Dna Damage Caused By Waterborne Metals Biology Essay

Among the aquatic pollutants, heavy metals are of particular concern due to their diversified and toxic ill-effects to the fish. In an aquatic ecosystem, fish are exposed to a assortment of metals, in mixture signifier, instead than a individual metal. Therefore, the present work will concentrate on the finding 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 harm, growing and their bio-accumulation in fish variety meats ( liver, kidney, bosom, gill and musculuss ) . The tolerance bounds in footings of 96-hr LC50 and deadly 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 yearss and during this period peripheral red blood cell DNA harm will be assessed by utilizing Comet check. The concentration and clip dependent metals bio-accumulation forms in the organic structure variety meats ( liver, kidney, bosom, gill and musculuss ) of each fish species will besides be studied during growing trail of 180 yearss. All the experiments with fish will be conducted at changeless hardness ( 225mgL-1 ) , pH ( 7.50 ) and temperature ( 30A°C ) of H2O.

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The physico-chemical variables of H2O viz. temperature, dissolved O, pH, Na, K, entire hardness, C dioxide, Ca, Mg and electrical conduction will be analyzed, daily, to set up their relationship with the growing public presentation of fish. Probit analyses method with 95 % assurance interval, will be employed to gauge the acute toxicity ( 96-hr LC50 and deadly concentrations ) for each intervention.

RCBD statistical design with three reproductions for each intervention and species will be employed to see statistical differences among different variables defined for this survey will be analyzed by utilizing Tukey ‘s / Student Newman-Keul trials. The relationships among defined variables will be established by utilizing Correlation and Regression analyses methods.


Outline for PhD Degree in Zoology

Title: Deoxyribonucleic acid harm caused by waterborne metals and their effects on growing and bio-accumulation in fish

Date of Admission: 05-10-2010Probable Duration: 12 Calendar monthsDate of Initiation: 01-05-2012


Student Ummara Batool( 2006-ag-287 )Supervisor Prof. Dr. Muhammad Javed

Supervisory Committee:

Prof. Dr.

Muhammad Javed ( Chairman )Dr. Sajid Abdullah ( Member )Prof. Dr. Munir A. Sheikh ( Member )


The uninterrupted release of metals and their compounds into the natural aquatic home grounds of Pakistan due to both natural and anthropogenetic activities, has accelerated the impairment of natural ecosystems due to their bio-magnification in the nutrient concatenation ( Jabeen, 2012 ) . Over the old ages, worlds have been fouling the aquatic environment without cognizing the inauspicious effects of assorted pollutants, including heavy metals, in the planetary ecosystem ( Idzelis et al. , 2010 ) .

The hint metals are indispensable for normal physiological procedures ( Wepener et al. , 2011 ) but abnormally high concentrations would go toxic to the aquatic beings, including fish ( Javed, 2012 ) . The metals above the allowable bounds may impact the bioenergetics of fish ( Abdullah et al. , 2007 ) taking to unnatural growing and mortality while sub-lethal concentrations may take to behavioural, biochemical and histological alterations in fish ( Wang, 2002 ; Amin et al. , 2003 ; Javed, 2004 ; Javed 2012 ) . Furthermore, metallic ion pollution, through nutrient concatenation, can do terrible negative impacts on human wellness ( Palaniappan et al. , 2009 ) .

Therefore, monitoring of metals in the variety meats of commercial fish species is of import to measure the possible hazards associated with the ingestion of this contaminated fish ( Watanabe et al. , 2003 ) . Fish are at the top of aquatic nutrient concatenation may roll up big sums of heavy metals from H2O, deposits and plankton ( Rauf and Javed, 2007 ) . Heavy metals are taken up through different variety meats of the fish because of the affinity between them. In this procedure, many of the metals get concentrated at different degrees in the fish variety meats ( Rao and Padmaja, 2000 ; Bervoets et al. , 2001 ) . Fish is an first-class beginning of quality proteins but due to their inclination to roll up heavy metals from H2O, it would go a beginning of contaminated nutrient for the human ( Carvalho et al. , 2005 ) .

Polluted Waterss normally contain elevated degrees of assorted metals. The accretion of certain metals can impact the consumption of other metals by the fish. These interactions among assorted metals are the ground of competitory metals uptake from the environment and their differential distribution in assorted tissues of fish. Therefore, the effects of assorted metal mixtures on the fish may besides differ. The effects may be interactive, counter or linear ( Jezierska and Witeska, 2001 ; Palaniappan and Karthikeyan, 2009 ; Kwong and Niyogi, 2009 ) . The toxic effects of assorted metals, in a mixture signifier is largely linear because metals in a mixture can impact the fish even though the single concentrations of the metals are below their ecotoxicological benchmark degrees and therefore should be taken into history in ecological hazard appraisals ( Voie and Mariussen, 2010 ) . The efficiency of consumption of metals, by the fish, from the contaminated H2O and nutrient differ in relation to taint gradients of nutrient, H2O and deposit ; ecological demands and metamorphosis of heavy metal and some other factors like temperature, salt and pH ( Balu et al. , 2001 ; Kishe and Machiwa, 2003 ; Canli and Atli, 2003 ) .

