Research Article

Changes in Blood Parameters of Clarias gariepinus Sub-adults Exposed to 2, 4-Dimethyl Amine  

Fidelis Bekeh Ada , Kenneth Igbang Sunday
Department of Fisheries and Aquatic Sciences, Faculty of Agriculture and Forestry, Cross River University of Technology, Obubra Campus, Cross River State, Nigeria
Author    Correspondence author
International Journal of Aquaculture, 2018, Vol. 8, No. 11   
Received: 20 Mar., 2018    Accepted: 23 Apr., 2018    Published: 11 May, 2018
© 2018 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Herbicide application for the control of weeds in rice fields has come to stay in Nigeria. A herbicide commonly applied is 2, 4-Dimethyl Amine. It became pertinent to investigate its effects on the haematology and possible lethality on Clarias gariepinus. Sub-adults of Clarias gariepins measuring 906.7 ± 39.3 mg in weight 164.7 ± 10.2 mm in length were exposed to different concentration of 2, 4-Dimethyl Amine in a 96 hour bioassay. The physicochemical parameters of the water, behavioural and biological responses of the fish and the haemstological analysis of the exposed fish were evaluated. Mortalities were observed as, LC50 in 24 hours (22.5 mg/L), 48 hours (18.0 mg/L), 72 hours (16.5 mg/L) and 96 hours (15.0 mg/L). Other results showed that temperature of the water was not influenced by the herbicide. The pH, dissolved oxygen and temperature of the water were not statistically different (p>0.05) between control and treatment groups. The conductivity showed differences between the control and the treated groups. Total white blood cells counts and other components of the white blood cells were not significantly (p>0.05) more populated than the non exposed groups. The red blood cells parameters like haemoglobin, mean cell volume, erythrocytes sedimentation rate and mean cell haemoglobin concentration were significantly (p>0.05) lower in exposed groups. This showed that the herbicide had a negative impact on the oxygen carrying ability of the red blood cell.

Clarias gariepinus sub adults; 2, 4-Dimethyl Amide; Haematology; Water quality; Behavioural and biological responses


Clarias gariepinus is important in Nigerian Aquaculture. Offem et al. (2010) said it is the second most culture fish in Nigeria in the 2000s. Presently, Clarias gariepinus is famous in Aquaculture because of its taste, high price command in the market, and availability of seeds since it can reproduce or at least initiate reproduction in captivity. This places it in Nigerian Aquaculture higher than its relative, Heterobranchus species that cannot reproduce in captivity (Freund et al., 1995; Offem et al., 2008; Anetekhai, 2013). Tilapia that was preferred for farming between 2005 and 2008 (Offem et al., 2010) is however reducing as the cat fishes are taking over this position. The guest for using tilapia in Nigerian Aquaculture is reducing possibly due to explanation offered by The Daily Trust of 10 March 2016, Adeyemo et al. (2005); which says that prominent among the factors scaring farmers away from tilapia production are lack of reliable source of parent stock, taste, market and low technical capacity among fish farmers across the country. It is therefore nearly impossible for a farmer who is just starting and has no breeding facilities to engage in tilapia farming. Its importance is reflected in the views of Nasar et al. (2002) in FAO, who described it as an equivalent of the guinea pig because of the amount of work done on it (Gabriel et al., 2007; Ayoola and Ajani, 2008; Olufayo, 2009; Ada et al., 2011, Ayotunde et al., 2015. Niaraland (2016) described it as equivalent of the cockerel in poultry because of its hardiness in the culture environment. Nairaland (2016) has agreed that Heterobranchus has a limitless size and grows very fast. But he pointed that the number of stunted individuals is higher in proportion compared to Clarias which is active and equally grows fast. Heterobranchus could grow up to 1 meter and weigh 20 kg at full size (Holden and Reed, 1972; Azeroual et al., 2017). But Heterobranchus is not the most cultured species of fish in Nigeria due to none availability of both broods and seeds. Clarias gariepinus farms accounts for 70.0% of fish culture in most farms in Nigeria.


With these qualities, Clarias could help to alleviate the problem of inadequate supply of animal protein in Nigeria if both the cultured and feral stocks are properly managed. Such management aspects include its protection from anthropogenic toxic substances introduced into aquatic environments. Most of these substances are deliberately added to water, though not aiming at killing fish. Fish become the victims as Olufayo (2009) and Gupta and Singh (2011) pointed that they are the most sensitive to pollutants.


