Introduction

Biomarkers such as serum chemistry and parameters of oxidative balance are good indicators of an overall biological status providing information on the effects of contaminants on the organism [1]. One of the most important environmental pollutants affecting health are trace metals [2,3,4,5,6,7,8,9,10], pesticides [11,12,13,14], pharmaceuticals [15,16,17], and endocrine disruptors [18, 19]. Monitoring of the presence and/or levels of contaminants in the organism under natural conditions and testing their associations to the physiological status is important for understanding their possible toxic effects and health risk on animals and humans. Particularly, trace metal investigation is still one of the main focuses of toxicological studies over the past years. Environmental contamination by trace metals is a consequence of anthropogenic activities connected to the industry, agriculture, and waste management [12, 20,21,22,23].

Measurement of blood serum chemistry parameters is necessary for the diagnosis of the health status in aquatic animals living in potentially polluted areas. Toxic effects of trace metals are generally connected to changes in the levels of biochemical parameters monitored in many studies [4, 21, 24,25,26,27].

A variety of chemicals may cause oxidative stress as a consequence of increased levels of reactive oxygen species (ROS) affecting the mitochondrial function followed by alterations to the enzymatic and/or endogenous antioxidants in blood [10] and tissues [8, 28,29,30]. Oxidative stress is a sensitive endpoint for metal toxicity due to alterations of target tissues or inhibition of enzymes containing thiol groups. As such, a combined exposure to trace metals can affect the antioxidant ability of blood [1]. Trace metals accumulated in aquatic animals are potentially redox active, which may suggest an imbalance between ROS production and antioxidant mechanisms of the fish [23, 31]. The main antioxidant enzymes are superoxide dismutase, glutathione peroxidase, and catalase [10, 23, 32]. Glutathione as endogenous antioxidant is also an integral part of the defenses against oxidative stress [33, 34] and primarily prevents the oxidation of water soluble proteins [35]. Furthermore, non-enzymatic antioxidants include uric acid and albumin. Biomarkers of oxidative stress (malondialdehyde as an index of lipid peroxidation and protein carbonyls as product of protein oxidation) are used as indicators of oxidative damage [14, 22, 36, 37].

The aim of the present study was to investigate seasonal and sex interactions between trace element/metal content in the blood serum and serum biochemical parameters as well as oxidative status markers in freshwater fish common carp.

Material and Methods

Experimental Design, Animal Management, and Blood Sampling

This study was realized during spring and summer of 2015. Fishes were bred by semi-intensive method of farming (university experimental pond in Kolíňany—West Slovak Lowland—Slovak Republic; 48°21′14.6″N 18°13′03.2″E) (Fig. 1). Fish stocking was realized at the beginning of March 2015. Catching of the fish was realized at beginning of May (spring season) and at the end of July (summer season) 2015. The freshwater fish (Cyprinus carpio) were caught by seine net. In total, 38 fishes were collected. After catching, the animals were transferred in polyethylene bags to the laboratory in 20 min for blood collection. Fish were manipulated by a competent person in accordance with the provisions of the national law, approved by the Ethics Committee of the Slovak University of Agriculture in Nitra, protocol number 48/2013. After standard ichthyology evaluation (standard length and weight measurements, age determination by scales—Table 1), blood sampling was realized. For comparison with other authors, which presented length of fish as total length (TL), the transformation equation is given: TL = 9.28564 + 1.16322 × SL (r = 0.9876, r2 = 95.76%, p < 0.001).

Fig. 1
figure 1

The University fish pond Kolíňany (West Slovak Lowland—Slovak Republic; 48°21′14.6″N 18°13′03.2″E)

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Table 1 Characteristics of analyzed fish
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Blood samples (n = 38) were taken in spring (n = 19) and summer (n = 19) seasons from male (n = 17) and female (n = 21) individuals. Blood was collected from aorta ventralis (Nomina Anatomica) from each fish. The samples were allowed to coagulate at room temperature. Subsequently, the samples were centrifuged for 20 min at 3000 rpm. Blood serum was collected and stored at − 20 °C until analyses at the Department of Animal Physiology.

