INTRODUCTION The ever increasing population in the world today has led to the mounting of sewage, refuse and industrial waste everywhere. This waste has no place where it could be discarded and now it has found its way into the water bodies polluting the water and thereby creating a threat to the marine flora and fauna. In the environment, organisms are usually exposed not just to a single pollutant but rather to a mixture of these chemicals.
Mumbai is one of the busiest metropolitans in the country today housing over a million people alone and the garbage generated daily needs to be dumped somewhere. Mumbai, the capital of economic development, has been under deep stress due to industrial, commercial and population growth. Infrastructural facilities are not adequate to cater to the needs of the people. The environmental status of the coastal region around Mumbai has deteriorated due to wastewater discharges (Dhage et. l 2005). Toxic discharges by illegal means now find its way into the creeks and lakes which are often flushed out into the sea. The escalating accumulation of heavy metals is a hazard to life underwater and is amplifying the mortality rate of the fishes and other aquatic fauna. This buildup of waste in the waters not only affects the aquatic life but also mankind both directly and indirectly. Fish is considered as one of the main protein sources of food for humans.
Water pollution leads to affecting fish with toxic metals resulting from different sources like accidental spillage of chemical wastes, periodic precipitation contaminated with air borne pollutants, discharge of industrial or sewerage effluents, agricultural drainage and domestic wastewater. Toxicity with heavy metals to humans as a result of fish contamination has led to many studies on heavy metals in fish (Rashed 2000). Contamination of aquatic ecosystems with heavy metals has been receiving increased worldwide attention due to their harmful effects on human health and other organisms in the environment.
Most of the studies dealing with toxic effects of metals deal with single metal species, while the aquatic ecosystems are typically exposed to mixtures of metals. (Demirak et. al 2005; Hill et. al 2005; Karadede and Unlu 1999) Hence, in order to provide data supporting the usefulness of freshwater fish as indicators of heavy metal pollution, it has been proposed in the present study to investigate the bioaccumulation of these heavy metals in selected organs of these fish (Palaniappan et. al 2008).
The presence of toxic materials in ecosystems is presently related with increased concentrations of heavy metals ions which enter the water sources with sewage waters. Heavy metals tend to accumulate in advanced organisms through bio-magnification effects in the food chain. Thus they can enter into human body, and accumulate in the human tissues to pose chronic toxicity (Qiao-qiao et. al 2007). The bioaccumulation of metals in fish is an index of the pollution status of the water and is a useful tool in studying the biological role of the metals present at elevated levels in the fish.
When fish are exposed to waters containing metals they tend to take up the dissolved metal ions and accumulate them in the various organs which would ultimately be fatal to the fish in certain cases. Accumulation of these toxic metals usually occur in the muscles, gills, liver, kidney etc. at different amounts which could be determined by analyzing the tissues separately in order to ascertain the total uptake and accumulation of these metals in the organs.
There is an urgent need to define a safe amount of chemicals to control pollution and to protect humans from slow poisoning through fish consumption. To protect the aquatic fauna especially food fishes, some criterion should be developed for the safe disposal of waste. Besides the study of the physicochemical and biological conditions of the affected area, laboratory experiments are necessary to determine the toxicity of these metals which can provide biological information to control the water quality.
Bioassays are now being recognized as the best measure to determine the toxicity limit and establishing a safe concentration of the toxicant which can be tolerated by the fish. The purpose of the acute toxicity test with fish species is to help in the assessment of the possible risk to similar species in natural environments, as an aid in the determination of possible water quality criteria for regulatory purposes, and for use in correlation with acute testing of other species for comparative purposes.
Toxicity studies also help in studying the harmful effects of these metals on other vertebrates too; on the whole it indicates to what extent these toxic elements can harm the biota and proves a threat to the environment. (Sprague 1969) Zebrafish has been a prominent model vertebrate in a variety of biological disciplines (Hallare et. al 2004; Hill et. al 2005; Johnson et. al 2007; Krone et. al 1997a; Lele et. al 1996; Wu et. al 2009). Substantial information gathered from developmental and enetic research, together with near-completion of the zebrafish genome project, has placed zebrafish in an attractive position for use as a toxicological model. There is a clear potential for zebrafish to provide valuable new insights into chemical toxicity, drug discovery, and human disease using recent advances in forward and reverse genetic techniques coupled with large-scale, high-throughput screening (Hill et. al 2005; Lele et. al 1996).
There is a level of concentration of heavy metals that could be present in the water and would not be toxic to the fish. Since the zebrafish is a well known model for toxicological research, exposing it to a variety of heavy metals at varying known concentrations in the water sample would help us determine the best possible concentration at which the fish would survive thereby being able to standardize the degree of the metal accumulation in the water that would not be lethal to the fish.
The zebrafish, being freshwater fish, are exposed to waters containing heavy metals, and as a result tend to live under stress conditions which could have an effect or even alter to an extent their normal body functions. Assessment of the early changes induced by exposure to the different stress conditions could be observed both externally as well as internally. External examinations would include behavior, reproduction, motility, colour of the gills, feeding pattern, etc. There are a group of proteins known as stress proteins that are expressed when the fish tend to live in any kind of stress conditions.
