrome accumulation in MAs in autumn, melanin in MAs in fish captured in the spring season, and number of splenic MAs containing combination of melanin and lipochrome was noted. Volumes and number of MAs containing combination of hemosiderin and lipochrome increased in spleen of fish captured in autumn from both Zletovska River and Kriva River, most likely due to the contribution of hemosiderin and lipochrome, respectively. Values measured for the various pigments in splenic MAs in fish captured from Bregalnica River, were overall closer to the values measured for fish captured from Kriva River. Notably, melanin and lipochrome are more likely to be found in fish from waters influenced by municipal wastewater (organic pollution) and hemosiderin in fish spleen from water influenced by mining activity (heavy metals pollution)., (© 2020 Wiley Periodicals, Inc.)

Prussian carp (Carassius gibelio) is an invasive freshwater fish known for its high tolerance to aquatic pollution. Our aim was to try to clarify its tolerance to increased exposure to metals/nonmetals, by determining their cytosolic distributions among peptides/proteins of different molecular masses (MM), which form a part of the fish protective mechanisms. The applied approach consisted of fractionation of gill and hepatic cytosols of Prussian carp from the Croatian river Ilova by size-exclusion high performance liquid chromatography, whereas Cd, Cu, Zn, Fe, Mo, and Se analyses were done by high resolution inductively coupled plasma mass spectrometry. The results indicated high detoxification of Cd by its binding to metallothioneins (MTs) in both fish organs. In addition, binding to MTs was observed for Cu in both organs and for Zn in the liver, whereas clear Zn binding to MTs in the gills was not recorded. Zinc in the gills was predominantly bound to proteins of higher MM (50-250 kDa) and to biomolecules of MM below 2 kDa. Predominant Fe binding to proteins of MM of ~400 kDa (presumably storage protein ferritin) was observed in the liver, whereas in the gills Fe was mainly associated to proteins of MM of ~15-65 kDa (presumably hemoglobin oligomers). Maximum Mo and Se elutions in the liver were noted at 235 kDa and 141 kDa, respectively, and in the gills below 10 kDa. The striking difference was observed between two organs of Prussian carp, with predominant metal/nonmetal binding to high MM proteins (e.g., enzymes, storage proteins) in the liver, and to very low MM biomolecules (<10 kDa) in the gills (e.g., antioxidants, metallochaperones, nonprotein cofactors). Such metal/nonmetal distributions within the gills, as the first site of defense, as well as association of several metals to MTs, indicated highly developed defense mechanisms in some organs of Prussian carp., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of the present study was to assess physico-chemical water parameters, granulometric sediment characteristics and concentrations of trace and macroelements in the water and sediments of the Ilova River. Samplings were conducted at three sampling sites (near villages Maslenjača, Ilova and Trebež) along the Ilova River, differing in the source and intensity of the anthropogenic influence. This study indicated disturbed environmental conditions, most pronounced in the downstream part of the river (Trebež village) impacted by the activity of fertilizer factory. Water from the Ilova and Maslenjača villages was of good quality, whereas COD, nitrates and phosphates exceeded the good quality levels in Trebež village. Trace and macroelement concentrations in water were mostly below thresholds set by environmental quality standards at all locations, but levels of Al, As, Cd and Ni were few times higher in Trebež village than at other locations. Metal contamination assessment of sediments (trace and macroelement concentrations, contamination and enrichment factor, pollution load index) confirmed deteriorated environmental quality in Trebež village. However, the overall assessment performed in this study revealed that anthropogenic impact was still not particularly strong in the Ilova River ecosystem. Nevertheless, the observed water and sediment characteristics serve as a warning and suggest that stricter protection measures should be initiated, including continuous monitoring and comprehensive quality assessment of the downstream part of the Ilova River, especially because it is a part of the protected area of the Lonjsko Polje Nature Park.

Metals play crucial physiological roles, but they can also cause irreparable toxic effects through binding to important cellular biomolecules in aquatic organisms. The aim of this study was to determine the exact molecular masses and to identify several selected metal-binding biomolecules in hepatic and gill cytosols of Vardar chub (Squalius vardarensis Karaman, 1928). Methods applied for the achievement of this goal were SEC-AEC-HPLC for two-dimensional separation of cytosolic biomolecules, HR ICP-MS for metal measurements, and mass spectrometry (MALDI-TOF-MS and LC-MS/MS) for biomolecule mass determination and identification. The analyzed biomolecules included: Fe-binding biomolecules, which were identified as hemoglobin subunit β in the liver (molecular masses of ∼15 kDa), and hemoglobin subunits α and β in the gills (molecular masses of ∼11 kDa, ∼13 kDa and ∼15 kDa); heat-stable Cd-binding biomolecules, which were identified as MT isoforms MT-I and MT-II (molecular mass of ∼6.0 kDa in both liver and gills, and an additional 4.9 kDa isoform in the gills); and heat-stable Mo-binding biomolecules of molecular masses equal to 3.3 kDa (in the gills) and 8.5 kDa (in the liver). An important finding of this study was the obvious presence of different isoforms of the same biomolecules in the liver and gills. This was, among others, manifested through the absence of Zn binding to MTs only in the gills, indicating that the same type of biomolecule can be responsible for different functions in different organs. Thus, for better understanding of metal behaviour in aquatic organisms, it is crucial to identify cellular metal-binding biomolecules and their functions.

