Hooking the scientific community on thorny-headed worms: interesting and exciting facts, knowledge gaps and perspectives for research directions on Acanthocephala.

Although interest in Acanthocephala seems to have reached only a small community of researchers worldwide, we show in this opinion article that this group of parasites is composed of excellent model organisms for studying key questions in parasite molecular biology and cytogenetics, evolutionary ecology, and ecotoxicology. Their shared ancestry with free-living rotifers makes them an ideal group to explore the origins of the parasitic lifestyle and evolutionary drivers of host shifts and environmental transitions. They also provide useful features in the quest to decipher the proximate mechanisms of parasite-induced phenotypic alterations and better understand the evolution of behavioral manipulation. From an applied perspective, acanthocephalans' ability to accumulate contaminants offers useful opportunities to monitor the impacts - and evaluate the possible mitigation - of anthropogenic pollutants on aquatic fauna and develop the environmental parasitology framework. However, exploring these exciting research avenues will require connecting fragmentary knowledge by enlarging the taxonomic coverage of molecular and phenotypic data. In this opinion paper, we highlight the needs and opportunities of research on Acanthocephala in three main directions: (i) integrative taxonomy (including non-molecular tools) and phylogeny-based comparative analysis; (ii) ecology and evolution of life cycles, transmission strategies and host ranges; and (iii) environmental issues related to global changes, including ecotoxicology. In each section, the most promising ideas and developments are presented based on selected case studies, with the goal that the present and future generations of parasitologists further explore and increase knowledge of Acanthocephala.

Title: Accrocher la communauté scientifique à des vers à la tête pleine d'épines : faits intéressants et passionnants, lacunes dans les connaissances et perspectives pour des orientations de recherche sur les Acanthocéphales. Abstract: Bien que l'intérêt pour les acanthocéphales semble n'avoir atteint qu'un petit nombre de chercheurs dans le monde, nous montrons dans cet article que ce groupe de parasites est composé d'excellents organismes modèles pour étudier les questions en suspens en biologie moléculaire et cytogénétique, écologie évolutive et écotoxicologie. Leur ascendance partagée avec les rotifères en fait un groupe idéal pour explorer les origines du mode de vie parasitaire et les moteurs évolutifs des changements d'hôtes et des transitions environnementales. Ils présentent également des caractéristiques intéressantes pour l'étude des mécanismes proximaux sous-tendant les altérations phénotypiques induites par les parasites, et ainsi mieux comprendre l'évolution de la manipulation comportementale. D'un point de vue appliqué, la capacité des acanthocéphales à accumuler les contaminants offre des opportunités utiles pour surveiller les impacts - et évaluer les possibilités d'atténuation - des pollutions anthropiques sur la faune aquatique et développer le domaine de la parasitologie environnementale. Cependant, l'exploration de ces pistes de recherche passionnantes nécessitera de relier des connaissances fragmentaires en élargissant la couverture taxonomique des données moléculaires et phénotypiques. Dans cet article, nous présentons l'état actuel de la recherche sur les acanthocéphales selon trois axes principaux : (i) la taxonomie intégrative (y compris les outils non-moléculaires) et la phylogénie à des fins d'analyse comparative ; (ii) l'écologie et l'évolution des cycles de vie, des stratégies d'exploitation des hôtes et de transmission ; (iii) les questions environnementales liées aux changements globaux, y compris l'écotoxicologie. Dans chaque section, nous soulignons les besoins et les opportunités, en espérant que cela incitera une nouvelle génération de parasitologues à s'intéresser aux acanthocéphales.

First insight in element localisation in different body parts of the acanthocephalan Dentitruncus truttae using TEM and NanoSIMS.

