Abstract: The water samples were collected from the 22 km segment III of Yamuna River from Okhla barrage. This segment receives water from 17 sewage drains of Delhi, Western Yamuna Canal (WYC), upper Ganga canal via Najafgarh drain and Hindon cut canal. Hence, the water samples collected were used to determine the presence of Chromium, Nickel and Lead through Atomic Absorption Spectrophotometry. The concentration of these heavy metals were much above the maximum permissible limits set by WHO. This was bound to have its influence on the riverine flora and fauna. To evaluate this, two popularly consumed fish species such as Channa striatus and Heteropneustes fossilis were caught and the bioaccumulation of these heavy metals were estimated in different organs (liver, kidney, gill and muscle). It was found that Cr accumulated the most in these organs (gill being most influenced) in both the species. The accumulation of all these heavy metals were above MPL set by World Health Organisation (WHO) and Food and Drug Administration (FDA). Histopathology was also conducted where heavy damages were observed in both liver and kidney of both the species.
INTRODUCTION
Water is most precious natural resource that exists on our planet and is essential for survival but the development of modern technology and rapid industrialization are responsible for aquatic pollution. Discharged waste from industries into the water bodies disturbs the flora and fauna (Gad, 2009; Veena et al., 1997; Ashraf, 2005; Farombi, et al., 2007; Wong et al., 2001). High concentration of heavy metal in water has been reported in different water bodies of India due to heavy metal contamination (Rajaganapathy et al., 2011; Ambedkar and Muniyan, 2011; Chattopadhyay, et al., 2002), particularly due to anthropogenic activities (domestic, industrial and agriculture) by human (Gumgum et al., 1994; Jordao et al., 2002; Nimmo et al., 1998). This stands true for Yamuna barrage as well. Heavy pollution in river water is attributed to effluent discharges from small and large industries, automobile wastes and surface run-offs from adjoining areas. Tremendous efforts have been put by Indian Government yet the effluents have brought river water quality far below the limits prescribed by EPA. The study was undertaken because rapid industrialisation and colonisation of Okhla region has increased the pollution level of river Yamuna at alarming rate. Now pollution of this river has increased drastically affecting human life, cattle and aquatic species and has reached far below bathing standards (NCT, 2005).
Water quality from various sources have been studied all over world (Ekeanyanwu et al., 2010; Mohamed, 2008; Montaser et al., 2010; Ozturk et al., 2009) but in India it is restricted to Southern region only (Ambedkar and Muniyan, 2011; Mahadev and Gholami, 2010; Sreedhara Nayaka et al., 2009; Murugan et al., 2008). Studies till now have been focussed on determining the water quality of Okhla barrage but there is no data on its impact on fish biology in general and bioaccumulation and histopathology in particular. The data so presented by us is useful to create awareness so that some preventive measures be implemented by the Government to protect the natural fauna and thus ultimately the health of local population. Fishes form major biota to be studied as it comes in direct contact with polluted water. They possess comparatively higher trophic level thus are used as bio-indicator of heavy metal concentration (Lopes et al., 2001; Svobodova et al., 2004; Vinodhini and Narayanan, 2009). Also, these fishes owing to their palatability are captured and are sold in local markets of Okhla region which in long term may consequently bio-accumulate in humans as well. Accumulation of chemical pollutants is known to adversely affect the liver, kidney, muscle and other tissues of fish (Canli et al., 1998; Javed, 2004; Olowu et al., 2010; Puttaiah and Kiran, 2007) which can be visualised through histopathology (Akan et al., 2009; Al-Attar, 2007; Nair et al., 1984). Liver is major target organ for xenobiotics and thus, is frequently cited as the site of parenchymal damage following exposure to various chemical agents (Gingerich, 1982; Montaser et al., 2010). Kidney is severely affected by different toxic chemical which is evident in form of pathological changes such as necrosis of hematopoietic tissue, vacuolation of tubular cells, dilation of glomerular capillaries and degeneration of epithelial cell linings (Abdel-Baki et al., 2011; Kumar and Pant, 1981). Such detrimental effect of heavy metal contamination was also seen in the study with high level of bioaccumulation and marked histopathological evidences. The present study was carried out on naturally affected fish species as it avoided unnecessary killing and induction of healthy fishes with heavy metals and thus avoided ethical issues for the use of animals.
