The evaluation of metal’s toxicity in freshwater is one of the imperative areas of research and there is an emergent concern on the development of techniques for detecting toxic effects in aquatic animals. Oxidative stress biomarkers are very useful in assessing the health of aquatic life and more in depth studies are necessary to establish an exact cause effect relationship. Therefore, to study the effectiveness of this approach, a laboratory study was conducted in the fish
Heavy metals play a crucial role in various biological functioning of aquatic organisms and remain present in trace amount in the body, that is, not exceeding 1
Aquatic system is an ultimate sink of heavy metal pollutants and since aquatic animals tend to accumulate heavy metals from various sources including sediments, soil erosion and runoff, air depositions of dust and aerosol, and discharges of waste water [
The hexavalent chromium when present in excess amount induces toxic effects in the cells [
The evaluation of toxic effects of metals in terrestrial and aquatic ecosystems is one of the imperative areas of recent research and there is an emergent concern on the development of technique for detection of toxic effects in aquatic animals [
Following acclimatization, short term test of acute toxicity over a period of 96 hrs was performed on the fishes following the renewal of bioassay. LC50 values were determined by EPA Probit Analysis Program [
Two biometric parameters were calculated, the condition factor (CF) and the hepatosomatic index (HSI). The condition factor of each fish was calculated using the method of Salam and Davies [
To calculate the enzyme activity, total protein content was determined in different tissues under study based on Biuret method [
Catalase was assayed according to the method of Sinha [
Known amount of tissue was homogenized in phosphate buffer. The reaction mixture includes 0.1 mL of hydrogen peroxide, 1.95 mL phosphate buffer, and 0.05 mL tissue extract. Change in absorbance was recorded at 240 nm and the enzyme activity was expressed as mmol−1 H2O2 min−1 mg−1 protein.
SOD activity was determined according to the method of Beauchamp and Fridovich [
The reaction mixture containing 1.95 mL phosphate buffer, 0.25 mL riboflavin, 0.25 mL methionine, 0.25 mL Na2EDTA, 0.25 mL NBT, and 0.05 mL tissue extract was pipetted in 4 glass tubes. Another set of 4 tubes was prepared adding 0.05 mL of phosphate buffer instead of enzyme extract. Three tubes from each set were then placed on shaker at 25°C in fluorescent light for 15 minutes and the last one was kept in dark at 25°C (reference sample, in darkness free radicals are not generated). After the incubation period the change in the absorbance was measured at 560 nm using respected dark-incubated sample as reference for test samples for each series. The SOD activity was expressed in terms of relative enzyme activity U/mg protein.
Glutathione reductase (GR; EC 1.6.4.2) activity was assayed as described by Carlberg and Mannervik [
Aquatic organisms have the ability to absorb and accumulate heavy metals that can pose a long term effect on the health of the organism and probably on the ecosystem. Bioaccumulation pattern of hexavalent chromium in different fish tissues was studied. The procedures adopted for heavy metal analysis in fish tissues are based on the method 3052 of EPA [
All statistical analyses were performed with SPSS statistical program. All experimental data were expressed as mean ± standard deviation (SD). Significant differences between experimental and control groups were compared by One-Way ANOVA (analysis of variance) followed by Least Significant Difference (LSD) (
While conducting LC50 assays some selective behavioral changes were also noticed as a function of hexavalent chromium and have been presented in Table
Behavioral changes in the fish
S.N. | Behavioral changes |
---|---|
1 | Surfacing and darting movement |
2 | Copious mucus secretion |
3 | Aggregation of fishes near aerator |
4 | Lethargic movement within 20 minutes after exposure |
5 | Increase in opercular movement in order to breathe faster |
6 | Irregular and burst swimming-sudden rapid and forward movements |
7 | Nip and nudge movement-biting and moving towards another fish |
8 | Fin flickering-contraction and extension of dorsal fin |
Figure
Effect of hexavalent chromium on HSI of
Condition factor indicates changes in energy storage and metabolism due to environmental stressors. In this parameter, length and weight relationship are commonly used as indicators of robustness. Significant reduction was noticed in condition factor of chromium treated fishes (Figure
Effect of hexavalent chromium on CF of
Figures
(a)–(d) Effect of hexavalent chromium on catalase activity in liver (a), muscle (b), gills (c), and brain (d) of
To verify the presence of oxidative stress, SOD was also analyzed in liver, muscle, gills, and brain of the fish (Figures
(a)–(d) Effect of hexavalent chromium on superoxide dismutase activity in liver (a), muscle (b), gills (c), and brain (d) of
The effects of Cr(VI) exposure on glutathione reductase activity in fish liver, muscle, gills, and brain are presented in Figures
(a)–(d) Effect of hexavalent chromium on glutathione reductase (GSSG-R) activity in liver (a), muscle (b), gills (c), and brain (d) of
The present study was planned to investigate accumulation of heavy metal chromium with respect to short term and chronic exposure. Fishes exposed to 1/3rd of LC50 were kept in 5 different groups and accumulation patterns of the metal in fish body organs were investigated after 1, 2, 3, 4, and 15 days.