In order to assess the extent of existing anthropogenic influence on biota of the vulnerable karst ecosystem of the Krka River, multi-biomarker approach was applied in the intestinal tissue of brown trout Salmo trutta Linnaeus, 1758. Biomarkers of the general stress (total cytosolic proteins), oxidative stress (malondialdehyde), antioxidant capacity (catalase activity, total glutathione) and of an exposure and effect of contaminants, especially metals (metallothionein) and organophosphorous pesticides and metals (acetylcholine esterase activity) were compared in the intestine of fish from the reference site (river source) and downstream of the technological and municipal wastewater impacted site (town of Knin) in two seasons, October 2015 and May 2016. Biological response was additionally evaluated by metal/metalloid concentrations in intestinal cytosol. Site-specific differences were observed as significantly higher As, Ca, Co, Cu, Se and Sr concentrations in intestinal cytosol of fish from the contaminated compared to the reference site. Significant seasonal differences existed for Ni, Cd, Mo, Cs and Na, with higher levels in autumn, following the trend of most of the dissolved metal levels in the river water. Impact of improperly treated wastewaters was also confirmed by significantly increased levels of glutathione, total proteins and Foulton condition indices, with 1.5, 1.13 and 1.12 times higher average values in fish from that site compared to the river source, respectively. The other biomarkers showed similar trend and pointed to specific biological changes regarding oxidative stress or metal exposure in fish from the anthropogenically impacted site, especially in autumn, but without significant differences. Thus, the anthropogenic impact still seems to be only moderate, although cytosolic metals and most of the biomarkers in fish intestine were confirmed as initial indicators of pollution impact, which pointed to the need of continuous monitoring of the Krka River in order to protect this natural karst world phenomenon., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Freshwater contamination with mining waste can result with high concentrations of toxic metals in the water and in fish organs. In North-Eastern Macedonia, several rivers (e.g., Zletovska, Kriva) are exposed to acid mine drainage from active Pb/Zn mines. Previous studies confirmed high concentrations of dissolved metals in their water. This study was performed in liver and gills of Vardar chub (Squalius vardarensis Karaman, 1928) from three Macedonian rivers (Bregalnica, Kriva and Zletovska) in spring and autumn 2012. The aim was to establish if increased exposure to certain metals have resulted with their increased bioaccumulation. The concentrations of 19 elements were measured in cytosolic tissue fractions, to obtain information on metabolically available metal species. The following ranges of cytosolic concentrations of highly toxic elements were measured in the Vardar chub liver (in μg/L): Cd, 1.18-184; Cs, 0.25-25.4; Tl, 0.02-5.80; Pb, 0.70-61.1. Their ranges measured in the gills (in μg/L) were the following: Cd, 0.24-59.2; Cs, 0.39-24.4; Tl, 0.01-1.00; Pb, 0.65-87.2. Although the water of the mining impacted Zletovska River was highly contaminated with several essential metals, especially with Mn and Zn, the majority of essential elements (Na, K, Ca, Mg, Co, Cu, Fe, Mn, Mo, and Zn) did not reflect the exposure level. In contrast, seven nonessential elements reflected the level of exposure in the water. Significantly increased hepatic and gill concentrations of Cs, Rb, Sr, and Tl were detected in Vardar chub from the Zletovska River compared to the other two rivers, of Cd and Pb in the Zletovska and Kriva River compared to Bregalnica, and of V in the Bregalnica River compared to Zletovska and Kriva rivers. Observed significant metal bioaccumulation, in particular of highly toxic elements, as a consequence of exposure to water contaminated with mining waste points to necessity of intensified supervision of mining impacted rivers., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In the present study novel histopathological approach, using fish intestine as a sensitive bioindicator organ of pollution impact in the freshwater ecosystem, was proposed. Histopathological alterations were compared between native brown trout (Salmo trutta Linnaeus, 1758) from the reference (Krka River spring) and pollution impacted location (influence of technological/municipal wastewaters and agricultural runoff near the Town of Knin) of the karst Krka River in Croatia. In brown trout from both locations, severe parasitic infestation with acanthocephalan species Dentitruncus trutae was found, enabling evaluation of acanthocephalan infestation histopathology, which indicated parasite tissue reaction in a form of inflammatory, necrotic and hyperplastic response that extended throughout lamina epithelialis mucosae, lamina propria, and lamina muscularis mucosae. New semi-quantitative histological approach was proposed in order to foresee alterations classified in three reaction patterns: control tissue appearance, moderate (progressive) tissue impairment and severe (regressive and inflammatory) tissue damage. The most frequent progressive alteration was hyperplasia of epithelium on the reference site, whereas the most frequent regressive alterations were atrophy and necrosis seen on the polluted site. Furthermore, histopathological approach was combined with micromorphological and macromorphological assessment as an additional indicator of pollution impact. Among 15 observed intestinal measures, two biomarkers of intestinal tissue damage were indicated as significant, height of supranuclear space (hSN) and number of mucous cells over 100 μm fold distance of intestinal mucosa (nM), which measures were significantly lower in fish from polluted area compared to the reference site. Obtained results indicated that combined histological and morphological approach on fish intestinal tissue might be used as a valuable biological tool for assessing pollution impact on aquatic organisms. Therefore, semi quantitative scoring and multiparametric morphological assessment of intestinal tissue lesion magnitude should become a common approach to handle environmental pollution impact., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mediterranean karst aquifers are sensitive systems vulnerable to contamination, exhibiting high rates of diversity and endemicity. In the present pilot-study, we aimed to detect the most suitable bioindicators of contaminant accumulation and mobilization within a Mediterranean karst river (Krka River, Croatia), whose lowermost sections belong to a designated protection area (national park). To meet our goal, we sampled water, drift and benthos (macroinvertebrates and periphytic microfauna) at the two Krka River sites, located upstream and downstream from town Knin and its urban influences. We compared: 1) environmental conditions (water physico-chemical parameters, trace- and macro-element concentrations); 2) abundance and diversity of periphyton and macroinvertebrate taxa constituting benthos; and 3) macroinvertebrate benthos-drift relationships between the two sites. Despite higher values of all measured physico-chemical parameters, and most trace- and macro-element concentrations at the urban-influenced site, the concentrations of contamination indicators (i.e., COD, nutrients, metals) at both sites were generally low. This is likely a result of specific "self-purification ability" of the Krka River, mediated by relatively high contaminant retention potential of the underlying tufa (i.e., calcareous) and/or macrophyte substrates. Between-site differences in water quality further affected the spatial variation of macrozoobenthos, drift, and periphytic microfauna. We suggest that increased COD and orthophosphate concentration, and macrophyte presence at the urban-influenced site, supported higher densities and diversity of benthic organisms dominated by eurivalent (i.e., contamination-tolerant) taxa. The most numerous macroinvertebrate taxa in benthos were amphipod Gammarus balcanicus and the representatives of the endemic Dinaric karst taxa - gastropods Emmericia patula and Radomaniola curta germari, and another amphipod Echinogammarus acarinatus. Although we expected to observe significantly increased drift at the urban-influenced site due to the degraded environmental conditions, it was not observed. The observed benthos-drift patterns suggest that freshwater amphipods (i.e., gammarids), which were found most numerous in drift, could be considered as the most suitable bioindicators of a contaminant (i.e., metal) accumulation and mobilization within karst aquifers comparable to Krka River., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cytosolic distributions of nonessential metals Cd and Tl and seven essential elements among compounds of different molecular masses were studied in the liver of brown trout (Salmo trutta) from the karstic Krka River in Croatia. Analyses were done by size exclusion high performance liquid chromatography and high resolution inductively coupled plasma mass spectrometry. Common feature of Cd and Tl, as highly toxic elements, was their distribution within only two narrow peaks. The increase of cytosolic Cd concentrations was reflected in marked increase of Cd elution within low molecular mass peak (maximum at ∼15 kDa), presumably containing metallothioneins (MTs), which indicated successful Cd detoxification in brown trout liver under studied exposure conditions. Contrary, the increase of cytosolic Tl concentrations was reflected in marked increase of Tl elution within high molecular mass peak (maximum at 140 kDa), which probably indicated incomplete Tl detoxification. Common feature of the majority of studied essential elements was their distribution within more peaks, often broad and not well resolved, which is consistent with their numerous physiological functions. Among observed associations of essential metals/nonmetal to proteins, the following could be singled out: Cu and Zn association to MTs, Fe association to storage protein ferritin, and Se association to compounds of very low molecular masses (<5 kDa). The obtained results present the first step towards identification of metal-binding compounds in hepatic cytosol of brown trout, and thus a significant contribution to better understanding of metal fate in the liver of that important bioindicator species., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Concentrations of 23 trace elements (TEs; essential: Co, Cu, Fe, Mn, Mo, Se, V, Zn; non-essential: Ag, Al, As, Ba, Cd, Cr, Cs, Li, Ni, Pb, Rb, Sr, Ti, Tl, U) in whole soft tissues of Noah's Ark shell (Arca noae) were determined monthly during one year (March 2013-February 2014) at two sampling sites in the central part of the Eastern Adriatic Sea. Our aim was to detect the influence of reproductive cycle and changes in the environmental factors on the variabilities of TEs' contents. Higher concentrations of Pb, Ba, V, Mo, Mn and Fe were found at potentially contaminated site in Pašman channel, whereas higher concentrations of Tl, Ni, Li, Cr, Cd, Ti and Se were found at reference site in Nature Park Telašćica. Since several bioaccumulated TEs were associated to mean gonadal index, in TEs monitoring in A. noae, animal gonadal status has to be considered., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Exceptional natural value of karst rivers, as well as their potential as a source of drinking water supply, contributes to the importance of developing strict environmental protection. Although most of its watercourse is proclaimed national park, Krka River is impacted by technological and municipal wastewaters, which are released without proper purification only 2 km upstream of the park border. In order to assess water ecological status of the Krka River, vulnerability of karst ecosystem and potential threat to the Krka National Park, spatial and temporal variability of physico-chemical and microbiological water parameters, as well as concentrations of 25 total dissolved metals/metalloids were evaluated. All parameters indicated deteriorated water quality near the wastewater influences, which was for certain metals and bacterial counts even comparable to other world rivers of technological/rural catchment. Downstream location pointed to purification processes in karst river, although nutrients and conductivity were still in a range below good water quality and Al, Co, Fe, Li, Mn, Ni, Sr, Ti, and Zn levels remained comparable to their levels in the pollution impacted area, especially in autumn, indicating season-dependent processes. Presented data provided background status and identified pollution influences in the karst riverine system which indicated potential risk for protected area and a need for comprehensive water quality assessment.

