ConCentrations of metals and traCe elements in different tissues of nine fish speCies from the međuvršje reservoir ( West morava river Basin , serBia )

Element concentrations in selected fish species from different trophic levels were analyzed. The following fish species were analyzed: common nase (Chondrostoma nasus), roach (Rutilus rutilus), freshwater bream (Abramis brama), barbel (Barbus barbus), Prussian carp (Carassius gibelio), chub (Squalius cephalus), European perch (Perca fluviatilis), wels catfish (Silurus glanis) and northern pike (Esox lucius). Fish were collected from the Međuvršje Reservoir (West Morava River Basin, western Serbia) during 2012, and samples of liver, muscle and gills were analyzed for As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Li, Mn, Mo, Ni, Pb, Sr, and Zn using inductively coupled plasma optical emission spectrometry (ICP-OES). The liver and gills had the highest measured element concentrations, with Cu, Zn, Fe and Sr being the most prominent. The bioaccumulation of metals was species-specific, with the accumulation exhibiting the following trends: Prussian carp > northern pike > freshwater bream > European perch > chub > common nase > barbel > roach > wels catfish; Li>Pb>Z n>Cu>Fe>Ba>Mn>Sr>Cr>As>Mo>Ni>B, in all examined species; for Co, Cd and Hg, the bioaccumulation factor (BAF) was 0. Results of this study point to the tissue-specific differences in element concentrations, and to distinct differences between fish species regarding the accumulation patterns: common nase, with the highest accumulation observed in the liver, and Prussian carp, with the highest accumulation observed in the gills.


introduCtion
Contamination of aquatic ecosystems (e.g.lakes, rivers, streams) with heavy metals and trace elements is a serious problem receiving world-wide attention [1,2].Metals from natural and anthropogenic sources are environmentally ubiquitous, released in and transported by water, and taken up by aquatic organisms.These elements enter aquatic ecosystems by atmospheric precipitation, soil and rock erosion, and through anthropogenic sources, such as industrial effluents, traffic, mining wastes and agriculture [3,4].Metals are a serious threat because of their toxicity, persistence, capacity for bioaccumulation and biomagnification in the food chain [5].Certain trace elements such as Fe, Cu, Zn, and Mn are essential in small amounts, but can be toxic and adversely affect aquatic life when present above certain concentrations.Hg and Cd are non-essential metals that are toxic even in traces [6].Metal accumulation analyses are the best indicator of aquatic ecosystem pollution [2].Aquatic organisms have the ability to accumulate metals from various sources.The toxic effects of metals range from complete loss of biota to effects on reproduction rate, growth and behavior of organisms [7].
Fish are sensitive to increases in concentrations of different contaminants, such as metals and organic pollutants in water.The accumulation level of metals in fish organs and tissues depends on the fish species, their age patterns, and on the physical and biochemical characteristics and chemical status of their environment [8].
In this work, we analyzed 16 elements in the liver, muscle and gills of nine fish species from different trophic groups: phytophagous (examined in common nase), benthivorous (barbel, freshwater bream), omnivorous (Prussian carp, roach, chub, European perch) and piscivorous species (wels catfish and northern pike).Fish feeding preferences and meal size are factors that determine the trend of element accumulation in fish tissues among species [9].Sampling was carried out in 2012 in the Međuvršje Reservoir in western Serbia that receives large amounts of untreated industrial and communal waters, with the water quality in the reservoir influenced by various pollutants.The main objectives of the study were to highlight the importance of species and tissue selection in biomonitoring by comparing the accumulation patterns among different fish tissues and species, and comparing element concentrations in fish tissues with their concentrations in the water.

materials and methods study area
A field study was carried out in the Međuvršje Reservoir (West Morava River Basin); coordinates: N -43 o 54'43.07";E -20 o 14'12.71";277 m a.s.l.(Fig. 1).The reservoir was formed in 1953 after the construction of a 31-m high dam built for water-level control and management [10].The reservoir is located at the exit of the Ovčar-Kablar Gorge.The length of the reservoir is 9.3 km, surface area 1.5 km 2 , maximum width 272 m, maximum depth12 m (directly below the dam).The quality of the water is influenced by numerous contaminants in the catchment area.Within the 3165-km 2 catchment area, there is intense emission of industrial, urban and rural wastewater.Neither the settlements nor most of the industrial plants in the area possess facilities for wastewater purification.

sample preparation
Sampling of nine fish species (10 individuals per species) was carried out in June and August in 2012.Collection of fish samples was performed using a set of standing gill-nets with a mesh diameter of 10-60 mm, as well as by electrofishing (HONDA 1.2 кW, 6 А).The total weight (g) and total body length (cm) of each fish specimen were measured.Fish species were determined according to Simonović [11].Samples of muscle, liver and gills were removed and frozen until analysis.Water samples were collected at a depth of 20-30 cm below the water surface in 50-ml polyethylene demineralized containers, and conserved with 0.25 ml of concentrated HNO 3 solution.Until analysis the water samples were stored in a fridge (kept at 4ºC).

