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Background and purpose: Pigmented macrophage aggregates (PMAs) are a part of the immune system of fish and are considered as valuable biomarkers of the impact of water pollution on fish health.Materials and methods: Immunomodulatory effect of water contaminated with high levels of metals on spleen and splenic PMAs of Vardar chub (Squalius vardarensis) (n=129) was examined in two seasons, spring, and autumn 2012. For this purpose, three rivers were chosen: the Zletovska River, heavily contaminated with metals, the Kriva River, moderately contaminated with metals combined with high level of organic matter, and the Bregalnica River, without heavy metal contamination. Spleen masses and splenosomatic indices (SSI) were used for determining the general impact of water contamination on the immune system of Vardar chub. Moreover, volumes and numbers of PMAs within spleen tissue were determined stereologically.Results: The isolated effect of high exposure to metals caused the reduction of spleen mass and/or SSI in the fish from the Zletovska River. The combined effect of exposure to metals and high levels of nutrients in the fish from the Kriva River referred to stimulation of PMAs response, namely increased volumes of splenic PMAs. The existence of seasonally dependent differences was noted since the observed variability in PMAs between rivers occurred only in the autumn.Conclusions: Splenic PMAs in the Vardar chub can be used as an additional biomarker of metal pollution, but with caution, since enhancement of the volumes and numbers of MACs depends on the exposure level of heavy metals, on the presence of the other contaminants, as well as on the physiological condition of the fish.

Background and purpose: Pigmented macrophage aggregates (PMAs) are a part of the immune system of fish and are considered as valuable biomarkers of the impact of water pollution on fish health. Materials and methods: Immunomodulatory effect of water contaminated with high levels of metals on spleen and splenic PMAs of Vardar chub (Squalius vardarensis) (n=129) was examined in two seasons, spring, and autumn 2012. For this purpose, three rivers were chosen: the Zletovska River, heavily contaminated with metals, the Kriva River, moderately contaminated with metals combined with high level of organic matter, and the Bregalnica River, without heavy metal contamination. Spleen masses and splenosomatic indices (SSI) were used for determining the general impact of water contamination on the immune system of Vardar chub. Moreover, volumes and numbers of PMAs within spleen tissue were determined stereologically. Results: The isolated effect of high exposure to metals caused the reduction of spleen mass and/or SSI in the fish from the Zletovska River. The combined effect of exposure to metals and high levels of nutrients in the fish from the Kriva River referred to stimulation of PMAs response, namely increased volumes of splenic PMAs. The existence of seasonally dependent differences was noted since the observed variability in PMAs between rivers occurred only in the autumn. Conclusions: Splenic PMAs in the Vardar chub can be used as an additional biomarker of metal pollution, but with caution, since enhancement of the volumes and numbers of PMAs depends on the exposure level of metals, on the presence of the other contaminants, as well as on the physiological condition of the fish.

The early effects of metal contamination in freshwater ecosystem can be seen at the molecular and cellular level of organisms exposed even to low metal concentrations and thus they can serve as “early warning” indicators. Accordingly, the aim of this study was to obtain the information on Cd, Zn and Mo distributions among various cytosolic biomolecules in the gills of Vardar chub (Squalius vardarensis Karaman) exposed to metals in mining and agriculturally contaminated Macedonian rivers. To achieve this goal, cytosolic fractionation, before and after heat-treatment, was performed by size exclusion high performance liquid chromatography, metal measurements by high resolution inductively coupled plasma mass spectrometry, and biomolecule identification by two techniques of mass spectrometry (MALDI-TOF and liquid chromatography-mass spectrometry, LC-MS). Cadmium was eluted within two peaks with maxima at 90 and 20 kDa. The first peak contained heat-sensitive proteins, based on its almost complete disappearance after the heat treatment. The second peak was confirmed to contain heat-stable proteins, most probably metallothioneins (MTs), based on the same elution time as MT standard. Zinc was eluted within one peak with maximum at 90 kDa, which almost completely disappeared after the heat-treatment, confirming the heat-sensitive nature of comprised biomolecules. Association of Zn with heat-stable MTs was not observed. Cytosolic Mo was eluted within single peak with maximum at 7 kDa, which contained heat-stable biomolecules, and which was confirmed to have molecular mass of 3.3 kDa, according to MALDI-TOF analysis. Heat-stable biomolecules involved in binding cytosolic metals Cd and Mo were further identified by LC-MS.

Many proteins contain essential metal Fe in ionic form, either within their own structures or bound to their active sites (del Castillo Busto et al., 2010). Iron distribution profiles among cytosolic biomolecules were determined for hepatic and gill cytosols of Vardar chub (Squalius vardarensis Karaman) from Macedonian rivers. Application of size-exclusion high performance liquid chromatography (SEC-HPLC) coupled offline with high resolution inductively coupled plasma mass spectrometry (HR ICP-MS) resulted with Fe separation in two peaks, the first one corresponding to biomolecules of 230-630 kDa, and the second one corresponding to biomolecules of 24-51 kDa. The first peak most likely corresponds to ferritin, the major Fe storage protein (450 kDa), and was more prominent in liver than in gills, which is in accordance with more significant role of liver in Fe storage. To obtain more information about biomolecules comprised by the second peak, which was observed in both studied organs, we have used anion exchange chromatography (AEX-HPLC) coupled offline with HR ICP-MS, as a second step of separation. Cytosolic biomolecules of 24-51 kDa from both organs were further separated according to their ionic strength, and clearly distinguishable Fe peaks were observed at elution times from 10th to 14th minute. The last step was application of two mass spectrometry techniques for the identification of Fe-binding biomolecules of 24-51 kDa which were purified by two-dimensional chromatographic separation. By use of MALDI-TOF, three protein peaks were obtained, corresponding to molecular masses of 15 kDa, 32 kDa and 47 kDa in the liver, whereas in the gills we have obtained two peaks corresponding to 11 kDa and 14 kDa. Application of liquid chromatography- mass spectrometry system (LC-MS) enabled identification of α and β subunits of haemoglobin (Hb, 15-16 kDa) in both organs, whereas hepatic peaks of higher molecular masses probably referred to Hb dimers and trimers.

Freshwater pollution with heavy metals may influence the immune system of fish, leading to impairment of their health or even to increase of 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, in Vardar chub (Squalius vardarensis Karaman) 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 0.56-1.68%. 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.

Metalotioneini (MT) su citosolski proteini odgovorni za homeostazu esencijalnih metala, poput Zn i Cu, i detoksikaciju neesencijalnih metala, poput Cd. Cilj ovoga istraživanja bio je potvrditi vezanje ova tri metala na MT u citosolu jetre vardarskog klena (Squalius vardarensis Karaman) uzorkovanog u listopadu 2012. godine u Krivoj reci, onečišćenoj rudarskim otpadom, u sjeveroistočnoj Makedoniji. Primijenjeni analitički pristup sastojao se od sljedećih koraka: (1) usporednog frakcioniranja citosolskih proteina iz jetre klena i standarda MT na osnovu njihove molekulske mase primjenom visokodjelotvorne tekućinske kromatografije s isključenjem po veličini (SE-HPLC), koje je bilo praćeno određivanjem koncentracija odabranih metala u tako dobivenim proteinskim frakcijama pomoću spektrometra masa s induktivno spregnutom plazmom (ICP-MS) ; (2) frakcioniranja proteina prema molekulskoj masi pomoću ultrafiltera, sa svrhom odvajanja proteina molekulske mase >30 kDa, koje je bilo praćeno daljnjim frakcioniranjem tih proteina pomoću anionsko- izmjenjivačke kromatografije (AEX-HPLC) te određivanjem raspodjele profila metala u tako dobivenim frakcijama primjenom ICP-MS-a ; (3) analiziranja proteinskih frakcija dobivenih dvodimenzionalnim frakcioniranjem, koje sadrže metale Zn, Cu i Cd, pomoću masene spektrometrije (ESI-Q-TOF MS) s ciljem potvrđivanja prisutnosti MT. Primijenjeni analitički pristup omogućio je identifikaciju MT u proteinskim frakcijama citosola jetre klena koje sadrže Zn, Cu i Cd, što je u skladu s ulogom MT u homeostazi i detoksikaciji metala. Analitički pristup koji se zasniva na kombiniranoj primjeni različitih analitičkih metoda, poput dvodimenzionalnog razdvajanja proteina (SE-AEX-HPLC) povezanog s određivanjem metala (ICP-MS) i identifikacijom proteina (ESI-MS) predstavlja brz, osjetljiv i pouzdan alat za prepoznavanje citosolskih proteina koji vežu metale.

