The stress on natural resources is increasing day by day due to increasing human population and rapid industrialization thus, their maintenance and conservation remains the ultimate challenge for the mankind. Environmental pollution (i.e. land, water and air) tends to be a devastating issue being faced by human life over the last few decades. However, aquatic contamination is trickier to be measured than terrestrial and air contamination (Barton 2003). The heavy metal contamination of the aquatic water bodies has become a universal problem during recent years, because of their indestructible nature and toxic effects on organisms (MacFarlane and Burchett, 2000).
Heavy metals contamination is of prime concern among the various environmental pollutants, due to their potential lethal effect and ability to bioaccumulate in aquatic environments (Censi et al 2006).
The quality of groundwater, being the foremost factor in influencing its suitability for drinking, domestic, irrigation and industrial purposes, is equally significant as its quantity. The concentration of chemical components determining the quality of groundwater is critically inﬂuenced by geological formations and anthropogenic activities.
Punjab, the north-western state of India faces a serious problem of contamination of groundwater especially the southwestern region of the state which includes the districts i.e. Bathinda, Barnala, Faridkot, Ferozepur, Mansa, Moga, Muktsar and Sangrur due to geogenic and anthropogenic activities. According to report by Government of India Planning Commission in 2013, the southwestern zone of Punjab has high contents of fluorides, nitrites and heavy metals like uranium (U), arsenic (As), lead (Pb), nickel (Ni), zinc (Zn) and chromium (Cr) far above the safe limits.
High ﬂuoride content, i.e., more than 10 mg/L has been observed in Barnala, Bathinda, Fazilka, Muktsar and Sangrur districts. The Muktsar district of Punjab is fronting a serious challenge of water logging and salinization. The existence of heavy metals and pesticide residues in aquatic environment leads to harmful consequences on plant and animal life (Ghaleno et al 2015).
Heavy metals have been recognized as potent biological poisons because of their persistent nature, toxicity, tendency to get accumulated in organisms and undergo biomagnification. The term ‘heavy metal’ refers to any metal or metalloid that has relative atomic density greater than 4g/cm³ or 5g/cm³ and is toxic even at very low concentrations (Lenntech et al 2004). Heavy metals are environmentally ubiquitous. Heavy metals are easily dissolved, transported through water and are readily taken up by aquatic biota. Due to extremely toxic nature, potential to persist for long time in environment and nonbiodegradable nature in the food chain, heavy metals are chief constituents of principal group of water toxins causing cellular toxicity, carcinogenicity and mutagenicity in organisms (More et al,2003), (Authman et al 2013).
In the study of heavy metal contamination in an aquatic environment, fishes are the valuable model because they travel freely among the diverse trophic levels among the aquatic fauna (Chopra et al, 2001), (Palaniappan et al,2009).Most of the heavy metals when present in trace amounts are imperative for physiological roles in fish to regulate biochemical processes. However, persistence of heavy metals in the form of mixtures exceeding the permissible restrictions results in biochemical and histological alterations in fish (Bu-Olayan and Thomas 2004). Heavy metals are considered to be disturbing the immune responses of fish resulting in population decrease, pathogenicity and high mortality rate (More et al 2003).
In aquatic system, heavy metals such as cadmium (Cd), copper (Cu), lead (Pb), mercury (Hg), and zinc (Zn) are most significant pollutants upsetting the aquatic environment and are tremendously hazardous for the well-being of fish (Authman et al 2015). Fish fauna integrate heavy metal impurities by different ways like intake of particulate matter suspended in water, ion-exchange of dissolved heavy metals across lipophilic membranes (gills) and adsorption on surface of tissues and membrane (Alam et al, 2002). Different studies from both field and laboratory works disclosed that accumulation of heavy metals in a tissue is mainly dependent on water concentrations of metals and exposure period; along many other environmental factors such as water temperature, pH, hardness, salinity, alkalinity, oxygen concentration and dissolved organic carbon also play noteworthy part in metal build-up and toxicity to fish ( Jitar et al 2014). Ecological needs, age and size of organism, their life cycle, feeding habits, and the season of capture were also found to affect experimental results from the tissues (polat et al, 2015) (Omar et al ,2014).
