SUSCEPTIBILITY OF DIFFERENTIAL LEUCOCYTE COUNTS OF THE FRESH WATER CAT FISH, Clarias batrachus TO LEAD
V.K. Verma.1, N. Gupta.2* and D. K Gupta.3
1.Department of Environment and Science, Future Institute of Engineering and Technology, Bareilly
2.C.S.J.M. University Kanpur, U.P. (India) *
3Department of Biotechnology, Bareilly College, Bareilly
[Corresponding Author*: email@example.com]
Received: 11-04-2019 Accepted: 02-12-2019
Expansion of industrial and domestic activities has resulted in increasing heavy metals deposition in natural water. The presence and loading of heavy metals deposition in water is a serious issue with respect to environmental pollution. The present communication provides a brief account of the lethal effects of lead (Pb) on Differential Leucocyte Count (DLC) of the fresh water catfish, Clarias batrachus. Acclimatized fish were treated with 0.5 ppm (Group B), 1.00 ppm (Group C), and 3.00ppm (Group D), of Pb during 7, 14, 21 and 28 days time interval and their values compared with the control (Group A) group. Significant changes in DLC were observed in all concentrations of lead. The small lymphocytes were found to decrease in lead treated groups as compared to the control ones. The lowest decrease in the numbers of small lymphocytes was 36% during 28 days exposure in Group B and C. Large lymphocytes reduced significantly to 22% during 28 days exposure in Group D. The neutrophils significantly increased in all the exposure groups reaching to a maximum of 28% during 28 days exposure of two groups (Group C and Group D). The value of monocytes also increased in all treated groups during final exposure time (28 days) showing the same value (6%) but the maximum number of monocytes (8%) were observed in Group C during 21 days time interval. Maximum number of eosinophils (6%) were recorded in Group B (21 and 28 days) while the numbers of basophils observed also showed minor fluctuations. The results of the present study indicate that Pb is highly toxic and causes undesirable effects on the haematological profile of living fish. Maximum 166.6 percentage increase was found in monocytes exposed in Group C treated with 1.00 ppm of Pb for 21 days exposure period.
Key words: Clarias batrachus, leucocytes, lead, pollution
Study of several researches have shown that heavy metal toxicity caused several haematological and biochemical changes in animals in vivo as well as in vitro conditions. The toxicity of heavy metals have been shown by several workers during the last decade (Adeyemo et al., 2010; Abedi et al., 2013; Hedayati and Ghaffari, 2013; Thangam et al., 2014; Moosavi and Shamushaki, 2015; Mehta, 2017; Kaur et al., 2018). Among all heavy metals, lead is the most important toxic heavy element in the environment. Due to its important physio-chemical properties, its use can be retraced to historical times. Globally, it is an abundantly distributed, important yet dangerous environmental chemical (Mahaffey, 1990; Gabriel et al., 2007). Several reports have indicated that Pb can cause neurological, hematological, gastrointestinal, reproductive, circulatory, immunological, histopathological and histochemical changes, all of them being related to the dose and time of exposure to Pb (Mirhashemi et al., 2010). Fish accumulate toxic chemicals such as lead nitrate directly from water and diet, and contaminant residues may ultimately reach concentrations hundreds or thousands of times above those measured in the water, sediments and food (Labonne et al., 2001; Godwin et al., 2003; Osman et al., 2007).
Blood parameters have been commonly used to observe and follow fish health, since variations in blood tissue of fish are caused by environmental stress (Shah and Altindag, 2005).Various workers have shown that the use of haematological parameters as indicators of metal toxicity can provide information on the physiological response of fish due to the close association of the circulatory system with the external environment (Gupta et al., 2002; Adyemo et al., 2010). Fish live in very intimate contact with their environment, and are therefore very susceptible to physical and chemical changes which may be reflected in their blood components (Wilson and Taylor, 1993). Furthermore, haematological indices are of different sensitivity to various environmental factors and chemicals (Lebedeva et al., 1998; Vosyliene, 1999a, b). Several researches have focused on adverse effects on fishes (Adakole, 2012; Ikeogu et al., 2013; Tenekhy, 2015; Mohiseni et al., 2016; Raja and Puvaneshwari, 2017; Samual et al., 2018). Inspite of recent interest in the immune system of fish species, very little attention has been paid to the structural features of fish blood cells (Esteban et al., 2000).The use of immune system parameters in order to assess alterations in fish experiencing heavy metal exposure and the interest generated in defense mechanisms stem from the need to develop health management tools to support a rapidly growing aquaculture industry (Adyemo et al., 2010, Dutta et al., 2015). The aim of present study was to evaluate the sublethal effects of lead on the alteration in nuclear morphology of leucocytes of the fresh water catfish Clarias batrachus.
MATERIALS AND METHODS
Clarias batrachus (n= 64) were collected from different fish markets of Bareilly district and acclimatized for 7 days to laboratory conditions. 16 healthy and large sized fishes (weighing 30-80 gms) were selected for each experimental group. One control and 3 chronic levels of lead-exposed groups were designed for the experiments as follows:
Group ‘A’-Control group.