Heat shock proteins are implicated in cellular protection and it is suggested that they may play a role in normal growth and development. It is believed that heat shock proteins protect the cells from heat induced lethality during the elevation of temperature and probably restore cell functions during recovery from stress. (Misra et. al 1988) Heat-shock proteins which are usually measured in times of stress can also be determined to observe the rate of stress at which the model organism is subjected. The most abundant heat shock protein is a 70kDa protein.
A variety of heat shock proteins including Hsp47, Hsp 70, Hsp 90alpha and Hsp 90beta and the heat shock transcription factor Hsf 1 have been identified and cloned from zebrafish (Graser et. al 1996; Krone et. al 1997a,b; Rabergh et. al 2000). Other internal observations would include the activity of various enzymes like Catalase, Glutathione-S-Transferase, Superoxide Dismutase, Acteylcholinesterase etc. Bioaccumulation of heavy metals in the various organs and tissues of the fish could also be studied and reported.
Once the standardizing of the levels of the heavy metals that the zebrafish would be able to survive in is complete, then the zebrafish could then be subjected to a number of polluted water samples from around Mumbai so as to reveal the heavy metal content of these waters and its toxicity and determine the danger it poses to the aquatic ecosystem. RESEARCH HYPOTHESIS Toxicity in fish has been studied by various scientists to determine the lethal levels that these toxicants could result in and also to determine the accumulation of these metals in the tissues of the fish.
Zebrafish has been gaining popularity as a model organism in recent years for studies of vertebrate development and gene function. Research with zebrafish has allowed advances in the fields of developmental biology, oncology, toxicology, reproductive studies, genetics, neurobiology, environmental sciences, stem cell and regenerative medicine and evolutionary theory (Lele and Krone 1996). Hence as per the expected aim of the project the use of zebrafish as a model organism in the toxicology study would help determine the tolerant values of heavy metals that the freshwater fish could survive in.
The study also includes the examination of the effects of heavy metals and the heat shock protein measurement during the developmental stages of the zebrafish and the effect of these heavy metals on the eggs and developing fry. The eggs of the zebrafish are transparent and the inner developmental changes occurring within the egg can be monitored and studied on a regular basis. The measure of heat shock proteins would help to determine the stress induced as well as the stress that the fish can survive in. The activity of various enzymes like Catalase, Glutathione S Transferase, Acetylcholinesterase, etc. re usually altered in times of stress and this activity will also be studied. This overall study would help determine the water quality from different areas around Mumbai and would help find out waters which are highly contaminated with heavy metals and thus a hazard. This information could then be used to protect the rich aquatic flora and fauna found in these areas which requires immediate attention. AIM To study the toxicity of heavy metals such as cadmium, chromium, copper, cobalt, nickel, zinc, lead etc. n the freshwater Danio rerio and the developmental stages of the eggs, to measure heat shock proteins like Hsp70 in the embryos and eggs of the zebrafish, to analyze the bioaccumulation of these metals in the various tissues of the adult fish, to determine the activities of enzymes such as Glutathione S Transferase, Catalase, Acetylcholinesterase, Superoxide Dismutase etc. in the fish and also to assess the water quality of various samples collected from around Mumbai using zebrafish as model organisms. OBJECTIVES 1.
To study the changes occurred in the developmental stages of the zebrafish eggs under the light microscope at various intervals of development when the eggs are under stress. The eggs are transparent and the changes can be easily studied. The effect of different concentrations of various heavy metals on the eggs and the fry and the changes developed due to stress in the stages of growth will be also observed and reported. 2. Heat shock proteins (Hsp) are a class of functionally related proteins whose expression is increased when cells are exposed to elevated temperatures or other stress.
Production of high levels of heat shock proteins can also be triggered by exposure to different kinds of environmental stress conditions, such as infection, inflammation, exercise, exposure of the cell to toxins. Heat shock proteins like Hsp70 will be measured to determine the stress rate. 3. To study the toxicity of various heavy metals on the adult zebrafish and determine the bioaccumulation of these metals in various tissues of the organism when subjected to various concentrations of the metal and cocktail of heavy metals of varying concentrations. . To investigate the activity of various enzymes like catalase, glutathione-S- Transferase, superoxide dismutase etc. in the model organism when exposed to stress conditions. 5. To review the water quality of various samples from in and around Mumbai for the presence of heavy metals by using zebrafish as the model organism. This will help determine the grade of contamination of these waters with heavy metals which is a threat to the aquatic ecosystem. MATERIALS AND METHODS 1.
The zebrafish, both males and females will be cultured in different tanks until they get acclimatized to the water. 2. Once acclimatized, they will be kept to breed to obtain transparent eggs in large numbers. The changes seen under stress in the developmental stages of the eggs will then to be studied under the light microscope until they hatch to obtain fry. 3. Some of the eggs will be subjected to varying concentrations of heavy metals and the changes developed will be examined and reported. . Proper care and treatment is required to grow the fry. The fry and adult fish will then be subjected to various heavy metals individually as well as in combinations. The metals will be added in different concentrations and the changes observed will be reported for a range of concentrations. 5. Bioaccumulation of these metals in selected tissues of the fish when exposed to varying concentrations will also be investigated to determine which tissue shows the highest accumulation of the metals.
This will be carried out by dry ash analysis of the tissue and standard protocols for particular metal estimation will be done. 6. Due to exposure of the model organism to varying concentrations of heavy metals, they tend to live in stress and hence bring about change in the activity of different enzymes like Catalase, Glutathione-S-Transferase, Superoxide Dismutase, etc. will also be estimated during this study. 7. Heat shock proteins like Hsp70 of the eggs will be measured by Western Blot analysis at varying concentrations of the heavy metals so as to determine the stress rate. . Moreover, on standardizing the heavy metal toxicity of the zebrafish, these fish will then be exposed to various water samples collected from different regions in Mumbai to assess the water quality and furthermore determine the various heavy metals present and determine whether they are present in optimal concentrations or if exceed the standard levels which poses a threat to mankind besides the aquatic flora and fauna. Dry Ash Analysis The main purpose of the ash analysis is to estimate the amount of metal imbibed by the fish when exposed to the heavy metal environment.
By incinerating living organisms the carbon present gets converted to carbon dioxide, hydrogen to water which evaporates thus destroying all organic compounds, and the ash left behind contains all the inorganic substances present in the fish. The ash is further treated with minimum amount of concentrated hydrochloric acid. This solution is then used to estimate the metal accumulation in the fish. Cadmium estimation Dithizone method: Cadmium ions under suitable conditions react with dithizone to form a pink to red colour that can be extracted with chloroform.
Chloroform extracts are measured photometrically and the cadmium concentration is obtained from a calibration curve prepared from a standard cadmium solution treated in the same manner as the sample. Chromium estimation Colorimetric method: This procedure measures only hexavalent chromium. Therefore, to determine total chromium, convert all the chromium to the hexavalent state by oxidation with potassium permanganate. The hexavalent chromium is determined colorimetrically by reaction with diphenylcarbazide in acid solution. A red-violet colour is produced.
The reaction is very sensitive. To determine total chromium, digest the sample with sulphuric-nitric acid mixture and then oxidize with potassium permanganate before reacting with the diphenylcarbazide. Copper estimation Dithioethyl carbamate method: Copper in solution forms a very light straw coloured complex with the carbamate, which is extracted out in the organic phase with isoamyl acetate. The optical density of the organic phase is measured at 430nm. Citric acid acts as a scavenger and chelates out any other metal ions present.
Liquor ammonia serves to maintain the pH at around 10. Lead estimation Dithizone method: An acidified sample containing microgram quantities of lead is mixed with ammonical citrate-cyanide reducing solution and extracted with dithizone in chloroform to form a cherry red lead dithizonate. The colour of the mixed colour solution is measured photometrically. Nickel estimation Heptoxime method: After preliminary digestion with nitric-sulphuric acid mixture, iron and copper are removed by extraction of the cupferrates with chloroform.
Nickel is separated from other ions by extraction of the nickel heptoxime complex with chloroform, re-extracted in to the aqueous phase with hydrochloric acid and determined colorimetrically in the acidic solution with heptoxime in the presence of an oxidant. The same procedure could be carried out by replacing heptoxime with dimethylglyoxime. Zinc estimation Zinc is separated from other metals by extraction with dithizone and is determined by measuring the colour of the zinc-dithizone complex in carbon tetrachloride.
Specificity in the separation is achieved by extracting from a nearly neutral solution containing bis(2-hydroxy-ethyl) dithiocarbamyl ion and cyanide ion, which prevent moderate concentrations of cadmium, copper, lead and nickel from reacting dithizone. Catalase estimation Kinetic assay: This assay measures the breakdown if hydrogen peroxide by catalase to form water and oxygen. It is a continuous assay that measures the optical density of hydrogen peroxide for each concentration of catalase over a stipulated period of time.
The rate of breakdown of hydrogen peroxide by catalase is measured as the slope of the graph of the time and absorbance at 240nm. This rate is in direct correlation with the amount of catalase present in the sample. Glutathione-S-Transferase estimation Glutathione-S-Transferase activity will be measured colorimetrically by using a single substrate 1-chloro-2,4-dinitrobenzene(CDNB). Standard readings are prepared and absorbance is read at 340nm to plot a graph through which the amount of Glutathione-S-Transferase can be determined in the ash sample of the fish. OUTCOMES
The outcome of the project will be the study of the effect of heavy metals on the developmental stages of the zebrafish, the heat shock protein measure, the standardization of the heavy metal toxicity on zebrafish eggs, fry and the adult fish. In addition the assessment of Mumbai water quality for the presence of toxic metals would also be carried out which is a current issue and needs to be dealt with soon. REFERENCES Demirak, A. , Yilmaz, F. , Tuna, A. L. and Ozdemir, N. (2005). Heavy metals in water, sediment and tissues of Leuciscus cephalus from a stream in southwestern Turkey. Chemosphere 63:1451-1458.
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