Clinical Biochemistry Analyses

Blood serum concentrations of calcium (Ca), magnesium (Mg), total protein (TP), glucose (Glu), urea, cholesterol (Chol), triglycerides (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), total bilirubin (Bili), and total protein (TP) were measured using DiaSys (Diagnostic Systems GmbH, Holzheim, Germany) commercial kits and the semi-automated clinical chemistry analyzer Randox RX Monza (Randox Laboratories, Crumlin, UK) [6]. Sodium (Na), potassium (K), and chloride (Cl) ions were analyzed using an EasyLite analyzer (Medica, Bedford, MA, USA) provided with an ion-selective electrode [7, 38].

Assessment of the Oxidative Status

ROS production in each sample was assessed by the chemiluminescence assay using luminol (5-amino-2,3-dihydro-1,4-phthalazinedione; Sigma-Aldrich) as the probe [39]. The test samples consisted of luminol (2.5 μL, 5 mmol/L) and 100 μL of sample. Negative controls were prepared by replacing blood with 100 μL of PBS (Dulbecco’s Phosphate Buffer Saline without calcium chloride and magnesium chloride; Sigma-Aldrich). Positive controls included 100 μL PBS, 2.5 μL luminol, and 50 μL hydrogen peroxide (H2O2, 30%; 8.8 M; Sigma-Aldrich). Chemiluminescence was measured using the Glomax Multi+ Combined Spectro-Fluoro Luminometer (Promega Corporation, Madison, WI, USA) [36, 37]. The results are expressed as relative light units (RLU)/s/g protein.

An improved enhanced chemiluminescence antioxidant assay using horseradish peroxidase conjugate and luminol was used to study the total antioxidant capacity (TAC) of the sample [40]. Five to one hundred micromoles per liter of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Sigma-Aldrich) was used as the standard, while a signal reagent consisting of 0.1 mol/L Tris-HCl (Sigma-Aldrich), 12 mol/L H2O2 (Sigma-Aldrich), 41.8 mmol/L 4-iodophenol (Sigma-Aldrich), and 282.2 mmol/L luminol (Sigma-Aldrich) was used to induce the chemiluminescent reaction. Chemiluminescence was measured on 96-well plates in 10 cycles of 1 min using the Glomax Multi+ Combined Spectro-Fluoro Luminometer (Promega Corporation). The results are expressed as micromoles of Trolox Eq. per gram of protein.

Superoxide dismutase (SOD), glutathione peroxidase (GPx), and d-3-hydroxybutyrate (BHB) activity was measured using the Randox commercial kits (Randox Laboratories, Crumlin, Great Britain) and the semi-automated analyzer Randox RX Monza (Randox Laboratories, Crumlin, UK) [36]. The results are expressed as units per gram of protein (SOD, GPx) and micromoles per gram of protein (BHB).

Catalase (CAT) activity was quantified according to Beers and Sizer [41] by monitoring the decrease of hydrogen peroxide (H2O2) at 240 nm [36]. The values are expressed as units per milligram of protein.

Reduced glutathione (GSH) was evaluated by the Ellman method [42]. GSH concentration is expressed as milligrams per gram of protein.

Albumin (Alb) concentration was measured using the ALB BioLa Test (PLIVA-Lachema, Brno, Czech Republic) commercial kit. Albumin binds with Bromo Cresol Green (BCG) at pH 4.2 causing a shift in absorbance of the yellow BCG dye. The blue-green color formed is proportional to the concentration of albumin, when measured photometrically at 578 nm with the help of the Genesys 10 spectrophotometer (Thermo Fisher Scientific Inc.). Albumin concentration is expressed as grams per gram of protein.