Budući da su prirodne koncentracije prijelaznih metala u rijekama vrlo niske, svaki dodatni unos utječe na njihovu koncentraciju i time mijenja prirodnu ravnotežu u vodenom okolišu. Metali nisu biorazgradivi te njihov unos predstavlja dodatnu prijetnju vodenim ekosustavima jer se mogu akumulirati u sedimentu ili živim organizmima (1). Procjena biološkog učinka metala obuhvaća i praćenje biomarkera izloženosti metalima, metalotioneina (MT). Ovi niskomolekulski citosolski proteini sudjeluju u održavanju homeostaze esencijalnih (Zn, Cu) i detoksifikaciji toksičnih metala (Cd, Hg, Ag), a njihova pobudna sinteza ukazuje na izloženost metalima (2). Pouzdana i osjetljiva elektrokemijska metoda određivanja MT u biološkim uzorcima je diferencijalna pulsna voltametrija (DPV). Prikazano istraživanje provedeno je tijekom jeseni, kao sezoni fiziološkog mirovanja nakon mrijesta Vardarskog klena (Squalius vardarensis L.), u makedonskim rijekama Zletovska i Kriva koje su onečišćene otpadnim vodama rudnika olova i cinka (rudnici Zletovo i Toranica) te Bregalnici koja nije izložena utjecaju rudarskih aktivnosti. Cilj istraživanja je procijeniti izloženost i biološke učinke metala praćenjem koncentracija metala i MT u citosolu probavila riba te koncentracija metala u riječnoj vodi. MT su određeni DPV, metodom po Brdički, koja se koristi za određivanje proteina koji sadrže tiolne skupine, uključujući MT (3). Mjerenje je provedeno na 797 Computrace (Metrohm) uređaju s troelektrodnim sustavom (radna elektroda (viseća živina kap), protuelektroda (platinska žica) i referentna elektroda (Ag/AgCl/3M KCl)) u amonijačnoj otopini kobaltove(III) soli (1M NH4Cl + 1M NH4OH, pH=9, 5 i 6 x10-4 M Co(NH3)6Cl3). Metoda se temelji na određivanju visine katalitičkog vala vodika pri potencijalu Ep = -1, 48 V, koji je proporcionalan koncentraciji MT. Kao kalibrant je korišten MT zečje jetre (Ikzus Proteomics) otopljen u 0.25 M NaCl. Na temelju toplinske stabilnosti, MT su određeni u toplinski obrađenom citosolu probavila klena. Koncentracije metala su izmjerene masenim spektrometarom visokog razlučivanja s induktivno spregnutom plazmom (HR ICP-MS). Statistički značajno povišene koncentracije MT (mg g-1) zabilježene su u riba iz Zletovske i Krive (3, 9 i 3, 3) koje su izložene utjecaju zagađenja iz rudnika Pb/Zn u odnosu na referentnu Bregalnicu (2, 1). Većina izmjerenih metala u riječnoj vodi i citosolu probavila Vardarskog klena također ima povišene koncentracije u rijekama pod utjecajem rudnika. Primjena DPV u praćenju promjena MT u biološkim uzorcima dokazana je kao osjetljiva metoda koja se koristi u biomonitoring studijama procjene izloženosti okoliša metalima.

Macrophage aggregates (MACs) are pigment- containing cells accumulations that are presented in fish spleen, kidney and liver. Their number, size and pigment content may change depending on environmental degradation and therefore can be used as bioindicators of water quality. There are some indications that metals have inhibitory effect on fish immune system, but little is known of the effect of complex mixture of pollutants on MACs pool in fish spleen. In this study quantitative influence of heavy metals pollution on the MACs amount in the spleen tissue will be evaluated for the first time. For this study female (n=26) and male (n=30) Vardar chub (Squalius vardarensis) were collected at three rivers: Bregalnica, Zletovska and Kriva Reka, with difference in pollution impact ; organic, heavy metals, and mixture of both, respectively. Using stereological technique, relative (Vv) and total volume (V) of the MACs were estimated as well as the amount of the pigments within MACs. The results showed that when V and Vv of MACs and the contributing pigments in the spleen were estimated, male fish appeared more impacted by pollution compared to female and also showed significant decrease of V of MACs in Zletovska compared to Bregalnica. Moreover, statisticaly lower value of Vv and V of MACs containing melanin/lipofuscin pigments were recorded in Zletovska when compared to the two other sites. In comparison, Bregalnica, which has mainly organic pollution impact, had highest values of V of MACs compared with other two rivers. Male fish from Kriva Reka showed low V of MACs, with the lowest amount of MACs which contains haemosiderin/lipofuscin pigments. Female fish showed lowest Vv of MACs in Kriva Reka which can be attributed to the significantly lower Vv for the lipofuscin containing MACs in the same locality. In conclusion, pigment content, as well as the amount of MACs in the chub can change concerning the type of pollutants. Metals reduce the appearance of the MACs pool, mostly through reduction of lipofuscin and melanin/lipofuscin pigments, and probably induce immunosuppression in these cells.

