Since it has been introduced in European freshwaters in the 1970s, Prussian carp has been economically important for fisheries in Serbia (Smederevac-Lalić et al. 2011) as well as in Croatia, so estimating the human health risk resulting from consumption of this fish species is very important. Syasina et al. (2012) distinguished Prussian carp as one of the most resistant species to the effects of pollution, while Djikanović et al. (2016) comparing metal accumulation in liver, muscle, and gills of nine fish species from the Međuvršje Reservoir (Serbia) pointed out Prussian carp as the most effective accumulator of the most metals. Hence, we detected PTEs in the muscle and gills of this species caught from three different freshwater ecosystems – isolated and non-flooded ponds near the South Morava River, the South Morava River in Serbia, and Kopačko Lake (periodically flooded by the Danube) in Croatia, each with different physicochemical parameters, to determine if they affect the concentration of the elements in fish tissues. Data on physical-chemical parameters T (C°), pH, EC (µS), DO (mg l− 1), DO%, BOD (mg l− 1), NO3-N (mg l− 1), NH4-N (mg l− 1), PO4 (mg l− 1), N-TOT (mg l− 1), P-TOT (mg l− 1) measured once in parallel with fish sampling are presented in Table S1.
The weight of the specimens examined ranged from 45.20 ± 6.33 to 565.62 ± 176.44 g while the length ranged from 13.97 ± 0.65 to 30.34 ± 0.73 cm (Table 1). The highest CF was recorded for specimens from the South Morava River (1.94 ± 0.17) and the lowest CF was calculated for specimens from the isolated ponds (1.51 ± 0.05). There were significant differences between the isolated ponds and Kopačko Lake, as well as between the isolated ponds and South Morava River based on the CF values. As Froese (2006) stated, the condition factor is a measure of the general health of fish and it reflects the recent feeding conditions and environmental quality. Since the values of CF in our study were greater than one, it can be concluded that the general health of fish was good. In studies Laflamme et al. (2000), Rajotte and Couture (2002), and Zhelev et al. (2018) CF decline was observed at highly contaminated locations. Concerning the location of the isolated ponds (near a highway) and their origin (created by sand and gravel excavation during the construction of the highway) (Stamenković et al. 2019), as well as the high anthropogenic pressure and lack of connection with the river and freshwater, the lowest CF value in this ecosystem was logical. However, all the mean CF values for the fish analyzed in this study were lower than the findings for the same fish species in Lake Eğirdir (CF 2.401 for males and 2.594 for females) in study Balik et al. (2004), but higher than for the specimens from Ilova River in Croatia (CF 1.59 in autumn and 1.31 in spring) in study Mijošek et al. (2021).
Table 1
Weight, length, and condition factor with the standard deviation for the freshwater Prussian carp (Carassius gibelio) per sampling site
Site
|
Number of specimens
|
Weight (g)
|
Length (cm)
|
Condition
factor
|
Isolated ponds
|
15
|
45.20 ± 6.33
|
13.97 ± 0.65
|
1.51 ± 0.05
|
South Morava River
|
10
|
543.80 ± 55.47
|
30.34 ± 0.73
|
1.94 ± 0.17
|
Kopačko Lake
|
10
|
565.62 ± 176.44
|
27.07 ± 3.55
|
1.83 ± 0.19
|
The highest PTEs values in muscle were recorded for IP – As, Co, Hg, Ni, Pb, Se, and Sn; for KL – Al, Cu, and Fe and for SMR – Cd, Cr, Mn, and Zn (Table 2). In both the muscle tissue and gills, the highest concentrations of Zn (muscle 7.978 ± 1.537 to 8.878 ± 1.799 mg kg− 1 ww; gills 31.168 ± 3.100 to 72.23 ± 118.19 mg kg− 1 ww) and Fe (muscle 3.813 ± 2.288 to 7.067 ± 3.834 mg kg− 1ww; gills 70.66 ± 60.98 to 178.30 ± 105.02 mg kg− 1ww) were recorded for all specimens. The lowest concentrations of Co (in muscle not detected (ND) at two sampling sites; in gills 0.019 ± 0.041 to 0.300 ± 0.390 mg kg− 1 ww) and Pb (in muscle 0.094 ± 0.099 to 0.20 ± 0.022 mg kg− 1 ww and in gills ND at two sampling sites) were recorded. The highest concentrations of essential elements Zn and Fe in this study are in accordance with our previous research (Milošković et al. 2018) and studies Syasina et al. (2012), Skorić et al., (2012), etc. Djikanović et al. (2016) pointed out, among nine fish species, Prussian carp from Međuvršje Reservoir with the highest concentrations of Fe and Zn were observed in all tissues. Goyer (1997) stated that the involvement of Zn and Fe in the regulation of key enzymatic detoxification processes may be the reason for the high concentrations of these elements in fish tissues. It is generally believed that fish actively regulate the essential element concentrations in their tissue. Bervoets and Blust (2003) emphasized that the levels of Zn and Fe in the tissue were influenced by several chemical and physiological processes in addition to the environmental concentrations. The concentration of Pb in both the muscle tissue and the gills was low, in fact, it was lower than in Prussian carp from Hutovo Blato nature reserve in Bosnia and Herzegovina (Has-Schön et al. 2008) and the Amur River basin (Syasina et al. 2012).