Uric acid quantification was characterized by the oxidation of the substance leading to H2O2 and allantoin formation. The resulting H2O2 was detected by reacting with N-ethyl-N-(2-hydroxy-3-sulphopropyl)-m-toluidine and 4-aminoantipyrine. The absorbance is subsequently measured at 550 nm. BioLa Uric Acid commercial kit (PLIVA-Lachema, Brno, Czech Republic) and the Genesys 10 spectrophotometer (Thermo Fisher Scientific Inc.) were used for the assay. Uric acid concentration is expressed as micromoles per gram of protein.

Carbonyl group quantification was performed through the traditional 2,4-dinitrophenylhydrazine (DNPH) method [43]. Protein carbonyls are expressed as nanomoles per gram of protein [44].

Lipid peroxidation (LPO) expressed through malondialdehyde (MDA) production was measured with the help of the TBARS assay, modified for a 96-well plate and ELISA reader [36, 37]. MDA concentration is expressed as micromoles per gram of protein.

Protein concentration was quantified using the DiaSys Total Protein (Diagnostic Systems GmbH, Holzheim, Germany) commercial kit and the semi-automated clinical chemistry photometric analyzer Randox RX Monza (Randox Laboratories, Crumlin, UK) [6]. The measurement is based on the Biuret method, according to which copper sulfate reacts with proteins to form a violet blue color complex in alkaline solution, and the intensity of the color is directly proportional to the protein concentration when measured at 540 nm.

Detection of Trace Metals

The concentrations of trace metals (arsenic [As], cadmium [Cd], chromium [Cr], copper [Cu], iron [Fe], manganese [Mn], nickel [Ni], lead [Pb], selenium [Se], strontium [Sr], and zinc [Zn]) were analyzed in blood serum samples of fishes by inductively coupled plasma optical emission spectrometry (ICP-OES) and mercury (Hg) was determined by cold-vapor atomic absorption spectroscopy (CV-AAS).

Pre-analytical Procedure for ICP-OES Analysis

High-purity chemicals were used for all operations. For elemental analysis, the fish serum samples were kept at − 20 °C until analysis. The defrosted samples (1 mL) were mineralized (wet mineralization) in the high-performance microwave digestion system Ethos UP (Milestone Srl, Sorisole, BG, Italy) in a solution of 5 mL HNO3 (TraceSELECT®, Honeywell Fluka, Morris Plains, USA) and 1 mL of H2O2 (30%, for trace analysis, Merck Suprapur®). Samples, and blank sample, were digested according to preloaded method “animal tissue” developed by manufacturer for assuring the best result. The method consists of 15-min heating to 200 °C, keeping this temperature for 15 min and 15 min of active cooling. The digests cooled to 50 °C were filtered through the Sartorius filter discs (grade 390) (Sartorius AG, Goettingen, Germany) into the volumetric flask and filled up with ultrapure water to a volume of 50 mL.

ICP-OES Analysis

Analysis of the elements (As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, Se, Sr, and Zn) was carried out using inductively coupled plasma optical emission spectrophotometer (ICP-OES 720, Agilent Technologies Australia (M) Pty Ltd.). Detections limits (μg/L) of measured elements were follows: As 1.50, Cd 0.05, Cr 0.15, Cu 0.30, Fe 0.10, Mn 0.03, Ni 0.30, Pb 0.80, Se 2.00, Sr 0.01, Zn 0.20. Details of the instrumental operating conditions are listed in Table 2. In the experiment, Multielement standard solution V for ICP (Sigma-Aldrich Production GmbH, Switzerland) was used. The validity of the whole procedure was checked by processing of duplicate samples against the certified reference material (CRM–ERM CE278K, Sigma-Aldrich Production GmbH, Switzerland).

Table 2 Operating parameters for the determination of elements by ICP-OES
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CV-AAS Analysis

Total mercury content (Hg) was determined directly in the defrosted blood serum samples by a selective mercury analyzer AMA-254 (Altec, Praque, Czech Republic) based on CV-AAS.

