Determination of Some Selected Heavy Metals in Water, Oreochromis Niloticus and Labeobarbus Intermedius Samples from Abaya and Chamo Lakes

The fish organs and water was collected from three sampling sites of Lakes Chamo and Abaya. A procedure consuming 12 mL mixture of HNO -H O for gill, kidney, liver and 1:2.5 mLmixture of HNO -HCl for 3 2 2 3 water samples for digestion of was developed. Concentrations of sixmetals in water and their accumulation in the edible tissue of Tilapia (Oreochromis niloticus)and Barbus (Labeobarbus intermedius) fish species were determined using Flame Atomic Absorption Spectroscopy. The results revealed that theaverage heavy metal concentrations in water samples were; Zn 0.017, Cu 0.077, Fe 7.50, Cr0.033, Cd 0.264 and Pb 1.731 mg/L respectivelty. The highest concentrations of iron (Fe) andlead (Pb) were observed at Chamo Park and Sile sites, respectively. The average contents ofheavy metals in fish samples were; Zn 0.062, Cu 0.047, Fe 2.270, Cr 0.041, Cd 0.045 and Pb1.78 mg/Kg in both fish species. The highest accumulations of iron (Fe) and lead (Pb)concentration were observed in liver and gill of Tilapia fish from Chamo Lake. There was no significant difference between the two lakes in accumulating trace metals.


INTRODUCTION
Heavy metals have wide environmental dispersion Heavy metals are extremely persistent in the the living organisms and have overall potential to be toxic environment. They are non-biodegradable and non-even at relatively low levels of exposure. Heavy metals thermo degradable and therefore readily accumulate to enter the fish through gills, skin, oral in food and water. In toxic levels [1]. They are released into the environment the fish body, the metal is transported through the blood from natural as well as anthropogenic activities.
stream and either stored, transformed or eliminated in the Ethiopian Rift Valley Lakes which comprises of seven liver, kidney or the gills [3,4]. principal Lakes are an important area for commercial At high concentration, they are dangerous because fisheries. The Lakes are also used for recreation, irrigation they accumulate faster in living things such as fish than and industrial purposes [2]. Despite the growing they are broken down. influences from natural and anthropogenic origins, there Fish consumption can be an important route of exists a general belief that presumes absence of human exposure to a variety of heavy metals such as permanent alteration or contamination of these Lakes. mercury, lead, cadmium and copper as fish is often at the However, rivers that flow into some of these Lakes are top of aquatic food chain and may, therefore, concentrate heavily loaded with contaminants of natural and large amounts of some heavy metals from the water [3]. anthropogenic origin such as discharges from factories The consumption of contaminated fish causes acute and and domestic sources [1,2]. chronic effects to humans [5]. This situation has arisen as with the tendency to accumulate in selective tissues of a result of the rapid growth of population, increased 5°3'19" and 6° 45'11" North latitude and 37°18'55"and urbanization and expansion of industrial activities, 38°7'55" East longitude. These lakes are located within exploration and exploitation of natural resources, the Main Ethiopian Rift Valley (MER), which extends from extension of irrigation and other modern agricultural the Southern Afar to the Konso highland in the southern practices as well as the lack of environmental regulations Ethiopia [7]. [6].