Table 2
The average element concentrations and standard deviation in the muscle tissue and gills of Prussian carp expressed in mg kg− 1ww (different letters in superscript denote significant differences in element concentrations among the study sites, p < 0.05) and the maximum permitted concentrations (MPCs) in fish meat for utilization in the human diet, established by the European Union (EU) and FAO
Elements
|
Tissue
|
Serbia
|
Croatia
|
MPC
|
Isolated
ponds
|
South Morava
River
|
Kopačko
Lake
|
MPC
EU (2006)
|
MPC
FAO (1983)
|
Al
|
muscle
|
0.147 ± 0.373a
|
0.174 ± 0.298a
|
0.720 ± 1.828a
|
|
|
gills
|
41.84 ± 80.93a
|
5.269 ± 1.670b
|
4.064 ± 2.940b
|
|
|
As
|
muscle
|
0.221 ± 0.255a
|
0.077 ± 0.081a
|
0.177 ± 0.176a
|
|
0.1-4.0
|
gills
|
0.32 ± 0.29a
|
0.999 ± 0.267b
|
0.747 ± 0.432a, b
|
Cd
|
muscle
|
0.076 ± 0.015a
|
0.087 ± 0.023a
|
0.074 ± 0.013a
|
0.05
|
0.05
|
gills
|
0.085 ± 0.015a
|
0.243 ± 0.054b
|
0.189 ± 0.078b
|
Co
|
muscle
|
0.0014 ± 0.0030
|
ND
|
ND
|
|
|
gills
|
0.019 ± 0.041a
|
0.184 ± 0.084b
|
0.300 ± 0.390b
|
Cr
|
muscle
|
0.5880 ± 0.1803a
|
0.842 ± 0.124b
|
0.680 ± 0.168a, b
|
|
|
gills
|
0.880 ± 0.389a
|
0.858 ± 0.182a
|
1.075 ± 0.979a
|
Cu
|
muscle
|
0.297 ± 0.152a
|
0.551 ± 0.145b
|
0.587 ± 0.358a, b
|
|
30
|
gills
|
0.729 ± 0.136a
|
3.001 ± 0.290b
|
3.071 ± 1.210b
|
Fe
|
muscle
|
3.813 ± 2.288a
|
5.892 ± 1.515a
|
7.067 ± 3.834a
|
|
|
gills
|
70.66 ± 60.98a
|
153.61 ± 79.03b
|
178.30 ± 105.02b
|
Hg
|
muscle
|
0.178 ± 0.175a
|
0.059 ± 0.067a
|
0.063 ± 0.070a
|
0.5
|
0.5
|
gills
|
ND
|
0.001 ± 0.002
|
0.0004 ± 0.0009
|
Mn
|
muscle
|
0.350 ± 0.236a
|
0.727 ± 0.126b
|
0.724 ± 0.471a, b
|
|
|
gills
|
5.08 ± 5.90a
|
1.377 ± 0.290a
|
1.334 ± 0.59a
|
Ni
|
muscle
|
0.098 ± 0.042a
|
0.080 ± 0.016a
|
0.085 ± 0.025a
|
|
|
gills
|
0.128 ± 0.160a
|
0.001 ± 0.002b
|
0.008 ± 0.024b
|
Pb
|
muscle
|
0.094 ± 0.099b
|
0.020 ± 0.022a, b
|
0.020 ± 0.043a
|
0.3
|
0.5
|
gills
|
0.038 ± 0.056
|
ND
|
ND
|
|
|
Se
|
muscle
|
0.205 ± 0.059a
|
0.162 ± 0.041b
|
0.162 ± 0.063b
|
|
|
gills
|
0.241 ± 0.065a
|
0.712 ± 0.135b
|
0.642 ± 0.277b
|
Sn
|
muscle
|
0.029 ± 0.024b
|
0.001 ± 0.001a
|
0.003 ± 0.004a
|
|
|
gills
|
0.219 ± 0.268a
|
0.155 ± 0.008a
|
0.154 ± 0.009a
|
Zn
|
muscle
|
7.978 ± 1.537a
|
11.030 ± 1.155b
|
8.878 ± 1.799a, b
|
|
100
|
gills
|
40.59 ± 60.60a
|
31.168 ± 3.100a
|
72.23 ± 118.19a
|
ND − not detected |
The Kruskal-Wallis ANOVA test (p ˂ 0.05) confirmed significant differences in the concentrations of PTEs in the fish gills and muscle tissues among the three groups of sites. Besides, the Mann-Whitney post hoc test revealed that there were no significant differences for six concentrations of PTEs in the gill tissue (Cr, Hg, Mn, Pb, Sn, and Zn) in the groups. The most significant differences in the element concentrations in the gills were observed between the first (isolated ponds) and the second group (South Morava River) as well as between the first (isolated