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.

Heavy metal(loid) effect on multi-biomarker responses in apex predator: Novel assays in the monitoring of white stork nestlings.

The goal of the present study was to investigate differences in biomarker responses related to metal(loid)s in white stork (Ciconia ciconia) nestling's blood from continental Croatia. To achieve this, a battery of biomarkers that can be affected by environmental pollutants, including metal(loid)s, was assessed (esterase activity, fluorescence-based oxidative stress biomarkers, metallothionein levels, glutathione-dependent enzyme activity). The research was conducted during the white stork breeding season in diverse areas (a landfill, industrial and agricultural sites, and an unpolluted area). White storks' nestlings near the landfill exhibited reduced carboxylesterase (CES) activity, elevated glutathione (GSH) concentration, as well as high Pb content in the blood. Increased As and Hg concentrations in blood were attributable to environmental contamination in agricultural area and an assumed unpolluted area, respectively. Furthermore, agricultural practices appeared to affect CES activity, as well as elevate Se levels. In addition to the successful implementation of biomarkers, present research showed that agricultural areas and a landfill are areas with increased metal(loid) levels possibly causing adverse effects on the white storks. This first-time heavy metal and metalloid analyses in the white stork nestlings from Croatia point to the necessary monitoring and future assessments of pollution impact to prevent irreversible adverse effects.

Metal(loid) exposure assessment and biomarker responses in captive and free-ranging European brown bear (Ursus arctos).

We investigated the level of five non-essential metal(loid)s (As, Cd, Hg, Tl, Pb) and nine essential metals (Mg, Ca, Mn, Fe, Co, Cu, Zn, Se, Mo) in hair and blood components of captive and free-ranging European brown bear populations in Croatia and Poland. Metal(loid) associations with biomarkers of oxidative stress (superoxide dismutase, SOD; glutathione-peroxidase, GSH-Px; malondialdehyde, MDA) and metal exposure (metallothionein, MT) were estimated in this top predatory mammal. Lead was the most abundant non-essential metal(loid) in both blood and hair, with 4 of 35 individuals having blood levels over 100 µg/L. A positive association was found between Pb level and SOD activity in blood. Free-ranging bears had higher blood SOD activity, Mn, Zn and Cd levels, hair Co, Cd, Tl and Pb compared to captive individuals, while the opposite was true for Mg and hair Ca thereby reflecting habitat and diet differences. With increasing age, animals showed lower levels of SOD activity and certain essential metals. Females had higher SOD activity and blood levels of some essential metals than males. Hair showed a higher Fe and Co level when sampled during the growth phase and was not predictive of non-essential metal(loid) blood levels. The established metal(loid) baseline values will enable future risk assessment in both captive and wild European brown bear populations.

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.

Effects of heavy metal pollution on pigmented macrophages in kidney of Vardar chub (Squalius vardarensis Karaman).

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

Assessment of metal exposure, ecological status and required water quality monitoring strategies in small- to medium-size temperate rivers.

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

Serum metallothionein in patients with testicular cancer.

PURPOSE: Metallothioneins (MTs) have been disclosed as a useful diagnostic factor for tumour progression and drug resistance in a variety of malignancies. Increased levels of MT in blood serum have been found in patients with several types of cancer, but there is no available information on serum MT levels in patients with testicular germ cell tumour (TGCT). The aim of the study was to determine MT levels in serum of patients with TGCT and to evaluate the portion of platinum (Pt) that binds to MT after cisplatin administration since MTs could be involved in drug resistance. METHODS: Concentration of total MT was determined in serum of 25 men with newly diagnosed TGCT by differential pulse voltammetry. The fractionation of serum was carried out by size exclusion high-performance liquid chromatography (SE-HPLC), while concentration of Pt in collected fractions was determined by inductively coupled plasma mass spectrometry. RESULTS: Concentration of serum MT was significantly higher in TGCT patients than in healthy volunteers. The results of SE-HPLC analysis showed that only a small amount of Pt was bound to proteins in the area of MT elution. CONCLUSIONS: Significant increase in MT levels in individuals with TGCT indicates certain health problem and, in combination with other commonly used diagnostic tools, could improve early diagnosis.