The detection limit for mercury was 1.5 ng/L [6].

Statistical Analyses

Obtained data were checked for normality using a Kolmogorov-Smirnov test before statistical analyses. Mann-Whitney non-parametric test was used to assess the differences in the investigated parameters and metal concentrations between seasons and genders. The Spearman R correlation coefficient was used to measure the association between the trace elements concentrations and all investigated parameters in the blood serum. The minimum significance level was P < 0.05. Statistical analyses were performed using STATGRAPHICS Centurion (© StatPoint Technologies, Inc., USA) and GraphPad Prism 3.02 (GraphPad Software Incorporated, San Diego, California, USA).

Results

In the present study, we assessed the relationship between trace metal content and biochemical/oxidative status markers in freshwater fish blood. Mean and median seasonal/gender concentrations of obtained trace metals in the blood serum are summarized in Fig. 2 (mean, median, 25–75%, min-max) and P values of Mann-Whitney test are shown in Table 3. The general scheme of descending concentrations of trace metals in blood serum samples was follows: Zn > Fe > Cu > Sr > Cr > Ni > Mn > Pb > Se > As > Cd > Hg. Zn was the most accumulated element (4.42–119.64 mg/L). Significant differences among season and sex were not detected for concentrations of As, Cd, Cr, Cu, Fe, Hg, Ni, Pb, and Se. Mn (P < 0.05) and Zn (P < 0.01) were significantly higher in spring season. Mean concentration of Sr was significantly higher in female fish (P < 0.01). Overall, the trace element content was higher in spring season, except Hg, Ni, Se, and Sr, but non-significantly.

Fig. 2
figure 2

Trace elements concentrations (mg/L resp. μg/L) measured in blood serum of C. carpio. Data are presented using box-whisker plot (mean, median, 25–75%, min.-max.)

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Table 3 Results (P values) of Mann-Whitney test for the concentrations of trace metals, oxidative status markers, and serum chemistry parameters among season and gender
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Mean and median seasonal/gender concentrations of oxidative status markers are presented in Fig. 3. Compared to fishes collected during different seasons, we observed that the activities of ROS, SOD, CAT, GPx, PC, MDA, and bilirubin were significantly increased in spring season and the activities of TAC, GSH, UA, and albumin were significantly higher in summer season. GPx and PC activities were significantly higher in male fish (Table 3).

Fig. 3
figure 3

Oxidative status markers measured in blood serum of C. carpio. Data are presented using box-whisker plot (mean, median, 25–75%, min.-max.)

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Median concentrations of serum chemistry parameters are presented in Fig. 4. Glucose, AST, and cholesterol contents were significantly higher in summer season (P < 0.05). Cholesterol level was significantly higher in female fish (P < 0.05). Finally, higher significant value of CK was found in male fish (P < 0.05).

Fig. 4
figure 4

Serum chemistry parameters measured in blood serum of C. carpio. Data are presented using box-whisker plot (mean, median, 25–75%, min.-max.)

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The Spearman R correlation coefficients were used to assess the relationships between the serum chemistry/oxidative stress parameters and the trace metal concentrations in the blood serum. Statistically significant correlations during seasons are shown in Table 4.

Table 4 Statistically significant correlations between trace metal concentrations and investigated blood serum parameters (oxidative status markers and serum chemistry parameters) in different seasons
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In the spring season, positive statistically significant correlations between the Co, Sr, and Zn concentrations and Ca and Mg were found. Moreover, a statistically significant positive correlation was detected between K and Fe resp. Mn; Cd, Fe, and Mn were negatively correlated with Na and Cl. Positive (statistically significant) correlations were found between Ni and ALP, Hg and Chol, As negatively correlated with bilirubin. Concentration of ROS negatively correlated with Cd, Cu, Ni, and Sr in spring season. Ni was also positively correlated with TAC and UA and was negatively correlated with SOD and MDA. Hg was negatively correlated (statistically significant) with TAC and was positively correlated with MDA and BHB. The analysis also revealed significant negative associations between Cu and MDA resp. SOD and positive association between Cu and GSH. Negative and significant relationships occurred between Cd and SOD, and Pb and BHB. Significant, negative correlation was seen between Fe and albumin in spring season.