ponds) and third group (Kopačko Lake) (see Table 2). The specimens from isolated ponds can be singled out as having the highest values of Al and Ni and the lowest values of As, Cd, Co, Cu, Fe and Se in the gills in Prussian carp. On the other hand, any differences were much less visible in the muscles. The Mann-Whitney post hoc test revealed that only the concentrations of elements Cr, Cu, Mn, Sn, and Zn were significantly different between specimens from the isolated ponds and South Morava River, and Pb and Sn between the isolated ponds and Kopačko Lake (see Table 2). Bury et al. (2003) stated that Cu, Zn, and Cd mainly enter the fish through the food, but the higher concentrations of these elements in the gills of the fish in this study from all three sampling sites could be explained with high concentrations of those elements in the water. The highest concentrations of Pb, Se, and Sn and the lowest concentrations of Cr, Cu, Mn, Zn, Pb, Se, and Sn were recorded in the muscle tissue of the fish from the isolated ponds. Since fish muscle is not the primary tissue for the accumulation of PTEs, only concentrations of Hg, Pb, and Ni were accumulated in high amounts in the muscle tissue at all three sites. Likewise, in a study by Has-Schön et al. (2008), the highest concentration of Hg was detected in the muscle tissue of Prussian carp (concentrations in the same range at the values in our study; 0.142–0.151 mg kg− 1 ww), but high concentrations of Pb were not detected in the muscle than in the gills. In a study by Syasina et al. (2012), higher levels of contamination by PTEs were detected in Prussian carp than in other species tested and the highest concentrations of Hg were detected in the muscle tissue. Concentrations of Pb, Cd, and Hg (0.052 ± 0.023, 0.017 ± 0.004 and 0.87 ± 0.006 µg/g dry weight, respectively) in Prussian carp from the Danube in Croatia in study Zrnčić et al. (2013) were higher, while the concentration of As (0.031 ± 0.011µg/g dry weight) was much lower than in Prussian carp from Kopačko Lake in this study. Prosi (1981) stated that the type of chemical, metabolic properties of the tissues, and the degree of environmental pollution affect the bioaccumulation levels in fish. Fish gills are responsive to the water quality since they are coming into immediate contact with the surrounding water (Syasina et al. 2012). When fish are exposed to high levels of elements in water, they typically exhibit elevated concentrations in the gills (Reynders et al. 2008), thus reflecting the concentration of elements in the water (Rao and Padmaja 2000). Particularly when there is a deficiency of trace elements in the food but a high concentration in the water, uptake from the water plays a considerable role (Kamunde et al. 2002). On the other hand, in low-level, chronic diffuse pollution, metal uptake in fish is predominantly from the food (Dallinger and Kautzky 1985).