Acanthocephalans, intestinal parasites of vertebrates, are characterised by orders of magnitude higher metal accumulation than free-living organisms, but the mechanism of such effective metal accumulation is still unknown. The aim of our study was to gain new insights into the high-resolution localization of elements in the bodies of acanthocephalans, thus taking an initial step towards elucidating metal uptake and accumulation in organisms under real environmental conditions. For the first time, nanoscale secondary ion mass spectrometry (NanoSIMS) was used for high-resolution mapping of 12 elements (C, Ca, Cu, Fe, N, Na, O, P, Pb, S, Se, and Tl) in three selected body parts (trunk spines, inner part of the proboscis receptacle and inner surface of the tegument) of Dentitruncus truttae, a parasite of brown trout (Salmo trutta) from the Krka River in Croatia. In addition, the same body parts were examined using transmission electron microscopy (TEM) and correlated with NanoSIMS images. Metal concentrations determined using HR ICP-MS confirmed higher accumulation in D. truttae than in the fish intestine. The chemical composition of the acanthocephalan body showed the highest density of C, Ca, N, Na, O, S, as important and constitutive elements in living cells in all studied structures, while Fe was predominant among trace elements. In general, higher element density was found in trunk spines and tegument, as body structures responsible for substance absorption in parasites. The results obtained with NanoSIMS and TEM-NanoSIMS correlative imaging represent pilot data for mapping of elements at nanoscale resolution in the ultrastructure of various body parts of acanthocephalans and generally provide a contribution for further application of this technique in all parasite species.

Pollution impact on metal and biomarker responses in intestinal cytosol of freshwater fish.

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.

A preliminary study of the cultivable microbiota on the plastic litter collected by commercial fishing trawlers in the south-eastern Adriatic Sea, with emphasis on Vibrio isolates and their antibiotic resistance.

Mediterranean Sea is the sixth largest area of marine litter accumulation in the world, and plastic pollution is a growing problem in its Adriatic sub-basin. The aim of the present study was to evaluate the cultivable microbiota associated with plastic litter collected by commercial fishing trawlers in the south-eastern Adriatic Sea in comparison with microbiota in seawater and sediment. Plastic litter in the sea contains an autochthonous microbiota that is different from that of the surrounding seawater and sediment. Vibrio abundance was higher on plastic litter than in surrounding seawater and sediment. All isolated Vibrio showing resistance to ampicillin and vancomycin, while resistance to other antibiotics depended on the isolated species. Overall, this study provides for the first time information on the cultivable microbiota associated with plastic litter collected by commercial fishing trawlers and provides a data base for further studies.

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.

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.

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 effect of different pollutants exposure on the pigment content of pigmented macrophage aggregates in the spleen of Vardar chub (Squalius vardarensis Karaman, 1928).

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).

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.

Mining waste as a cause of increased bioaccumulation of highly toxic metals in liver and gills of Vardar chub (Squalius vardarensis Karaman, 1928).

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.

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.

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.

Malondialdehyde concentrations in the intestine and gills of Vardar chub (Squalius vardarensis Karaman) as indicator of lipid peroxidation.

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.

Does the Serum Metallothionein Level Reflect the Stage of Testicular Germ Cell Tumor?

Increased levels of metallothionein (MT) have recently been found in the blood serum of men with newly diagnosed testicular germ cell tumors (TGCT). In light of previously published results, the aim of this study was to investigate the difference in serum MT levels among patients with different stages of TGCT and compare MT with commonly used markers (α-fetoprotein, ß-human chorionic gonadotropin and lactate dehydrogenase). The concentration of total MT was determined in the serum of 25 men with TGCT (seminoma or non-seminoma) by differential pulse voltammetry. Serum samples were obtained prior to chemotherapy, after two cycles of chemotherapy and 1 year after chemotherapy. A statistically significant difference in MT levels in patients with different stages of TGCT was observed in the serum of patients with non-seminoma obtained before chemotherapy. Although not significant, an increase in serum MT levels commensurate with the disease stage increase was also observed in patients with seminomatous TGCT. The results indicate that, in combination with the existing markers, MT could be useful for the identification of the histological type of tumor and stage of the disease before biopsy diagnosis.

Investigation of the soluble metals in tissue as biological response pattern to environmental pollutants (Gammarus fossarum example).

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, Crnomerec 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.