Sustavno praćenje stanja okoliša mjerenjem bioloških promjena u indikatorskih organizama naziva se biomonitoring. S obzirom da se za razliku od organske tvari ne mogu razgraditi u okolišu, metali jednom unešeni u okoliš tu i ostaju te predstavljaju najopasnija zagađivala. Biomonitoring studije izloženosti metalima obuhvaćaju mjerenje metala u vodi i sedimentu te u indikatorskim organizmima. Ribe, kao organizmi na vrhu hranidbenog lanca u vodenom ekosustavu, su često korišteni indikatorski organizmi. Ribe unose metale putem škrga, probavila i kože, ali se neselektivno uz esencijalne metale akumuliraju i toksični. Jedna od ranih promjena na staničnoj razini organizma (biomarkeri) koja ukazuje na izloženost organizma metalima je pobudna sinteza metalotioneina (MT), citosolskih proteina odgovornih za homeostazu esencijalnih (Zn, Cu) i detoksifikaciju toksičnih metala (Cd, Hg, Ag) (1). Među različitim tehnikama mjerenja MT, elektrokemijska metoda diferencijalna pulsna voltametrija (DPV) predstavlja osjetljivu metodu određivanja MT u biološkim uzorcima. S obzirom da osim metala na koncentraciju MT utječu i drugi faktori, poput biometrije, fiziološkog stanja riba te abiotičkih faktora, istraživanje je provedeno u sezoni mrijesta i ubrzanog metabolizma Vardarskog klena (Squalius vardarensis L.) u makednoskim rijekama pod utjecajem otpadnih voda rudnika olova i cinka, Zletovska (rudnik Zletovo) i Kriva (rudnik Toranica). U riječnoj vodi su izmjereni metali, a u citosolu probavila klenova metali i MT kao biomarkeri izloženosti metalima te uspoređeni s referentom postajom, rijekom Bregalnicom na kojoj nema utjecaja otpadnih voda rudnika. DPV se temelji na difuzijom kontroliranom putovanju analita do površine živine elektrode. Depolarizacijski potencijal omogućava identifikaciju iona prisutnih u otopini, a mjerenjem difuzijske struje može se izračunati njihova koncentracija. Metodu je razvio češki kemičar Jaroslav Heyrovský te za nju 1959. dobio Nobelovu nagradu. Njegov student, Rudolf Brdička, je u svom radu 1933. otkrio da proteini s –SH skupinama daju karakterističan val i upravo se metoda po Brdički koristi za određivanje MT. Frakcija MT se izolira toplinskom obradom na 85°C, a mjerenje se provodi u otopini amonijačnog pufera (1M NH4Cl + 1M NH4OH), pH=9.5 koja sadrži 6x10-4 M Co(NH3)6Cl3 pri sljedećim mjernim parametrima na uređaju 797 Computrace (Metrohm): početni/završni potencijal: -0.9/-1.65 V ; amplituda/trajanje naponskog pulsa: 0.02502 V/0.057 s ; vrijeme ponavljanja promjene pulsa: 0.5 s ; brzina promjene potencijala: 0.005 Vs-1 (2). U proljetnom su periodu gotovo svi od 26 izmjenenih metala u vodi i citosolu povišeni u riba iz Zletovske i Krive u odnosu na Bregalnicu na kojoj nema rudarske aktivnosti. Biološki učinak ovih metala potvrđen je i značajno povišenim koncentracijama MT (mg g-1) u riba iz Zletovske i Krive (3, 1 i 2, 7) u odnosu na Bregalnicu (2, 0). DPV je potvrđena kao pouzdana metoda mjerenja MT u nativnim populacijama indikatorskih organizama.

