Among the aquatic pollutants, heavy metals are of special concern due to their diversified and toxic ill-effects to the fish. In an aquatic ecosystem, fish are exposed to a variety of metals, in mixture form, rather than a single metal. Therefore, the present work will focus on the determination of acute toxicity of Co, Pb, Cr and their mixtures on three fish species (Catla catla, Cirrhina mrigala and Labeo rohita) and their sub-lethal chronic effects on peripheral DNA damage, growth and their bio-accumulation in fish organs (liver, kidney, heart, gill and muscles). The tolerance limits in terms of 96-hr LC50 and lethal concentration for each fish species will be determined for selected metals and their mixtures. Each fish species will be exposed to 1/3rd, 1/4th, 1/5th and 1/6th of LC50 for 180 days and during this period peripheral erythrocyte DNA damage will be assessed by using Comet assay. The concentration and time dependent metals bio-accumulation patterns in the body organs (liver, kidney, heart, gill and muscles) of each fish species will also be studied during growth trail of 180 days. All the experiments with fish will be conducted at constant hardness (225mgL-1), pH (7.50) and temperature (30Â°C) of water. The physico-chemical variables of water viz. temperature, dissolved oxygen, pH, sodium, potassium, total hardness, carbon dioxide, calcium, magnesium and electrical conductivity will be analyzed, daily, to establish their relationship with the growth performance of fish. Probit analyses method with 95% confidence interval, will be employed to estimate the acute toxicity (96-hr LC50 and lethal concentrations) for each treatment. RCBD statistical design with three replications for each treatment and species will be employed to see statistical differences among different variables defined for this study will be analyzed by using Tukey's / Student Newman-Keul tests. The relationships among defined variables will be established by using Correlation and Regression analyses methods.
UNIVERSITY OF AGRICULTURE, FAISALABAD DEPARTEMENT OF ZOOLOGY AND FISHERIES
Synopsis for PhD Degree in Zoology
TITLE: DNA damage caused by waterborne metals and their effects on growth and bio-accumulation in fish
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Date of Admission : 05-10-2010
Probable Duration : 12 Months
Date of Initiation : 01-05-2012
Student Ummara Batool
Supervisor Prof. Dr. Muhammad Javed
Prof. Dr. Muhammad Javed (Chairman)
Dr. Sajid Abdullah (Member)
Prof. Dr. Munir A. Sheikh (Member)
NEED FOR THE PROJECT
The continuous release of metals and their compounds into the natural aquatic habitats of Pakistan due to both natural and anthropogenic activities, has accelerated the deterioration of natural ecosystems due to their bio-magnification in the food chain (Jabeen, 2012). Over the years, humans have been polluting the aquatic environment without knowing the adverse effects of various pollutants, including heavy metals, in the global ecosystem (Idzelis et al., 2010). The trace metals are essential for normal physiological processes (Wepener et al., 2011) but abnormally high concentrations would become toxic to the aquatic organisms, including fish (Javed, 2012). The metals above the permissible limits may affect the bioenergetics of fish (Abdullah et al., 2007) leading to abnormal growth and mortality while sub-lethal concentrations may lead to behavioral, biochemical and histological changes in fish (Wang, 2002; Amin et al., 2003; Javed, 2004; Javed 2012). Furthermore, metallic ion pollution, through food chain, can cause severe negative impacts on human health (Palaniappan et al., 2009). Therefore, monitoring of metals in the organs of commercial fish species is important to assess the possible risks associated with the consumption of this contaminated fish (Watanabe et al., 2003). Fish are at the top of aquatic food chain may accumulate large amounts of heavy metals from water, sediments and plankton (Rauf and Javed, 2007). Heavy metals are taken up through different organs of the fish because of the affinity between them. In this process, many of the metals get concentrated at different levels in the fish organs (Rao and Padmaja, 2000; Bervoets et al., 2001). Fish is an excellent source of quality proteins but due to their tendency to accumulate heavy metals from water, it would become a source of contaminated food for the human (Carvalho et al., 2005).