The correlation analysis in summer season showed the significant positive relationship between Mg and Cr; Cu, Fe, and Hg; Ca and Zn; Na and Cu; K and As; Cl and Ni; and Mn and Urea resp. ALP, Cu, and cholesterol. Concentrations of CK were positively correlated with Cr and Hg; TG was also positively correlated with Cu and Hg. Negative significant correlations between Ni and Na, TP, Chol, and TG were found in summer season. Glucose level showed a negative correlation with Cr and Hg. During summer season, the concentration of Cu positively correlated with ROS, MDA, and BHB activities; Sr concentrations positively correlated with both TAC and GSH activities; Hg concentration positively correlated with ROS activity; Mn concentration positively correlated with GSH activity. The negative correlations during summer season in oxidative status markers were obtained between the concentration of Fe and bilirubin, Sr and MDA, and Cu and UA.

Discussion

Findings obtained in the present study describe the interactions between environmental pollutants and physiological status of freshwater fish during spring and summer season. Various trace elements in polluted environments may have different effects on living organisms; therefore, it is necessary to monitor and test such associations between them. Most of the studies demonstrate bioaccumulation of metals in different tissues [9, 22, 45,46,47,48,49,50,51,52] of aquatic animals. Still, there are very few blood studies of aquatic animals [24, 26]. Reports have demonstrated ecotoxicology interactions between trace metals and serum chemistry parameters [21, 53], or oxidative stress markers [5, 8, 29, 54] in aquatic animals.

Seasonal effect was confirmed for Mn and Zn and sex effects were observed only in case of Sr. Seasonal variations of trace metals bioaccumulation in aquatic animals were confirmed in many studies [3, 5, 55, 56]. Other studies showed sex differences in metal tissue bioaccumulation, such as Zn in the liver and skin of Lethrinus lentjan [57] or Hg in the muscle of Silurus glanis [58]. Secondly, we observed seasonal and gender differences for serum chemistry and oxidative stress markers. The serum biochemical values were consistent with values found in other fresh water fish [12, 13, 59]. Giarratano et al. [5] emphasized on a significant seasonal effect on oxidative stress markers (lipid radical content, MDA, α-tocopherol, total thiol groups, and metallothioneins) and trace metals content (Cd, Pb, Cu, Zn, Ni, Cr, Al) in the tissue of Neohelice granulata.

Health Status Observation

Serum chemistry parameters were within the reference range [60], except for decreased values of Cl− and increased contents of K+, TP, Glu, Chol, and ALP. Changes in the levels of serum glucose and total protein can be observed in case of liver failure [27] and nephrotoxicity [1]. Decreased chlorides are associated with stress, extreme temperatures, and infection as well as trace metal toxicity [60]. Gopal et al. [26] described the effects of Cu, Hg, Ni, and Pb on the blood protein biochemistry of C. carpio. Their results revealed a same tendency for all metals: an initial increased mobilization followed by a steady depletion. However, it should be noted that lethal and sub-lethal concentrations of respective trace metals were used. Increased blood glucose content (hyperglycemic conditions; enhanced glycogenolysis) influenced by trace metals (Cu, Ni, Fe, Mn, Zn) was observed in fish (Mastacembelus armatus) living in water contaminated by wastewater from a thermal power plant [61]. On the other hand, Fırat and Kargın [4] tested individual and combined effects of Cd and Zn on the serum chemistry parameters of freshwater fish (Oreochromis niloticus), but their results had an opposite tendency for glucose, for TP, and partly for cholesterol (decreasing content against higher levels of Cd and Zn in spring season). Fırat and Kargın [4] as well as Öner et al. [27] also confirmed associations between metals (Cd and Zn) and liver enzymes ALT and AST (increasing after exposure) which is comparable with our levels of ALT, but not for AST. We observed positive correlation between Ni levels as well as an increased ALP content in spring season and between Mn and ALP in summer season. Increased blood levels of liver enzymes are the main indicator to inform us about the liver and cellular damage caused by metal poisoning [62, 63]. ALT and AST activities were comparable to other studies on common carp [64]; however, the ALP activity was several-fold higher in our study, which could be explained by pathological processes such as liver impairment, kidney dysfunction, and bone disease [63]. The enzymes ALT and AST are used as biomarkers to detect hepatotoxicity, while ALP indicates bile duct epithelial damage [65].

