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The present work will focus on the growth responses of metal mixture stressed fish under semi-intensive pond culture systems. The effects of sub-lethal concentrations of metals mixture (Zn+Pb+Mn) on the growth performance of Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cirrhina mrigala, Labeo rohita and Catla catla will be elucidated. The growth performance of 90-day metals (mixture) stressed fish will be monitored in terms of wet weights (g), fork and total lengths (mm), condition factors (K), feed intake (g) and feed conversion ratios and compared with the unexposed (control) fish. Fish will be grown in ponds under composite culture system for a period of 210 days. Fortnightly data on all the growth parameters and physico-chemistry of pond's water viz. pH, temperature, dissolved oxygen, phosphates, nitrates, sodium, potassium, calcium, magnesium, total ammonia, total hardness, total alkalinity, electrical conductivity, light penetration and dry weights of planktonic biomass will be collected and analyzed statistically by using Factorial Experiment with two replications for each treatment (RCBD) and Tukey's / Student Newman-Keul tests. Correlation and regression analyses will also be performed to find-out statistical relationships among various parameters defined for this study.
NEED FOR THE PROJECT
Tremendous increase in human population and establishment of industries have resulted in the discharge of wastewater, containing heavy metals and their compounds, into the natural aquatic habitats in Pakistan (Rauf et al., 2009). The environmental pollutants tend to accumulate in organisms and are persistent because of their chemical stability (Wepener et al., 2001). Heavy metals are the major aquatic pollutants that are regarded to be cytotoxic, mutagenic and carcinogenic (Muley et al., 2000; More et al., 2003). Heavy metals are introduced into the environment by a wide spectrum of natural sources such as volcanic activeties, erosion and anthropogenic ones, including industrial wastes as well as domestic sewage (Jabeen and Javed, 2011). Some of these metals like lead, nickel, cadmium and mercury are toxic to living organisms even at quite low concentrations while others such as copper, iron, zinc and manganese are biologically essential and natural constituents of the aquatic ecosystems and become toxic only at very high concentrations. Aquatic pollution could affect the aquatic organisms and even human health directly and indirectly by consuming the aquatic animlas (Cohen et al., 2001; Storelli et al., 2006; Karadede-Akin and Unlu, 2007).
Both industrial and domestic sewage wastes are finally dumped into the natural resources of water like rivers, ponds, lakes and seas that have been polluted with different types of solid and liquid wastes (Garg et al., 2009). In aquatic ecosystems, heavy metals have received attention of the toxicologists due to their toxicity and accumulation in aquatic biota (Javed, 2004). Contamination of metals may cause devastating impacts on the ecological balance of the natural water bodies and hence disturbing the diversity of aquatic organisms (Vosyliene and Jankaite, 2006; Farombi et al., 2007; Hayat and Javed, 2008). For the estimation of trace metal's (Co, Cr, Cu, Fe, Mn, Ni, Sr and Zn) pollution in freshwater ecosystems, fish are considered as one of the most indicative factors (Rashed, 2001). Heavy metals even in low amounts can have negative impacts on fish life, causing different types of disturbances in its health and wellbeings (Vosyliene and Jankaite, 2006). Any disturbance can result in reduced metabolic rate and hence cause reduction in fish growth (Sarnowski, 2003). Growth has also been used as a factor to monitor and predict the toxic effects of metals on fish (Javed, 2006). Toxicity of metals can reduce the food consumption by the fish and hence caused reduction in its assimilation that ultimately affected the fish growth (Vincent et al., 2002; Hussain et al., 2010). Low concentrations of heavy metals can cause a chronic stress, which may not kill individual fish, but lead to poor growth and thus reduce their ability to compete for food and habitat (Laflamme et al., 2000; Hussain et al., 2010; 2011a).
The growth of freshwater animals requires certain concentrations of zinc but its elevated levels become deleterious. It could cause acid-base disturbances, ionoregulation, disruption of gill tissues, growth retardation, hypoxia and even death in fish (Hogstrand et al., 1994). Lead occurs in aquatic bodies in a wide range of physical and chemical forms that could affect the behavior of fish adversely, even at low concentrations. Most of lead in the environment is in inorganic form and exists in several oxidation states (Jackson et al., 2005). It is the most stable ionic species present in the environment, instead, the toxicity of lead depends upon many factors including fish age, pH and hardness of the water (Nussey et al., 2000). Lead exposure to the fish can adversely affect the fish growth and release of digestive enzymes (Jain et al., 1996). Manganese is an essential nutrient for all organisms including fish and plays critical physiological role as a constituent of several metalo-enzymes and as a coactivator of many other enzymes (Maage HYPERLINK "../../../../../Abbas/Desktop/02 may 12/manganese one sentence.htm#bib29"et al.HYPERLINK "../../../../../Abbas/Desktop/02 may 12/manganese one sentence.htm#bib29", 2000). Excess concentrations of manganese, however, can have negative effects including impacts on carbohydrate metabolism, effects on immune response and neurotoxicity (Barnhoorn HYPERLINK "../../../../../Abbas/Desktop/02 may 12/manganese one sentence.htm#bib3"et al.HYPERLINK "../../../../../Abbas/Desktop/02 may 12/manganese one sentence.htm#bib3", 1999; Hernroth HYPERLINK "../../../../../Abbas/Desktop/02 may 12/manganese one sentence.htm#bib24"et al.HYPERLINK "../../../../../Abbas/Desktop/02 may 12/manganese one sentence.htm#bib24", 2004; Newland, 1999;Â Gunter HYPERLINK "../../../../../Abbas/Desktop/02 may 12/manganese one sentence.htm#bib22"et al.HYPERLINK "../../../../../Abbas/Desktop/02 may 12/manganese one sentence.htm#bib22", 2006). An elevated concentration of manganese may also have a chronic harmful effects on the growth of fish (Partridge and Lymbery, 2009).
Major carps could act as appropriate monitoring organisms to predict bioavailability of water-borne metals in freshwater habitats (Palaniappan and Karthikeyan, 2009). Catla catla, Labeo rohita and Cirrhina mrigala are the most successful species of polyculture in Pakistan due to suitable climate for their culture on one hand and their consumer preference on the other. The populations of these indigenous fish species have dramatically been reduced in the inland waters of Pakistan, due to harmful effects of metals to decline their growth potential (Javed, 2004). However, heavy metals are present in the form of mixtures in the aquatic habitats affecting the fish growth and facundity. Therefore, it is important to investigate the growth responses of metals mixture stressed major carps in earthen ponds under semi-intensive culture system to predict their growth responses in the natural habitats. This work will help conservation of indigenous fish species in freshwaters of Pakistan.
REVIEW OF LITERATURE
Stasiunaite (1999) reported possible effects of heavy metal mixture (HMM) on hatching, survival and growth of early-life history stages of rainbow trout (Oncorhynchus mykiss). The eggs of rainbow trout were exposed to HMM concentrations of between 12.5% and 200% from fertilisation to near complete yolk absorption, for 70 days. The concentrations of metals in the model mixture tested were taken as 100%: Cu: 0.02, Cr: 0.02, Ni: 0.05, Zn: 0.06, Pb: 0.03, Fe: 0.3, Cd: 0.003 and Mn: 0.09 mg L-1. Gross malformations of the vertebral axis (bent tails, lordosis), retarded growth and development in newly hatched larvae were observed. The MATC (Maximum Acceptable Toxicant Concentration) for growth was estimated to be between 100% and 200% concentrations of HMM (141.4% HMM).
Vosyliene et al. (2003) investigated the effects of a heavy metal model mixture (HMMM) of seven metals viz. Cu, Zn, Ni, Cr, Pb, Cd and Mn on the developmental stages of rainbow trout, Oncorhynchus mykiss. Results suggested that, the long-term exposure to sublethal concentrations of HMMM induced significant changes in the development of embryo, growth of larvae and morpho-physiological, physiological and haematological parameters of adult fish. Growth parameters and respiratory responses of fish were found to be the most sensitive to even low concentrations of HMMM.
Ansari et al. (2006) derived relationships of body size (length and weight) with metals i.e., zinc, iron, copper, manganese and cadmium in freshwater wild Puntius chola. It was observed that all metals, except copper, showed significantly positive correlation (P<0.001) with total body weight or total body length. All metals, except cadmium, were found to increase in direct proportion to an increase in body weight gain of fish.
Hussain et al. (2010) examined the growth performance of Cirrhina mrigala stressed with sub-lethal mixture of Fe+Zn+Pb+Ni+Mn in earthen ponds, for a period of 6 months. It was concluded that, during semi-intensive culture system in ponds, the unstressed Cirrhina mrigala showed non-significantly better growth performance in terms of wet weight, fork and total lengths increments as compared to the metals mixture stressed fish.