Polluted waters usually contain elevated levels of various metals. The accumulation of certain metals can affect the uptake of other metals by the fish. These interactions among various metals are the reason of competitive metals uptake from the environment and their differential distribution in various tissues of fish. Therefore, the effects of various metal mixtures on the fish may also differ. The effects may be synergistic, antagonistic or additive (Jezierska and Witeska, 2001; Palaniappan and Karthikeyan, 2009; Kwong and Niyogi, 2009). The toxic effects of various metals, in a mixture form is mostly additive because metals in a mixture can affect the fish even though the individual concentrations of the metals are below their ecotoxicological benchmark levels and thus should be taken into account in ecological risk assessments (Voie and Mariussen, 2010). The efficiency of uptake of metals, by the fish, from the contaminated water and food differ in relation to contamination gradients of food, water and sediment; ecological needs and metabolism of heavy metal and some other factors like temperature, salinity and pH (Balu et al., 2001; Kishe and Machiwa, 2003; Canli and Atli, 2003). Moreover, the tendency of various tissues to accumulate heavy metals also differs, depending on their biochemical characteristics (Calta and Canpolt, 2006). The heavy metals may also induce cellular and histological alterations and thus leading to genetic modifications (Tkatcheva et al., 2000). Metals can potentially disrupt the endocrine system of the animals also (Farkas et al., 2002). The uptake and bioconcentration of metals determine the extent of the physiological disturbances that are related with the physical and chemical composition of surrounding medium (Wepener et al., 2001). The toxic effects of heavy metals, in fish, may influence individual growth rates, reproduction and mortality (Hayat et al., 2007). Fish show highly variable or decreased growth rates in polluted waters. The effects of various metals on the growth of fish are related to their stress response (Voie and Mariussen, 2010). Growth along with bio-accumulation of metals in various fish organs is considered quantitative indicators of fish habitat quality (Gilliers et al., 2006).
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Cobalt has essentially important biochemical functions in living organisms at the levels, which allow the enzymes systems to function without interference. Cobalt has been recognized as essential constituent in the diet of fish, which is a component of vitamin B12 associated with nitrogen assimilation, erythrocyte maturation and the production of hemoglobin (Yousafzai and Shakoori, 2008). However, elevated levels of Co in the aquatic environments may become toxic to the fish (Mukherjee and Kaviraj, 2009). Production of active oxygen species (Co(II) ions and Co metal) and inhibition of DNA repair (Co(II) ions) appear to be the predominant modes of action in the genotoxic activity of cobalt (Lison et al., 2001) also able to induce single strand breaks and DNA protein cross-links (Baldwin et al., 2004). Lead is one of the persistent and cumulative pollutants of the aquatic environment and is harmful to the living organisms even at low concentration (Burden et al., 1998). It has numerous commercial applications due to its physical properties and relative chemical inertness. Exposure of fish to lead affects adversely the body weight gain, release of digestive enzymes and accumulation of free radicals due to alteration of the oxidative processes of cells and effects on repair mechanisms in which lead has been implicated as a co-carcinogen (Fracasso et al., 2002). Chromium is known to cause mutagenic, genotoxic and carcinogenic effects in living organisms (Mount and Hockett, 2000; Matsumoto et al., 2006; Yilmaz et al., 2010). It is an essential trace metal of human and some animals but it causes lethal and sub-lethal effect in most animals including fish (Akan et al., 2009). It is also reported to induce oxidative DNA damage, sister chromatid exchange and also cytotoxicity (Figgitt et al., 2010; Rudolf et al., 2009)
Various toxicants in polluted water are capable of interacting with DNA, hence causes genotoxic effects like DNA strand breaks, base modifications and cross-linkages. The loss of DNA integrity induces chromosomal aberrations, mutations and developmental defects in vertebrates. Therefore, it is important to assess the effect of genotoxic compounds for environmental monitoring and fish health (Frezilli et al., 2004). Several authors used the nuclear abnormalities such as lobed, blebbed and notched nuclei as possible indicator of genotoxicity in aquatic organisms (Aylan and Garcia-Vazquaz, 2000; Cavas and Ergene-Gozukara, 2003). DNA is actually the target site for most of carcinogenic and mutagenic compounds so damage in aquatic organisms is linked with tumor, retardation, developmental defects and decrease in viability of embryos and larvae (Galindo et al., 2002; Yang et al., 2006). Metal genotoxicity is generally linked to the formation of reactive oxygen species (Soto-Reyes et al., 2005). Oxidative stress on fish increases when rate of reactive oxygen species production exceeds the rate of its decomposition by antioxidant defense and repair system leading to the oxidation of key cell components like proteins, fatty acids and DNA. Oxidation of DNA with reactive oxygen species can produce strand breaks which represent a major category of oxidative damage to DNA (Cabiscol et al., 2000).
The single cell gel electrophoresis or comet assay is a genotoxicity test which detects the DNA damage, caused by alkylating, oxidizing and interacting agents (Tice et al., 2000). This assay is very sensitive, rapid and reliable for detecting double strands and single strand breaks in DNA. Blood is important parameter to detect the DNA damage in fish, exposed to different contaminants by using comet assay because peripheral erythrocytes reflects the overall health of fish. Carps are the most successful species of composite culture in Pakistan. They have assumed popularity among public and private sector farmers. The density of major carps in the natural waters has alarmingly been declined due to discharges of untreated industrial and sewage waters into the rivers. Ultimately, the fish production of Pakistani inland waters has reduced due to population depletion and decline in growth potential of these cyprinids (Javed, 2004). A number of studies concerning single metal exposure have been done on various fish species (Abdullah et al., 2007; Hayat et al., 2007; Naz et al., 2008; Parveen and Javed, 2010; Javed and Saeed, 2010). However, in the natural waters metals exist in the form of various combinations and no work has been done on growth responses and genotoxicity of fish, exposed to mixtures of different metals. Therefore, the present project is planned to achieve following objectives:
Determination of acute toxicity of selected metals (Co, Pb, and Cr) and all their mixtures to the fish, Catla catla, Cirrhina mrigala and Labeo rohita.
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Determination of concentration and time dependent bio-accumulation patterns of metals and their mixtures in the organs (liver, kidney, heart, gills and muscles) of selected fish species during acute (LC50 and Lethal concentrations) and chronic exposures.
Assessment of DNA damage in peripheral erythrocytes of fish during chronic metal stressed period by using Comet assay.
REVIEW OF LITERATURE
The toxic effects of heavy metal on living organism, in aquatic ecosystem varies depending upon their concentrations and length of exposure period (Calta and Canpolt, 2006; Cao et al., 2010). 96-hr LC50 and lethal concentration values of aluminium for different age groups (60, 90 and 120-day) of Labeo rohita, Catla catla and Cirrhina mrigala were determined by Azmat et al. (2011). Metal accumulation in the organs of three fish species was also determined. Results showed that 60-day fish was most sensitive to aluminium during this acute exposure while 120- day fish was least sensitive. Analysis of metal accumulation showed that concentration of aluminium varies between the species as well as organs, where kidney and liver showed significantly higher tendency of metal accumulation. Abdullah et al. (2011) studied heavy metals accumulation patterns in Labeo rohita, Cirrhina mrigala and Catla catla during acute (96-hr LC50 and lethal concentrations) exposure of zinc, lead, manganese and nickel. All fish species showed significantly higher tendency of manganese storage in their organs followed by zinc, nickel and lead, respectively. Among fish species, Cirrhina mrigala exhibited higher tendency of metals accumulation than that of Labeo rohita and Catla catla.
Heavy metals are frequently present as mixtures of essential and non-essential elements in natural water bodies and therefore evaluation of their toxic effects individually does not offer a realistic estimate of their impacts on biological processes (Palaniappan and Karthikeyan, 2009; Firat and Kargin, 2009). Pandey et al. (2008) studied the effects of a mixture of four heavy metals viz. Cd, Cu, Fe and Ni on the gills of freshwater fish, Channa punctata using environmentally relevant concentrations. The results indicated that low concentrations of metals could lead to functional alterations and interference with fundamental processes. The effect of heavy metal mixture of Cd, Cr, Cu, Pb, Ni, Mn and Zn was investigated at all stages of the development (embryos, larvae and adults) of Oncorhynchus mykiss by Vosyliene et al. (2003). The concentration of mixture used was based on average annual concentrations of these metals in waste water discharged from Ignalina Nuclear Plant into Lithuanian lake. The growth parameters of the fish were found to be most sensitive to low concentrations of heavy metal mixtures. The toxicity of heavy metals in a mixture was found to be additive.
Effect of copper and cadmium on DNA damage, in terms of binuclei and micronuclei, in peripheral blood erythrocytes, liver and gill epithelial cells of Carassius gibelio, Cyprinus carpio and Corydoras paleatus were investigated by Cavas et al., 2005. The results demonstrated that tissues of each fish species showed genotoxicity and differential sensitivity towards the heavy metal exposure. DNA damage induction by cadmium chloride in climbing perch Anabas testudineus was investigated by using comet assay (Ahmad et al., 2010). Genotoxicity was measured in different tissues viz. liver, kidney and gill by calculating percentage of DNA in comet head and tail. Liver showed higher DNA damage, followed by kidney and gill tissues.
Acute and sub-chronic genotoxic impacts on Prochilodus lineatus, exposed to aluminum for 15 days, were evaluated by Bruno et al., 2010. Results showed that fish peripheral erythrocytes showed higher DNA damage after 6 and 96-hr aluminum exposure. Zhang et al. (2008) investigated genotoxic effect of heavy metals Cd, Pb, Zn and all their possible mixtures, on Misgurnus anguillicaudatus, by using comet assay. Their results showed significant time and dose dependent relationship between the heavy metal exposure and DNA damage. Among metals Zn showed highest percentage of DNA damage followed by Cd and Pb, while among all treatments Cd+Pb+Zn mixture caused severest damage of DNA.
Farkas et al. (2002) investigated the relationship between growth and metal concentration in the organs of Abramis brama L. (common bream) populating Lake Balaton. The bioaccumulation of Cd, Hg, Cu, Pb and Zn were determined in liver, gills and muscles of the fish. The gills showed higher concentrations of Cd, Zn, Cu and Pb whereas higher concentrations of Hg were measured in muscles. They found a positively significant correlation between the heavy metal load and instantaneous growth rate of the fish. The distributions of heavy metals viz. Pb, As, Cr, Cd, Cu, Hg and Zn were analyzed in liver, kidney, muscles, gills, spleen, testes and ovaries of adult common carp (Cyprinus carpio), grown in ponds. Results demonstrated differential affinities of metals for different organs. The study revealed that gonads and meat of pond carp were safe from contamination with the metals investigated in the Czech Republic (Celechovska et al., 2007). The time-integrated uptake and distribution of Cu, Zn and Fe mixture in the organs of freshwater teleost, Tilapia sparrmanii was studied by Wepener et al. (2001). The concentration of metal mixture used in bioassay was relevant to that found in Olifant river of South Africa. After metal mixture exposure, the organs viz. liver, plasma and gills were sampled at different time-intervals (from zero hour to four weeks). The results showed that gills were the initial site of accumulation and particular characteristics of metals in mixture form affect the interaction between metals and gill surface. Copper accumulation in the gill was greater than Fe and Zn, while the Zn accumulation was limited to liver and plasma.
The accumulation patterns of different heavy metals differ significantly among fish species. Yousafzai et al. (2010) compared accumulation patterns of heavy metal viz. Cu, Cd, Cr, Ni, Pb and Zn in the organs viz. liver, gills, skin, intestine and muscles of two species of fresh water fish viz. Labeo dyocheilus and Wallago attu in their natural ecosystem. Results showed significantly higher bioaccumulation of Zn and least was that of Cd in both species. Relative abundance of heavy metals in different organs showed a significantly higher burden in skin of Wallago attu, while liver accumulated higher metals concentration in Labeo dyocheilus. Idzelis et al. (2010) investigated the accumulation of heavy metal mixtures (Zn+Ni+Cu+Cr+Cd+Pb) in the tissues of Noemacheilus barbatulus (stone loach) and Oncorhynchus mykiss (rainbow trout). Fish were exposed to Maximum Permitted Concentrations (MPC) and accumulation of all metals was determined by using atomic absorption spectrophotometer. Both investigated species accumulated heavy metals with similar general intensity. The results showed highest concentrations of Pb and Cd in the organs of fish. The results urged for the constant control of heavy metal amounts in the tissues of fish. The accumulation capacities of different organs also differ within same and different species. The concentrations of heavy metal in edible portions and some other organs (gill, liver and intestine) of selected freshwater fish species were found to differ significantly in both tissues and species and frequently exceed MPC (Uysal 2011).
Hypothesis: Three species of fish viz. Catla catla, Cirrhina mrigala, and Labeo rohita will respond differently in terms of their sensitivity and tolerance limits to the selected metals that would cause significant impacts on growth performance, bioaccumulation patterns and DNA integrity.
PLAN OF WORK
The proposed research work will be conducted at the Fisheries Research Farms, Department of Zoology and Fisheries, University of Agriculture, Faisalabad. The fingerlings of three fish species viz. Catla catla, Cirrhina mrigala and Labeo rohita will be brought to the laboratory and acclimated in cemented tanks for 10 days. After acclimation, the stocks of each fish species will be divided into 8 groups for stress experiments (seven treatments and one for control). The toxicity of individual water-borne Co, Pb, Cr and their following treatment combinations will be tested against control for their toxic and genotoxic impacts on fish growth and bioaccumulation patterns.
Co - Pb
Co - Cr
Pb - Cr
Co - Pb - Cr
PHASE-I: Acute Toxicity Tests
Laboratory tests will be conducted in glass aquaria at constant water temperature (300C), pH (7.25) and total hardness (225mgL-1). 10 individuals (180-day age) of each fish species will be placed, separately, in glass aquaria for stress experiments. Acute toxicity test for each treatment will be performed in terms of 96-hr LC50 and lethal concentrations. Chemically pure chloride compounds of aluminum, lead and chromium will be dissolved in deionized water and stock solutions will be prepared for required metals and their mixtures concentrations. The concentrations of metal mixtures in each aquarium will be increased gradually and total test concentrations be maintained within seven hours. For each species, there will be a control without any metal stress. Constant air will be supplied to all the test media with an air pump fixed with a capillary system. The test media will be checked on daily basis for the maintenance of desired metal mixture concentrations in each aquarium. During the whole stress period, fish mortality and physico-chemical variables of water viz. carbon-dioxide, dissolved oxygen, pH, potassium, sodium, temperature, total hardness and total ammonia will be checked at 12-hr intervals by following the methods described by A.P.H.A. (1998).
PHASE-II: Assessment of DNA Damage and Metal Accumulation Patterns During Chronic Exposure
50 individuals of each fish species will be exposed to 1/3rd, 1/4th, 1/5th and 1/6th of 96-hr LC50 values for each treatment to investigate the growth performance, bioaccumulation patterns and DNA damage during 180 days exposure period. The fish will be fed, to satiation, twice a day with the feed having 32% digestible protein and 3.00 Kcalg-1 of energy. The growth performance of each fish species will also be monitored on weekly basis in terms of wet weights (g), fork and total lengths (mm), condition factors (K), feed intake (g) and feed conversion efficiency. Three fishes of each species will be dissected and their body organs viz. liver, kidney, heart, gills and muscles will be analyzed for metal concentration (ÂµgL-1) through Atomic absorption Spectrophotometer (Analyst 400) after every 30 days. Fish peripheral erythrocytes will be used to assess DNA damage caused by metallic toxicants viz. Co, Pb, Cr and all their mixtures during chronic exposure period after 30, 60, 90, 120, 150 and 180 days by using Comet assay test (Singh et al., 1988)
COMET ASSAY MATHODOLOGY
Fish blood sample will be taken from caudal vein. Heparin sodium salt will be used to stabilize the fish blood. Blood sample will be diluted with 1 ml of PBS. 60 Âµl of sample will be mixed with 200 Âµl of 0.65% low melting point (LMP) agarose. 70 Âµl of this mixture will be then layered on the slides precoated with 0.5% normal melting point (NMP) agarose and immediately covered with a cover slip and then kept for 10 minutes in a refrigerator to solidify. After gently removing the cover slips, the slides will be coated with a third layer of 90 Âµl low-melting point agarose and covered with slip again.
After solidification of the gel, cover slips will be removed and the slides immersed in cold lysing solution (2.5 M NaCl, 100 mM Na2-EDTA, 10 mM Tris, pH 10 with 10% DMSO and 1% Triton X-100 added fresh) and refrigerated at 4 0C for 2 hours. The slides will be then placed on a horizontal electrophoresis box side by side. The tank will be filled with fresh electrophoresis solution (1 mM Na EDTA, 300 mM NaOH and pH 13.5) to a level approximately 0.25 cm above the slides. The slides will be left in the solution for 20 minutes to allow the unwinding of DNA strands. Electrophoresis will be performed using the same solution at 25 V, 300 mA for 25 minutes. The slides will be neutralized gently with 0.4 M Tris buffer at pH 7.5 and DNA stained with 75Âµl ethidium bromide (20Âµg/ml). Two hundred cells (100/replicate) will be scored at 400x magnification. Cells with no DNA damage will have intact nucleus without a tail, whereas the cells with DNA damage will show comet like appearance. The length of DNA migration in the comet tail is an estimate of DNA damage. The cells with no head or dispersed head will be regarded as apoptotic cells and will not be included in the analysis.
Analysis of slides: The DNA damage will be quantified by visual classification of cells into five categories "comets" corresponding to the tail length, Undamaged: Type 0; Low level damage: Type I; Medium level damage: Type II; High level damage: Type III; Complete damage: Type IV. The extent of DNA damage will be exposed as the mean percentage of cells with medium, high and complete damaged DNA, which will be calculated as the sum of cells with damage Types II, III and IV. From the arbitrary values assigned to the different categories (from Type=0 to Type IV=4) a genetic damage index (GDI) will be calculated for each subject. DNA damage will be quantified for each cell, by using the following formula:
Comet tail length = Maximum total length - Head diameter
Probit analyses method (Hamilton et al., 1977) with 95% confidence interval will be used to estimate the acute toxicity test for each treatment combinations. The statistical differences among different treatments and parameters of growth, metals accumulation patterns in fish body organs and 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 defined for this study.