Many bioindicators have not yet been well characterized regarding their tendency to bind trace elements by different cytosolic biomolecules in response to trace element exposure. Accordingly, our principal aim was to define the cytosolic distributions of Cd, Co, Cu, Fe, Mn, Mo, Se, and Zn among the biomolecules of different molecular masses in liver and gills of Vardar chub (Squalius vardarensis Karaman), a representative fish species of Macedonian rivers, and to determine distribution changes which occur as a consequence of increased exposure to specific trace elements. Additionally, we aimed to confirm the presence of heat-stable biomolecules in chub hepatic and gill cytosols. Distribution profiles were obtained by separation of cytosols and heat-treated cytosols using size-exclusion high performance-liquid chromatography, and by offline determination of trace element concentrations using high resolution inductively coupled plasma-mass spectrometry. Distribution profiles of trace elements were mainly characterized by several peaks encompassing different ranges of molecular masses, as a sign of incorporation of trace elements in various biomolecules within hepatic and gill cytosols. Especially interesting finding was probable binding of Fe to ferritin, which was especially pronounced in the liver, as a sign of important liver function in Fe storage. Furthermore, association with heat-stable proteins, metallothioneins (MT), was indicated for Cd, Cu, and Zn in the hepatic cytosol, as well as for Cd in the gill cytosol, whereas a sign of Zn-MT association was not observed in the gills. The presence of Mo- and Se-binding heat-stable compounds of very low molecular masses (<10kDa) in the cytosol was determined for both liver and the gills. Trace elements under all studied conditions were found associated to the same biomolecules, and only their proportions associated to specific cytosolic compounds have changed as a consequence of their increased bioaccumulation in the liver and gills of Vardar chub., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Total and cytosolic concentrations of twenty metals/metalloids in the liver of brown trout Salmo trutta (Linnaeus, 1758) were studied in the period from April 2015 to May 2016 at two sampling sites on Croatian river Krka, to establish if river water contamination with metals/metalloids downstream of Knin town has influenced metal bioaccumulation in S. trutta liver. Differences were observed between two sites, with higher concentrations of several elements (Ag, As, Ca, Co, Na, Se, Sr, V) found downstream of Knin town, whereas few others (Cd, Cs, Mo, Tl) were, unexpectedly, increased at the Krka River spring. However, total metal/metalloid concentrations in the liver of S. trutta from both sites of the Krka River were still mainly below previously reported levels for pristine freshwaters worldwide. The analysis of seasonal changes of metal/metalloid concentrations in S. trutta liver and their association with fish sex and size mostly indicated their independence of fish physiology, making them good indicators of water contamination and exposure level. Metal/metalloid concentrations in the metabolically available hepatic cytosolic fractions reported in this study are the first data of that kind for S. trutta liver, and the majority of analyzed elements were present in the cytosol in the quantity higher than 50% of their total concentrations, thus indicating their possible availability for toxic effects. However, the special attention should be directed to As, Cd, Cs, and Tl, which under the conditions of increased exposure tended to accumulate more within the cytosol. Although metal/metalloid concentrations in S. trutta liver were still rather low, monitoring of the Krka River water quality and of the health status of its biota is essential due to a trend of higher metal/metalloid bioaccumulation downstream of Knin town, especially taking into consideration the proximity of National Park Krka and the need for its conservation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pollution with heavy metals may influence the immune system of fish, leading to impairment of their health or even increase their mortality. The fish kidney is one of the first fish organs to be affected by water contamination. Amounts of kidney macrophages (MACs), which are involved in fish immune response, as well as the qualitative and quantitative changes in the pigmented MACs in fish kidney, are used as biomarkers of pollution. Therefore, in this study, we have evaluated relative and total volumes of trunk kidney pigmented MACs, and analyzed the pigments accumulated within them. Fish were sampled from two mining impacted rivers, Kriva and Zletovska, highly contaminated with heavy metals, and from one reference river, Bregalnica, in spring and autumn of 2012. We have observed that main pigments found in kidney MACs of Vardar chub were melanin and lipofuscin/ceroid, as well as that relative volumes of MACs ranged from 0.56 to 1.68%. Moreover, the results showed that relative volumes of pigmented MACs were higher in metal contaminated rivers, especially in autumn season in the Zletovska River, concurrently with extremely high metal exposure. In addition, condition factors and kidney somatic indices were found significantly lower in the Zletovska River in both seasons, autumn and spring, possibly also as a consequence of high water pollution. Our data confirm that increase in relative volumes of pigmented MACs may serve as warning sign of potential heavy metal pollution in aquatic environment., (© 2017 Wiley Periodicals, Inc.)

