Detection on Electroplating Effluent-Induced Cytopathological Alterations and DNA Damage in the Blood Samples of Cirrhinus mrigala (Hamilton, 1822)

Research Article

Austin J Environ Toxicol. 2021; 7(2): 1041.

Detection on Electroplating Effluent-Induced Cytopathological Alterations and DNA Damage in the Blood Samples of Cirrhinus mrigala (Hamilton, 1822)

Sudhasaravanan R1, Binukumari S1*, Rajeshkumar S1, Mohan Kumar M1,2, Mukherjee R1,2 and Selvaraj J1,3

¹Department of Zoology, Kongunadu Arts and Science College (Autonomous), Affiliated Bharathiar University, India

²Shree PM Patel Institute of PG Studies and Research in Science, Sardar Patel University, India

³Department of Biochemistry, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Science, Saveetha University, India

*Corresponding author: Binukumari S, Department of Zoology, Kongunadu Arts and Science College, College of Excellence, G. N Mills Post, Coimbatore- 641 029, Tamil Nadu, India

Received: September 20, 2021; Accepted: October 14, 2021; Published: October 21, 2021


In the present study, fresh waterfish Cirrhinus mrigala has been used as a model organism to understand the mechanistic link between electroplating industrial effluent (EIE) and oxidative stress in aquatic ecosystems. To this end, the fishes were exposed to industrial effluent, in the tank water for 10, 20 and 30 days under controlled laboratory conditions. Various hematological parameters (Total RBC and WBC, Hemoglobin, MCV, MCH, PCV), biochemical metabolites (Glucose, protein, Cholesterol) and markers of genotoxicity (comet assay and micronuclear assay) were assessed. Fishes exposed for 30 days showed marked increase (p <0.001) in RBC with concomitant decrease in total WBC. There is significant (p <0.001) decline in indices of blood cells such as Hb, MCV, MCH and PCV. Results from comet assay showed significantly (p < 0.05) increased frequency genotoxicity in erythrocytes of as evident from observed increase in tail length in fishes exposed to EIE for 30 days. Overall, the results of this study, clearly demonstrated concentration dependent response of electroplating industrial effluent on freshwater fish Cirrhinus mrigala on haematological, biochemical and genotoxic markers. The results obtained herein suggests EIE as potential xenobiotics contributing towards ecotoxic and genotoxic effect in fishes.

Keywords: Electroplating industrial effluent (EIE); Cirrhinus mrigala; Hematological; oxidative stress; Genotoxicity


Fish serves as an excellent model organism in understanding aquatic toxicology involving diverge nature of toxicants [1,2]. Electroplating Industrial Effluent (EIE) has redox potential to suppress immune system and induce oxidative stress in fishes [3,4]. Genotoxic potential of electroplating effluenthas been reported in humans, rodents and fish cell lines [5-8]. In the current scenario, the assessment of the genotoxicity of electroplating effluentin terrestrial and aquatic ecosystems has emerged as major thrust area of research and there is increased trend to develop methods for detection of genotoxic effects for ecotoxicological applications [9]. Though, many methods like micronucleus test, chromosomal aberrations and DNA damage assays have been used for assessing genotoxicity of various chemicals in different animals, the DNA damage (comet assay) protocol is known to be simple, sensitive, more reliable and cost effective, and has been used to investigate the genotoxic potential of toxicants in the environment [10]. Influx of unwanted substances like toxic metals, into water bodies causes physical, chemical and biological changes and ecological imbalance. Heavy metal, released by both natural and anthropogenic process induces oxidative stress. This heavy metal induced oxidative stress, is not only prevalent in aquatic ecosystems worldwide, but has been reported in many fresh water species even within Indian subcontinent [11-13]. Apart from oxidative stress, hematological tests are important diagnostic tools and valuable indicators of disease or stress due to pollutants and environmental fluctuations. Since Electroplating Industrial Effluent (EIE) is involved in creating heavy metal pollution by direct discharge of effluent into water bodies, piscine haematology can be very useful in assessing the health status and changing environmental conditions. As such, hematological alterations in fishes have taken shape as an important tool in studying both general physiological states of fishes as well as environmental quality [14,15]. Fish erythrocyte is distinct from mammalian erythrocytes because they possess a nucleus and their interpretation in the morphological changes is an important bioindicator of pollution. Various abnormalities like bilobed, notched, binucleated and lobed nuclei also serves as an indicators of genotoxicity [16]. According to Matsumoto et al. [17,18] the comet assay is sensitive to be used for monitoring quality of the contaminated water with effluents containing heavy metals. In view of the background information wherein hematological parameters including oxidative stress has a significant role in fishes exposed to EIE and scarce scientific data on the genotoxic potential of electroplating effluent in aquatic animals, the present inventory was taken up with the following objectives: (1) To investigate the effects of sub lethal concentration of electroplating effluent on oxidative stress, hematological, serum biochemical and genotoxicity of the fresh water fish, Cirrhinus mrigala on short and long term exposure. (2) To unravel the relationship, if any between oxidative stress and DNA damage.