The mine waste was recognized as one of the most serious threats for freshwater ecosystems, and it still represents one of the biggest environmental concerns in Macedonia. Some of the reasons for its toxicity are the acidity and high content of trace metals, which could lead to significant loss of biodiversity. Macedonian most significant mineral deposits are Pb and Zn ores, the exploitation of which is carried out in active mines in the north-eastern part of the country, such as Zletovo in Probishtip and Toranica in Kriva Palanka. Two rivers were chosen for this study, the Zletovska River, which receives the waste from the mine Zletovo, and the Kriva River, which receives the waste from the mine Toranica, whereas a location at the Bregalnica River was chosen as non-impacted sampling site. The impact of mining on the surface water quality was studied by measuring physico-chemical parameters, nutrient concentrations, and the concentrations of dissolved macro and trace elements. The sampling was performed in spring (May/June) and autumn (October) of 2012. A number of physico-chemical parameters (temperature, pH, redox potential, conductivity, and dissolved oxygen) were measured on site with a portable multiparameter instrument, whereas the other parameters (alkalinity, turbidity, total dissolved solids, chemical oxygen demand, total hardness, and concentration of nutrients and other anions) were measured in the river water samples in the laboratory by standardized ISO, APHA and EPA methods. The measurements of both trace and macro elements were performed on high resolution inductively coupled plasma mass spectrometer. The Bregalnica River was characterized mainly by slightly alkaline pH and weak contamination with As, Ba, Fe, Mo, Ti, U, V, nitrates and phosphates, which could be connected to agricultural activities, whereas the impact of the mines was not observed. Interesting finding for the Bregalnica River was several degrees higher water temperature compared to the other two rivers, in both sampling periods. The cause of that increase was possibly the mixing of the river water with the hot water from the reservoirs of the nearby situated geothermal system Kezhovica-Ldzhi. Contrary, both Zletovska and Kriva rivers revealed a clear impact of Pb/Zn mines on water quality. The sampling location at the Zletovska River had slightly acidic water, the highest conductivity, total dissolved solids, total hardness, the concentration of CaCO3, the concentrations of macro elements (Na, K, Ca, Mg), as well as sulphates and chlorides. Both sulphates and conductivity are useful indicators of acid mine drainage contamination, because they remain increased even when pH approaches neutral values due to large dilutions. In the Zletovska River increased concentrations of Cd, Co, Cs, Cu, Li, Mn, Ni, Rb, Sn, Sr, Tl, and Zn were also found, among which Cd and Ni belong to priority toxic substances, and Cd even exceeded the environmental quality standards set by the European Water Framework Directive for inland surface waters. Due to low water level, especially high concentrations of several elements were measured in autumn (e.g. Cd: 2.0 μg L-1 ; Mn: 2.5 mg L-1 ; Zn: 1.5 mg L-1). The Kriva River water had slightly alkaline pH and continuous slight increase of several trace elements characteristic for agricultural activities (Ba, Fe, Mo and V). In the Kriva River increased concentrations of metals which could be associated with the impact of the nearby mine, such as Cd (0.270 μg L-1) and Pb (1.85 μg L-1), were found only in spring, possibly due to sediment resuspension during higher water discharge, indicating probable sediment contamination by mining waste. The results obtained in this study revealed severe impact which the active mines Zletovo and Toranica have on the water quality of the rivers receiving their waste (Ramani et al., 2014). The need for the continuous monitoring of those rivers should be emphasized, as well as the necessity to investigate possible detrimental effects of mining waste on the aquatic organisms.