MATERIALS AND METHODS
Study area: The sampling site (Yamuna barrage from where Yamuna River leaves Delhi laden with city’s biological and chemical waste) lies in Delhi in North India, at an altitude 216 km above sea level. The 22 km segment III of Yamuna River from Okhla barrage (Fig. 1) (28o 32’ 49” N longitude and 77°18’ 48” E latitude), receives water from 17 sewage drains of Delhi and also from Western Yamuna Canal (WYC) and upper Ganga canal via. Najafgarh drain and Hindon cut canal. The categories of industries discharging waste water into Yamuna water includes pulp and paper, sugar, distillaries, textiles, leather, chemical, pharmaceuticals, oil refineries, thermal power plants food etc.
Collection of water and fish specimens: The systematic water sampling was adopted to get unbiased profiling of the river. Zonal sampling was done from four sides of barrage with three replica of each zone. Simultaneously, fish (murrel, Channa striatus and catfish, Heteropneustes fossilis) samples were also collected with the help of local fisherman. Physico-chemical parameters (temperature, pH and dissolved Oxygen) were recorded at the site itself. Water samples were stored for the analysis of Heavy metals as per standard method of sampling techniques, (APHA, AWWA and WEF 1999).
Sample analyses: Fishes (12 specimen) obtained (Length 20-25 cm; Weight 220-280 g) were dissected and organs (liver, kidney, gill and muscle) were removed and collected.
| Fig. 1: | Map showing sampling location of Okhla barrage of Yamuna River, India |
Wet digestion of the tissue was conducted to evaluate the bioaccumulation of heavy metals according to the methods proposed by Vanloon (1980) and Du Preez and Steyn (1992). Heavy metals (Cr, Ni and Pb) were analyzed using Atomic Absorption Spectrophotometer and results are expressed in μg g-1 (for tissue samples) and mg L-1 (for water samples). All the chemicals (Metal stock solutions, Sulphuric acid, Nitric acid and Perchloric acid) used were of analytical grade.
Bioaccumulation factor: Bioaccumulation factor was evaluated for the three heavy metals in four different tissues of the fishes. Bioaccumulation Factor (BAF) is calculated as the ratio of concentration of a pollutant (heavy metal) accumulated in the tissue of organism with respect to the concentration of that pollutant (heavy metal) in water body (Authman and Abbas, 2007). The BAF was calculated using the formula:
Histopathology: Live fishes were considered for histology. Soon after catching, the fishes were dissected and the organs were fixed in Bouins fluid for 24 h. Tissue processing, blocks preparation and staining of the slides were done as per described by Gray (1954). The prepared slides (5 μm sections) were observed under the microscope (Nikon 80i) at 400X and examined for histopathological alterations.
Statistical analysis: Parameters (mean and standard deviation) were calculated for the data obtained for physiochemical properties of water and for the presence of Heavy metals in water and fish samples. One-way analysis of variance (ANOVA) was applied to compare the difference amongst the means. p-values less than 0.05 were considered significant. As post hoc test for multiple comparisons, Duncan’s new Multiple Range Test (MRT) was applied (Duncan, 1955).