A lipid peroxidation product, malondialdehyde (MDA), was studied in Vardar chub (Squalius vardarensis Karaman) as an indicator of oxidative stress, using native fish from three rivers in northern Macedonia: the mining-impacted Zletovska and Kriva rivers and the agriculturally impacted Bregalnica River. MDA concentrations were measured in the intestine in the spring and autumn of 2012 and in the gills in autumn. The aims of the study were to establish the type of contamination which provokes a more pronounced MDA increase, as well as the organ which more reliably reflects the occurrence of oxidative stress. MDA levels in the intestine in spring amounted to 3.29-155.8 nmol g -1 and in autumn to 4.85-111.1 nmol g -1 , whereas MDA concentrations in the gills in autumn were 7.69-147.5 nmol g -1 . Stronger influence of organic contamination on development of oxidative stress was observed in both organs, as seen from higher median MDA concentrations in autumn in fish from the highly pesticide-contaminated Bregalnica River (gills 78.4 nmol g -1 ; intestine 23.5 nmol g -1 ) compared to the highly metal-contaminated Zletovska River (gills 15.9 nmol g -1 ; intestine 17.4 nmol g -1 ). The response of the gills to contamination was twice stronger than that of the intestine. The majority of fish from the pesticide-polluted river had increased MDA in the gills, in contrast to only sporadically increased MDA in the intestine. Our results indicated that development of oxidative stress strongly depends on the selected fish organ and that the gills seem to be a better choice for monitoring oxidative stress than the intestine, due to their continuous and direct exposure to polluted river water.

Target screening of 463 organic contaminants in surface water using ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) with direct injection was performed in spring of 2015 in northern Macedonia, at six sampling sites in four rivers belonging to Vardar basin: Kriva, Zletovska, Bregalnica and Vardar. The aim of the study was to differentiate between various types of organic contamination characteristic for different types of anthropogenic activities, such as mining, agriculture, and urbanization. Depending on the studied river, 9-16% of analyzed compounds were detected. The highest total levels of organic contaminants were recorded in agriculturally impacted Bregalnica River (1839-1962ngL -1 ) and Vardar River downstream from the city of Skopje (1945ngL -1 ), whereas the lowest level was found in the mining impacted Zletovska River (989ngL -1 ). The principal organic contaminants of the Bregalnica River were herbicides (45-55% of all detected compounds; 838-1094ngL -1 ), with the highest concentrations of bentazone (407-530ngL -1 ) and molinate (84-549ngL -1 ), common herbicides in rice cultivation. The main organic contaminants in the other rivers were drugs (70-80% of all detected compounds), with antibiotics as a predominant drug class. The highest drug concentrations were measured in the Vardar River, downstream from Skopje (1544ngL -1 ). Screening of surface water by UHPLC-QTOF-MS was proven as a practical tool for fast collection of comprehensive preliminary information on organic contamination of natural waters, which can present a significant contribution in the monitoring and preservation of good ecological status of freshwater ecosystems., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Commercially important bivalve Noah's Ark shell (Arca noae Linnaeus, 1758) represents a high-quality seafood product, but the data on levels of metal contaminants that could pose a human health risk and also on some essential elements that are important for health protection are lacking. This study examined the concentrations of Cd, Pb, Cr, Ni, Cu, Co, and Zn in the soft tissue of A. noae from harvesting area in the central Adriatic Sea, to survey whether heavy metals are within the acceptable limits for public health and whether tourism could have an impact on them. The concentrations of analysed metals varied for Cd: 0.15-0.74, Pb: 0.06-0.26, Cr: 0.11-0.34, Ni: 0.09-0.22, Cu: 0.65-1.95, Co: 0.04-0.09, and Zn: 18.3-74.7 mg/kg wet weight. These levels were lower than the permissible limits for safe consummation of seafood, and only for Cd, some precautions should be taken into account if older shellfish were consumed. Increase of Cd, Cr, and Cu in shell tissue was observed during the tourist season at the site closest to the marine traffic routes, indicating that metal levels in shellfish tissue should be monitored especially carefully during the peak tourist season to prevent eventual toxic effects due to increased intake of metals, specifically of Cd.

The study of metal bioaccumulation in the gills of European chub (Squalius cephalus) was conducted in September 2009 at the medium-sized rural river Sutla, characterized by agricultural and municipal type of water contamination. The concentration ranges were established for the first time in the soluble, metabolically available fractions of chub gills for 12 metals, which are environmentally extremely relevant and yet only seldom studied, as follows in a decreasing order: K, 225-895 mg L(-1); Na, 78-366 mg L(-1); Ca, 19-62 mg L(-1); Mg, 13-47 mg L(-1); Rb, 164-1762 μg L(-1); Sr, 24-81 μg L(-1); Ba, 13-67 μg L(-1); Mo, 1.3-16 μg L(-1); Co, 0.7-2.7 μg L(-1); Li, 0.4-2.2 μg L(-1); Cs, 0.2-1.9 μg L(-1); and V, 0.1-1.8 μg L(-1). The concentrations of Fe (1.6-6.4 mg L(-1)) and Mn (16-69 μg L(-1)) were also determined and were in agreement with previous reports. By application of general linear modelling, the influence of different abiotic (metal exposure level) and biotic parameters (fish sex, age, size and condition) on metal bioaccumulation was tested. It was established that bioaccumulation of many metals in fish depended on various physiological conditions, wherein Ba could be singled out as metal exhibiting the strongest association with one of biotic parameters, being significantly higher in smaller fish. However, it was also undoubtedly demonstrated that the concentrations of three metals can be applied as reliable indicators of metal exposure even in the conditions of low or moderate water contamination, such as observed in the Sutla River, and those were nonessential elements Li and Cs and essential element Fe. The results of our study present an important contribution to maintenance of high ecological status of European freshwaters, through enrichment of knowledge on the bioaccumulation of various metals in gills of European chub as frequently applied bioindicator species in monitoring of water pollution.

