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Metallothionein and total protein concentrations in gills and liver of Vardar chub (Squalius vardarensis) as biomarkers of water contamination in three rivers in Macedonia

Metallothionein (MT) and total cytosolic protein (TP) concentrations were determined as biomarkers of water contamination in gill and hepatic cytosols of Vardar chub (Squalius vardarensis) caught in three rivers in north-eastern Macedonia, one agriculturally impacted (Bregalnica) and two mining impacted (Zletovska and Kriva), in spring (May/June) of 2012. MT concentrations were determined in heat-treated (10 min at 85˚C) cytosols by differential pulse voltammetry on 797 VA Computrace (Metrohm). TP concentrations were determined on the spectrophotometer/fluorometer Infinite M200 (Tecan) at 750 nm wavelength using the method according to Lowry. Comparison of the results obtained for two organs indicated generally higher concentrations of analyzed proteins in the liver. However, for TPs differences were statistically significant only at the Bregalnica River (p<0.01), and for MTs at Bregalnica and Zletovska River (p<0.001). The analysis of spatial variability indicated comparable concentrations of TPs in chub gills from Bregalnica, Zletovska and Kriva rivers (20.2, 20.8, and 21.3 mg mL-1, respectively), whereas in the liver statistically significant (p<0.001) concentration decrease was observed at the Zletovska River (19.3 mg mL-1) compared to other two rivers (Bregalnica: 22.2 mg mL-1 ; Kriva: 21.8 mg mL-1). TPs are regarded as nonspecific biomarkers of pollution, and their synthesis could be induced or supressed due to exposure to pollutants. Therefore, TP concentration decrease in chub liver at the Zletovska River was possibly a consequence of low river water quality, characterized by increased concentrations of number of trace elements, sulphates, chlorides, and other changes of physico-chemical water properties (Ramani et al., 2014). Contrary to TPs, MT concentrations were comparable in chub liver from all three rivers (317.8, 312.0, and 332.8 μg mL-1, respectively), and differed in the gills, with statistically significantly higher concentrations in the gills of chub from the Kriva River (310.8 μg mL-1) compared to other two rivers (Bregalnica: 215.1 μg mL-1 ; Zletovska: 202.5 μg mL-1). Since MTs represent inducible biomarkers of metal exposure, the association of their concentrations with three known MT inducers, Cd, Zn and Cu (measured in gill and hepatic cytosols by high resolution inductively coupled plasma mass spectrometry) was also analyzed. In the gills, in all three rivers, positive correlations were obtained with Cd (r ranged from 0.264 to 0.777) and Zn (r ranged from 0.690 to 0.873), whereas in the liver the association between MTs and metal concentrations was much weaker, and referred only to essential elements Zn and Cu. Based on these results, apparently gill MTs were more sensitive to changes in metal exposure than hepatic MTs, which were maintained constant despite increased exposure to Cd (Ramani et al., 2014), as well as increased hepatic Cd concentrations (Bregalnica: 4.0 ng mL-1 ; Zletovska: 34.4 ng mL-1 ; Kriva: 34.5 ng mL-1). However, gill MTs were increased only at the Kriva River, whereas Cd concentrations in the gills were increased in both Zletovska and Kriva rivers (19.3 and 17.8 ng mL-1, respectively), compared to the Bregalnica River (0.5 ng mL-1). Therefore, it could be hypothesized that possibly accumulation of other metals, in addition to Cd, in chub gills could have caused increase of MT concentrations at the Kriva River. For example, cytosolic Pb concentrations were much higher in the gills of chub from the Kriva River (34.0 ng mL-1), compared to both Bregalnica and Zletovska rivers (3.7 and 8.2 ng mL-1, respectively). In addition, some other factor could have influenced MT concentrations in the chub gills from the Kriva River, such as a month later sampling campaign (in June) compared to the other two rivers (in May). Analysis of association with biometric parameters (total chub mass, condition index (CI), and hepatosomatic index (HSI)), indicated only week negative correlations of TPs in liver with chub mass (r ranged from -0.229 to -0.440), CI (r ranged from -0.086 to -0.404) and HSI (r ranged from -0.237 to -0.494), in some cases even statistically significant. Since TPs were the lowest in the liver of chub from the Zletovska River, where also the smallest chub specimens were caught, this association cannot explain the differences between sites. On the other hand, MT concentrations in gills showed positive correlation only with chub mass (r ranged from 0.137 to 0.678), statistically significant at Zletovska River. The chub specimens from the Kriva River were indeed bigger than from the Zletovska River, but of comparable size as from the Bregalnica River, and thus, the fish size solely could not be the cause of gill MT increase at the Kriva River. TPs in gills and MTs in liver, which did not exhibit spatial variability, also did not exhibit association with biometric parameters. Analysis of differences between chub sexes, indicated higher concentrations of TPs in both organs in males than females, whereas MTs showed opposite trend with higher concentrations in females. For both parameters, differences were more pronounced in the gills, and in some cases were even statistically significant. However, at the Kriva River, characterized by the highest gill MTs, sampled chub comprised only 33% of females, which was much lower percentage compared to other two rivers (Bregalnica: 47% ; Zletovska 60%), and thus chub sex could not be the reason for observed spatial differences in gill MTs. In a conclusion, in spring period, TP concentrations in the chub liver possibly could be used as a nonspecific indicator of water pollution, whereas MT concentrations in the chub gills possibly could be applied as indicator of increased metal accumulation in that organ. However, further investigation is needed to discern all the factors and metals which could influence MT concentrations.

metallothioneins ; total cytosolic proteins ; chub ; liver ; gills

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