Furthermore, the inclination of assorted tissues to roll up heavy metals besides differs, depending on their biochemical features ( Calta and Canpolt, 2006 ) . The heavy metals may besides bring on cellular and histological changes and therefore taking to familial alterations ( Tkatcheva et al. , 2000 ) . Metallic elements can potentially interrupt the hormone system of the animate beings besides ( Farkas et al. , 2002 ) .

The consumption and bioconcentration of metals determine the extent of the physiological perturbations that are related with the physical and chemical composing of environing medium ( Wepener et al. , 2001 ) . The toxic effects of heavy metals, in fish, may act upon single growing rates, reproduction and mortality ( Hayat et al. , 2007 ) . Fish show extremely variable or reduced growing rates in contaminated Waterss. The effects of assorted metals on the growing of fish are related to their emphasis response ( Voie and Mariussen, 2010 ) . Growth along with bio-accumulation of metals in assorted fish variety meats is considered quantitative indexs of fish home ground quality ( Gilliers et al. , 2006 ) .

Cobalt has basically of import biochemical maps in life beings at the degrees, which allow the enzymes systems to work without intervention. Cobalt has been recognized as indispensable component in the diet of fish, which is a constituent of vitamin B12 associated with nitrogen assimilation, erythrocyte ripening and the production of haemoglobin ( Yousafzai and Shakoori, 2008 ) . However, elevated degrees of Co in the aquatic environments may go toxic to the fish ( Mukherjee and Kaviraj, 2009 ) . Production of active O species ( Co ( II ) ions and Co metal ) and suppression of DNA fix ( Co ( II ) ions ) appear to be the prevailing manners of action in the genotoxic activity of Co ( Lison et al. , 2001 ) besides able to bring on individual strand interruptions 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 life organisms even at low concentration ( Burden et al. , 1998 ) . It has legion commercial applications due to its physical belongingss and comparative chemical inertness. Exposure of fish to take affects adversely the organic structure weight addition, release of digestive enzymes and accretion of free groups due to change of the oxidative procedures of cells and effects on fix mechanisms in which lead has been implicated as a co-carcinogen ( Fracasso et al. , 2002 ) . Chromium is known to do mutagenic, genotoxic and carcinogenic effects in life beings ( Mount and Hockett, 2000 ; Matsumoto et al.

, 2006 ; Yilmaz et al. , 2010 ) . It is an indispensable hint metal of homo and some animate beings but it causes deadly and sub-lethal consequence in most animate beings including fish ( Akan et al. , 2009 ) . It is besides reported to bring on oxidative DNA harm, sister chromatid exchange and besides cytotoxicity ( Figgitt et al. , 2010 ; Rudolf et al. , 2009 )Assorted poisons in contaminated H2O are capable of interacting with DNA, hence causes genotoxic effects like DNA strand interruptions, base alterations and cross-linkages.

The loss of DNA unity induces chromosomal aberrances, mutants and developmental defects in craniates. Therefore, it is of import to measure the consequence of genotoxic compounds for environmental monitoring and fish wellness ( Frezilli et al. , 2004 ) . Several writers used the atomic abnormalcies such as lobed, blebbed and notched nuclei as possible index of genotoxicity in aquatic beings ( Aylan and Garcia-Vazquaz, 2000 ; Cavas and Ergene-Gozukara, 2003 ) .

Deoxyribonucleic acid is really the mark site for most of carcinogenic and mutagenic compounds so damage in aquatic beings is linked with tumour, deceleration, developmental defects and lessening in viability of embryos and larvae ( Galindo et al. , 2002 ; Yang et al. , 2006 ) . Metal genotoxicity is by and large linked to the formation of reactive O species ( Soto-Reyes et al. , 2005 ) .