The concentrations of PTEs affected the MPI values (Fig. 2). In the study Đikanović et al. (2016) Prussian carp was distinguished from others by higher MPI values. Our results pointed out the muscle exposed to the lower pressure of PTEs pollution than the gills. Kopačko Lake distinguished with the highest values of MPI for muscles (0.24) and isolated ponds with the highest values of MPI for gills (0.8). The reason for higher values of MPI for gills may be that the gills are in direct contact with pollutants in the water and the primary site of element uptake (Klavins et al., 2009). Based on above statement, we can conclude that the overall concentrations of PTEs are the highest in the water of isolated ponds. On the other hand, many authors reported muscle as less loaded with elements (lower MPI) (Subotić et al. 2013a; Djikanović et al. 2016, etc.). If we consider the statement by Subotić et al. (2013a, b) that the weight of the fish affects the MPI value for muscle tissue, the reason for the lower value for muscle tissue in the isolated ponds is comprehensible.
The RDA showed that concentrations of the elements in the gill tissues were significantly correlated with the environment (Monte Carlo F = 4.301, p < 0.001) (Fig. 3). The model obtained showed that 57.3% (R2adj = 0.573, p < 0.001) of the element concentrations in the fish tissue were driven by environmental attributes (listed in Table S1). The lengths of the environmental variable arrows reflect how much variance was explained by that factor, and the direction of the arrows for a particular environmental factor indicates an increasing concentration of that factor. The element arrows pointing in approximately the same direction as the environmental factor arrows indicate a high positive correlation (the longer the element line, the stronger the relationship). According to the angles between these arrows, the concentration of Al was positively correlated with T, pH, DO, and DO%, and negatively correlated with PO4-P, NH4-N, and NO3-N. In comparison, the concentration of Mn and Zn increased when the values of BOD, PTOT, and NTOT increased. Finally, Cu and Fe were slightly influenced by the environmental gradient. The other element concentrations did not show visible dependence on the physicochemical parameters of the water, as shown in Fig. 3.
In contrast, the RDA based on element concentrations in the fish muscles indicated no significant relationship with the environment (Monte Carlo F = 1.409, p = 0.178), providing a model that revealed that only 14.28% (R2adj = 0.1428, p = 0.178) of the element concentrations in the muscle tissue can be explained by environmental parameters (Fig. 3).
According to Chai et al. (2019), sources of pollution and different hydrological characteristics may be the main factors affecting the pathways of PTEs. The inorganic quality of natural water is affected by physicochemical parameters, such as pH, conductivity, and dissolved oxygen (Yılmaz and Doğan 2008; Chintala et al. 2013). The exposure period, the concentration of the chemical, environmental temperature, or the pH potentially affects the amounts of contaminants in aquatic organisms (Terra et al. 2008; Copa et al. 2012). Based on the above claims, we found that only the concentrations of Al, Mn, Zn, and to a lesser extent Cu and Fe in the gills were significantly correlated with the physicochemical parameters of the environment. Ghosh and Adhikari (2006) notified that increasing water hardness and acidified freshwaters reduce metal toxicity. In our study only the concentration of Al in gills was positively correlated to the pH in water. There were no significant relationships between the PTEs in the muscle tissue and environmental variables, which is in accordance with Zrnčić et al. (2013) who stated that it is not possible to correlate the measured element concentrations (Pb, Cd, Hg, As) in muscles in different fish species with water properties in Danube River in Croatia. Those results are in accordance with Syasina et al. (2012), who stated that, since Prussian carp is a bottom feeder, the dietary habits of this species subject it to the intensive influence of PTEs that accompanies food resources.
Fish samples from all the sites examined had concentrations of Cd above the MPC prescribed by the EU (2006) and FAO (1983) in both the muscle and gills (Table 2). Since EU legislation does not prescribe any MPC for As, and FAO prescribes MPC at a range of 0.1-4 mg kg− 1, we singled out Prussian carp from the isolated ponds and Kopačko Lake as having concentrations of As slightly above the MPC (Table 2). The negative impact of PTEs on human health was analyzed only by comparing the concentrations of these elements with the appropriate MPCs in several earlier studies dealing with pollution in aquatic environments (Milošković et al. 2016; Djikanović et al. 2016; Jovanović et al. 2018, etc.). We estimated elevated concentrations of Cd at all sampling sites, and of As in fish from the isolated ponds and Kopačko Lake. This information is useful but often very restricted because it does not consider the effect of intraspecific variations in the human population (exposure rate, human weight, meal size, etc.) or long-term exposure to pollutants (Nikolić et al. 2019). Because As and Cd are very toxic to humans, this information is doubtless useful as an early warning, especially for fishing activities in these areas.