sample analysis
In the laboratory, the samples were dried using a GAMMA 1-16 LSC plus Freeze Dryers Rotational-Vacuum-Concentrator (Germany), and sample portions between 0.2 and 0.4 g dry weight were subsequently processed in a microwave digester (Speed wave MWS-3; Berghof Products Instruments GmbH, Eningen, Germany), using 6 ml of 65% HNO 3 and 4 ml of 30% H 2 O 2 (Merck Suprapur) at a food temperature program (100-170 o C).
The potential presence of analyzed elements was resolved using a number of blank samples.After reaching room temperature, the digested samples were diluted with distilled water to a total volume of 25 ml.The analysis was performed by inductively All elemental concentrations were expressed as µg g -1 dry weight (dw).Metal concentrations in fish meat (i.e., muscle samples) were also recalculated to the wet tissue weight (WW) and compared with the maximum allowed concentrations (MAC) in fish meat for utilization in the human diet established by the European Union (EU) and the national legislation.According to EU legislation [12], MAC for Cd, Hg and Pb are 0.05, 0.50 and 0.30 µg g -1 w/w, respectively.National legislation prescribes MAC for As, Cd, Hg, Pb, Cu, Fe and Zn in fish meat at 2.0, 0.1, 0.5, 1.0, 30.0, 30.0 and 100.0 µg g -1 w/w, respectively [13].

statistical analysis
To compare the total metal content in fish species and their tissues, the metal pollution index (MPI) was used, obtained using the equation [14]: MPI = (As x B x Ba x Cd x Co x Cr x Cu x Fe x Hg x Li x Mn x Mo x Ni x Pb x Sr x Zn) 1/16 .When the elemental concentration was equal to zero (not detected), the value equal to half of the spectrometer sensitivity (ICP-OES) for the corresponding element was used.Assessment of the differences among groups was performed by ANOVA two-factor analysis (StatSoft, Inc. 2007).Relationships between fish size, weight and trace element concentrations in different tissues were assessed by Spearman's non-parametric correlation test.The bioaccumulation factor (BAF), the ratio of the concentrations of the chemicals in the organism (CB) to that in the water (CWT), was calculated according to the equation: BAF= CB/CWT [15].

results
The concentrations of 16 metals in muscle, liver and gills of nine selected fish species from different trophic levels are presented in Table 1.The lowest concentrations of all analyzed metals were found in muscle tissue (Table 1).The highest concentrations in liver were found for As, Cd, Cu and Fe in common nase, freshwater bream, European perch, northern pike, wels catfish, for B in common nase, northern pike, European perch, wels catfish, for Mo in common nase, roach, Prussian carp, barbel, chub, for Co in European perch, wels catfish, for Hg in chub, European perch, for Zn in common nase, freshwater bream, European perch and for Li in wels catfish.In the gills, the highest concentrations of Sr, Pb, Ba, Cr, Mn, Ni were observed in all species except northern pike, of Li in all species except wels catfish, of Hg in common nase, roach, Prussian carp, freshwater bream, barbel, northern pike and wels catfish, of Co in roach, Prussian carp and freshwater bream, of B in roach, Prussian carp, freshwater bream, barbel and chub, of Mo in freshwater bream, European perch, wels catfish and northern pike (Table 1).The three most abundant elements were Zn, Cu, and Fe (Table 1).
According to comparison of tissues, different fish species and different tissues of the same species, the MPI values (ANOVA two-factor analysis) of fish species significantly differed (p=0.000001) (Fig. 2b); the MPI values of tissues significantly differed (p=0.000001) (Fig. 2c), and the MPI value of tissues and fish species also significantly differed (p=0.000001) (Fig. 2a).The MPI value was highest in the gills in most of the analyzed species.Prussian carp, common nase and freshwater bream were distinguished from others by their higher MPI values (Table 2).There were only a few significant correlations between the overall elemental accumulation and fish size and weight: As and Prussian carp weight correlation coefficient R= -0.6, p<0.05;As and freshwater bream total length R=0.553, p<0.05;Cr and chub weight and total length, R=-0.613 and R=-0.578, respectively, p<0.05;Pb and roach weight and total length R=-0.553, p<0.05;Mo and wels catfish weight R=-0.685, p<0.05.There were no differences in the distribution of Fe, Li, Mn and Sr among tissues.Two fish species  The BAF was not calculated for Cd, Co and Hg because these metals were not detected in the water.The highest BAF values were observed in the gills of the majority of analyzed fish species (Table 3).
In three different tissues of the nine studied fish species almost all of the 16 metals and trace elements were detected.The concentrations of the metals in muscle did not exceed the MAC prescribed by the EU regulations [12], international standards [16] and the Regulation of the Republic of Serbia [13] (Table 4).