Oxidative emphasis on fish additions when rate of reactive O species production exceeds the rate of its decomposition by antioxidant defence and fix system taking to the oxidization of cardinal cell constituents like proteins, fatty acids and DNA. Oxidation of Deoxyribonucleic acid with reactive O species can bring forth strand interruptions which represent a major class of oxidative harm to DNA ( Cabiscol et al. , 2000 ) .The individual cell gel cataphoresis or comet check is a genotoxicity trial which detects the DNA harm, caused by alkylating, oxidising and interacting agents ( Tice et al. , 2000 ) . This check is really sensitive, rapid and dependable for observing dual strands and individual strand interruptions in DNA. Blood is of import parametric quantity to observe the DNA harm in fish, exposed to different contaminations by utilizing comet assay because peripheral red blood cells reflects the overall wellness of fish.

Carps are the most successful species of composite civilization in Pakistan. They have assumed popularity among public and private sector husbandmans. The denseness of major carps in the natural Waterss has alarmingly been declined due to discharges of untreated industrial and sewage Waterss into the rivers. Ultimately, the fish production of Pakistani inland Waterss has reduced due to population depletion and diminution in growing potency of these cyprinids ( Javed, 2004 ) . A figure of surveies refering individual metal exposure have been done on assorted 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 Waterss metals exist in the signifier of assorted combinations and no work has been done on growing responses and genotoxicity of fish, exposed to mixtures of different metals. Therefore, the present undertaking is planned to accomplish following aims:


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.Determination of concentration and clip dependent bio-accumulation forms of metals and their mixtures in the variety meats ( liver, kidney, bosom, gills and musculuss ) of selected fish species during ague ( LC50 and Lethal concentrations ) and chronic exposures.Appraisal of DNA harm in peripheral red blood cells of fish during chronic metal stressed period by utilizing Comet check.


The toxic effects of heavy metal on life being, 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 deadly concentration values of aluminum for different age groups ( 60, 90 and 120-day ) of Labeo rohita, Catla catla and Cirrhina mrigala were determined by Azmat et Al. ( 2011 ) . Metallic accretion in the variety meats of three fish species was besides determined. Consequences showed that 60-day fish was most sensitive to aluminium during this acute exposure while 120- twenty-four hours fish was least sensitive.

Analysis of metal accretion showed that concentration of aluminum varies between the species every bit good as variety meats, where kidney and liver showed significantly higher inclination of metal accretion. Abdullah et Al. ( 2011 ) studied heavy metals accretion forms in Labeo rohita, Cirrhina mrigala and Catla catla during ague ( 96-hr LC50 and deadly concentrations ) exposure of Zn, lead, manganese and nickel. All fish species showed significantly higher inclination of manganese storage in their variety meats followed by Zn, Ni and lead, severally.

Among fish species, Cirrhina mrigala exhibited higher inclination of metals accretion than that of Labeo rohita and Catla catla.Heavy metals are often present as mixtures of indispensable and non-essential elements in natural H2O organic structures and therefore rating of their toxic effects separately does non offer a realistic estimation of their impacts on biological procedures ( 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 fresh water fish, Channa punctata utilizing environmentally relevant concentrations. The consequences indicated that low concentrations of metals could take to functional changes and intervention with cardinal procedures. The consequence of heavy metal mixture of Cd, Cr, Cu, Pb, Ni, Mn and Zn was investigated at all phases of the development ( embryos, larvae and grownups ) of Oncorhynchus mykiss by Vosyliene et Al.

( 2003 ) . The concentration of mixture used was based on mean one-year concentrations of these metals in waste H2O discharged from Ignalina Nuclear Plant into Lithuanian lake. The growing parametric quantities 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 linear.Consequence of Cu and Cd on DNA harm, in footings of binuclei and micronuclei, in peripheral blood red blood cells, liver and gill epithelial cells of Carassius gibelio, Cyprinus carpio and Corydoras paleatus were investigated by Cavas et al. , 2005. The consequences demonstrated that tissues of each fish species showed genotoxicity and differential sensitiveness towards the heavy metal exposure. DNA harm initiation by Cd chloride in mounting perch Anabas testudineus was investigated by utilizing comet assay ( Ahmad et al.

, 2010 ) . Genotoxicity was measured in different tissues viz. liver, kidney and gill by ciphering per centum of Deoxyribonucleic acid in comet caput and tail. Liver showed higher DNA harm, followed by kidney and gill tissues.Acute and sub-chronic genotoxic impacts on Prochilodus lineatus, exposed to aluminum for 15 yearss, were evaluated by Bruno et al. , 2010.

Result showed that fish peripheral red blood cells showed higher DNA harm after 6 and 96-hr aluminium exposure. Zhang et Al. ( 2008 ) investigated genotoxic consequence of heavy metals Cd, Pb, Zn and all their possible mixtures, on Misgurnus anguillicaudatus, by utilizing comet check. Their consequences showed important clip and dose dependent relationship between the heavy metal exposure and DNA harm. Among metals Zn showed highest per centum of DNA harm followed by Cd and Pb, while among all interventions Cd+Pb+Zn mixture caused severest harm of Deoxyribonucleic acid.