RESULTS AND DISCUSSION
Physico-chemical properties of river and bioaccumulation in fish: The physico-chemical properties of Yamuna river water, Delhi are given in Table 1. The River water temperature ranged between 22.8-23°C, the pH was found to be slightly alkaline (7.32-7.78). Dissolved oxygen concentration (1.5-3.7 mg L-1) was below normal for fish health. Lead (Pb) (0.4 mg L-1), Nickel (Ni) (0.39 mg L-1) and Chromium (Cr) (0.28 mg L-1) were present in levels beyond the recommended values set by WHO. This had its toll on fish health as the bioaccumulation of heavy metals (Cr, Ni and Pb) (μg g-1 wet weight) in different tissues of fish C. striatus was detected and is given in (Table 2). Amongst the heavy metals Cr (gills (51.91 μg g-1) followed by kidney (42.01 μg g-1), liver (15.31 μg g-1) and muscle (5.51 μg g-1) accumulated the most in all the organs. It was followed by Pb (kidney (21.49 μg g-1)>gill (19.03 μg g-1)>liver (13.45 μg g-1) and muscle (3.16 μg g-1)]. The least accumulated heavy metal was Ni [gill (9.09 μg g-1)>kidney (8.71 μg g-1)>liver (4.05 μg g-1), and muscle (1.45 μg g-1)]. In H. fossilis the pattern observed was comparable to C. striatus (Table 3). Cr accumulated the most [gill (157.64 μg g-1)> liver (94.82 μg g-1)> kidney (21.21 μg g-1)> muscle (18.51 μg g-1)] in all the organs followed by Ni [gill (42.4 μg g-1), followed by kidney (1.57 μg g-1)>muscle (1.2 μg g-1)> liver (0.56 μg g-1)) and Pb [gill (20 μg g-1) followed by muscle (2.21 μg g-1)> kidney (1.63 μg g-1) and liver (0.45 μg g-1)]. Gill was the most influenced organ in both the fish species.
Bioaccumulation factor: The heavy metal accumulation in water and values accumulated in tissues were used to calculate Bioaccumulation Factor (BAF) (Table 4 for C. striatus) and (Table 5 for H. fossilis).
| Table 1: | Physico-chemical properties of Yamuna river, Delhi |
| Table 2: | Bioaccumulation of heavy metals (Cr, Ni and Pb) in (μg g-1) wet weight in different tissues of fish C. striatus |
| *Average heavy metal load (mean of all the four organs studied) in order to compare with workers who have taken the average values/fish as a whole, Data is shown as yVx±SD, whereas V = Mean value of twelve replicates, x = Superscript of different letters which are statistically significant for the accumulation of different heavy metals within a tissue and y = Subscript of different letters which are statistically significant for a heavy metal accumulation within tissues, at the p = 0.05 level | |
| Table 3: | Bioaccumulation of heavy metals (Cr, Ni and Pb) in μg g-1 wet weight in different tissues of fish H. fossilis |
| *Average heavy metal load (mean of all the four organs studied) in order to compare with workers who have taken the average values/fish as a whole, Data is shown as yVx±SD, whereas V = Mean value of twelve replicates, x = Superscript of different letters which are statistically significant for the accumulation of different heavy metals within a tissue and y = Subscript of different letters which are statistically significant for a heavy metal accumulation within tissues, at the p = 0.05 level | |
| Table 4: | Bioaccumulation Factor (BAF) in C. striatus tissue samples |
| Table 5: | Bioaccumulation Factor (BAF) in H. fossilis tissue samples |
The values observed during the present study have been compared with the recommended values for the three heavy metals (Cr, Ni, and Pb) and they were found to be above the Maximum permissible limit (MPL) of WHO (1985) and FEPA (1999) (Table 6).
Histopathological effects: Histological changes in the two species due to the exposure to the heavy metals are evident from the Fig. 2-5.