Lake Abaya is 60 kilometers long and 20 kilometers The aim of this study is to determine the wide, with a surface area of 1162 Km . It has a maximum concentrations of heavy metals viz. Cr, Cd, Fe, Pb, Cu and depth of 13.1 m and is at an elevation of 1285 m. Lake Zn in water and fish species from Chamo and Abaya Chamo is one of the three large lakes in Ethiopia next to Lakes. The study was necessary as a large number of Lake Tana and Abaya. It is located in the Great Rift Valley people consume the fish species in this area, but there is Lake at an elevation of 1,235 meters. It is just to the south no literature report on the levels of heavy metal of Lake Abaya and the city of Arba Minch and East of the concentrations in water, Tilapia  and liver parts were dried in an electric oven at 100-105 °C Scientific, USA). Buffer solutions of pH 4 and 7 were used until constant weight was obtained. The samples were for pH meter calibration and potassium chloride was used crushed using a clean mortar and pestle to produce for conductivity meter calibration. powdered forms. A homogenized 0.5 g of each grounded Sample Collection balance. Each of 0.5 g of powder sample was then Fish Sampling and Drying: The selected fish species transferred into a digestion flask into volume 12 mL of a were randomly collected with the help of local fishermen mixture of Nitric acid (70%, Spectrosol) and hydrogen within the sampling stations by using plastic nets. The peroxide (35%, Riedel-de Haen) prepared in a 1:1 (v/v) freshly collected fish varieties were two types namely ratio was added. The suspended mixture was digested for Tilapia (Oreochromisneloticus) and Barbus one hour, until a clear solution was obtained [9]. After (Labeobarbusintermedius). First the scales of the fish that, it was allowed to cool and then filtered through were removed with plastic knife and the selected body Whatman filter paper No.42. Finally, the filtrate so parts (gills, kidney and liver) were removed carefully and obtained was diluted to 50 mL in a volumetric flask with separately. At the sites, gill, kidney and liver samples were deionized water and get a side for later analysis of the removed from different fishes of the same species and heavy metals content using Flame Atomic Absorption washed with the Lake water, distilled water; HNO Spectrophotometer. 3 containing distilled water and was combined to form a single composite sample and placed in labeled plastic Digestion of Water Samples: Digestion of water took bags. Immediately, the plastic bags were closed with place after composite samples were prepared from the two masking tape, frozen in icebox and transported to the Lakes. First of all, mark in about 20 mL of the beaker was laboratory for drying and further treatment. made using marker. According to the Methods developed performed at the internal of 50 cm with 50 m distance from mL). Then the beaker was removed and cooled. Each of first point to internal part by using boat in order to get the digested water samples was filtered through Whatman representative sample and 50 cm depth was considered in filter paper No.42 in to a 100 mL volumetric flask and filled order to exclude the dust materials as well as oily liquids up to the mark with deionized water by addition of 2 mL of suspended above the Lake and collected in to the nitric acid to get a clear solution [8].

Method Validation for Metal Analysis
Recovery Test: Recovery is one of the most commonly Preparation of Stock Standard Solution: Working used techniques utilized for validation of the analytical standard solutions of lead (Pb), iron (Fe), copper results and evaluating how far the method is acceptable (Cu), chromium (Cr), zinc (Zn) and cadmium (Cd) were for its intended purpose. In present study due to the prepared from the stock standard solutions containing absence of certified reference material for fish and water in 1000 ppm of element in 2 N nitric acid. Calibration and our laboratory, the validity of the digestion procedure, measurement of elements were done by using precision and accuracy of FAAS was assured by spiking AtomicAbsorption Spectrophotometer. The calibration fish and water samples with standard of known curves were prepared for each element individually concentration. The spiked and non-spiked fish and water applying linear correlation by least square method. A samples were digested following the same procedure blank reading was also taken and necessary corrections employed in the digestion of the respective samples and were made during the calculation of concentration of analyzed in similar condition. Then the percentage various elements.
recoveries of the analytes were calculated by Kiflom and recovery for heavy metals analysis were within 80-120%. According to the instrument operation manual to attain its better sensitivity, the working standards were then Heavy Metal Analysis: After calibration of the instrument aspirated one after the other into the flame atomic the samples were aspirated into the FAAS instrument absorption spectrometer and its absorbance was according to standard method [13]. Concentrations of Fe, recorded. Calibration curves were plotted with different Zn, Pb, Cd, Cu and Cr in the extracted water and different points for each metal standard solution using absorbance parts of fish sample were estimated by using FAAS against concentrations (mg/L). Immediately after Agilent Technology (Model-AT-240). The concentration calibration using the standard solutions, the sample of Cd, Cr, Zn, Cu, Fe and Pb were determined in both the solutions were aspirated into the FAAS instrument and water and fish samples. The samples were analysed in direct reading of the metal concentrations were recorded.