Farkas et Al. ( 2002 ) investigated the relationship between growing and metal concentration in the variety meats of Abramis Brama L. ( common bream ) dwelling Lake Balaton. The bioaccumulation of Cd, Hg, Cu, Pb and Zn were determined in liver, gills and musculuss of the fish. The gills showed higher concentrations of Cd, Zn, Cu and Pb whereas higher concentrations of Hg were measured in musculuss. They found a positively important correlativity between the heavy metal burden and instantaneous growing rate of the fish. The distributions of heavy metals viz. Pb, As, Cr, Cd, Cu, Hg and Zn were analyzed in liver, kidney, musculuss, gills, lien, testicles and ovaries of grownup common carp ( Cyprinus carpio ) , grown in pools.

Consequences demonstrated differential affinities of metals for different variety meats. The survey revealed that sex glands and meat of pool carp were safe from taint with the metals investigated in the Czech Republic ( Celechovska et al. , 2007 ) . The time-integrated consumption and distribution of Cu, Zn and Fe mixture in the variety meats of fresh water teleost, Tilapia sparrmanii was studied by Wepener et Al. ( 2001 ) . The concentration of metal mixture used in bio-assay was relevant to that found in Olifant river of South Africa. After metal mixture exposure, the variety meats viz.

liver, plasma and gills were sampled at different time-intervals ( from zero hr to four hebdomads ) . The consequences showed that gills were the initial site of accretion and peculiar features of metals in mixture signifier affect the interaction between metals and gill surface. Copper accretion in the gill was greater than Fe and Zn, while the Zn accretion was limited to liver and plasma.The accretion forms of different heavy metals differ significantly among fish species. Yousafzai et Al.

( 2010 ) compared accretion forms of heavy metal viz. Cu, Cd, Cr, Ni, Pb and Zn in the variety meats viz. liver, gills, tegument, bowel and musculuss of two species of fresh H2O fish viz.

Labeo dyocheilus and Wallago attu in their natural ecosystem. Consequences showed significantly higher bioaccumulation of Zn and least was that of Cd in both species. Relative copiousness of heavy metals in different variety meats showed a significantly higher load in tegument of Wallago attu, while liver accumulated higher metals concentration in Labeo dyocheilus.

Idzelis et Al. ( 2010 ) investigated the accretion 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 accretion of all metals was determined by utilizing atomic soaking up spectrophotometer.

Both investigated species accumulated heavy metals with similar general strength. The consequences showed highest concentrations of Pb and Cd in the variety meats of fish. The consequences urged for the changeless control of heavy metal sums in the tissues of fish.

The accretion capacities of different variety meats besides differ within same and different species. The concentrations of heavy metal in comestible parts and some other variety meats ( gill, liver and bowel ) of selected fresh water fish species were found to differ significantly in both tissues and species and often exceed MPC ( Uysal 2011 ) .Hypothesis: Three species of fish viz. Catla catla, Cirrhina mrigala, and Labeo rohita will react otherwise in footings of their sensitiveness and tolerance bounds to the selected metals that would do important impacts on growing public presentation, bioaccumulation forms and DNA unity.


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 research lab and acclimated in cemented armored combat vehicles for 10 yearss. After acclimatization, the stocks of each fish species will be divided into 8 groups for emphasis experiments ( seven interventions and one for control ) .

The toxicity of single water-borne Co, Pb, Cr and their undermentioned intervention combinations will be tested against control for their toxic and genotoxic impacts on fish growing and bioaccumulation forms.

Treatment combinations

Co – LeadCo – ChromiumPb – ChromiumCo – Pb – Chromium

PHASE-I: Acute Toxicity Trials

Lab trials will be conducted in glass fish tank at changeless H2O temperature ( 300C ) , pH ( 7.25 ) and entire hardness ( 225mgL-1 ) . 10 persons ( 180-day age ) of each fish species will be placed, individually, in glass fish tank for emphasis experiments. Acute toxicity trial for each intervention will be performed in footings of 96-hr LC50 and deadly concentrations.

Chemically pure chloride compounds of aluminium, lead and Cr will be dissolved in deionized H2O and stock solutions will be prepared for needed metals and their mixtures concentrations. The concentrations of metal mixtures in each fish tank will be increased bit by bit and entire trial concentrations be maintained within seven hours. For each species, there will be a control without any metal emphasis. Constant air will be supplied to all the trial media with an air pump fixed with a capillary system. The trial media will be checked on day-to-day footing for the care of coveted metal mixture concentrations in each fish tank. During the whole emphasis period, fish mortality and physico-chemical variables of H2O viz. carbon-dioxide, dissolved O, pH, K, Na, temperature, entire hardness and entire ammonium hydroxide will be checked at 12-hr intervals by following the methods described by A.P.