Mining activity can seriously affect the quality of surface water, especially regarding the levels of trace elements, which present a serious threat to the aquatic organisms because of their toxicity, persistence and bioaccumulation. The aim of this study was to assess bioavailability of metals and metalloids in the river water based on their accumulation in two target organs (liver and gills) of Vardar chub (Squalius vardarensis) from three rivers in north-eastern Macedonia, agriculturally impacted Bregalnica River and two mining impacted rivers, the Zletovska and the Kriva River. For the assessment of metal accumulation, 12-30 chub specimens were caught per site in spring (May/June) and autumn (October) of 2012. Metal/metalloid concentrations were determined by high resolution inductively coupled plasma mass spectrometer in the soluble cytosolic fractions of gills and liver, obtained after tissue homogenization and centrifugation at 50 000xg. Under conditions of low metal exposure, following ranges of metal/metalloid concentrations were measured in the chub liver: 1000 μg L-1 for Cu, Fe, and Zn. In chub gills, concentration ranges slightly varied, as follows: 1000 μg L-1 for Fe and Zn. The comparison between two organs indicated higher basal concentrations of several elements in the liver: As, Cu, Mn, Mo, Se, Tl, and V were higher 2, 30-50, 3-7, 8, 2-3.5, 2, and 9 times, respectively, in both seasons, whereas Fe was 9 times higher only in autumn. Higher concentrations in the gills were found for Sr (13 times) in both seasons and for Fe and Zn (2 times) only in one season (in spring and autumn, respectively). As a result of increased exposure to metals in the river water, increased bioaccumulation was observed for several elements in both liver and gills of Vardar chub in both seasons, specifically for V in the Bregalnica River, and for Cs, Rb, Sr, and Tl in the Zletovska River. Increased exposure to Cd was evident in both seasons in the Zletovska River, but only in spring in the Kriva River, and accompanied by adequate increase of cytosolic Cd concentrations in both organs. In the Kriva River, dissolved Cd concentrations decreased in autumn and resulted with decrease of cytosolic Cd concentrations in both organs. Increased exposure to Pb in the river water was observed in the Kriva River in spring and in the Zletovska River in autumn, and followed by increase of cytosolic Pb concentrations in both organs. Decrease of Pb concentrations in the river water resulted with partial decrease of cytosolic Pb concentration in both organs. Among listed elements, more pronounced accumulation of Cd, Rb, Tl and V was observed in the liver, of Pb and Sr in the gills, while accumulation of Cs was comparable in both organs. Contrary to above listed elements, several elements did not exhibit signs of bioaccumulation, even under conditions of extremely high exposure in the river water, indicating existence of regulation mechanisms for those elements in chub organism. For example, despite increased concentrations of dissolved Fe in the Bregalnica River and dissolved Mn and Zn in the Zletovska River in both seasons, as well as dissolved Fe in the Kriva River in autumn, increased cytosolic concentrations of these elements were generally not found in the cytosols of chub liver and gills. Only in the Zletovska River, cytosolic concentrations of Mn in spring and Zn in autumn were found increased in the gills (3 times and 30%, respectively) compared with other two rivers, at exposure level of 350 μg L-1 of dissolved Mn and 1.5 mg L-1 of dissolved Zn. However, at much higher exposure level of dissolved Mn in autumn (2.4 mg L-1), increased bioaccumulation of Mn was not observed. In order to exclude chub physiology as a cause of differences in metal/metalloid concentrations between three rivers, we have analyzed their association with the fish size (mass range: 13.86-307.89 g), sex (43% females, 57% males) and season, using the data obtained at less impacted Bregalnica River. The significant associations with chub mass and sex were determined for several elements, but only for cytosolic Ba concentrations in the liver and for cytosolic Mn concentrations in the gills they were determined in both seasons. Negative correlations with chub mass and higher concentrations in males were obtained for Ba, whereas positive correlations with the chub mass and higher concentrations in females were obtained for Mn. Seasonal differences were also observed, with 2-3 times higher concentrations of Cu and Sr in both organs in spring and Fe and Zn in the gills in spring and autumn, respectively. Elements which have been found to accumulate in chub liver and gills due to increased exposure to metals in water (i.e. Cs, Rb, Tl, Cd, Pb, and V) generally showed no association with the chub size, sex or season. Integrated approach to estimation of metal pollution of river water requires linking of data on concentrations of dissolved metals in water with data on metal accumulation in target organs of indicator organisms. In the cytosol of liver and gills of Vardar chub (Squalius vardarensis), increased concentrations of Cd, Cs, Pb, Rb, Sr, Tl, and V were determined in association with increased concentrations of these elements in the river water, whereas, at the same time, their dependence on fish physiology was not established. This indicates the possibility of applying cytosolic concentrations of these metals in chub liver and gills in investigative monitoring as indicators of increased biological availability of metals in the river water.