triplicates and the blank determinations in triplicates were Analytical Method Validation Method Detection Limit: Three replicate blank samples Statistical Analysis: The analysis of variance ANOVA were digested following the same procedures utilized for was used to examine the significance level of all digesting the fish and water samples. Each blank was parameters measured. Least Significant Difference (LSD) assayed for its metal contents (Cr, Cd, Zn, Fe and Pb) by test was used for means comparison. The level of FAAS. The SD of the three replicate blanks was significance for the t-test and means comparison was at calculated to determine the MDL [10]. Method detection p<0.05. The concentrations of all metals in fish and water limit (MDL) was then calculated according to equation sample were expressed in mg/L. indicated below [10,11].
where Lake Chamo ranged from 7.66±0. 15-8.52±0.18 at the site of yB= mean of the replicate blank Sile and kulfo inlets, respectively, whereas the pH of Lake SD= Standard deviation of the blank Abaya ranged from 7.29±0.65-8.09±0.48 at the site of Bilate Tarekegn [8] also run in the same manner during the analysis.

RESULTS AND DISCUSSION
Physicochemical Variables of Water Samples: The pH of

Physicochemical Parameters
Lake Chamo Lake Abaya  and Lante inlets, respectively. The values of pH has lowering of the amount of drainage water pouring into the relatively decreased in the lake when compared with the lake. The lower values are due to direct effect of dilution value reported from previous researches done by by drainage water especially in areas facing the drains. Alemayehu [14] (pH=8.5) and Elizabeth et al. [15] (8.8) for the Lake Hawassa. These may reveal that there is a Method of Detection Limit: Method of detection limit is notable increment in the organic matter load of the lake defined as the minimum concentration of analyte that can system. The pH of the Lake is within permissible limits of be measured. In other words, it is the lowest analyte FDRE, MoWR and WHO [16, 17] for drinking, recreation, concentration that can be distinguished from statistical agricultural and aquatic life water use (6.5-9). The pH variations in a blank, which usually correspond to the values investigated in the present study are relatively low signal of blank three times the standard deviation of the when compared with other lakes, which may be blank (3 blank, where = standard deviation of the recognized to the decomposition of organic matter in the blanks) [10]. As shown in Table 2, the method detection course of time.
limits of the investigated elements by FAAS along with Conductivity is a measure of the ability of aqueous their instrument detection limits arepresented, solution to carry an electric current that depends on the respectively. presence and total concentrations of ions, their mobility, valance and on the temperature. The mean levels of Calibration Curves: Calibration curves for Cr, Cd, Zn, Pb, electrical conductivity of both lakes were recorded by Fe and Cu were obtained by using suitable standard conductometry using an instrument of model 4310. In all solutions prepared from stock solutions. Calibration sampling sites the EC values of Lake Chamo ranged from standards for the elements analyzed were prepared in 773.67±6.03 to 843.67±7.02 ìS/cm at the inlet of Kulfo and concentration ranges expected for the analytes in the Sile sites, respectively; whereas the EC values of the Lake samples analyzed. In addition, the calibration standards Abaya were ranged from 477±7.00 to 525.67±9.61 ìS/cm at were prepared by taking into consideration the optimum Lante and Alge, sites respectively. All these values are by working ranges of the elements. The determination far below the WHO [17] guideline value recommended for coefficient (R ) values that are closer to the absolute value drinking purpose (1500 ìS/cm) as well as the EPA of 1 indicate that there is a strong relationship between guideline value of (1000 ìS/cm). Therefore, the EC values the variables being determined whereas values closer to for both lakes can be assessed under excellent classes for zero (0) indicate that there is no linear relationship all livestock, poultry and irrigating purposes betweenabsorbance and concentration [4]. The (FAO[18],<1000 ìS/cm). The observed variations of these determination coefficients of the elements were values however may be accounted for the changes in the