Among various body organs, the liver of the fish
(a)–(d) Bioaccumulation of hexavalent chromium after short term exposure (24–96 hrs) in liver (a), muscle (b), gills (c), and brain (d) of
Bioaccumulation of hexavalent chromium after long term exposure (15 days) in liver, muscle, gills, and brain of
Behavior provides a unique perspective linking the physiology and ecology of an organism and its environment [
Fishes are especially susceptible to environmental variations and respond more sensitively to pollutants than mammals. The fish liver has been shown to be a very interesting model for studying the interactions between environmental factors and hepatic structure and function. Hepatosomatic index (HSI) has been frequently used as a biomarker for examining fish exposed to environmental toxicants. HSI values are generally elevated in vertebrates experiencing induction of hepatic microsomal P-450 for detoxification of the pollutants [
In the present study (Figure
The CF is a measure of the fattiness of the fish and this is based on the ratio between body weight and length: 100 × body weight (g)/length (cm3), which allows comparisons to be made between populations living under different conditions [
The overall decrease in CF in the present study in
The antioxidant enzymatic system protects organisms from the toxic effects of the activated oxygen species and helps to maintain cellular homeostasis by removing ROS. The use of antioxidant profiles, particularly as a function of heavy metal exposure, is of high toxicological relevance. The level of certain biomarkers of oxidative stress was evaluated in
The biochemical profiles for catalase, GSSG, and SOD in different tissues are represented in Figures
Cr(VI) is reduced intracellularly to the reactive intermediates Cr(V) and (IV) and ultimately to the more stable Cr(III), by cellular reductants [
Among antioxidant enzymes, SOD is considered as the first line of defence against oxygen toxicity, due to its inhibitory effects on oxyradical formation [
One very unique and interesting finding was observed in the present study that the SOD increased from very initial period of stress (i.e., 24 hrs) and decreased or returned to normalcy during 96 hrs in all the tissues except brain, whereas CAT increased its maximum level during 48 hrs to 72 hrs and did not return to normalcy even in 96 hrs (except muscle). Therefore, it may be concluded based on the results that SOD takes the lead in order to detoxify the oxyradicals followed by corresponding increase in CAT activity and both are time-dependent.
We also observed that SOD and CAT activity in brain was strongly induced by Cr(VI) even during 96 hrs of treatment, which could be due to the flux of superoxide radicals, resulting in increased H2O2 in the cells [
In free radical scavenging and other cellular metabolism, reduced glutathione and GSH-related enzymes play a major role [
In our study, we did not find significant elevations in glutathione reductase activity in liver, muscle, and gills tissues. Experiment conducted on Zebra fish as a function of norfloxacin by Bartoskova et al. [
Thus, the increase in catalase, SOD, and GR activity observed in the current study suggests that Cr(VI) is capable of inducing oxidative stress to the fish
Knowledge of concentrations of heavy metal in different tissues of fish is important with respect to nature of management and human consumption of fish. Metal accumulation in the tissues of fish varies according to the rates of uptake, storage, and elimination. This means that metals which have high uptake and low elimination rates in the tissues of fish are expected to be accumulated to higher levels.
Amongst all the tissues studied (liver, muscle, gills, and brain), the highest concentration of chromium (4.56 ± 1.45
The liver exhibited highest tendency to accumulate chromium metal, whereas the most consumed part of the fish, that is, muscle, showed least tendency. Liver and gills of fish species, namely,
The results of the present study clearly indicate that heavy metal chromium causes oxidative stress in fishes. Increased CAT, SOD, and GR activities in all organs might suggest the crucial role of these enzymes in cell protection against the deleterious effects of chromium and development of adaptive response to chromium toxicity. The current results also contribute to improving our knowledge about possible development of oxidative stress induced by exposure to chromium metal in aquatic organisms and indicate a possible role for antioxidant systems in the prevention of induced damage. Bioaccumulation of chromium varied between the different tissues and the liver, being the storage and detoxification organ, accumulated highest level during short term exposure followed by gills. The gills had the highest metal concentrations during long term exposure, due to their intimate contact with the environment and their importance as an effecter of ionic and osmotic regulation. Muscle accumulated much less chromium. Thus, it is proposed to include these parameters in biomonitoring program in areas potentially polluted with metals to assess the health of the ecosystem.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by the Grant sanctioned by DBT, Delhi under Biocare Women Scientist Award Scheme (BT/Bio-CARe/07/176/2010-11) and the first author thankfully acknowledges DBT, Delhi for the financial assistance. The authors are thankful to the Director, CSIR-NEERI, Nagpur for providing all the laboratory facilities. We are also thankful to the Head, EIRA Division, CSIR-NEERI, Nagpur and Prof. R. C. Sinha, Chief Executive, CENC, Patna University for their kind support and constant encouragement. The authors are also thankful to Mr. Arvind Kulkarni, College of Fishery Science, Telankhedi, Nagpur, for providing the healthy fishes during the period of study.