The impact of fish spawning on metal and protein levels in gastrointestinal cytosol of indigenous European chub.

The study presented here reports for the first time cytosolic metal and protein levels in the gastrointestinal tissue of field-collected European chub (Squalius cephalus), living in low metal-contaminated river section. In two sub-cellular fractions (untreated cytosol and heat-treated cytosol), isolated from the whole gastrointestinal tract of indigenous chubs collected during spawning (April/May 2006) and post-spawning period (September 2006), seasonal or gender related differences of trace metals and proteins were determined. In both fractions, cytosol and heat-treated cytosol, metal levels decrease as follows: Zn>Fe>Cu>Mn>Cd and are significantly higher during the spawning period. Besides that, the level of heat-sensitive proteins (metalloenzymes) in cytosolic fractions is also significantly higher during the spawning period in both male and female specimens. Higher condition indices of chubs in the spawning period imply higher feeding activity, energy reserves and recent growth of indigenous chubs. Metallothionein levels, determined in the heat-treated cytosol, are comparable in gastrointestinal tract of feral chubs collected in both seasons and confine the background metallothionein levels, on average 3mgg(-)(1) wet tissue. Chub spawning has been identified as a confounding factor, having an impact on cytosolic metal and protein levels, presumably due to enhanced food supply (higher condition indices) and fish spawning (higher gonadosomatic indices in some male specimens).

Reduction of cisplatin-induced nephrotoxicity by pyrazolone compounds, derivatives of tetrahydroindazolonedicarboxylic acid.

The structural requirements relevant for protective efficacy against cisplatin-induced renal toxicity was studied for seven newly synthesized pyrazolone compounds. Since tetrahydroindazolonedicarboxylic acid (HIDA) has shown potential nephroprotective efficacy, our study involved HIDA derivatives with specific modifications of functional groups. Pyrazolone compounds comprised four types of structural modifications: an HIDA regioisomer derivative (compound 1), compounds with modifications at the pyrazolone ring (compounds 2, 3 and 4) or the dicarboxylic moiety (compounds 5 and 6), and a compound without a cyclohexane moiety (compound 7). The best nephroprotective efficacy was found for compound 1, as reflected in the lowering of cisplatin-induced levels of blood urea nitrogen (BUN) by 86.3-89.3%, depending on cisplatin administration time. The alicyclic pyrazolonedicarboxylic acid (compound 7), characterized by free rotation of attached moieties due to the lack of a cyclohexane moiety, also showed good protection (lowering of cisplatin-induced BUN levels by 29.5-81.7%, depending on cisplatin administration time). Lower nephroprotective activity was found for compounds 2 and 3, with N- and O-substituted pyrazolone rings, and for the cyano derivative 5, while compounds without a carboxylic and pyrazolone moiety (compounds 6 and 4, respectively) did not show a nephroprotective effect. Therefore, carboxylic and pyrazolone moieties play an important role in the interaction with cisplatin and represent relevant functional groups required for nephroprotective efficacy in pyrazolone compounds.

Age- and tissue-dependent metallothionein and cytosolic metal distribution in a native Mediterranean fish, Mullus barbatus, from the Eastern Adriatic Sea.

The levels of metallothionein (MT), a biomarker of metal exposure, and of cytosolic metals (Zn, Cu, Cd), known as MT inducers, were investigated as variables of age (1 to 8 years) and tissue mass (liver, kidney, brain) of red mullet (Mullus barbatus). Within the age from 1 to 8 years the most significant increase is evident for cytosolic Cd in liver (43-fold) and in kidney (5-fold). MT and essential metals are constant with age or slightly increased. Over the growth period, statistically significant MT and metal increase is evident only between 1 and 6-8 years old specimens, while for Cd in liver and kidney cytosol significant increase already exists at 4 years old specimens. Metal distribution in all tissues follows the order: Zn>Cu>Cd, with even 500-800 times lower Cd levels than essential metal levels. Consequently, MTs follow the levels of essential metals, Zn and Cu, indicating MT involvement in homeostasis of essential metals. In contrast to kidney and brain, hepatic MT levels are not age-dependent. Inclusion of hepatic MT measurements and the associated cytosolic metals will be useful in the assessment of long-term metal effects in demersal fish M. barbatus.