determinedusing prepared standards concentration water level as a result of high rate of evaporation and versus their corresponding absorbance. In the current study the precision of the results were the analyte determination. This was performed by evaluated by the standard deviation and relative standard carefully spiking the standard solution prepared, in to deviation of the results of three replicate measurements water and fish samples to evaluate the extent to which the (n = 3). These parameters are useful in estimating standard added could be recovered after determination indeterminate error. The results of the present analysis are through sample preparation. The percent recovery was reported with the corresponding standard deviation and calculated after small and known amounts of the heavy relative standard deviation. As shown in Tables 3, the metals from the stock solutions were added or spiked in to percent relative standard deviation determined was less the water and fish samples to bedigested and after than 10 for all samples which indicate acceptable absorbance of both spiked and non-spiked samples were variability of the analytical results [20]. read. Results were obtained through calculation. Method validation is the process of providing that analytical Levels of Heavy Metals in Water Sample: The dissolved method is acceptable for its intended purpose. As shown metal concentrations in Chamo and Abaya Lake water in Figure 1 below, the percentage recovery for the water were measured at the six sampling sites (Sile, Chamo Park, samples lie in the range of 85.00 -95.35 % for Lake Kulfo, Bilate, Lante and Alge) during the dry season. The Chamo, 83.00 -92.7 % for Abaya Lake, which are within concentrations of dissolved heavy metals in Chamo and the acceptable range for each metal.
Abaya lakes were found to be above the instrumental For fish samples, the recoveries obtained from Lake detection limit (FAAS) in all sampling sites. The average Chamo varied from 87.00-98.50 and 87.00 -95.50 % in concentration (mg/L) of heavy metals in water sample Tilapia and Barbus fish respectively. Although, the from both study sites are shown in Table 3. Statistical recoveries obtained from Lake Abaya varied from 87.00-analysis shows that the metal concentrations in the water 95.50 % and 88.5-93.5 % in Tilapia and Barbus fish samples differed significantly (p<0.05). However, there respectively. These values were within the acceptable was no significance difference in each elements in water range of 80 to 120% for the elements indicating good samples collected in different sampling occasions accuracy for the analysis procedure [19]. (p<0.05).

Evaluation of Analytical Data: Errors in analytical results
were within the same range as background values for are most often expressed using accuracy and precision.
reports in literature by WHO [17]. The concentration The precision of an analytical procedure expresses the (mg/L) of zinc in the area of investigation for lake closeness or agreement between a series of measurements water varies from 0.011±0.001 to 0.021±0.002; 0.011±0.001 obtained from multiple sampling of the same to 0.024±0.001 mg/L from Chamo and Abaya lakes. homogeneous sample under the established conditions The results show that there was no significant difference whereas the accuracy of an analytical procedure in the concentration of zinc between Sile and Kulfo site, The concentrations of each heavy metal in both lakes Heavy Metals in water sample from Lake Abaya Heavy metals in water sample from Lake Chamo Chamo Park and Kulfo site, but there is the significance As shown in Table 3 above the level of Cr difference between Sile and Chamo Park at (P<0.05). As concentration varies from 0.014±0.001 to 0.035±0.003 mg/L shown in Table 3, there was no significant difference in were observed in the site of Alge and Bilate from Abaya the concentration of zinc between Alge and Alge Lante Lake, respectively. The levels of Cr concentration in Lake 1 2, 1 and Bilate sites. The concentration of zinc in Lake Abaya Chamo were ranged from 0.018±0.001 to 0.048±0.004 mg/L. 2 was slightly higher than zinc concentration from Lake The highest concentration of Cr (0.048±0.004 mg/L) was Chamo. This is may be due to the agricultural activity detected in the water sample from Kulfo site while the around the Lake Abaya was more dominated than Lake lowest concentration (0.018±0.001 mg/L) was observed Chamo.