One such pollutant is 2, 4-Dimethyl Amine, an herbicide introduced into aquatic environment to control weeds in rice farms. Knowing well that rice and fish are environmentally related, for they both have affinity for riparian environment (Ada et al., 2013). Therefore, chemicals used in weed control in rice farms may not only annihilate weed plants but also non-target organisms. It is observed to cause neurotoxicity, reproductive and developmental toxicities (U.S. EPA, 2005). It interferes with cellular metabolism by disrupting acetyl-coenzyme A (CoA) and uncoupling of oxidative phosphorylation due to either the disrupted CoA activity or cellular membrane damage (Akobundu, 1987; Hussein et al., 1996). In plants, it is a secondary plant growth regulator and is used to selectively kill plants. It kills plants by causing their mitotic cells to divide uncontrollably. According to NPIC (2011) ‘abnormal increase in cell wall plasticity, biosynthesis of protein and production of ethylene occur in plant tissues following exposure resulting in uncontrolled cell division’. The herbicide which is a salt, dissociates to acid form in water (NPSC, 2011) As Deshmukh (2016) pointed out, blood parameters in fish are influenced by many factors such as temperature, salinity, oxygen, hydrogen ion concentration of the water which affects the respiratory metabolism. The influence of agrochemical on fish blood has been intensively investigated by scientists. These include the works of Ayoola and Ajani (2008) that investigated the histopathological Effects of Cypermethrin on juvenile African cat fish; Cengiz et al. (2001) investigated the Histopathological Effects of Thiodin on the liver and gut of mosquito fish, Gambusia afini; Gabriel et al. (2007) studied the haematology and gill Pathology of Clarias gariepinus exposed to refined Petroleum oil, Kerosene under laboratory conditions; Agbon et al. (2002) carried out acute toxicity of Diazonon to Rotifers, Cyclops, Mosquito larvae and fish; Kori-Siakpere et al. (2007) studied acute Haematological effects of Sub lethal levels of Paraquat on the African Catfish. Though Dimethyl amine is more frequently used in aquatic environment, more work on its effects has been carried out in terrestrial organisms than in aquatic organisms. It is shown not to be very toxic to rat with the LD50 values: 736 mg/kg (mouse, i.p.); 316 mg/kg (mouse, p.o.); 698 mg/kg (rat, p.o.); 3900 mg/kg (rat, dermal); 240 mg/kg (guinea pig or rabbit, p.o.) (NIEHS, 2008).


This work was therefore carried out to determine the lethal concentration of 2, 4-Dimethyl Amine and to observe the haematologicel changes that occur in Clarias gariepinus during exposure. Assessment of the influence of 2, 4-Dimethyl Amine on the water quality parameters as well as measurement of the effects of 2, 4-Dimethyl Amine on the condition factor, gonadosomatic and hepatosomatic indices were carried out.


1 Materials and Methods

The study was carried out in the wet Laboratory of the Department of Fisheries and Aquatic Science, Cross River University of Technology, Obubra Campus. 600 apparently healthy looking fish were obtained from Institute of Oceanography, University of Calabar. The fish were batch weighed using a digital scale (Model EK-5350 made by BIBBY scientific Ltd, UK) and acclimated, prior to the experiment, in a plastic holding tank. These fish weighed 906.7±39.3 mg and length 164.7±10.2 mm (Gabriel et al., 2007). During this period, the fish were fed 5% of their total body weight, with 2 mm COPPENS industrial feed. The rations were divided into two parts, and feeding was done at 9:00 am and 5:00 pm daily (Olatayo, 2004). Thirty liters of stream water was measured into each transparent plastic aquarium of lengths, widths and heights of 52 cm, 38 cm and 30 cm, respectively (Ada et al., 2016).


1.1 Toxicity experiment

A total number of 10 fish was randomly selected and stocked in each aquarium (APHA, 1981; Cengiz et al., 2001; Olatayo, 2004; Ayoola and Ajani, 2008; Ayotunde et al., 2015; Ada et al., 2017). A static bioassay method was used in this experiment. There was no replacement of the stock solution throughout the experiment. There was no aeration of water during the range finding test.


1.2 Range finding test to find sub-lethal concentration

1.2.1 Preparation of 2, 4-Dimethyl Amine

In computing the amount of pesticides needed in this experiment, the following formula was used:



Where V = volume of water in the aquarium (30 L)

CF = conversion factor (=1.0 mg/L)

PPM = the desired concentration of chemical/pesticides required in the aquaria expressed as part per million

AI = active ingredient, strength of chemical expressed as a Decimal (100 divided by % active = 100/WP value)


1.2.2 Observation of water quality parameters

Temperature: Mercury in glass thermometer was used to measure temperature. The thermometer was inserted in the aquarium for a period not less than three minutes, it was removed and the Mercury level in the glass was read off as the temperature of the water in °C. Dissolved Oxygen concentration was measured electronically using 970 JENWAY DO2 meter (made by BIBBY scientific Ltd., UK) to the nearest 0.1 mg/L. pH: pH was also measured using electronic method. The pH meter model 3505 JENWAY (made by BIBBY scientific Ltd., UK) was used to determine the pH. The Conductivity was measured using a DDS-307 conductivity meter.


