Search

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 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.

Fish are frequently selected as the organisms of choice in aquatic biomonitoring due to their top position in the biotic communities and their sensitivity to low concentrations of environmental pollutants. Among other pollutants, metals represent a serious threat to the aquatic environment, since they are not biodegradable and can accumulate in the sediments and biota, including fish. For normal cellular metabolism, fish require essential metals which may be taken up from water, food and/or sediments in varying degrees. Metals are taken up to fish through skin, gills and gastrointestinal tract. In the last decade there is a growing concern that dietborne metal uptake may be of equal or greater importance than the waterborne for native fish. Accordingly, in the present field study gastrointestinal tissue was selected as an indicator organ for evaluating dietary metal exposure of freshwater fish Vardar chub (Squalius vardarensis) in two mining impacted rivers (Zletovska and Kriva) and in less contaminated river (Bregalnica). High contamination of the aquatic systems by the waste from active mines can seriously affect the quality of surface water, with the regard to metal/metalloid levels, and consequently influence the aquatic life. The exposure of Vardar chub to dietborne metal levels in three differently impacted rivers by Pb/Zn active mines Toranica and Zletovo, situated in the north-eastern Macedonia, was assessed by measuring 22 trace and 4 macroelements in sub-cellular gastrointestinal tissue fraction, cytosol, in order to determine the levels of bioavailable metal form. Cell cytosol was isolated from posterior part of gastrointestinal tissue by homogenisation at 4°C in 5 volumes of homogenising buffer (100 mM Tris/Base, pH=8.1) containing 1 mM DTT as a reducing agent and 0.5 mM PMSF and 0.006 mM leupeptin as inhibitors of proteolitic activity. The homogenates were centrifuged at 50000xg for 2 hours (4°C), resulting in water soluble cytosolic fraction (S50), which was 10 times diluted for determination of trace and 100 times for determination of macroelements. Metal measurements were carried out by high resolution inductively coupled plasma-mass spectrometer (HR ICP-MS). The sampling campaigns were performed in spring (May/June) and in autumn season (October), which coincided with chub spawning and post-spawning period. Significantly higher gastrointestinal metal concentrations in the mining impacted Kriva River compared to the Bregalnica River in both seasons were found for Se, in the mining impacted Zletovska River for Li, Rb, Mo, Cs, Tl, U, Co, Ni, Cu, Sr, As, Ca, while the highest V, Mn, Zn and Ba concentrations were found in the agriculturally contaminated Bregalnica River. Although dietborne metal levels usually do not directly reflect total dissolved metal concentrations in water, spatial variability of gastrointestinal cytosolic metal levels were in accordance with higher Ba and V total dissolved concentrations in the Bregalnica water and Co, Cs, Cu, Li, Ni, Rb, Sr, Tl and Ca in the Zletovska water. Moreover, seasonal differences of metal concentrations in water were followed by gastrointestinal metal variability, which was evident as higher Pb levels in Bregalnica in spring, higher Tl and Sr in Zletovska in autumn, and higher Cd and Pb levels in the Kriva River in spring. In addition, significant correlation of cytosolic Pb and chub total length and body mass (r=0.5, p

In the past decades, the interrelation between parasites and contaminants has gained increasing interest, especially in aquatic ecotoxicology. Fish intestinal parasites, acanthocephalans, were reported to effectively accumulate metals in concentrations that are orders of magnitude higher than those in the commonly used indicator organisms like fishes, bivalves or crustaceans. Acanthocephalans lack a gastrointestinal system and consequently, depend on host micronutrients and highly absorb essential metals from the fish intestine, as elements of physiological importance. Due to their competition for essential elements, non-essential elements are also absorbed by the parasites, even more effectively than essential metals. Accordingly, attempts have been made to utilize acanthocephalans as biological indicators of metal exposure in environmental risk assessment studies. Application of acanthocephalans as bioindicators in metal exposure assessment of mining impacted rivers in the north-eastern Macedonia involved parasites hosted by Vardar chub (Squalius vardarensis) dwelling in the Zletovska and Kriva River impacted by Pb/Zn active mines, and in the less contaminated Bregalnica River. Sample preparation involved acid digestion of acanthocephalans in a drying oven at 85°C for 3.5 hours using 1.5 ml of concentrated HNO3 and 0.5 ml H2O2, followed by measurement of 24 trace and 4 macroelements by high resolution inductively coupled plasma-mass spectrometer (HR ICP-MS). For the purposes of calculation of bioconcentration factors (BCFs), cytosolic gastrointestinal metal levels were multiplied with tissue dilution (6 fold) and BCFs were determined as the ratio of the element concentration in the parasites to that in host gastrointestinal tissue (BCF = C[parasite]/C[host intestine]). Two acanthocephalan species, Pomphorhynchus laevis and Acanthocephalus sp., were found in Vardar chub, but only from Bregalnica River and not from two mining affected rivers. Such occurrence of acanthocephalans only in moderately contaminated river disables their application as bioindicators of environmental pollution in mining impacted rivers. It follows that absence of acanthocephalans might be considered as a first sign of highly polluted aquatic environment. In the Bregalnica River, prevalence in both sampling periods, spring (May/June) and autumn (October), was comparable, i.e. around 70% of infected chub specimens. In spring, all measured metal concentrations were higher in P.laevis than Acanthocephalus sp., statistically significantly for 14 trace elements, while in autumn only for 7 trace elements. The data on BCF confirmed higher metal accumulation in P. laevis than Acanthocephalus sp., which was evident in both seasons for all measured metals, except for Sr, Na, Mg and Ca. Estimation of metal accumulation based on BCF indicated in both acanthocephalan species and in both seasons 10-45 times higher Al, Pb, Sr, Tl, Li, and Ba, 3-20 times higher Cr, Ti, Ca, Cu, and Mn, and 1-6 times higher Cd, Ni, V, U, Fe, Mg, As, and Na concentrations in acanthocephalans than in fish gastrointestinal tissue. In contrast, Se, Rb, Mo, Cs, Co, Zn and K were not accumulated by parasites. Although involvement of acanthocephalans in metal exposure assessment gives valuable data, which might serve as a sensitive bioindicator of bioavailable metal levels in the freshwater ecosystem, acanthocephalan-chub system unfortunately cannot be applied as an indicator of acute or chronic metal exposure in mining impacted rivers. Evaluation of possible protective role of acanthocephalans against metal accumulation in chub, regarding high metal uptake capacity of acanthocephalans, pointed to significantly higher Rb, Cs, Tl, Pb, Mn and Cu levels in uninfected compared to chub infected with acanthocephalans in spring, while in autumn only for Cd and Tl. It follows that acanthocephalans can reduce levels of some metals in the gastrointestinal tissue of their host, related to the uptake of bile-bound metals by parasites and reduction of metal levels available for reabsorption by the host. Our data suggest that in metal exposure assessment acanthocephalans should be considered as confounding factors, due to their ability to modify metal concentrations in fish gastrointestinal tissue.