Many natural freshwater ecosystems, especially in the north eastern Macedonia, are polluted with heavy metals, which are released by active mines. Long-term exposure to high levels of dissolved metals might result in increased metal bioaccumulation in organs of aquatic organisms, and consequently might cause various sub-toxic and toxic effects. The aim of this study was to assess the health of Vardar chub (Squalius vardarensis) inhabiting mining impacted rivers Zletovska and Kriva, in comparison with chub from the reference Bregalnica River. It was done by use of indicators of tissue damage (histopathology of liver and gonads) and general indicators of exposure to environmental stressors (condition factor, organo-somatic indices and external/internal macroscopic lesions). Histological assessment of gonads revealed good reproductive health in all three rivers, indicating high tolerance of gonads to contaminant exposure. Contrary, several external/internal lesions were more pronounced in chub from severely metal contaminated Zletovska River. Prevalence of hepatic lesions was also higher in mining impacted rivers (in Kriva, 70%; in Zletovska, 59%) compared to Bregalnica River (38%). The spectrum of histological lesions observed in chub liver varied from non-specific minor degenerative conditions, such as lymphocyte infiltration, fibrosis, parasites, granulomas and lipidosis, to extensive and/or more severe changes such as bile duct proliferation, necrosis, megalocytosis, light-dark hepatocytes and hepatocytes regeneration. The results of histopathological investigation for all three rivers showed clear signs of water contamination, especially prominent in mining influenced rivers. More research efforts should be devoted to study of environmental conditions and metal contamination in the mining impacted rivers worldwide, especially of their effects on health of local ichthyofauna., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In the present study, Gammarus fossarum was used to investigate the bioaccumulation and toxic effects of aquatic pollutants in the real environmental conditions. The novelty of the study is the evaluation of soluble tissue metal concentrations in gammarids as indicators in early assessment of metal exposure. In the Sutla River, industrially/rurally/agriculturally influenced catchment in North-Western Croatia, physico-chemical water properties pointed to disturbed ecological status, which was reflected on population scale as more than 50 times lower gammarid density compared to the reference location, Črnomerec Stream. Significantly higher levels of soluble toxic metals (Al, As, Cd, Pb, Sb, Sn, Sr) were observed in gammarids from the Sutla River compared to the reference site and reflected the data on higher total dissolved metal levels in the river water at that site. The soluble metal estimates were supplemented with the common multibiomarker approach, which showed significant biological responses for decreased acetylcholinesterase activity and increased total soluble protein concentrations, confirming stressed environmental conditions for biota in the Sutla River. Biomarker of metal exposure, metallothionein, was not induced and therefore, toxic effect of metals was not confirmed on molecular level. Comparable between-site pattern of soluble toxic metals in gammarids and total dissolved metal levels in water suggests that prior to biomarker response and observed toxic impact, soluble metals in tissue might be used as early warning signs of metal impact in the aquatic environment and improve the assessment of water quality., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The present study was undertaken to obtain a better understanding of the seasonal variability of total dissolved metal/metalloid levels and physicochemical parameters within small- to medium-size freshwater ecosystems in temperate climate region. The research was conducted in four seasons in the Sutla River, medium-size polluted, and the Črnomerec Stream, small-size unpolluted watercourse in Croatia. In the Sutla River, characterized by the rural/industrial catchment, physicochemical parameters and total dissolved metal concentrations of 21 trace and 4 macro elements were analysed downstream of the point source of pollution, the glass production facility, indicating for the first time their variability across four seasons. Based on dissolved oxygen, total dissolved solids, nutrient concentrations, conductivity and total chemical oxygen demand, quality status of the Sutla River was good, but moderate to poor during summer, what was additionally confirmed by the highest levels of the most of 25 measured metals/metalloids in summer. Comparison with the reference small-size watercourse, the Črnomerec Stream, indicated significant anthropogenic impact on the Sutla River, most evident for Fe, Mn, Mo, Ni, Pb, Rb and Tl levels (3-70-fold higher in the Sutla River across all seasons). Generally, presented results indicated significant decrease of the water quality in the anthropogenically impacted small- to medium-size watercourses in summer, regarding physicochemical water parameters and total dissolved metal/metalloid concentrations, and pointed to significant seasonality of these parameters. Confirmed seasonality of river ecological status indicates that seasonal assessment represents a prerequisite for proper classification of the water quality in small- to medium-size temperate rivers.