Interactions between blood serum minerals (Ca, Mg, Na, K, Cl) and trace metals were confirmed in spring (Sr, Zn, Cd, Fe, Mn) and summer (Zn, Cr, Cu, Fe, Hg, Ni, As) seasons. Relation between Pb and Ca is probably the best known. Lead as a potential neurotoxicant is able to mimicry calcium. Lead competes with calcium for binding sites on calcium-regulated proteins [66, 67]; and it can also start the disruption of calcium transport [68]. The physiological investigation of Cd poisoning in fish showed a slight decrease of blood potassium and an increase of blood plasma magnesium [69], while copper exposure reflected a decrease in the plasma sodium, potassium, calcium, and chloride concentrations [70], though these relationships were not confirmed in our study, except Cu and Na relationship in summer season.

The correlation analysis also showed some other relationships between the tested parameters and trace metals. Blood urea concentration was significantly affected by manganese in summer season. Öner et al. [27] confirmed a significant increase of blood urea nitrogen (BUN) in Cd, Cu, and Cr exposed fish (Oreochromis niloticus). Increased BUN is associated with gill and kidney disease or failure [21, 71].

Oxidative Status Markers

In response to environmental pollutants, oxidative stress can provide valuable information regarding the internal environment. The production of reactive oxygen species (ROS) can cause oxidative damage at the cellular level. Generally, trace metals generate and promote ROS (such as hydrogen peroxide or the peroxide radical, superoxides, and nitric oxide) overproduction in the cell [72]. Inversely, cells own specific defense mechanisms to protect against ROS-mediated oxidative damage. The antioxidant systems are classified into two major groups. Enzymatic antioxidants (SOD, CAT, GPx) that act as the body’s first line of defense by catalyzing ROS conversion to less reactive or inert species, and non-enzymatic (endogenous) antioxidants (GSH, UA, Alb), which provide a secondary defense against ROS ([10, 23, 31,32,33,34]). Environmentally induced oxidative stress in aquatic animals was described in several studies, specifically in relation to the trace metal content [3, 5, 10, 22].

Bocchetti et al. [73] tested the effect of various sampling periods to oxidative stress in aquatic organisms (Tapes philippinarum and Mytilus galloprovincialis) and showed a significant seasonal impact on the activities of CAT, GST, CAT, and GPx, which is comparable with our results (TAC, CAT, and GPx activities exhibited significant differences among seasons). Moreover, the ROS, SOD, GSH activity, and concentrations of PC, MDA, UA, albumin, and bilirubin in the blood were also significantly affected by season.

We observed many significant correlations between metals and oxidative status markers in both seasons (Table 4). The effect of Cu and Pb content on the CAT activity, Al, and Ni content on higher MDA concentrations in burrowing crab (Neohelice granulate) were confirmed by Giarratano et al. [5]. Shi et al. [74] also found that nickel toxicity is associated with ROS generation with a subsequent lipid peroxidation, and alkyl and alkoxyl radical production.