Exposure of fish to contaminants resulted in increase in Reactive Oxygen Species (ROS) leading to impairment of normal oxidative metabolism and finally to oxidative stress (Lushchak 2011). Laboratory studies confirmed that the measurement of changes in the expression of activities of certain antioxidant enzymes can be explored in an early warning system of toxicant exposure (Livingstone 2003, Lushchak 2011). Antioxidant enzymes such as Catalase (CAT), Glutathione S-Transferase (GST), Glutathione Peroxidase (GPx) and Superoxide Dismutase (SOD) are well-thought to be latent biomarkers and are often used as screening tools for the assessment of the effects of environmental stress as there are typical changes in the activity of these antioxidant enzymes as well as the levels of malondialdehyde (MDA) produced due to oxidative stress. Oxidative damage primarily occurs through production of reactive oxygen species (ROS) and can damage lipids, proteins, and DNA contributing to loss of activity and structural integrity of enzymes and may activate inflammatory processes (¨Ozyurt et al, 2004). In most cases, the abnormal generation of ROS, which can result in significant damage to cell structure, is considered as an important signal of oxidative damage (Barzilai,2004). Oxidative stress is induced as a result of the three factors: (a) an increase in oxidant generation, (b)a decrease in antioxidant protection, and (c)failure to repair oxidative damage (Das et al, 2010). Thus, examining the change in activity of antioxidant enzymes such as SOD, CAT, and GR shall be an effective method of denoting oxidative stress and changes in their activity and other biomarkers could be the possible tools in aquatic toxicological research.
Histopathology of fish tissues is also an unfailing monitoring tool, allowing the assessment of the effect of environmental stressors. It is one of the most dependable indicators of the health damage brought by the water contaminants in aquatic organism (Lushchak 2011). The major advantage of using such markers in environmental monitoring is that it allows examining specific vital organs including liver, gills and kidney that are responsible for fundamental functions such as accumulation and biotransformation of xenobiotics, excretion and respiration in fish (Gernhofer,2001)
Gills are the most important organ of the fish, play multifunctional role in performing dynamic functions such as osmoregulation, acid-base balance, respiration and excretion of nitrogenous wastes (Evans et al., 2005). The environmental contaminants such as heavy metals damaged the gills of fish by being attached to the mucus layer, entered into the gills and lead to changes in the ultrastructure and morphology of fish gills (Athikesavan et al 2006).
Liver key-functioning enzymes (aminotransferases and phosphatases) are considered as potential biomarkers of effects in toxicological studies showing the state of liver injury (Mcgill , 2016). Fish liver is the main source of an antioxidant enzyme GPx and shows higher activity of this enzyme as compared to the other organs to overcome the oxidative stress caused by heavy metals (Murugan et al., 2008). The antioxidant enzyme, (GPx) showed higher activity in liver to overcome the oxidative stress caused by heavy metals as compared to the other organs (Murugan et al 2008). Saini recorded severe histopathological lesions such as infiltration of lymphonuclear cells, degeneration of hepatic parenchyma and deformation of hepatocytes, induced by heavy metals, in liver of L. rohita caught from natural freshwaters of Punjab.
Histological alternations in different tissues (gills and liver) have been used as highly sensitive biomarkers for xenobiotic induced toxic effects on the fish health (Paithane et al, 2012).
Histopathological abnormalities have been reported in kidney such as damaged renal tubules, shrinkage of tubules and tubule lumen and degeneration of tubules and hematopoietic tissues of different fish species on exposure to several pollutants such as heavy metals, insecticides, pesticides (Daskalov et al 2000, Lliopoulou and Kotsani 2001). Saini noticed histological changes such as, vascular degeneration, congestion of blood vessels and degeneration of tubules in the kidney of fish L. rohita procured from freshwaters of Punjab.
There are many biotic and environmental factors that influence the response of fish antioxidant defence system on exposure to field contaminants. The biotic factors such as reproductive and metabolic status of fish and environmental conditions, such as food availability, oxygen level, temperature of water, salinity, photoperiod, etc are seasonal parameters affecting the antioxidative response of fish (Parihar et al 1997, Buet et al 2006, Da Rocha et al 2009). Hypoxic conditions, temperature, increase and acidification usually render the fish more susceptible to intoxication, while increase in mineral contents (hardness & salinity) reduces metal toxicity (Witeska & Jezierska, 2003).
Labeo rohita (rohu), belonging to the Cyprinidae family, is the most important fish among three major Indian carps. It inhabits freshwaters of South and South-East Asia. It is widely consumed fish in Asia due to its high meat quality and it is also suitable for evaluation of the level of water contamination (Ramani et al 2002). India is the second largest producer of L. rohita after Bangladesh due to its high growth potential and high consumer preferences. The antioxidant potential of fish during different seasons depend on the physico-chemical properties and presence of contaminants in water therefore affecting the physiology of fish which may cause alterations in protein quality. Thus, proposed studies have been planned for the assessment of oxidative stress in muscles, gills, liver and kidneys of L. rohita inhabiting aquatic water bodies in Shri Muktsar Sahib district of Punjab during different seasons. For the detailed study following objectives were explored: