Institute of Animal Science

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Evaluation of histopathological alterations in the gills of Vardar chub ( Squalius vardarensis Karaman) as an indicator of river pollution
(Elsevier BV, 2015-08)
Barišić, Josip
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Dragun, Zrinka
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Ramani, Sheriban
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Filipović Marijić, Vlatka
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Krasnići, Nesrete
Quanti!cation of histopathological alterations in the gills of Vardar chub (Squalius vardarensis Karaman) 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 classi!ed 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, speci!cally 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 con- tamination was more pronounced due to concentration effect. The pattern and severity of histopatho- logical alterations in the chub gills re"ected 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
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Mining waste as a cause of increased bioaccumulation of highly toxic metals in liver and gills of Vardar chub (Squalius vardarensis Karaman, 1928)
(Elsevier BV, 2019-04)
Dragun, Zrinka
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Tepić, Nataša
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Ramani, Sheriban
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Krasnići, Nesrete
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Filipović Marijić, Vlatka
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.
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Characterization and identification of selected metal-binding biomolecules from hepatic and gill cytosols of Vardar chub (Squalius vardarensis Karaman, 1928) using various techniques of liquid chromatography and mass spectrometry
(Oxford University Press (OUP), 2019-06-19)
Krasnići, Nesrete
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Dragun, Zrinka
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Kazazić, SnjeŽana
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Muharemović, Hasan
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Erk, Marijana
Metals play crucial physiological roles, but they can also cause irreparable toxic effects through binding to important cellular biomolecules in aquatic organisms. The aim of this study was to determine the exact molecular masses and to identify several selected metal-binding biomolecules in hepatic and gill cytosols of Vardar chub (Squalius vardarensis Karaman, 1928). Methods applied for the achievement of this goal were SEC-AEC-HPLC for two-dimensional separation of cytosolic biomolecules, HR ICP-MS for metal measurements, and mass spectrometry (MALDI-TOF-MS and LC-MS/MS) for biomolecule mass determination and identification. The analyzed biomolecules included: Fe-binding biomolecules, which were identified as hemoglobin subunit β in the liver (molecular masses of ∼15 kDa), and hemoglobin subunits α and β in the gills (molecular masses of ∼11 kDa, ∼13 kDa and ∼15 kDa); heat-stable Cd-binding biomolecules, which were identified as MT isoforms MT-I and MT-II (molecular mass of ∼6.0 kDa in both liver and gills, and an additional 4.9 kDa isoform in the gills); and heat-stable Mo-binding biomolecules of molecular masses equal to 3.3 kDa (in the gills) and 8.5 kDa (in the liver). An important finding of this study was the obvious presence of different isoforms of the same biomolecules in the liver and gills. This was, among others, manifested through the absence of Zn binding to MTs only in the gills, indicating that the same type of biomolecule can be responsible for different functions in different organs. Thus, for better understanding of metal behaviour in aquatic organisms, it is crucial to identify cellular metal-binding biomolecules and their functions.
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Malondialdehyde concentrations in the intestine and gills of Vardar chub (Squalius vardarensis Karaman) as indicator of lipid peroxidation
(Springer Science and Business Media LLC, 2017-07)
Dragun, Zrinka
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Filipović Marijić, Vlatka
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Krasnići, Nesrete
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Ramani, Sheriban
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Valić, Damir
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.
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Effects of heavy metal pollution on pigmented macrophages in kidney of Vardar chub (Squalius vardarensis Karaman)
(Wiley, 2017-08)
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Dragun, Zrinka
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Ramani, Sheriban
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Ivanova, Lozenka
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.

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