Treated sewage sludge is commonly used in agriculture as fertilizer. It is, therefore, necessary to determine possible detrimental influences of sludge application on soil contamination and accumulation of contaminants in tissues of terrestrial animals, which in the long run could also have undesirable effects on humans. With that aim, the study was performed using earthworm Eisenia fetida as test organism and semi-solid depot sludge from a wastewater treatment plant as exposure media. The concentrations of 26 metals/metalloids were determined in depot sludge, and their bioaccumulation was estimated in whole tissue of E. fetida, and for the first time in the soluble tissue fraction, which represents metal fraction available for metabolic requirements and toxic effects. Obtained results have revealed acceptable levels of several elements (Cd, Cr, Cu, Ni, Pb, Zn) in depot sludge, when compared to currently valid regulations, and only moderate accumulation of some elements (e.g. As, Ba, Cd, Co, Fe, Tl, V, and Zn) in earthworms, as a consequence of exposure to depot sludge. However, a concentration increase after exposure to depot sludge was observed in E. fetida for several elements (Cd, Mo, and Zn), which were present in lower concentrations in the exposure mixtures than in soil. Contrary, a concentration decrease was observed for Cs, Mn, and Rb, although they were present in higher concentrations in depot sludge than in soil. It was an indication of disturbance in metal homeostasis in earthworms, possibly caused by exposure to complex mixture of contaminants present in depot sludge. The cumulative effect of exposure to a number of various contaminants (inorganic, organic, microbiological and pharmaceutical), even if each of them was not present in very high concentrations, could have caused distress in earthworms exposed to depot sludge., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Quantification of histopathological alterations in the gills of Vardar chub (Squalius vardarensis Karaman) was performed in 2012 in rivers of north-eastern Macedonia, with the aim to examine the effects of water quality in the rivers (Zletovska and Kriva River-impacted by active Pb/Zn mines; Bregalnica River-contaminated by agricultural waste). The biological alterations in chub were classified as: circulatory disturbances, regressive and progressive changes, but their severity differed. Altogether the mildest changes were observed in the gills of chub from the Bregalnica River, a less polluted river, whereas mining impacted rivers were characterized by more severe alterations. In the gills of chub from the Zletovska River, which is highly contaminated with numerous metals, sulphates and chlorides, the highest lesion indices were found for the regressive changes of both epithelium and supporting tissue, with typical lesions referring to atrophy, thinning and lifting of epithelial cells, necrosis of epithelium and chloride cells, as well as deformations of lamellar cartilaginous base. Gill damages of chub from the Kriva River were overall milder compared to the Zletovska River, in accordance with pollution status. In the gills of chub from that river, progressive changes were more pronounced, specifically severe hyperplasia of mucous cells and epithelium in the interlammellar space, leading to fusion of lamellae, as well as hypertrophy of chloride cells. The comparison between seasons indicated higher intensity of progressive changes in all three rivers in autumn, when water level was very low, and consequently, water contamination was more pronounced due to concentration effect. The pattern and severity of histopathological alterations in the chub gills reflected differences in contamination levels and type of contaminants in different rivers and sampling periods, and thus have been proven as a valuable indicator of water quality., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The distribution of essential elements Co, Cu, Fe, Mn, Se, and Zn, and nonessential element Cd among cytosolic proteins of different molecular masses in the gills of European chub (Squalius cephalus) sampled in the moderately contaminated Sutla River in September of 2009, was studied after the protein separation by size exclusion high-performance liquid chromatography (SEC-HPLC), and the metal determination in the obtained fractions by high-resolution inductively coupled plasma mass spectrometry (HR ICP-MS). The aims of the study were to characterize the distribution profiles of metals within different protein categories in gills in the conditions of low metal exposure in the river water, and to compare them with the previously published hepatic profiles. The distribution profiles of analyzed metals were mainly characterized with several peaks. However, some observations could be emphasized: both Cu and Cd were eluted near metallothionein elution time; elution time of one of Co peaks could be associated with Co-containing compound cobalamin; increasing cytosolic Fe concentrations resulted in possible Fe binding to storage protein ferritin; both Mn and Zn had poorly resolved peaks covering wide ranges of molecular masses and indicating their binding to various proteins; both Zn and Se increased in protein fractions of molecular masses <5 kDa following their concentration increase in the gill cytosol; expected clear metallothionein peak was not observed for Zn. Comparison of gill profiles with previously published hepatic profiles revealed similar and in case of some elements (e.g., Co, Fe, Mn, and Se) almost identical distributions in both organs regarding elution times. On the contrary, heights of obtained peaks were different, indicating possible metal binding to the same proteins in the gills and liver, but in different proportions. The results obtained in this study can be used as a basis for comparison in monitoring studies, for identification of changes that would occur after exposure of chub to increased metal concentrations.

Mine waste is recognized as being one of the most serious threats for freshwater ecosystems, and it still represents one of the greatest environmental concerns in Macedonia. The aim of our investigation was to obtain an in-depth understanding of mining influence on freshwater systems from water contamination to effects on aquatic organisms. In this study, we assessed the impact of active lead (Pb)/zinc (Zn) mines Zletovo and Toranica on the water quality of three rivers in northeastern Macedonia (Bregalnica, Zletovska, and Kriva rivers) based on data collected in spring and autumn of 2012. The Bregalnica River, near Shtip, was characterized mainly by weak contamination with arsenic, barium, iron, molybdenum, titanium, uranium, vanadium, nitrate, and phosphate, as well as critical faecal pollution, which alltogether could be connected to agricultural activities; however, an impact of the mines was not observed. Contrary, both the Zletovska and Kriva rivers showed a clear impact of Pb/Zn mines on water quality. In the Zletovska River, increased concentrations of cadmium (Cd), cobalt, cesium, copper, lithium, manganese (Mn), nickel, rubidium, tin, strontium, thallium, Zn, sulphates, and chlorides were found, especially in autumn (e.g., Cd 2.0 μg L(-1); Mn 2.5 mg L(-1); Zn 1.5 mg L(-1)). In the Kriva River, increased Cd (0.270 μg L(-1)) and Pb (1.85 μg L(-1)) concentrations were found only in spring, possibly due to sediment resuspension during greater water discharge. The selected sampling sites on the Bregalnica, Zletovska, and Kriva rivers were confirmed as being appropriate locations for further studies of mining waste's impact on freshwater ecosystems, the first one as a nonimpacted site and the other two as possible areas of increased exposure of aquatic organisms to metals.

The assessment of general condition of fish in the moderately contaminated aquatic environment was performed on the European chub (Squalius cephalus) caught in September 2009 in the Sutla River in Croatia. Although increases of the contaminants in this river (trace and macro elements, bacteria), as well as physico-chemical changes (decreased oxygen saturation, increased conductivity), were still within the environmentally acceptable limits, their concurrent presence in the river water possibly could have induced stress in aquatic organisms. Several biometric parameters, metallothionein (MT), and total cytosolic protein concentrations in chub liver and gills were determined as indicators of chub condition. Microbiological and parasitological analyses were performed with the aim to evaluate chub predisposition for bacterial bioconcentration and parasitic infections. At upstream river sections with decreased oxygen saturation (~50%), decreased Fulton condition indices were observed (FCI: 0.94 g cm(-3)), whereas gonadosomatic (GSI: 2.4%), hepatosomatic (HSI: 1.31%), and gill indices (1.3%) were increased compared to oxygen rich downstream river sections (dissolved oxygen ~90%; FCI: 1.02 g cm(-3); GSI: 0.6%; HIS: ~1.08%; gill index: 1.0%). Slight increase of MT concentrations in both organs at upstream (gills: 1.67 mg g(-1); liver: 1.63 mg g(-1)) compared to downstream sites (gills: 1.56 mg g(-1); liver: 1.23 mg g(-1)), could not be explained by induction caused by increased metal levels in the river water, but presumably by physiological changes caused by general stress due to low oxygen saturation. In addition, at the sampling site characterized by inorganic and fecal contamination, increased incidence of bacterial bioconcentration in internal organs (liver, spleen, kidney) was observed, as well as decrease of intestinal parasitic infections, which is a common finding for metal-contaminated waters. Based on our results, it could be concluded that even moderate contamination of river water by multiple contaminants could result in unfavourable living conditions and cause detectable stress for aquatic organisms.