The concentrations of lead in this study were above the    of all an understanding of the mechanisms or modes of The area in the past has been affected by global climatic action for the chemical concern is important such that the change with a shift to a decrease in precipitation peaks appropriate fish organs are selected and sampled. and with consequent impact on the lakes [7]. In previous Selection of the appropriate fish organs can be specific to times, those lakes are known by its fish potential as a contaminants [23]. In the fish body, the metal is freshwater lake, contained freshwater fish species and transported through the blood stream and either stored, now its fish potential is decreasing time to time and at the transformed or eliminated in the liver, kidney or the gills same time studies showed the water salinity increased [21]. The levels of heavy metal concentrations recorded in progressively from past to present [7]. Generally, the the gills, kidney and livers of the two investigated fish distribution patterns of all heavy metals except Pb species from the two lakes are shown in Tables 4 and 5. concentrationin both lakes were below the permissible The mean±standard deviation, coefficient of variation and guidelines [17,18,21,22] due to these it has no effect on least significance difference ofheavy metal concentration the aquatic environment and human being who used in Tilapia and Barbus fish observed from ANOVA resultat these lakes.

Levels of Heavy Metals in Tilapia and Barbus Fish from
between the mean values of the various determination Chamo Lake: The distribution and accumulation of both experimental (statistical) and critical t-value were metals in fish organs depends on the metal type, considered. The number of degrees of freedom for finding interaction with natural factors, ability to bind metals in the t-value is (n +n )-2, where n and n are number of forms that are not biologically available, character of replicate measurements of sample A and sample B, mechanisms determining bioaccumulation of metals respectively. The pooled standard deviation value can be and also on the type of tissue, fish species, size and obtained by: age [23]. In the present study the levels zinc, copper, There are 4 degrees of freedom for each heavy metal iron, chromium, cadmium and lead in tissues of gill, measurement and the critical value at t = from table is 2.78 kidney and liver parts of Tilapia and Barbus fish sample (P= 0.05). The obtained value of t = 0, 0.743 for zinc and collected from Abaya and Chamo Lakes were cadmium determination was less than the critical value so investigated.
the null hypothesis is retained which is to mean that there

Levels of Heavy Metals in Tilapia and Barbus Fish from
cadmium between the two lakes. The calculated t-values Abaya Lake: The distribution of metals in fish organs of 26.421, 3.506, 10.070 and 34.005 for copper, iron, depends on the metal type, interaction with natural chromium and lead are greater than the critical t-value, factors, ability to bind metals in forms that are not here also the null hypothesis is rejected that there is a biologically available, excretion of metals and also on the significant difference (p=0.05) between the mean type of tissue, fish species, size and age [23]. The levels concentration with respect to copper, iron, chromium and of mean values in the gills, kidney and livers of the two lead between the two lakes.In a series of experiments on investigated fish species from the Abaya Lake are shown the determination of mean difference between selected in Tables 5 below. heavymetals (Zn, Cu, Fe, Cr, Cd and Pb) in Tilapia and As indicated in the Table above the metal Barbus fish samples from Chamo and AbayaLakes were concentrations in the fish species did not vary between obtained from the calculation of their mean value and the sampling sites and Cd, Zn, Cr, Cu concentrations did standard deviation.The experimental t-value of Zn and Fe not differ between fish species (p<0.05). However, Fe and were less than the critical t-values, this shows there is no Pb concentrations were highly significant (p<0.05) in significance difference (p=0.05) between the mean values tilapia than in barbus when the fish species are compared in gills of both fish species collected from Chamo between Chamo and Abaya Lakes.
Lake.But, the t-values of Cr, Cd and Pb were above the T-test for Sample Analysis: The mean differences difference in gill of the two fish species. Although,the between the values of samples obtained in this study experimental t-value of copper in the kidney was not were evaluated by the student's paired t-test at 95% significantly different (p=0.05) betweenthe values of fish confidence level. To find out the significance differences species.
1 2 1 2 4 is no significant difference in concentration of zinc and critical t-value,this indicates there was highly significance