The natural environment of many river ecosystems, especially in the north eastern part of Macedonia, is more and more polluted with heavy metals, most of which were released into the environment by the active mines in this region. It could be presumed with high certainty that long-term exposure to high levels of dissolved metals will result in increased levels of metals bioaccumulated in the tissues of aquatic organisms, and consequently cause various sub-toxic and toxic effects. In monitoring studies on long-term metal pollution, fish proved to be the most appropriate indicator organisms due to their long life span and top position in the aquatic food chain, as well as potential risk of human consumption of “metal-loaded” fish. Histopathology is one approach used to monitor toxicopathic tissue changes in fish. Several liver lesions are reliable biomarkers in assessing anthropogenic stress, being consistently associated with contamination exposure including metal contamination. Facing the above, our aim was to assess the current metal pollution impact on the health status of the local ichthyofauna, namely by studying toxicopathic changes in gonad and liver tissue. In this vein, we studied the gonads and liver of 158 chubs collected from three rivers: the Bregalnica River, as the site nonimpacted by mines and contaminated mainly with agricultural and domestic waste waters, as well as the Kriva River and the Zletovska River, two rivers suspected to be under high degree of metal pollution from active Pb/Zn mines Toranica and Zletovo, respectively. Fish were collected in two seasons, spring and autumn of 2012. Gonad and liver tissues were routinely dehydrated through ascending series of ethanol, cleared in xylene, infiltrated, and embedded in paraffin. The obtained 5 μm thin sections were stained with hematoxylin and eosin. Histological alterations in gonad tissues, in both males and females, were not detected in examined chub. Contrary, if all sites were considered, the spectrum of observed histological lesions in the liver (and prevalence of affected fish) varied from non-specific minor degenerative conditions, such as lymphocyte infiltration (19%), focal necrosis (12%), parasitic granuloma (2%), lipidosis (3%) to extensive and/or more severe changes such as: cholangiofibrosis (19%), bile duct proliferation (21%), megalocytosis (25%), light-dark hepatocytes (6%) and hepatocytes regeneration (3%). Although prevalence of light-dark hepatocytes and hepatocytes regeneration was low, these lesions, as well as megalocytosis, were previously established as histopathological biomarkers of contaminant exposure in both field and laboratory studies, in a variety of species. In general, if all lesions were considered, no differences in prevalence were found between locations and it varied from 45% in Bregalnica, 43% in the Kriva River and 41% in the Zletovska River. Significant differences were found only for lymphocyte infiltration between Bregalnica (13%) and the Kriva River (29%) and for hepatocytes regeneration between Bregalnica (0%) and the Zletovska River (4%). The results of histopathological investigation showed clear effects of water contamination on fish health in all three rivers, and not only in two mining impacted rivers. Some lesions observed in this study suggested environmental contamination as the most likely etiology. Although only a long-term monitor programme can provide definitive answers, the obtained data indicated that more research efforts should be devoted to the environmental conditions and metal contamination in the rivers in Macedonia, especially on their effects on local ichtyofauna health.

Anthropogenic activities increase discharge of different pollutants in the aquatic environment which cause deterioration of the environment. Mining activities release in the environment different inorganic species and metals, which has negative effects on the water quality, aquatic organisms and humans. Aquatic organisms are affected by metal concentrations not only in their surrounding water but also in their food. Microorganisms are also affected by changes in the environment and can also provide biological responses to the environmental disturbances. Microorganisms associated with aquatic organisms can thus be used to monitor the impact of mining activities on the aquatic environment because of the ubiquity of microorganisms in aquatic environments. The objectives of the present study were (i) to determine abundance of fish bacterial population and species diversity of bacterial community of Vardar chub (Squalius vardarensis) from three Macedonian rivers, (ii) to compare obtained bacterial communities, and (iii) to isolate and characterize the isolates using different methods of bacterial identification (MALDI and VITEK 2 Systems), and (iv) to evaluate whether they provide a good assessment of the potential detrimental effects of pollution on aquatic organisms. For that purpose, we sampled 68 fish from rivers: Zletovska (12), Bregalnica (30) and Kriva (26), and obtained samples for bacteriological analysis from the gills and internal organs (spleen, liver and kidney). According to bacterial analysis Kriva and Bregalnica Rivers are aquatic environments under high anthropogenic influence. In contrast, Zletovska River constitutes a less microbial polluted ecosystem. Bacterial analyses were negative for internal organs, whereas samples form the gills were only positive. Bacterial analysis of gills indicated that size of the bacterial population were higher in fish from the Bregalnica River (10.8 CFU/fish) in relation to Kriva (7.2 CFU/fish) and Zletovska River (7 CFU/fish). Contribution of bacterial genera to the bacterial population assemblage was higher in Zletovska River, with three genera: Pseudomonas (52%), Aeromonas (24%) and Acinetobacter (24%), more than in relation to Kriva River, two genera: Pseudomonas and Morganella, as well as to Bregalnica River with one genus Acinetobacter. This study reveals the influence of microbial water quality on diversity of fish bacterial populations in the rivers. In general, dominant genera Pseudomonas and Acientobacter, which are known as genera that can remove metals and have bioaccumulation potentials for metals from polluted aquatic environments, can provide a good assessment for the determination of the potential effects of metal pollution on the aquatic organisms.