Association of selected essential (Co, Cu, Fe, Mn, Mo, Se, and Zn) and nonessential (Cd, Pb) trace elements with cytosolic proteins of different molecular masses was described for the liver of European chub (Squalius cephalus) from weakly contaminated Sutla River in Croatia. The principal aim was to establish basic trace element distributions among protein fractions characteristic for the fish living in the conditions of low metal exposure in the water. The fractionation of chub hepatic cytosols was carried out by size exclusion high performance liquid chromatography (SE-HPLC; Superdex™ 200 10/300 GL column), and measurements were performed by high resolution inductively coupled plasma mass spectrometry (HR ICP-MS). Elution profiles of essential elements were mostly characterized by broad peaks covering wide range of molecular masses, as a sign of incorporation of essential elements in various proteins within hepatic cytosol. Exceptions were Cu and Fe, with elution profiles characterized by sharp, narrow peaks indicating their probable association with specific proteins, metallothionein (MT), and ferritin, respectively. The main feature of the elution profile of nonessential metal Cd was also single sharp, narrow peak, coinciding with MT elution time, and indicating almost complete Cd detoxification by MT under the conditions of weak metal exposure in the water (dissolved Cd concentration ≤0.3 μg L(-1)). Contrary, nonessential metal Pb was observed to bind to wide spectrum of proteins, mostly of medium molecular masses (30-100 kDa), after exposure to dissolved Pb concentration of ~1 μg L(-1). The obtained information within this study presents the starting point for identification and characterization of specific metal/metalloid-binding proteins in chub hepatic cytosol, which could be further used as markers of metal/metalloid exposure or effect on fish.

The possibility of direct measurement of trace elements in hepatic cytosol of European chub (Squalius cephalus) by high-resolution inductively coupled plasma mass spectrometry (HR ICP-MS) after cytosol dilution with Milli-Q water and subsequent acidification was investigated. Due to low detection limits of this procedure, determination of 13 elements (As, Cd, Co, Cu, Fe, Mn, Mo, Pb, Sb, Sn, Sr, V and Zn) was possible in the chub hepatic cytosol, exhibiting excellent measurement repeatability in duplicates. Some of these elements were also measured by HR ICP-MS in acid digested cytosols (Cd, Co, Cu, Fe, Mn, Mo, Sr, V and Zn). Good agreement of the results obtained after sample dilution and sample digestion indicated that complex organic matrix of hepatic cytosol did not affect measurement reliability. Cytosolic concentrations of 13 trace elements in the chub liver were quantified in the following order: Fe, Zn>Cu>Mn>Mo>Sr, V, Cd>Co>As, Pb>Sn>Sb. Unlike Cd, Cu, Fe, Mn and Zn for which the cytosolic concentrations were previously reported after measurement by AAS, cytosolic concentrations of eight additional trace elements characteristic for the liver of chubs inhabiting the low contaminated river water were reported here for the first time (in nanogrammes per gramme)-Mo, 136.8-183.6; Sr, 32.7-63.0; V, 17.5-69.0; Co, 24.3-30.7; As, 9.9-29.5; Pb, 5.8-35.6; Sn, 5.5-12.4; and Sb, 0.9-2.6. The simultaneous measurement of large number of trace elements in the cytosolic fractions of fish tissues, which comprise potentially metal-sensitive sub-cellular pools, could be beneficial as a screening tool in the monitoring of natural waters, because it would enable timely recognition of increased fish exposure to metals.

Purpose: To examine if chronic exposure of feral fish to elevated Pb concentrations in the river water (up to 1 μg L(-1)), which are still lower than European recommendations for dissolved Pb in surface waters (7.2 μg L(-1); EPCEU (Official J L 348:84, 2008)), would result in Pb accumulation in selected fish tissues., Methods: Lead concentrations were determined by use of HR ICP-MS in the gill and hepatic soluble fractions of European chub (Squalius cephalus) caught in the Sutla River (Croatia-Slovenia)., Results: At the site with increased dissolved Pb in the river water, soluble gill Pb levels (17.3 μg L(-1)) were approximately 20 times higher compared to uncontaminated sites (0.85 μg L(-1)), whereas the ratio between contaminated (18.1 μg L(-1)) and uncontaminated sites (1.17 μg L(-1)) was lower for liver (15.5). Physiological variability of basal Pb concentrations in soluble gill and hepatic fractions associated to fish size, condition, sex, or age was not observed, excluding the possibility that Pb increase in chub tissues at contaminated site could be the consequence of studied biotic parameters. However, in both tissues of Pb-exposed specimens, females accumulated somewhat more Pb than males, making female chubs potentially more susceptible to possible toxic effects., Conclusions: The fact that Pb increase in gill and hepatic soluble fractions of the European chub was not caused by biotic factors and was spatially restricted to one site with increased dissolved Pb concentration in the river water points to the applicability of this parameter as early indicator of Pb exposure in monitoring of natural waters.

The study on medium size river Sutla in Croatia indicated considerable water contamination at specific sites during the baseflow period, probably associated to low flow-rate (0.73-68.8 m3 s(-1)), and consequently low dilution capacity of this river. Various aspects of contamination were observed: increased conductivity to 1,000 microS cm(-1), decreased dissolved oxygen level to 50%, 4-5 degrees C increased water temperature, increased concentrations of several dissolved trace elements (e.g., maximal values of Li: 45.4 microg l(-1); Rb: 10.4 microg l(-1); Mo: 20.1 microg l(-1); Cd: 0.31 microg l(-1); Sn: 30.2 microg l(-1); Sb: 11.8 microg l(-1); Pb: 1.18 microg l(-1); Ti: 1.03 microg l(-1); Mn: 261.1 microg l(-1); and Fe: 80.5 microg l(-1)) and macro elements (e.g., maximal values of Na: 107.5 mg l(-1); and K: 17.3 mg l(-1)), as well as moderate or even critical fecal (E. coli: 4,888 MPN/100 ml; total coliforms: 45,307 MPN/100 ml; enterococci: 1,303 MPN/100 ml) and organic pollution (heterotrophic bacteria: 94,000 cfu/ml). Although metal concentrations still have not exceeded the limits considered as hazardous for aquatic life or eventually for human health, the observed prominent increases of both metal concentrations and bacterial counts in the river water should be considered as a warning and incentive to protect the small and medium size rivers from the future deterioration, as recommended by EU Water Framework Directive.