As shown in Table 3, there were no THQ values over 1 for the elements in the freshwater Prussian carp from the isolated ponds, South Morava River, or Kopačko Lake, indicating that there was no health risk. The lowest potential health risk was that of Cr, which may be ascribed to a higher oral reference dose of this element. Concerning all the THQ values, the highest HI (see Table 3) values were assessed for the human population in Serbia, mainly ascribed to the consumption of Prussian carp from the isolated ponds, with a value of 0.16, and the South Morava River (0.10). The lowest HI value was assessed for the human population in Croatia (0.07).
Table 3
THQs and HI for potentially toxic elements in Prussian carp from isolated ponds and the South Morava River in Serbia, and Kopačko Lake in Croatia and TR for As and Pb for the general population due to consumption of Prussian Carp
Study area/
Country of consumption
|
Al
THQS
|
As
THQS
|
Cd
THQS
|
Co
THQS
|
Cr
THQS
|
Cu
THQS
|
Fe
THQS
|
Hg
THQS
|
Mn
THQS
|
Ni
THQS
|
Pb
THQS
|
Se
THQS
|
Sn
THQS
|
Zn
THQS
|
HI
|
TR
(As)*
|
TR
(Pb)
|
Isolated ponds
|
Serbia
|
0.033
|
0.066
|
0.007
|
0.0004
|
0.00004
|
0.0007
|
0.009
|
0.032
|
0.00022
|
0.0004
|
0.002
|
0.004
|
0.0009
|
0.0024
|
0.16
|
9.9 × 10− 7
|
1.1 × 10− 8
|
South Morava River
|
0.039
|
0.023
|
0.0078
|
/
|
0.00005
|
0.0012
|
0.013
|
0.011
|
0.0005
|
0.0004
|
0.0004
|
0.003
|
0.00002
|
0.0033
|
0.10
|
3.5 × 10− 7
|
2.4 × 10− 9
|
Kopačko Lake
|
Croatia
|
0.01
|
0.032
|
0.004
|
/
|
0.00002
|
0.0008
|
0.01
|
0.007
|
0.0003
|
0.0002
|
0.0003
|
0.0018
|
0.00005
|
0.0016
|
0.07
|
4.3 × 10− 7
|
2.4 × 10− 9
|
*It was assumed that inorganic As was 3% of the total As
|
In this study, the highest TR values for inorganic As (9.9 × 10− 7) and Pb (1.1 × 10− 8) were recorded for Prussian carp from the isolated ponds in Serbia (Table 3). According to the result for TR obtained in this study, lower TR values were recorded for Pb than for As, as seen in study Nikolić et al. (2021). It is assumed that THQ value greater than one, and a TR above 1×10− 6 is of concern because there is a high risk of developing chronic systemic effects (assessed by THQ) or cancer (assessed by TR) due to the intake of the contaminants (USEPA 2000). The THQ value for each element in each sampling site was lower than the hazard quotient threshold of 1, which indicates that there is no significant non-carcinogenic health risk resulting from the intake of these elements, as in studies by Chien et al. (2002) and Zheng et al. (2007). Consequently, the HI for all the study sites was lower than 1, suggesting there was also no potential health risk from consumption of the Prussian carp meat. We presented the levels of inorganic toxic As from the measured levels of the total As since organic As compounds (such as arsenobetaine) are considered to be nontoxic and therefore not a threat to human health (ATSDR 1998). The inorganic form of As causes a number of diseases, such as skin, lung, bladder, and kidney cancers (Castro-González and Méndez-Armenta 2008; Sirot et al. 2009) so the calculated TR is very important for human health assessment. All of the TR values for As (3.5–9.9 ×10− 7) and Pb (2.42 × 10− 9 – 9.9 × 10− 8) were below 1×10− 6, as proposed Islam et al. (2014), suggesting there was no cancer risk for humans consuming fish from the sampling sites.
Based on the results of this research, it can be concluded that type of aquatic ecosystem in certain level affect the pollution status of the fish. This applies primarily to fish gills, which are an indicator of water pollution. Isolated ponds with no inflow of freshwater stand out as the most polluted, followed by Kopačko Lake with occasional floods. Flowing freshwater ecosystem South Morava River can be single out as at least polluted with PTEs.