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In the present study, essential and nonessential metal content and biomarker responses were investigated in the intestine of fsh collected from the areas polluted by mining. Our objective was to determine metal and biomarker levels in tissue respon sible 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 infuenced by the active mines Zletovo and Toranica, respectively. Biological responses were analyzed in Vardar chub (Squalius varda rensis ; Karaman, 1928), using for the frst time intestinal cytosol as a potentially toxic cell fraction, since metal sensitivity is mostly associated with cytosol. Cytosolic metal levels were higher in fsh under the infuence 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 fsh from mining afected 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 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.

Integrated chemical and biomarker approaches were performed to estimate if there is ongoing toxicity in the River Bregalnica, namely connected with the presence of metals. The study was performed in water, sediment, and barbel (Barbus peloponnesius), collected in two seasons, from two suspected polluted and one reference zones. The water analyses revealed higher mean values in polluted sites for most of the examined physicochemical parameters. Metal concentrations (Zn, Cu, Cd, Mn, Pb, and Fe) in water were more or less constant, whereas in sediment, they were higher at the two polluted locations. Condition factor (CF), as a general health indicator, revealed better overall condition in barbel from the reference site. In general, blood parameters revealed higher values in the polluted localities. Irrespective of sex and/or season, the frequency of micronuclei (MN) and vacuolated nuclei (VN) were with higher rates in polluted sites. Similarly, the frequencies of the leucocytes (Le), binuclei (BN), and irregularly shaped nuclei (ISN) were also significantly increased in the polluted localities, but they seemed prone to be influenced by sex and/or season. However, strong positive correlations between blood biomarkers and most water physicochemical parameters and metal in sediment were estimated. Our data support that the River Bregalnica's lower course receives significant genotoxic pollution, likely via metal industry effluents, agricultural runoff, and domestic sewage, and reinforced the utility of MN and other nuclear abnormalities as sensitive and suitable biomarkers for genotoxicity when used in monitoring studies.