H.A. ( 1998 ) .

PHASE-II: Appraisal of DNA Damage and Metal Accumulation Patterns During Chronic Exposure

50 persons of each fish species will be exposed to 1/3rd, 1/4th, 1/5th and 1/6th of 96-hr LC50 values for each intervention to look into the growing public presentation, bioaccumulation forms and DNA harm during 180 yearss exposure period. The fish will be fed, to repletion, twice a twenty-four hours with the provender holding 32 % digestible protein and 3.00 Kcalg-1 of energy. The growing public presentation of each fish species will besides be monitored on hebdomadal footing in footings of moisture weights ( g ) , fork and entire lengths ( millimeter ) , status factors ( K ) , feed consumption ( g ) and feed transition efficiency. Three fishes of each species will be dissected and their organic structure organs viz.

liver, kidney, bosom, gills and musculuss will be analyzed for metal concentration ( AµgL-1 ) through Atomic soaking up Spectrophotometer ( Analyst 400 ) after every 30 yearss. Fish peripheral red blood cells will be used to measure DNA harm caused by metallic poisons viz. Co, Pb, Cr and all their mixtures during chronic exposure period after 30, 60, 90, 120, 150 and 180 yearss by utilizing Comet assay trial ( Singh et al. , 1988 )


Fish blood sample will be taken from caudal vena. Heparin Na salt will be used to stabilise the fish blood. Blood sample will be diluted with 1 milliliters of PBS. 60 Aµl of sample will be assorted with 200 Aµl of 0.65 % low thaw point ( LMP ) agarose.

70 Aµl of this mixture will be so layered on the slides precoated with 0.5 % normal thaw point ( NMP ) agarose and instantly covered with a screen faux pas and so kept for 10 proceedingss in a icebox to solidify. After gently taking the screen slips, the slides will be coated with a 3rd bed of 90 Aµl low-melting point agarose and covered with faux pas once more.After hardening of the gel, screen faux pass will be removed and the slides immersed in cold lysing solution ( 2.5 M NaCl, 100 millimeter Na2-EDTA, 10 millimeter 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 so placed on a horizontal cataphoresis box side by side. The armored combat vehicle will be filled with fresh cataphoresis solution ( 1 millimeter Na EDTA, 300 millimeter NaOH and pH 13.

5 ) to a degree about 0.25 centimeters above the slides. The slides will be left in the solution for 20 proceedingss to let the unwinding of DNA strands. Electrophoresis will be performed utilizing the same solution at 25 V, 300 ma for 25 proceedingss. The slides will be neutralized gently with 0.

4 M Tris buffer at pH 7.5 and DNA stained with 75Aµl ethidium bromide ( 20Aµg/ml ) . Two 100 cells ( 100/replicate ) will be scored at 400x magnification. Cells with no DNA harm will hold integral nucleus without a tail, whereas the cells with DNA harm will demo comet like visual aspect. The length of DNA migration in the comet tail is an estimation of DNA harm.

The cells with no caput or dispersed caput will be regarded as apoptotic cells and will non be included in the analysis.Analysis of slides: The DNA harm will be quantified by ocular categorization of cells into five classs “ comets ” matching to the tail length, Undamaged: Type 0 ; Low degree harm: Type I ; Medium degree harm: Type II ; High degree harm: Type III ; Complete harm: Type IV. The extent of DNA harm will be exposed as the average per centum of cells with medium, high and complete damaged DNA, which will be calculated as the amount of cells with harm Types II, III and IV. From the arbitrary values assigned to the different classs ( from Type=0 to Type IV=4 ) a familial harm index ( GDI ) will be calculated for each topic. Deoxyribonucleic acid harm will be quantified for each cell, by utilizing the undermentioned expression:

Comet tail length = Maximum entire length – Head diameter

Statistical Analysiss

Probit analyses method ( Hamilton et al.

, 1977 ) with 95 % assurance interval will be used to gauge the acute toxicity trial for each intervention combinations. The statistical differences among different interventions and parametric quantities of growing, metals accretion forms in fish organic structure variety meats and H2O quality variables will be analyzed by utilizing Factorial Experiment ( RCBD ) and Tukey ‘s / Student Newman-Keul trials. Correlation and arrested development analyses will besides be performed to find-out relationships among assorted parametric quantities defined for this survey.

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