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Научни трудове на Съюза на учените в България - Пловдив. Серия В. Техника и технологии. Том XVII, ISSN 1311 -9419 (Print); ISSN 2534-9384 (Online), 2019. Scientific Works of the Union of Scientists in Bulgaria - Plovdiv. Series C. Technics and Technologies. Vol. XVII., ISSN 1311 -9419 (Print); ISSN 2534-9384 (Online), 2019
CHANGES IN BLOOD CELLS OF CARP (CYPMNUS CARPIO L.) UNDERTHE INFLNCREAONINCINAEINR CONCENTRAEIONS OF LEAD Desislava Arnaudova, Darinka Boyadjieva - Doychinova, Atanas Arnaudov PlovdivUniversity"P. Hilendarski", ^cukyofBiotogy, Bulgasia
Abstract
The impact of increasing concentrations of lead (Pb) on the morphofunctional characteristics of carp (Cyprinus carpio L.) erythrocytes and leukocytes was studied. In the erythrocytes nuclear changes were established in all studied treatment groups. Under the action of the highest concentration, the nuclear changes were expressed by the presence of round and swollen erythrocytes and cores. The fish leukocytes in the researched group which was treated with the highest concentration, the number of monocytes and lymphoblasts was found to be increased, probably due to increased tissue decay.
Key words: aquatic toxicology, biomonitoring, Cyprinus carpio, erythrocytes, leukocytes,
lead.
Introduction
Heavy metals have a negative impact on water ecosystems due to their high toxicity, constant activity and subsequent bioaccumulation (Ali et al., 2013).
Pursuant to Directive 2013/39/EU of the European Parliament lead (Pb) is classified as a priority pollutant. Lead is subject to monitoring and control by the Council of Europe due to its toxicity, stability, and affinity to bioaccumulation (Rougier, 1994; Bubb and Lester, 1996). Similarly to mercury, lead is not excreted through the excretory system but is accumulated in the body, primarily in the bones where it turns Ca3(PO4)2 into Pb3(PO4)2. The latter maintains toxic concentrations of lead in the blood (Pelova & Simeonova, 2004). Lead in food products is not permissible even in trace amounts due to the high toxicity of lead compounds and its ability to be accumulated in the body (Manolov, 1979). It has been proven that even low doses of lead impact the biochemical processes in red blood cells and slow down normal body development. The effects of chronic lead exposure are related to brain damage, kidney disorders, and malignant diseases (Robohm, 1986; Saxena, 2008).
Fish are invaluable as a test subject in studying the haematopoiesis and morphology of the different blood cells including red blood cells (RBC), as well as white blood cells (WBC) and platelets (Jill et al., 2010).
Haematological indices may show different sensitivity of tested fish to the impact of certain environmental factors and chemical compounds (Lebedeva et al., 1998).
The aim of this study is to establish the morphological and functional changes in erythrocytes and leukocytes in carp fish (Cyprinus carpio, Linnaeus, 1758) which occur under increasing concentrations of lead in the water.
Materials and methods
Young carp fish specimens from a single size group (100-150 mm) were used for the purposes of the experiment. They were divided into four test groups and one control group
comprising of ten fish each. They were grown in glass aquariums of 50 l each filled with dechlorinated water and equipped with aerator pumps. The fish were not fed during the experiment.
The fish in the tested groups were exposed over a period of 72 hours to lead nitrate Pb(NO3)2, where concentrations were prepared as 75%, 50% and 25% of the maximum permissible concentration (100% MPC) of the metal as specified in the Bulgarian legislation (Ordinance on the environmental quality standards for priority substances and other pollutants, 2013). The physical and chemical properties of the water - pH, temperature, and conductivity were measured at the 24th and 72nd hour using a combined pH-meter (HANNA instruments).
The test blood samples were obtained through cardiac puncture. EDTA was used to prevent haemocoagulation with a final concentration of 0.5% where blood films were prepared at the time when the samples were obtained. Blood films were stained using an express staining kit DKK-Color-200 (VIVA MT-Plovdiv). The microscopic test was conducted using an Olympus CX22LED microscope. Photographs of the microscopic test smears were taken using a digital camera Lumenera (Canada). The percentage ratio between normal morphology erythrocytes and erythrocytes with morphological and nuclear changes was determined. Each film featured not less than 100 erythrocytes. The more characteristic changes in the cytoplasm and the nucleus were described and photographed. In addition to erythrocyte morphology, the test determined the leukocyte formula (through differential count of not less than 100 leukocytes per sample).
The obtained results were processed statistically using the software product SAS-SPSS-2014. The reliability of the results was calculated applying the F-test.
Results and considerations
In the course of the experiment the physical and chemical parameters of the water (pH, temperature, conductivity, oxygen content) in control samples and in the test samples were identical. Levels were maintained within the range specified in table 1.