Materials and Methods

Experimental animals

C. mirgala, commonly known as Indian mrigal carp, was selected as an experimental animal model. The fish (average length 7.4 ± 0.54 cm average weight 9.2 ± 0.85gm) were procured from Aliyar fish farm, Tamil Nadu, India, and acclimatized to the laboratory conditions for 15 days. The fish were placed in glass aquaria with tap water (pH 7.2 ± 0.6, temperature 28.2 ± 0.5oC, dissolved oxygen 3.65 mg/L, conductivity 222 μs/cm, Total Suspended Solids (TSS) 105.6 mg/L and salinity 0%). No mortality was observed during the acclimatization. The fish were fed with aquarium flake food twice a day and 12 hr light and 12 hr dark photo period was maintained during the acclimatization.

Experimental design

After acclimatization for 2 weeks the fish were divided into four groups (n = 15). One group was served as control and the other three are exposed groups for three different time durations of 10, 20 and 30 days. Fishes were exposed to a mixture of electroplating effluent on fresh water containing 100% solution. The experiment was planned in such a way that the fish from all the groups were sacrificed on the same day. Level of Lipid Peroxidation (LPO), Reduced Glutathione (GSH) and activities of enzymic-antioxidants Superoxide Dismutase (SOD, Catalase (CAT) and Glutathione Peroxidase (GPx). The activities of lipid peroxidation (Abcam), reduced glutathione (Thermofisher), and superoxide dismutase, catalase and glutathione peroxidase (Sigma Aldrich) were assessed as per manufacturer’s instructions.

Haematological analysis

Hemoglobin was estimated by acid haematin method [19]. Red Blood Cells (RBC) and White Blood Cells (WBC) were counted using the improved Neubaurhaemocytometer [20]. Hence blood was diluted (1:200) with Hayem’s fluid [21]. Erythrocytes were counted in the loaded haemocytometer chamber and total numbers were reported as 106 mm3 (Wintrobe, 1967). White Blood Cells (WBC) were counted using animproved Neubaurhaemocytometer [20,22]. Blood was diluted (1:20) with Turk’s diluting fluid and placed in haemocytometer, 4 large (1 corner squares of the haemocytometer were counted under the microscope (Olympus Microsystems). The total number of WBC was calculated in mm3 x 103 [23]. The Mean Corpuscular Volume was calculated by using values of PCV% and Red Blood Cell counts and expressed in μm [24]. Blood cell was sucked into heparinized haematocrit capillary tube (7.5 cm length, 0.1 cm width). After sealing both the sides of the tube it was centrifuged in the microhaematocrit centrifuge at 6000 rpm for 2 min. From the volume of blood taken and cell volume after centrifugation, the PCV percentage was calculated employing standard method and formula [25]. Mean Corpuscular Haemoglobin (MCH in pg), was calculated as (Hb x 10)/ RBC.

Biochemical analysis

The blood samples were collected from caudal vein of the fish, C.mrigala. The blood samples (without EDTA) was centrifuged for 10 minutes at 4000 rpm, supernatant serum was decanted and stored at -2oC. Protein, glucose and cholesterol concentration was measured according to the Lowry et al., [26]; glucose test kit (AGAPPE Diagnostics, India) using GOD-PAP methodology [27] and serum cholesterol was analyzed using cholesterol test kit (AGAPPE Diagnostics, India) based on CHOD-PAP methodology [28] respectively.

Genotoxicity analysis

Blood was smeared on two clean glass slides, air dried for 24 h, then fixed in absolute methanol for 10 min. After fixing, the same slides were stained in 4% Giemsa for 10 min, air-dried and then prepared for permanent use. The Giemsa solution was centrifuge and filtered before staining to reduce precipitation that could interfere with the analyses. Micronuclei were identified and scored with an optical microscope (1000x). Two thousand erythrocytes were scored for each specimen (1000/slide) to determine the frequency of micronucleated erythrocytes. Micronuclei has to be smaller than onethird of the main nuclei, clearly separated from the main nuclei, and had to be no-refractive small nuclei (> 1/3 of the main nucleus) with intact cytoplasm [29]. The presence of other nuclear abnormalities in erythrocytes was also analyzed. The frequency of Micronuclei (MN) was calculated as (number of cells containing micronuclei X 1000) total number of cells counted.

Comet assay

Comet assay was performed according to the protocol that had been previously described by Singh et al., 1988 and modified by Tice et al., [30,31] Blood samples were diluted with 1 ml of PBS. 60 μl of the diluted and mixed with 200 μl of 0.65% Low Melting- Point (LMP), agarose 75 μlof the mixture were then layered on the slides precoatedwith 0.5% Normal- Melting Point (NMP) agarose and immediately covered with cover slip and then kept for 10 min in a refrigerator to solidify. After gently removing the cover slips, the slides were covered with a third layer of 90 μlow-melting-point agarose and covered with cover slips again. After solidification of the gel, cover slips were removed and slides were immersed in cold lysing solution (2.5 M NaCl, 100 mM Na2-EDTA, 10 mMTris, pH 10 with 10% DMSO and 1% Triton X-100 added fresh) and refrigerated at 4oC for 2 h. After lysis, the slides were placed on a horizontal electrophoresis box side by side. The mixture was incubated for 5min, and thecell suspension was transferred into another tube by avoiding debris. The number ofcells in the cell suspension were counted using hemocytometer and pelleted at 4oC. The pellet was suspended in 1ml of ice cold PBS at 1×105 cells/ml. Molten agarosewas prepared in a boiling water bath, cooled down to 37oC and mixed with isolatedcells in a 1:10 ratio in a eppendorf tube. Seventy-fiveμl of the mixture of agarose andcells were taken on comet slides. The comet slides were placed in dark for 10 min at 4oC to solidify the gel. After 10 min, the slides were placed in lysis solution containingdimethyl sulfoxide, for 30 min at 4oC. Then the excess solution was removed andthe slides were placed in alkaline solution to denature the DNA for 40 min at roomtemperature. After 40 min, the slides were subjected to electrophoresis in Tris Borateelectrophoresis buffer (TBE) with 1 volt/cm current between the two electrodes for10 min. After 10 min of electrophoresis, the slides were fixed with 70% ethanol for 5min. The slides were stained with syber green and air-dried. Control comet slides were prepared along with the exposed cells comet slides. The whole process was done under yellow light in order to minimize the UV light damage. The processed slides were examined for DNA damage using an epifluorescent microscope (OlympusBX51 TRF, USA). Blood samples of fishes were analyzed per treatment. In each of the fishes, a minimum of 75 individual cells per sample were screened, and a total of 225 individual cells (triplicate) were examined. The data were analyzed using a DELL computer equipped with a DNA damage analysis software(Loats Associates Inc., USA).

Statistical analysis

The data was analyzed using SPSS/PC+ Statistical package (version 11.5). Significant difference between control and experimental groups were determined using Duncan’s test were performed to determine if there were significant differences among and between treatment groups. Significant differences were considered at p <0.05.


Level of lipid peroxidation, glutathione and Enzymicantioxidants

Table 1 and 2 shows level of lipid peroxidation, glutathione content and alterations of antioxidant enzymes in erythrocytes and gills of C. mrigala exposed to Electroplating Industrial Effluent (EIE) for 10, 20 and 30 days. There is duration dependent increase in lipid peroxidation and depletion in glutathione with most pronounced effect seen on 30 day exposure. With regard to enzymic antioxidants, 10 day exposure showed significant (p<0.001) increase in the activities of SOD, CAT and GPx whereas both 20 and 30 day exposure showed decline consistently in both blood as well as gills. Oxidative stress was maximum in fishes exposed to EIE for 30 days.