The study of acidic/basic nature of metallothioneins and other metal-binding biomolecules in the soluble hepatic fraction of the northern pike (Esox lucius).

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.

Dynamics of drug contamination of the river-water in the rural, semirural and urban areas of the Mreznica River in Croatia during COVID-19 pandemic (2020-2021).

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 Mreznica 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.

Accumulation of metal(loid)s in the digestive gland of the mussel Unio crassus Philipsson, 1788: A reliable detection of historical freshwater contamination.

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 Mreznica 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.

Yesterday's contamination-A problem of today? The case study of discontinued historical contamination of the Mreznica River (Croatia).

The remnants of historical industrial contamination can be detected in many aquatic ecosystems worldwide even at present time. Mreznica 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 Mreznica 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.

Efficiency of metal bioaccumulation in acanthocephalans, gammarids and fish in relation to metal exposure conditions in a karst freshwater ecosystem.

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.

Metal-binding biomolecules in the liver of northern pike (Esox lucius Linnaeus, 1758): The first data for the family Esocidae.

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.

Disturbance in invasion? Idiopathic necrotizing hepatopancreatitis in the signal crayfish Pacifastacus leniusculus (Dana, 1852) in Croatia.

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.

First insight in trace element distribution in the intestinal cytosol of two freshwater fish species challenged with moderate environmental contamination.

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.

Gold and silver nanoparticles effects to the earthworm Eisenia fetida - the importance of tissue over soil concentrations.

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.

Comparison of intracellular trace element distributions in the liver and gills of the invasive freshwater fish species, Prussian carp (Carassius gibelio Bloch, 1782).

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.

The assessment of metal contamination in water and sediments of the lowland Ilova River (Croatia) impacted by anthropogenic activities.

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 Maslenjaca, Ilova and Trebez) 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 (Trebez village) impacted by the activity of fertilizer factory. Water from the Ilova and Maslenjaca villages was of good quality, whereas COD, nitrates and phosphates exceeded the good quality levels in Trebez 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 Trebez 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 Trebez 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.

Evaluation of multi-biomarker response in fish intestine as an initial indication of anthropogenic impact in the aquatic karst environment.

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.

Acute toxicity of selenate and selenite and their impacts on oxidative status, efflux pump activity, cellular and genetic parameters in earthworm Eisenia andrei.

Selenium (Se) is an essential element for humans, animals, and certain lower plants, but can be toxic at high concentration. Even though Se is potentially toxic, little information is available about the effects of Se on soil animals. The aim of this study was to assess the impact of different concentrations of two Se forms, selenate and selenite, on earthworm Eisenia andrei. In order to obtain comprehensive overview on the Se effects, different parameters were measured. Namely, acute toxicity, apoptosis, efflux pump activity, different enzymatic and non-enzymatic biomarkers (acetylcholinesterase, carboxylesterase, glutathione S-transferase, catalase, glutathione reductase and superoxide dismutase activities, lipid peroxidation level and GSH/GSSG ratio) and expression of genes involved in oxidative and immune response have been investigated. Additionally, measurement of metallothioneins concentration and concentration of Se in exposed earthworms has been also performed. The assessment of acute toxicity showed a greater sensitivity of E. andrei to selenite exposure, whereas Se concentration measurements in earthworms showed higher accumulation of selenate form. Both Se forms caused inhibition of the efflux pump activity. Decrease in superoxide dismutase activity and increase in lipid peroxidation and glutathione reductase activity indicate that Se has a significant impact on the oxidative status of earthworms. Selenate exposure caused an apoptotic-like cell death in the coelomocytes of exposed earthworms, whereas decreased mRNA levels of stress-related genes and antimicrobial factors were observed upon the exposure to selenite. The obtained data give insight into the effects of two most common forms of Se in soil on the earthworm E. andrei.

Changes in the tissue concentrations of trace elements during the reproductive cycle of Noah's Ark shells (Arca noae Linnaeus, 1758).

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 Pasman channel, whereas higher concentrations of Tl, Ni, Li, Cr, Cd, Ti and Se were found at reference site in Nature Park Telascica. Since several bioaccumulated TEs were associated to mean gonadal index, in TEs monitoring in A. noae, animal gonadal status has to be considered.

Benthos-drift relationships as proxies for the detection of the most suitable bioindicator taxa in flowing waters - a pilot-study within a Mediterranean karst river.

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.

Evaluation of architectural and histopathological biomarkers in the intestine of brown trout (Salmo trutta Linnaeus, 1758) challenged with environmental pollution.

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.

Cytosolic distributions of highly toxic metals Cd and Tl and several essential elements in the liver of brown trout (Salmo trutta L.) analyzed by size exclusion chromatography and inductively coupled plasma mass spectrometry.

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.

Total and cytosolic concentrations of twenty metals/metalloids in the liver of brown trout Salmo trutta (Linnaeus, 1758) from the karstic Croatian river Krka.

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.

Influence of technological and municipal wastewaters on vulnerable karst riverine system, Krka River in Croatia.

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.

Age and sex differences in genome damage between prepubertal and adult mice after exposure to ionising radiation.

The mechanisms that lead to sex and age differences in biological responses to exposure to ionising radiation and related health risks have still not been investigated to a satisfactory extent. The significance of sex hormones in the aetiology of radiogenic cancer types requires a better understanding of the mechanisms involved, especially during organism development. The aim of this study was to show age and sex differences in genome damage between prepubertal and adult mice after single exposure to gamma radiation. Genome damage was measured 24 h, 48 h, and 72 h after exposure of 3-week and 12-week old BALB/CJ mice to 8 Gy of gamma radiation using an in vivo micronucleus assay. There was a significantly higher genome damage in prepubertal than in adult animals of both sexes for all sampling times. Irradiation caused a higher frequency of micronuclei in males of both age groups. Our study confirms sex differences in the susceptibility to effects of ionising radiation in mice and is the first to show that such a difference occurs already at prepubertal age.