Histochemical research of enzymes involved in cellular digestion in the digestive tract of tub gurnard, Chelidonichthys lucerna.

The tub gurnard Chelidonichthys lucerna (Linnaeus, 1758), Triglidae, is an opportunistic, demersal carnivorous fish. Data on the digestive enzymes of tub gurnard have not been reported in the literature. Therefore, the aim of this research was to investigate the distribution and intensity of alkaline phosphatase, acid phosphatase, non-specific esterase, and aminopeptidase in the digestive tract of tub gurnard. To investigate data about those enzymes tissue samples of the esophagus, anterior and posterior part of the stomach, pyloric caeca, anterior, middle and posterior part of the intestine proper, and rectum were taken. Azo-coupling methods were used to detect the enzymatic reactions. The intensities of the reactions were measured using ImageJ software. Alkaline phosphatase, acid phosphatase, and non-specific esterase activities were found in all parts of the digestive tract. The brush border of the pyloric caeca and intestine proper were the main sites of alkaline phosphatase reaction, with intensity decreasing toward the posterior parts of the digestive tract. The high intensities of acid phosphatase were found in the epithelium of the anterior part of the stomach, pyloric caeca, anterior part of the intestine proper, and in the rectum. The intensity of non-specific esterase was mainly increased from the anterior to the posterior parts of the digestive tract. Aminopeptidase activity was found in the esophagus, pyloric caeca, and intestine proper. Our results suggest that the entire digestive tract of the tub gurnard is involved in the digestion and absorption of dietary components.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

Histopathology investigation on the Vardar chub (Squalius vardarensis) populations captured from the rivers impacted by mining activities.

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

Pomphorhynchus laevis (Acanthocephala) from the Sava River basin: New insights into strain formation, mtDNA-like sequences and dynamics of infection.

Here we report the genetic variability and presence of mtDNA-like sequences of Pomphorhynchus laevis from the chub, Squalius cephalus, caught at the sampling sites along the Sava River and its tributary the Sutla River in Croatia. Sequences of the cytochrome c oxidase subunit 1 (COI) gene of the recovered P. laevis specimens were used for haplotype network construction and phylogenetic analysis. These analyses showed that some specimens contained mitochondrial-like sequences, and they uncovered the existence of a Sava River basin strain different from known strains of P. laevis. This is the first time that P. laevis has been shown to contain mtDNA-like sequences, suggesting the need to exercise caution during COI analyses of P. laevis using universal primers. Highly conserved sequences of two nuclear markers, the ITS region and 18S rRNA, were not helpful for understanding genetic variability or differentiating strains. Furthermore, analysis of the dynamics of P. laevis infections in S. cephalus from the Sava and Sutla Rivers showed decreased prevalence and abundance at sites with inferior water quality, positive association of parasite abundance with fish size, and no clear association of parasite abundance with fish condition index or sex.

Assessment of general condition of fish inhabiting a moderately contaminated aquatic environment.

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