disCussion
The analysis of metal bioaccumulation in different tissues of fish with different diet requirements showed a high level of differentiation, as well as significant differences in the distribution of elements in the body.In many studies, the highest metal bioaccumulation was found in the liver, and this was consistent over a wide range of different fish species [2,[17][18][19][20].Muscle is generally considered a tissue with little potential for bioaccumulation and is a tissue with the lowest metal content [21,22].The liver is a metabolically active and elimi- native organ, due to the activities of metallothioneins, proteins with the ability to bind to specific metals, such as Cu, Cd and Zn, thereby reducing their toxicity and allowing for the accumulation of high metal concentrations [20,22,23].In our study, the highest concentrations of most of the analyzed metals were detected in the gills (Zn, Sr, Pb, Ni, Mn, Li, Cr, Ba, and B).When comparing MPI values between fish species and tissues, only the wels catfish and European perch had higher values of MPI in the liver than in the gills.This could be explained by the fact that the gills are the first organ to come into contact with metals and trace elements in the water.The Međuvršje Reservoir is greatly influenced by many pollutants from upstream sources (industrial, municipal and rural wastewater) [24].
Metal accumulation analyses in different fish tissues have been conducted in some localities of Serbia.An ecotoxicological investigation of common nase, freshwater bream and bleak in the Međuvršje Reservoir was conducted by Lazić et al. [25].In this study, the concentrations of selected metals, which were also examined in the present study, were close to the maximal allowed values, except for Hg, whose concentration was higher.During similar investigations [26] of tissues of Prussian carp in Old Begej (a Special Nature Reserve in the Vojvodina province), As and Li were not detected, Mn and Mo were not found in the muscle tissue and Cu was present only in the liver.The concentrations of Ba in Prussian carp gills were higher than in the present study, while the concentration of Mо was lower.Substantially higher concentrations of Zn and Fe, up to 10 times higher than those detected in fish from the Međuvršje Reservoir, were detected in the liver and gills [26].An examination of metal concentrations in tissues of barbel from the Belgrade section of the Danube River did not report the presence of Cr, Cd, Pb, Co, Ni and Li [27].In this section of the Danube ecotoxicological studies were also performed on freshwater bream and wels catfish.Seventeen selected elements were investigated: B, As, Ba, Cu and Mo were not detected in muscle, As, B and Ba were measured in the liver, As and Cu and As and Ba were not detected in the gills of freshwater bream and catfish, respectively [28].The level of Cr and Hg in the liver was higher in piscivorous fish (Sander lucioperca and wels catfish) [29], whereas in our study the concentrations of these metals were higher in omnivorous (Prussian carp) and benthivorous fish (barbel and freshwater bream).The concentrations of B and Fe were higher in the gills of omnivorous species, Lota lota and Cyprinus carpio [29], as was also observed in the present study.Muscle samples from Prussian carp from the Gruža Reservoir (West Morava River Basin) exhibited the highest tendency of element accumulation (Fe, Cd, and Cu) [30], whereas in the Međuvršje Reservoir this tendency was not observed for Cu, Zn, Fe and Li in the same species.Remarkably higher concentrations of Cu in Prussian carp liver in comparison to other analyzed tissues, were also registered by other authors [16,21,22].
The presence of metals and trace elements in fish is dependent on species, body size, physiological state, feeding patterns and tissue type [17].According to our results, common nase and Prussian carp differed from the other analyzed species by the high metal concentrations in their tissues.Therefore, these fish have the potential to be used as bioindicators for monitoring purposes.They are two of the species most frequently caught by anglers in the Međuvršje Reservoir.

25 *
ND indicates values below the detection threshold table 3. continued: were differentiated from other species by the tissue metal distribution: in common nase, As and Mo had the highest concentrations in all analyzed tissues, and Cd, Zn and Cu had the highest concentrations in the liver; in the Prussian carp, Pb, Ni, Li, Cr and B had the highest concentrations in all analyzed tissues, with the concentrations of Pb and Fe being the highest in the gills.

table 1 .
Concentrations of 16 metals and trace elements in different tissues of 9 selected fish species.All element concentrations are presented as µg g -1 dry weight (dw).
*ND indicates values below the detection threshold

table 2 .
Metal pollution index (MPI) based on the concentrations of 16 elements in the liver, gills and muscle of 9 fish species (means±standard deviation).

table 4 .
Concentrations in fish meat (i.e., muscle samples) recalculated to the wet tissue weight (ww) and compared with the maximum allowed concentrations (MAC) established by the European Union (EU), FAO and the national legislation (SER).