Ruas et al. [10] have observed oxidative status biomarkers, such as changes in the glutathione (GSH) content, activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and the levels of lipid peroxidation (LPO), in the blood of three cichlid fish (Oreochromis niloticus, Tilapia rendalli, and Geophagus brasiliensis) during two seasons (autumn, spring) from unpolluted and polluted (Cr, Cd, Cu, Zn, Mn, Fe pollution) sites. Their results showed a significantly increased activity of SOD, LPO, GSH, and LPO/CAT+GPx ratio in the polluted site, which may indicate the induction of oxidative stress. According to Javed et al. [23], antioxidant enzymes (SOD, CAT, GST) and lipid peroxidation in different tissues (liver and kidney) of fish (Channa punctatus) exposed to metals (Cu, Ni, Fe, Mn, Cr, Zn) showed a significant increase of their activity; however, the level of non-enzymatic parameters (GSH) decreased. In another study [28], the authors recorded a decreased activity of SOD and CAT and an increased MDA (lipid peroxidation) content in the liver of metal-exposed catfish. As such, we may suppose that if the first line of defense is activated immediately, the activity of antioxidant enzymes will increase. If the first antioxidant protection line is not rapidly involved, oxidative stress and subsequent lipoperoxidation can occur, resulting in an increased concentration of MDA in blood, cells, and tissues. In addition, oxidative stress can be the cause of protein oxidation, which can be expressed through an increased content of PC in the blood or tissues.

Correlation analysis of Abarikwu et al. [28] showed comparable results to ours, particularly the effect of nickel and cadmium on the activity of superoxide dismutase. Lead affected the function of various antioxidant enzymes; decreased activity of SOD, CAT, GPx, and GSH; and increased lipid peroxidation [75], which demonstrate induced oxidative injury. GSH is a sulfhydryl-rich tripeptide that is generally involved in the protection of cells against toxicants and in the metabolism of xenobiotics [76]. [33]) showed that the exposure of a fish population (Oreochromis niloticus) to polluted water (Cd, Cu, Cr, Pb, Zn) caused oxidative stress followed by the response of the glutathione metabolism. Glutathione molecules were depleted after metal exposure, which caused increased glutathione S-transferase activity and may reflect on the deterioration cell protection ability. Gopal et al. [26] observed a decreasing tendency of albumin content in blood serum of common carp following exposure of different concentrations of mercuric chloride, lead nitrate, copper sulfate, and nickel sulfate, which refers to liver disease and stress situations. De Oliveira et al. [22] evaluated several biomarkers in Anodontites trapesialis after 96 h of confinement downstream of a coal mine. Increased bioaccumulation of metals (Al and Fe) resulted in an increased lipid peroxidation and protein oxidation in gills, while SOD was not affected. Low level of uric acid as an endogenous antioxidant is less of a health concern; however, its increased levels may refer to several disturbances of the kidney [77].

Conclusions

Taken together, obtained data on C. carpio indicate that trace metals (especially Cd, Cr, Cu, Fe, Hg, Mn, Ni, Sr, Zn, As) affect blood serum chemistry parameters (Ca, Mg, Na, K, Cl, Urea, TP, Glu, ALP, Chol, TG, CK); however, there was not serious damage to health status, except for ALP which may indicate bile duct epithelial damage. The correlation analysis between oxidative status markers (ROS, TAC, MDA, SOD, GSH, UA, BHB, Alb) and trace metal (Cd, Cu, Ni, Sr, Hg, Pb, Fe, Mn) content confirmed statistically significant interactions in both seasons.

Nevertheless, the results indicate many other associations between monitored contaminants and physiological parameters. When aquatic ecosystems are polluted with contaminants such as trace metals, aquatic animals, especially fish, should be also contaminated through bioaccumulation, as these are in continual contact with polluted environment, suggesting that trace metals negatively influence the fish physiology.

Further studies are necessary for the bio-monitoring of environmental, ecological, and pollutant stress factors as ecological risk assessment in association of biomarkers in living organisms.