Hussain et al. (2011b) studied the growth responses of metals mixture (Fe+Zn+Pb+Ni+Mn) stressed Catla catla under semi-intensive pond culture system for 24 weeks. The results revealed that, in comparison to control, sub-lethal stress of mixture caused significantly lower growth to the fish than that of control (unstressed fish).
Naeem et al. (2011) determined the relationships of Cd, Cu, Mg, Mn and Zn concentrations with biological aspects as size and condition factor, of Oreochromis niloticus. The fish samples were collected from different locations in the river Indus near Ghazi Ghat area, Pakistan. Regressions were performed for both fish size and condition factor with metallic ion concentrations. The results indicated that, Mg and Zn were found highly correlated (P<0.001) with fish size (length and weight). Mn was found significantly (P<0.01) correlated while Cu and Cd were least significant (P<0.05) with total length, however, these were insignificant with weight increments. Condition factor showed significant (P<0.05) relationship with Mn concentration only.
The proposed research work will be conducted at the Fisheries Research Farms, Department of Zoology and Fisheries, University of Agriculture, Faisalabad. The fingerlings (90-day age) of five fish species viz. Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cirrhina mrigala, Labeo rohita and Catla catla will be brought and acclimatized to laboratory conditions for 10 days, in cemented tanks.
Stock of each fish species will be divided into two groups and kept separately in glass aquaria. Chemically pure chloride compounds of zinc, lead, and manganese will be dissolved in deionized water and stock solutions prepared for required metal mixture concentrations. The treated group of fish will be exposed sub-lethal (1/3rd of LC50) concentrations of metals mixture (Zn+Pb+Mn) in solution form, for 90 days, as determined by Javed and Yaqoob (2011). However, the control group of fish will be kept un-stressed in clean metal free water. The fish viz. Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cirrhina mrigala, Labeo rohita and Catla catla will be exposed to sub-lethal concentrations of 17.85, 18.84, 18.21, 21.03 and 18.59 mg L-1, respectively.
Sub-lethal stress of metals mixture will be given to the fish in glass aquaria at constant water temperature (28ËšC), pH (7.50) and total hardness (225 mg L-1). Individuals (90-day age) of each fish species will be kept in glass aquaria, separately, for stress experiments. Constant aeration will be provided to all the test media with an air pump fixed with a capillary system. The fish will be fed, to satiation, with the feed having 32% digestible protein and 3.00 Kcalg-1 of energy, twice a day. During sub-lethal metals mixture exposure period physico-chemical variables of water viz. temperature, pH, dissolved oxygen, carbondioxide, total hardness, total ammonia, potassium, sodium, calcium and magnesium will be analyzed twice a day by following the methods described in A. P. H. A. (1998).
Fish Growth Studies in Ponds
The metals mixture exposed and control (un-stressed) fish will be stocked in earthen ponds (0.012 ha), separately, at the rate of 65 fish per pond with the stocking density of 10, 10, 20, 40 and 15 percent for Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cirrhina mrigala, Labeo rohita and Catla catla, respectively. Both treated and control fish ponds will be fertilized with broiler droppings at the rate of 0.17g nitrogen per 100g fish weight daily, in order to promote the pond biota for fish consumption. The fish will also be provided supplementary feed, at the rate of three percent of their standing body weight initially and then decreased gradually. However, no feed will be offered to the fish at water temperature below 25ËšC. The fish will be grown in earthen ponds for 210 days. From each pond, random sampling (n=7) of each fish species will be done by using drag net, on fortnightly basis, and their wet weights (g), fork and total lengths (mm) will be measured and recorded. After recording the data, fish will be released back into their respective ponds.
The limnological parameters viz. water pH, temperature, dissolved oxygen, phosphates, nitrates, sodium, potassium, calcium, magnesium, total ammonia, total hardness, total alkalinity, electrical conductivity, light penetration and dry weights of planktonic biomass will be monitored, fortnightly, of each treated and control ponds. The growth parameters under study will include increase in fish wet weights, fork and total lengths, length-weight relationships, condition factors and feed conversion ratios and net yields at final harvest.
The statistical differences among different parameters of growth along with water quality variables will be analyzed by using Factorial Experiment (RCBD) and Tukey's / Student Newman-Keul tests. Correlation and regression analyses will also be performed to find-out relationships among various parameters, described for this study.