| Fig. 2(a-b): | Transverse section of liver from two fishes, C. striatus (a) Liver of control fish characterized by Nucleus (N), Hepatic Cell (HC), Bile Canaliculi (BC) and sinusoid (S), (b) Liver of infected fish features Tissue vacuolization (V), Pyknotic Nuclei (P) and Distorted Artery wall (DA) (400x) |
| Table 6: | Recommended values (Maximum permissible limit) of Heavy metals in fish (μg g-1) and water (mg L-1) compared to present study |
| *Presence in the edible part i.e., the muscle | |
| Fig. 3(a-b): | Transverse section of the kidneys from two fishes, C. striatus (a) Kidney of control fish showing Glomerulus (G), Renal Tubule (RT), Hematopoietic Tissue (HT), (b) Kidney of infected fish with marked Degenerated Renal tubules (DR), pyknotic nuclei (P), Haemorrhage (H) and Glomerular Degeneration (GD) (400x) |
Marked changes were observed in liver and kidney. Industrial effluents induced significant structural changes in C. striatus liver tissue (Fig. 2b) such as pyknosis and vacuolization of tissue. Ruptured liver tissue can also be seen in adjoining areas of distorted artery wall. Kidney morphology also changed markedly due to the presence of heavy metals in the water (Fig. 3b). Alterations are visible in the form of kidney tubules necrosis, pyknosis, hemorrhage and glomerular degeneration.
| Fig. 4(a-b): | Transverse section of the liver from two fishes, H. fossilis A. liver of control fish characterized by Hepatic cells (H), Sinusoids (S) and Nucleus (N), (b) Liver of infected fish with marked Tissue Vacuolization (V), Ruptured Vein (RV) and Haemorrhage (H) (400x) |
Liver of infected H. fossilis (Fig. 4b) shows vacuolization of the tissue, ruptured vein and hemorrhages. Similarly, the infected kidney due to the influence of heavy metal shows several changes (Fig. 5b). The variations observed were necrotic urinary tubules, vacuolization, hemorrhages and congested renal tubules.
Aquatic environment is loaded with wide range of pollutants. Puttaiah and Kiran (2007), studied the levels of heavy metals in Jannapura lake, Karnataka and they were in the order Pb>Cu>Zn>Cd>Ni>Co and their value exceeded Maximal Permissible Level (MPL) as per WHO standards. Seyhan River, Turkey (Canli et al., 1998); Lithuania fresh waters (Staniskiene et al., 2006) and Okumeshi River, Turkey (Ekeanyanwu et al., 2010) are few other heavy metal polluted rivers.
| Fig. 5(a-b): | Transverse section of the kidney from two fishes, H. fossilis (a) Kidney of control fish showing Renal tubule (RT), Glomerulus (G) and Hematopoietic Tissue (HT), (b) Kidney of infected fish with marked Necrosis (N), Tissue Vacuolization (V), Congested Renal Tubules (CR) and Hemorrhages (H) (400x) |
Heavy metals have become a serious issue now-a-days because of its persistent nature and for causing serious health complications. Presence of heavy metals in aquatic environment is dependent upon wide range of chemical, biological and environmental factors (Ajmal and Razi-ud-Din, 1988). This has been realized and work has been done on estimation of various heavy metals in different water bodies. But this work was conducted to study the presence of Cr, Pb and Ni in Yamuna Barrage, a zone used as source to supply fish to various markets. Heavy metals then accumulate in different tissues of the fish from three possible ways i.e., through body surface, gills and digestive tract (Pourang, 1995). Food may also be an important source of heavy metal accumulation in fish (Javed and Hayat, 1996) which may be attributed to the fact that the food sources of these fishes are polluted which leads to biomagnifications of these heavy metals in food chain (Javed, 2004). Heavy metals (Pb 0.4 mg L-1), Ni (0.39 mg L-1) and Cr (0.29 mg L-1)) estimated from the site selected were above the maximum permissible limit set by WHO (1985). This can exert toxic effects on human beings if is used for drinking and irrigation. The accumulation of these heavy metals was also observed. Cr ranged from 5.51-51.91 μg g-1 in the tissue of C. striatus (gill>kidney>liver>muscle), while between 18.51-157.64 μg g-1 in H. fossilis (gill>liver>kidney> muscle) and these values are higher than WHO and FEPA recommended limits of 0.15 μg g-1 in food fish. Other workers have reported lesser (0.8-1.07 μg g-1) Cr in various fish species such as Chrysichthys nigrodigatatus (taken as a whole) from Yamuna River in Delhi region (Sen et al., 2011). Comparable accumulation of Cr (3.7-26.9 μg g-1) was reported in tissue of H. fossilis obtained from the Yamuna River water (Ajmal and Razi-ud-Din, 1988). Whereas, fishes Wallago attu and Labeo dyocheilus obtained from water sources such as Kabul River, Pakistan showed higher accumulation of Cr (Yousafzai et al., 2010). The amount of Cr accumulation reported in different tissues was 600 μg g-1 (gills)>533.3 μg g-1 (muscle)>510 μg g-1 (liver) and 730.3 μg g-1 (gills) 647.3 μg g-1 (muscle)> 643.7 μg g-1 (Liver), respectively.