1.2.3 Biological and behavioural parameters

At the start, the fish were monitored to observe their behaviours every 30 minutes. Interval of observation was increased to every one hour after 12 hours for the remaining period of the range finding experiment. Death fish were immediately removed and preserves in formaldehyde (Ayoola and Ajani, 2008). A fish was said to be death when it could not respond to external stimulus. Behavioural characteristics that were watched out for included; erratic swimming, air gulping, rate of operculation, death, loss of balance, excessive mucus secretion, tail movement, moulting and barbell deformation (Ada et al., 2017).


1.3 Haematological analysis

Haematological analyses were done by the use of computerized, automated hematology analyzer (sysmex kx-21N™) (Ada et al., 2016). Although Baker et al. (2001) argued that the manual method is preferred; the manual method is also not devoid of human errors. This computerized method is fast and remove the problem of waiting to analyse the samples and parameters one after another. This can lead to changes in the samples.


2 Results

The LC50 24 hours (22.5 mg/L), 48 hours (18.0 mg/L), 72 hours (16.5 mg/L) and 96 hours (15.0 mg/L) of Clarias gariepinus sub-adults exposed to 2, 4-Dimethyl Amine were shown in Figure 1. Changes in total white blood cells counts (Figure 2) were observed to be statistically different among one another (α=0.05). But Lymphocytes counts, Blood protein concentrations (g/dl), ESR (%) and Granolocyte counts also (Figure 3; Figure 4; Figure 5 and Figure 6) respectively did not have significant differences between mean values (α=0.05). Concentration of haemoglobin (g/dl) (Figure 7) was statistically the same. Haematocrit (%), plasma albumen and mean platelet volume showed no significant difference between treatments (Figure 8). But there were significant difference among means in Mean cell volume (fl), Mean cell haemoglobin (pg), Mean cell haemoglobin concentration (g/dl) Platelet counts (Figure 9; Figure 10; Figure 11; Figure 12). Physicochemical properties of water exposed to different concentrations of 2, 4-Dimethyl Amine during acute toxicity assay of Clarias gariepinus showed that conductivity was different among treatments (Table 1). Means of parameters as dissolved oxygen, temperature and pH were not statistically different. Table 2 expressed the LC50 24 hours, 48 hours, 72 hours, 96 hours as well as the safe concentrations for these periods of time. The behavioral and biological responses of the fish to the toxin shown in Table 3.


Figure 1 The LC50 24 hours (22.5 mg/L), 48 hours (18.0 mg/L), 72 hours (16.5 mg/L) and 96 hours (15.0 mg/L) of Clarias gariepinus sub-adults exposed to 2, 4-Dimethyl Amine



Figure 2 Changes in total white blood cells counts in Clarias gariepinus sub-adults exposed to 2, 4-Dimethyl Amine (mg/L)

Note: The means were observed not to be statistically different among each other (α = 0.05)


Figure 3 Lymphocytes counts in blood of Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There were no significant difference between the mean values (α = 0.05)


Figure 4 Blood protein concentrations (g/dl) in Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There were no significant differences between mean values (α = 0.05)


Figure 5 Erythrocyte sedimentation rate of blood of Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There was no significant difference between the means (α = 0.05)


Figure 6 Granulocyte counts in Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There were no significant difference between mean values (α = 0.05)


Figure 7 Concentration of haemoglobin (g/dl) in Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There were significant differences between means (α = 0.05)


Figure 8 Results showing haematocrit (%) level in the blood of Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There were no significant differences between the means (α = 0.05)


Figure 9 Mean cell volume (fl) in blood of Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There were significant differences between the mean values (α = 0.05)


Figure 10 Mean cell haemoglobin (pg) in blood of Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There were significant differences between the means (α = 0.05)


Figure 11 Mean cell haemoglobin concentration (g/dl) in blood of Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There were significant differences between the means (α = 0.05)


Figure 12 Platelet level in blood of Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There were significant differences between the means (α = 0.05)


Table 1 Physicochemical properties of water exposed to different concentrations of 2, 4 Dimethyl Amine during acute toxicity assay of Clarias gariepinus

Note: Mean values of conductivity that carry the same letters were statistically the same while those with different letters were statistically different. Means of parameters as dissolved oxygen, temperature and pH were not statistically different.


