Interrelation between environmental conditions, acanthocephalan infection and metal(loid) accumulation in fish intestine: an in-depth study.

Metal(loid) bioaccumulation in acanthocephalans (Dentitruncus truttae) and intestines of fish (Salmo trutta) from the Krka River, influenced by industrial and municipal wastewaters, was investigated in relation to exposure to metal(loid)s from fish gut content (GC), water, and sediment to estimate potentially available metal (loid)s responsible for toxic effects and cellular disturbances in biota. Sampling was performed in two seasons (spring and autumn) at the reference site (river source, KRS), downstream of the wastewater outlets (Town of Knin, KRK), and in the national park (KNP). Metal(loid) concentrations were measured by ICP-MS. The highest accumulation of As, Ba, Ca, Cu, Fe, Pb, Se and Zn was observed mainly in organisms from KRK, of Cd, Cs, Rb and Tl at KRS, and of Hg, Mn, Mo, Sr and V at KNP. Acanthocephalans showed significantly higher bioaccumulation than fish intestine, especially of toxic metals (Pb, Cd and Tl). Metal(loid) bioaccumulation in organisms partially coincided to exposure from water, sediments and food, while in GC almost all elements were elevated at KNP, reflecting the metal(loid) exposure from sediments. Seasonal differences in organisms and GC indicated higher metal (loid) accumulation in spring, which follows enhanced fish feeding rates. Higher number of acanthocephalans in the intestine influenced biodilution process and lower concentrations of metal(loid)s in fish, indicating positive effects of parasites to their host, as supported by high values of bioconcentration factors. Fish intestine and acanthocephalan D. truttae were confirmed as sensitive indicators of available metal fraction in conditions of generally low environmental exposure in karst ecosystem. Since metal(loid) accumulation depended on ecological, chemical and biological conditions, but also on the dietary habits, physiology of organisms and parasite infection, continuous monitoring is recommended to distinguish between the effects of these factors and environmental exposure when assessing dietary associated metal(loid) exposure in aquatic organisms.

Long-term and seasonal trends of water parameters in the karst riverine catchment and general literature overview based on CiteSpace.

Although Europe is the continent with the highest proportion of karst areas, where hydrological systems are essential but extremely sensitive, data on the ecological status of karst riverine catchments are scarce. The aim of the present study was to assess the spatial and temporal (long-term and seasonal) variability of the physico-chemical and organic water parameters in the headwaters of the Krka River and its tributaries, as representatives of a typical karst ecosystem, situated in one of the largest karst areas in Europe, Dinarides in Croatia. It is affected in its upper reaches by improperly treated wastewaters, so anthropogenic influences and ecological status were estimated with the aim to present consequences of pollution exposure and importance of strict monitoring of such sensitive karst ecosystems worldwide. Results indicated degraded water quality, poor ecological status, and disturbed seasonal fluctuations at wastewater-influenced sites, primarily due to high levels of nutrients and organic matter. However, improvement was observed downstream in the Krka National Park, confirming the self-purification as important processes in dynamic karst rivers. Natural seasonality, observed at sites without wastewater influence, was mainly driven by fluctuations in water levels and primary production during the year. Literature analysis by CiteSpace pointed to scarce data on this topic worldwide (China and the USA account for 49% of all publications) and in Europe (34%). Therefore, such study is a valuable contribution in presenting the long-term and seasonal variability of ecological water parameters and in providing a more comprehensive understanding of the health of catchment under influence of multiple stressors.

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.

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 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.

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.