Ni ranged from 1.45-9.09 μg g-1 (gill> kidney> liver> muscle) in C. striatus and 0.56-42.40 μg g-1 (gill>kidney> muscle>liver) in H. Fossilis tissues and values are higher than WHO and FEPA recommended limits of 0.5-0.6 μg g-1, respectively in food fish. Studies conducted on fishes such as Mugil cephalus and Trachurus mediterraneus from Iskenderun Bay, Turkey (Yilmaz, 2003) exhibited lower accumulation of Ni ranging between 7.35 and 0.99 μg g-1 in various organs. Similarly, study conducted by OztUrk et al. (2009) in Avsar Dam Lake in Turkey also revealed the accumulation of Ni in muscle (1.27 μg g-1), gills (3.52 μg g-1) and liver (7 μg g-1) of Cyprinus carpio, which shows from Iskenderun Bay and Avsar Dam Lake are less polluted than present study. Whereas higher accumulation observed in L. dyocheilus [152 μg g-1 (gills)>117.7 μg g-1 (muscles)>111.7 μg g-1 (liver)] and W. Attu (122.7 μg g-1 (gills)>108 μg g-1 (liver)>106.7 μg g-1 (muscle)] obtained from river Kabul as reported by Yousafzai et al. (2010). But in all these studies, gill was most influenced organ as also been reported in the present study.
Accumulation of Pb found to be 3.16-21.49 μg g-1 (kidney>gill>liver>muscle) in C. striatus while 0.45-20 μg g-1 (gill>muscle>kidney>liver) in H. fossilis. These values are higher than WHO and FEPA recommended limits of 0.3 μg g-1 in food fish. Our values are comparable to those reported by (Ajmal and Razi-ud-Din, 1988), in fish H. fossilis obtained from five different sampling stations of Yamuna River from Delhi to Allahabad with concentration of Pb between 1.4-12.8 μg g-1. While, Sen et al. (2011) shown range from 0.47-24 μg g-1 in a catfish, Chrysichthys nigrodigatatus (fish as a whole) caught from river Yamuna (Delhi), which is comparable to the present study for catfish, H. fossilis. This shows that Yamuna river been contaminated with respect to the increasing load of Ni over the last decade. Kabul River of Pakistan showed the values for Pb accumulation as 528.7 μg g-1 (muscle)> 377 μg g-1 (liver)> 301.3 μg g-1 (gills) for L. dyocheilus and 623.3 μg g-1 (liver)> 599.3 μg g-1 (muscle)> 453.3 μg g-1 (gills) in W. attu, as reported by (Yousafzai et al., 2010) and thus reveals the fact that heavy metal loads are much higher than Yamuna Barrage. Their report also reveals higher accumulation of Pb in muscle and liver while in the present study Pb accumulated the most in gills. Studies conducted by Olowu et al. (2010) on Tilapia zilli and Chrysichthys nigrodigatatus to determine the influence of heavy metals (Fe, Zn and Ni) in fish tissue (head, trunk, tail, gills and intestine) and water sample from Epe [Fe(7.30)>Ni(0.69)>Zn(0.42)} and Badagry (Fe(6.65)> Zn(0.54)>Ni(0.10)) lagoons shows that Fe and Ni were present in highest concentration in the head and gills as water passes through these two organs. Preset study also depicted gill to be most influenced organ with highest bioaccumulation factor due to the fact gills have large surface area and branchial respiration leads to direct contact of heavy metal laden water to enter the gills.
