Quantification of histopathological alterations in the gills of Vardar chub (Squalius vardarensisKaraman) was performed in 2012 in rivers of north-eastern Macedonia, with the aim to examine the effects of water quality in the rivers (Zletovska and Kriva River -impacted by active Pb/Zn mines; Bregalnica River -contaminated by agricultural waste). The biological alterations in chub were classified as: circulatory disturbances, regressive and progressive changes, but their severity differed.Altogether the mildest changes were observed in the gills of chub from the Bregalnica River, a less polluted river, whereas mining impacted rivers were characterized by more severe alterations. In the gills of chub from the Zletovska River, which is highly contaminated with numerous metals, sulphates and chlorides, the highest lesion indices were found for the regressive changes of both epithelium and supporting tissue, with typical lesions referring to atrophy, thinning and lifting of epithelial cells, necrosis of epithelium and chloride cells, as well as deformations of lamellar cartilaginous base. Gill damages of chub from the Kriva River were overall milder compared to the Zletovska River, in accordance with pollution status. In the gills of chub from that river, progressive changes were more pronounced, specifically severe hyperplasia of mucous cells and epithelium in the interlammellar space, leading to fusion of lamellae, as well as hypertrophy of chloride cells. The comparison between seasons indicated higher intensity of progressive changes in all three rivers in autumn, when water level was very low, and consequently, water contamination was more pronounced due to concentration effect. The pattern and severity of histopathological alterations in the chub gills reflected differences in contamination levels and type of contaminants in different rivers and sampling periods, and thus have been proven as a valuable indicator of water quality.

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.

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.

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