Contamination of surface waters is a world-wide environmental problem. Anthropogenic activities like industry and mining can increased metal concentrations in the surface waters and may result in significant deterioration of water quality. Microorganisms are commonly used as biological indicators of surface water conditions. Microorganisms respond rapidly to changes in their environment due to their short generation time. Metal toxicity, acidic pH and low nutrient levels result in stressed microbial communities. High levels of heavy metals can affect the structure of microbial communities, resulting in decreased metabolic activity and diversity of microorganisms. Therefore, there is a need to monitor microbial water quality and to provide insight into their interactions with the impact of mining and the subsequent implications on the rivers ecosystem function. Two rivers were chosen for this study in autumn 2012, the Zletovska River, which receives waste from the Zletovo mine, and the Kriva River, which receives waste from the Toranica mine. The Bregalnica River was chosen as a non-impacted aquatic system. Microbiological analyses of river water were performed using defined substrate technology. Total coliforms bacteria were identified using Colilert test (IDEXX Laboratories., Westbrook, USA), whereas enterococci were identified using Enterolert-E test (IDEXX Laboratories). Total coliforms and enterococci were enumerated using Quantitray 2000 (IDEXX Laboratories), which used a 97-test-well system and provided the most probable number (MPN) of bacteria/100 mL. Analysis of the microbial water quality showed higher levels of bacterial indicators in the rivers Kriva and Bregalnica downstream from the big cities. Together with significantly higher number of bacterial indicators an increase in nitrate and phosphate was observed, indicating a particular anthropogenic impact on these rivers. A small number of bacterial indicators were found in the Zletovska River, indicating less anthropogenic influence, but it is not possible to say is that the effect of pollution from mining activities. A small number of bacterial indicators were found in the Zletovska River, indicating less anthropogenic influence, but it is not possible to conclude if that was the effect of pollution from mining activities. Based on these preliminary results from one sampling season, it can be concluded that the impact of large cities is significant on analyzed rivers from a microbiological point of view.

Histopathological alterations on the gills of Vardar chub (Squalius vardarensis) were studied in three rivers in Pb/Zn mining region in north-eastern Macedonia. The Zletovska and Kriva River are directly impacted by active Pb/Zn mines Zletovo and Toranica, respectively, whereas the Bregalnica River is moderately contaminated mainly by agricultural waste, and considered in this study as nonimpacted site. The analyses were performed for two seasons of 2012, spring (May/June) and autumn (October), and the gills of 6-10 chub of similar size and age were examined for each river in each season. Tissue samples were fixed in 4% neutral formaldehyde, and processed using a standard histological technique: dehydration in the ethanol series, embedding in paraffin, and serially sectioning at 2 μm. Sections were stained with haematoxylin and eosin. In all three rivers, the circulatory disturbances, regressive and progressive changes were found, but their severity differed. The signs of inflammation and neoplasm were not observed. The circulatory disturbances and regressive changes had the highest scores according to Bernet et al. (1999) at the Zletovska River, progressive changes were more pronounced at the Kriva River, whereas all changes were the mildest at Bregalnica. If importance of the changes was considered, the alterations described with importance factors 1-2 had higher scores at Zletovska and Kriva River than Bregalnica, but changes described with importance factor 3, e.g. necrosis, were especially pronounced only at the Zletovska River. The characteristic alteration on the gills of the chub from the Bregalnica River was hyperplasia and erythrocyte congestion throughout the entire lamella (aneurism). This progressive changes were observed in both seasons, with higher score in autumn. More severe lesions were observed in the gills of the chub from two mining impacted rivers. Typical lesions on the Zletovska River included thinning and lifting of epithelial cells, congestion and oedema, fusion of several secondary lamellae usually on the tips of secondary lamellae, acute necrosis of epithelium and calcified cartilage, as well as oedema and deformations of the lamellar cartilaginous base. In some areas the secondary lamellae had atrophied. On the Kriva River, moderate to marked gill damage was found, such as degeneration and necrosis, exfoliation and delaminating of respiratory epithelium. Extreme telangiectasia was also found. In both seasons hyperplasia and hypertrophy of mucous and epithelial cells appeared severe. 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 more pronounced due to preconcentration effect. All these findings were in agreement with observed water contamination by mining waste, which was continuously the worst in the Zletovska River, occassionaly at Kriva River, whereas the best water quality was found in the Bregalnica River (Ramani et al., 2014). Therefore, the histopathological alterations on the chub gills could be used as a reliable, although nonspecific, indicator of water quality.