In order to study the impact of these heavy metals on histopathology two organs i.e., liver and kidney are used as they are commonly studied organs by other workers as well. Teleost liver is major target organ for xenobiotics metabolism and thus, frequently cited as the site of parenchymal damage following exposure to various chemical agents (Gingerich, 1982). Microscopically, the parenchyma can be seen as a three-dimensional network of sinusoids composed of cuboidal hepatocytes with narrow spaces lined with, endothelial cells. While, kidney is involved in removal of wastes from blood (Fange, 1986) and is severely affected by different toxic chemical which is evident in form of pathological changes such as necrosis of hematopoietic tissue, vacuolation of tubular cells, dilation of glomerular capillaries and degeneration of epithelial cell linings (Kumar and Pant, 1981). The influenced liver of C. striatus exhibited tissue vacuolization, pyknosis and distorted artery wall which has been in comparison with control (Fig. 2a) shows normal arrangement of hepatocytes with nucleus, bile canaliculi and sinusoid. Liver of control H. fossilis (Fig. 4a) showing Normal histology of liver tissue with hepatic cells, nucleus and sinusoids while liver of infected fish is marked by vacuolization of the tissue, ruptured vein and hemorrhages. Kidney of C. striatus showed degenerated renal tubules, pyknosis, hemorrhages and glomerular degeneration as compared to its control (Fig. 3a) with normal histology showing glomerulus, renal tubules and hematopoietic tissue. The control kidney of H. fossilis (Fig. 5a) showed normal renal tubules, glomerulus and hematopoietic tissue while necrosis, tissue vacuolization, congested renal tubules and hemorrhages were evident in the kidney of infected H. fossilis. Such changes have been reported by other workers but from fishes inhabiting water bodies lying in Southern India. Similar to the present study, in other report hepatocytes showed marked cytoplasmic vacuolization and sinusoids, in most areas were distended and central veins appeared severely damaged due to marked swelling and degeneration of the endothelial lining cells (Radhakrishnan and Hemalatha, 2010). Acute toxicity impacts of hexavalent chromium on kidney of Channa punctatus showed hypertrophy of epithelial cells of renal tubules with reduced lumens, atrophy of the renal tubules, glomerular contraction in the Bowman's capsules and necrosis of hematopoietic tissues. The inter-renal cells of the head kidney exhibited distinct hypertrophy and vacuolization (Mishra and Mohanty, 2008). Thus, these changes influences fish health and therefore the population of the fish in this zone. This will ultimately influence the major protein source available to the population as well as can endanger the species surviving there.
CONCLUSION
To conclude, the present study generates data regarding the increasing pollution in the Yamuna Barrage and it confirms that it is having strong impact on fish health as these heavy metals are accumulating in different tissues of the selected fish species i.e., C. striatus and H. fossilis This is further supported by the deformities that occur in two major organs such as kidney and liver. The edible fish species thus is unfit for human consumption. Further studies can be conducted in order to study the impact of these heavy metals on the reproductive system as it will reveal the decline in the population of species inhabiting the site selected. Effective measures such as legislative provision and other waste management tools for environmental protection should be implemented for the well being of dwelling flora and fauna and ultimately the health of local populations.
ACKNOWLEDGMENTS
The author gratefully acknowledges the Chairman, Department of Zoology, AMU, Aligarh for providing facilities for undertaking the studies. The author is grateful to the Maulana Azad National Fellowship (GRANT No. F.40-3(M/S)/2009(SA-III/MANF), University Grant Commission, New Delhi, for financial assistance (awarded Junior Research Fellowship). Author is also thankful to the Instrumentation Unit, Department of Chemistry, AMU, Aligarh for technical support.