Group ‘B’-Exposed to low concentration (0.5ppm) of Pb
Group ‘C’-Exposed to intermediate concentration (1.00ppm) of Pb
Group ‘D’-Exposed to high concentration (3.00ppm) of Pb
Thin blood smears were prepared on clean slides, immediately air-dried and fixed in methanol for 2-5 minutes. The films were then stained with Giemsa (Qualigens) in phosphate buffer solution (pH 6.8), the solutions were mixed uniformly in the ratio of 1:7 by means of a soft brush, stained for 40 mins and washed in running tap water for 2-4 mins to drain the excess stain. They were allowed to stand on end to dry and mounted in DPX. The cells were counted using high power microscope in a strip running along the whole length of the film by means of manual / electronic blood cell counter. At least 100 cells were counted and the leucocytes differentiated by the morphological characteristics of the nucleus and characterized as small lymphocytes, large lymphocytes, monocytes, neutrophils, eosinophils and basophils according to Wintrobe (1981).
RESULTS AND DISCUSSION
Significant changes in different leucocyte counts (Figs 1-6) were observed in all concentrations of lead. The small lymphocytes decreased in lead treated groups as compared to the control ones. The lowest decrease in the numbers of small lymphocytes was 36% during 28 days exposure in Group B and C. Large lymphocytes reduced significantly to 22% during 28 days exposure in Group D. The maximum percentage fell to 13.6% during 28 days exposure period in Group B and C in small lymphocyte count (Fig. 7) and 33.3% during 28 days exposure period in Group D in large lymphocytes counts (Figs. 2, 8).
The neutrophils significantly increased in all the exposure groups reaching to a maximum of 28% during 28 days exposure of two groups (Group C and Group D) and the maximum (75%) changes occurred in Group C during 28 days exposure period (Fig. 4, 9). The value of monocytes increased in all treated groups during final exposure time (28 days) showing the same value (6%) but the maximum number of monocytes (8%) was observed in Group C during 21 days time interval, the percentage change rose to 133.33% during 21 days exposure period in Group D (Fig. 3, 10). Maximum numbers of eosinophils (6%) were recorded in Group B (21 and 28 days) (Fig. 5, 11). The number of basophils also showed minor fluctuations. The percentage changes were 100% in all the groups (B, C and D) after 28 days exposure to Pb (Fig. 12.) (Table 1).
Water pollution has become a global problem, the heavy metals have long been recognized as serious pollutants of the aquatic environment, they can enter water bodies through anthropogenic activities like mining, industrial, agricultural and domestic discharge in streams, ponds, lakes, river and ground water (Obasohan et al., 2008). Pb is not necessary for biological functions of living organisms even at low concentration. In the 18th and 19th century, lead was illegally added to wine both as a sweetener to make it appear fresh. (Mai, 2006). Its poisoning is believed to be primarily responsible for the collapse of the Roman Empire,in which lead acetate was used in wine and its prolonged use was considered to have caused as dementia, it also causes anaemia (Cohen et al. 1981). Now it is being discharged in aquatic systems mainly from petroleum, dye, paint, mining and other human activities, which have toxic effects and can bioaccumulate as well as biomagnify (Sorenson, 1991; Heath, 1995). Hematological changes have been considered to be good indicators of fish health and are strongly related to the way fish respond to environmental variations, thus they constitute an ideal tool for in vitro or in situ toxicological studies (Al-Akel and Shamsi, 2000; Hamidipoor et al., 2015; Seriani et al., 2015). The differential counting of blood cells is considered to be a suitable tool for assessing the health status of fish, and this approach has been employed in fish farms after drug administration and parasite infestations, as well as for other purposes (Dias et al., 2011; Alabbasi et al., 2017). It is believed that neutrophils and monocytes have phagocytic stir which might elucidate their increased proportion during infectious condition.Darvish et al. (2001) observed that neutrophils and monocytes increased in channel catfish exposed to KMnO4 solution and our findings fall on similar lines.
The differential counts (Rey and Guerrero, 2007; Tripathi, 2014) were examined at different lead concentrations and time intervals. All differential leucocytes were affected due to toxicity of exposed heavy metal, both the lymphocytes decreased at maximum concentration during exposure period. Shah (2006) on Tinca tinca and Adyemo (2007) on Clarias batrachus observed that the immune system weakened due to decrease in leucocytes but differential leucocytes like lymphocytes percentage increased and neutrophil values decreased corresponding to the increase in the concentration of lead. Mastan et al. (2008) and Adeyemo et al. (2010) also held similar views on lead-exposed fish, the differential leucocyte count deviating significantly from the normal values, lymphocytes and eosinophils increased while neutrophils and monocytes of Clarias batrachus and C. gariepinus decreased. When Rutilus rutilus were exposed to lead and cadmium for a period of 4 days, Pb reduced lymphocyte percentage and neutrophils and monocytes increased while the cadmium elevated the lymphocyte values (Akbari, 2010). Furthermore, Witeska et al. (2010) also observed that lead suppressed the immune system of fish. The results of Hedayati and Gaffari (2013) proved that the neutrophils and monocytes raised while lymphocytes decreased. Khalesi et al. (2017) studied the lethal effects of Pb and Cd on common carp, remaining unchanged in lymphocytes values but neutrophil percentage showed a more significant rise in response to both the heavy metals.
The results of the present study suggest that lead at environmentally comparable concentrations could induce differential leucocyte counts changes leading to significant reduction in lymphocytes and increase in neutrophils while the monocytes and eosinophils showed slight changes. Basophils did not record any significant changes due to lead in the differential leucocyte counts of Clarias batrachus.
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