Table 1. Physicochemical
properties in the experimental water tanks
Concentration of pH T0 C Dissolved Conductivity
lead oxygen (mg/L) (^S/cm)
Control 7.22±0.3 22.6±0.8 7.2±0.3 472±0.3
Pb (25%) 7.58±0.2 21.7±0.3 7.0±0.3 501±0.5
Pb (50%) 7.52±0.5 21.2±0.3 7.4±0.5 496±0.3
Pb (75%) 7.69±0.5 21.4±0.5 6.8±0.3 454±0.5
Pb (100%) 7.62±0.5 21.3±0.5 7.6±0.3 496±0.3
In normal erythrocyte cells the nucleus is oval and it is located in the centre of the cell surrounded by a homogeneous cytoplasm.
The morphological changes in blood cells treated with varying concentrations of lead have been described in figures 1, 2 and 3. At lead concentrations of 25%, morphological changes in the cell mainly took the form of changes in the structure of the nucleus. Results revealed an enlarged paler nucleus which may be a sign of karyolysis. At lead concentrations of 50% and 75% of MPC, the results revealed prominent vacuolisation of erythrocyte nuclei (Fig. 1).
Figure 1. Vacuolisation of erythrocyte nuclei of Cyprinus carpio treated with lead (Pb 50% - A and Pb 75 - B). X 600 At lead concentrations of 100% of the MPC the fibrous protein of the nuclear membrane begins to decompose and the diameter of the nucleus increases. This could lead to increase in the perinuclear space and thus to changes in the biochemical activity of the cell (Fig. 2).
Figure 2. Vacuolisation of erythrocyte nuclei of Cyprinus carpio treated with lead
(Pb 100%). X 600
Vacuolisation combined with changes in the diameter of the nucleus can be seen in the smears of all cells treated with lead.
Table 2 shows the morphological changes of erythrocyte cells in percentage terms after treatment of the tested fish with lead compounds in increasing concentrations.
Table 2. Characteristics of erythrocytes from Cyprinus carpio under the influence of _increasing concentrations of lead_
Lead-treated erythrocytes
Experienced groups Normal morphology cells (%) Morphological changes of erythrocyte cells (%) Nuclear changes (%)
Control 84.21±5.5 14.95±3.8 0.64±0.2
Pb (25%) 66.4±1.8378 23.6±2.0655 10.0±3.3993
Pb (50%) 61.0±2.1602 20.8±1.6865 18.2±3.5839
Pb (75%) 56.4±2.2705 23.8±2.7406 19.8±4.9396
Pb (100%) 42.4 ±3.2386 22.8±2.6997 34.8±4.6380
Samples from all tested concentrations showed statistically reliable decrease in the proportion of cells with normal morphology and a corresponding increase in the number of cells with morphological and nuclear changes (P<0.05).
White blood cell differentials were characterised mainly by the manifestation of monocytosis and the appearance of lymphoblasts and monoblasts in the peripheral blood of fish exposed to lead (Fig. 3). This indicates a certain activation of the immunocompetent cell generation process.
Figure 3. Morphology of leukocyte cells from Cyprinus carpio exposed to lead (100%). Lymphoblast (A), Monoblast (B). X 600
Table 3. Leukocyte formula under the conditions of increasing lead concentrations
Indicators Leukocytes
Experienced Lymphocyte Neutrophils Eosinophils Basophiles Monocytes
groups s (%) (%) (%) (%) (%)
Pb (25%) 87.6+5.3 11.8+2.0 0.2+0.01 - 0.4+0.05
Pb (50%) 85.6+5.7 13.8+2.4 0.1+0.01 - 0.5+0.1
Pb (75%) 79.4+6.6 19.4+3.0 0.3+0.08 0.1+0.01 0.8+0.1
Pb (100%) 70.0+8.8 23.8+3.7 0.4+0.1 0.1+0.01 5.7+0.2
Increase in lead concentrations leads to reduction in lymphocyte content in percentage terms and to increase in the percentage of neutrophils. The most significant changes could be seen in the proportion of monocytes. At lead concentrations of 75% and 100% of MPC the increase in monocyte percentage proportions is statistically reliable (P<0.05). However, in all test groups the white blood cell differential was of the lymphocyte type.
The morphological changes were similar to results obtained by other authors researching blood cells exposed to heavy metals (Velcheva et al., 2006; Arnaudova et al., 2008; Arnaudov et al., 2009; Georgieva et al., 2010). The monocytosis seen in the test groups treated with the highest lead concentrations (75% and 100% of MPC) is probably due to the increased tissue decomposition caused by the presence of heavy metals in the bodies of the fish.
In summary, it may be concluded that lead is a strong harmful factor whose effects become present at concentrations as low as 25% of MPC. This implies further research into this field.
Conclusions
1. In carp fish lead causes erythrocyte deformations with a clear upward trend proportionate to the increase in concentration.
2. Erythrocyte deformations due to lead exposure predominantly take the form of changes in the nucleus.
3. Leukocyte changes due to lead exposure occur only at the highest tested concentrations of the metal which cause monocytosis and the appearance of monoblasts and lymphoblasts in peripheral blood.
A cknowledgments
This study was supported by the NPD - Plovdiv University "Paisii Hilendarski" under Grant No NI15-BF-003, Integrated biological approaches for monitoring priority substances in water.
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