Background: Due to their popularity as a complementary therapy in many diseases, homeopathic products of animal, vegetable, mineral and chemical origin should be tested for the presence of contaminants to prevent eventual toxic effects., Objective: Thirty samples of homeopathic products were analyzed to estimate possible contamination with potentially toxic elements: Pb, Cd, As, Hg, Cr, Ni and Zn, and to assess human exposure to these metals/metalloid as a consequence of their consumption., Methods: Atomic absorption spectrometry was used to determine metal and metalloid concentrations., Results: Most tested products had very low metal/metalloid levels (below the limit of quantification of the method), but the metal/metalloid levels in the remaining products were in the following ranges (in microg g(-1)): Pb 0.33-1.29 (6 samples), Cd 2.78 (1 sample), As 0.22 (1 sample), Hg 0.02-0.12 (24 samples), Cr 0.40-10.27 (10 samples), Ni 0.43-55.00 (19 samples), and Zn 2.20-27.80 (11 samples). In the absence of regulatory standards for homeopathic products, the obtained results were compared to maximum allowable levels (MALs) as proposed by USP Ad Hoc Advisory Panel. Some analyzed preparations had metal levels above MALs (Pb: 2 samples; Cd: 1 sample; Ni: 2 samples). However, estimated cumulative daily intakes from tested homeopathic products were in all cases lower than permitted daily exposures for all dosage forms., Conclusion: The risk of bioaccumulation of metals/metalloid from the homeopathic medicines seems to be rather low, due to small quantities of those products prescribed to be applied per day, as well as insignificant metal contamination of the majority of tested products. However, the fact that particular formulations were contaminated by metals above MALs indicates potential risk and points to the necessity of regular monitoring of homeopathic products for metal contamination, due to their frequent and mostly unsupervised use., (2010 Elsevier Ltd. All rights reserved.)

Purpose: The aim of this study was to compare the level of metal contamination in two bays in the middle part of the Eastern Adriatic coastal zone in Croatia using the gills of mussels Mytilus galloprovincialis as indicator tissue. Despite the existing sources of contamination, previous studies with caged mussels only indicated moderate metal contamination of the Kastela Bay, contrary to the Trogir Bay in which marina and shipyard present a probable source of Cu- and Zn-contamination., Methods: The measurements of metallothioneins (MTs) and metals that induce MT synthesis (Cu, Zn, and Cd) were performed in the heat-treated gill cytosol and total proteins (TPs) in the untreated gill cytosol. MTs were determined by differential pulse voltammetry, Cu and Zn by flame atomic absorption spectrometry (AAS), Cd by graphite furnace AAS, and TPs by Bradford spectrophotometric procedure., Results: The results collected in four sampling campaigns (autumn periods from 2001 to 2004) indicated that MT levels in mussel gills (expressed on dry mass basis 2.3+/-0.3 mg g(-1)) were comparable with basal levels reported in the literature (2.5+/-0.8 mg g(-1)). Observed interindividual, temporal, and spatial MT variability could be associated with different confounding factors, such as the time of sampling, total protein concentration, and mussel size rather than cytosolic levels of Cu and Zn. Metal levels, expressed on wet mass basis, in the heat-treated gill cytosol ranged from 1.33 to 11.31 microg g(-1) for Zn, from 0.72 to 2.96 microg g(-1) for Cu, and from 0.036 to 0.100 microg g(-1) for Cd. The highest Zn level was measured at Vranjic (Kastela Bay)-the site influenced by untreated domestic wastewater, while somewhat increased Zn and the highest Cu levels were found at marina and shipyard locations (Trogir Bay). The highest Cd level was measured at Inavinil (Kastela Bay)., Conclusions: The observed association of gill MT levels with several biotic and abiotic factors limits its use as the biomarker of low-level metal exposure. Therefore, the use of the metal concentrations in the heat-treated gill cytosol of Mediterranean mussels should be considered for the assessment of the low-level metal contamination of coastal marine areas.

This study provides the preliminary data set for total dissolved trace metal concentrations in the surface water of the Sava River in Croatia and the assessment of Sava River water quality status. The highest levels of total dissolved metals were observed for Fe, Mn, and Zn (12.6 +/- 7.8 mirog L(-1), 3.44 +/- 3.95 mirog L(-1), and 2.27 +/- 1.53 mirog L(-1), respectively), the intermediate concentrations for Ni, Cu, and Cr (0.59 +/- 0.14 mirog L(-1), 0.54 +/- 0.14 mirog L(-1), and 0.32 +/- 0.06 mirog L(-1), respectively), and the lowest levels for Co, Pb, and Cd (0.064 +/- 0.022 mirog L(-1), 0.055 +/- 0.051 mirog L(-1), and 0.011 +/- 0.004 mirog L(-1), respectively). The results refer to the grab water samples taken at five sites in the period from March to June, 2006. For four trace metals (Mn, Pb, Zn, and Fe), the high temporal variability within one season was observed. It can present a problem for reliable evaluation of total dissolved concentrations of these metals in the river water, if the assessment is based on the occasional grab water sampling. The comparison of results obtained in this study with previously reported data for several unpolluted rivers indicated that Sava River water reflects a certain anthropogenic impact. However, according to the levels proposed by European regulations, it still can be classified as water containing total dissolved trace metals in concentrations not significantly above the natural level.

In autumn 2005, the site-specific variability of cytosolic metal and protein concentrations in gills of European chub from the Sava River could be mostly associated with gill mass variability. In spring 2006, the correlations of metals and proteins with gill mass were mainly nonsignificant, and their site-specific variability could be presumably associated with river water pollution. The spring cytosolic concentrations of Zn, Cu, and Mn did not differ significantly between the sites (medians: 8.37-11.34 microg ml(-1), 68.2-86.2 ng ml(-1), and 55.9-68.6 ng ml(-1), respectively). Increased cytosolic Cd concentrations were obtained at Oborovo and Lukavec Posavski (median: 4.01 ng ml(-1))-the sites influenced by pollution sources from two major urban areas-compared with the remaining sampling sites (median: 1.93 ng ml(-1)). Cytosolic Fe concentrations were almost two times higher at Jasenovac (median: 11.98 microg ml(-1)) compared with the concentrations at Sava-Zagreb (median: 6.72 microg ml(-1)). Labile Fe concentrations measured in river water with passive samplers indicated that cytosolic Fe concentrations in the spring possibly reflected water-borne Fe uptake. The spring cytosolic protein concentrations decreased from upstream (Otok Samoborski: 27.2 +/- 5.6 mg ml(-1)) toward the downstream sites (Lukavec Posavski: 14.8 +/- 2.8 mg ml(-1)), possibly because of the influence of organic pollution and water toxicity. The spring period seems to be a more appropriate season for the assessment of the river water pollution if chub gills are used as the target organ.