Table 2 The LC50 and safe concentrations of 2, 4-Dimethyl Amine to Clarias gariepinus sub-adults

Note: Safe concentrations are obtained from the formula of Koesomadinata (1980)


Table 3 Behavioural changes and biological responses in Clarias gariepinus sub-adults exposed to 2, 4-Dimethyl Amine herbicide (definitive experiment)

Note: N = No change in behaviour found; Y = Yes, change in behaviour found


3 Discussion

NPIC (2011) noted that the salt of this herbicide (2, 4-Dimethyl Amine) dissociates in water to form acid. It is noted that pH range outside optimal (6.5-9.0) does not favour normal metabolism in fish and so growth, reproduction and other life processes are hampered. U.S. EPA (2005) observed that the herbicide has low toxicity; this was reflected in the pH here being optimal (7.6 to 8.76) as shown in Table 1. Its low toxicity value is explicable from the fact that that herbicide is highly water soluble in the bodies of organisms and so it is excreted in urine. Observations shown in Table 3 revealed that at low concentration of the herbicide, there were no visible abnormal biological and behavioural changes in the fish. It is also recorded that some fish died at these low concentrations. The causes of death here, if attributable to the herbicide could said to be physiological. It is worthy of note that mortality at these concentrations where there were no visible biological abnormality may have affected those specimens that had extreme inability to adapt to changes in environmental situations such as exposure to 2, 4-Dimethyl Amine. This phenomenon is explained in statistical books for individuals falling out at the extremes of population distribution (Frank and Althoen, 1995). As was observed, some red blood cells parameters were lowered, which could result in reduction in oxygen carrying capacity. It is less toxic to aquatic organisms also because bodies of organisms do not make attempt to metabolise it as it is reported that the kidney is constantly excreting it from the body. In spite of the low toxicity of 2, 4-D, it was observed to be highly toxic in a synergistic manner in combination with Carbaryl (Jervais et al., 2008).


The results obtained from the toxicity experiment of Clarias gariepinus sub-adults exposed to varying concentrations of 2, 4-Dimethyl Amine for a period of four days showed significant differences in some blood parameters when compared with that of the control. For instance, there was an increase in the white blood cells counts of fish that increase with increase in the concentration of 2, 4-Dimethyl Amine. A similar increase was reported by Akinwole et al. (2014) who attributed the increase in white blood cells to a surge in leucocytes production in the haematopoietic tissue (kidney and maybe the spleen). Lymphocytes are responsible for the production of antibodies. They serve as protection against infections. Ada et al. (2011) also recorded a similar increase in the WBC of Oreochromis niloticus subjected to an increasing concentration of an herbicide (Butachlor), with a resultant effect of leukemia in the fish. The population of Lymphocytes (Figure 3) were not different between the treated and control groups, though there were absolute elevations in the population of the treated groups. Akinwole et al, (2014) had a similar report when Clarias gariepinus were subjected to different levels of Plumbago zeylanica extracts.


There were no significant differences in the Erythrocyte Sedimentation Rate among treatments (Figure 5). This agrees with Gabriel and Ugbomeh (2016) who after exposing Clarias gariepinus to different concentrations of Cypermethrin reported that the increase in the herbicide concentration might have affected the blood constituent which led to haemodilution. However, abnormalities observed in blood parameters of fish subjected to 2, 4-Dimethyl Amine or toxins, as recorded in this study, resulting to a decrease in values lower than the control indicates an anaemic condition. Gabriel and Ugbomeh (2016) stated that anaemic conditions could be as a result of inhibition of erythropoiesis, haemosynthesis and increase in the rate of erythrocyte destruction in haematopoietic organs.


There were no significant differences in the values of RBC, Haemoglobin, Heamatocrit and MCV with increase in concentration of 2, 4-Dimethyl Amine (Figure 7; Figure 10; Figure 11). The result evidentially showed that increase in Haematocrit led to corresponding increase in the Haemoglobin content as well. Observable decline was recorded in the values of MCH and MCHC when compared with the control, with increase in time and concentration of 2, 4-Dimethyl Amine (Figure 9; Figure 11).


The decline in the values of Haemoglobin level in the present study could be an indication of a reduction in haemoglobin synthesis. Yekeen and Fawole (2011) observed reduction in haemoglobin when they exposed Clarias gariepinus to endosulfan. Reduction in the Haemoglobin level leads to a reduction in the oxygen carrying capacity. Endosulfan, according to them, may have interfered with haemoglobin synthesis pathway.


Polycythemia may be caused by hypoxia or splenic contraction in excited animals or those that have been exercised (Akinwole and Oguntuga, 2014).


The Albumin level (Figure 12) reduced apparently in fish treated with 2, 4-Dimethyl Amine when compared with those in control but were not significantly different. Akinwole and Oguntuga (2014) observed progressive decrease across treatments and attributed such reduction to their possible utilization for metabolic purpose. They also stated that reduction could be attributed to cells destruction and subsequently resulting to damage in the protein synthesis machinery.


Figure 13 and Figure 14 showed that the platelets were reducing in both population and corpuscle size with increase in concentration of the herbicide. It is deducible that this herbicide can render the fish’s blood incapable not listed in reference list of clotting (Akinwole et al., 2014). Gabriel and Ugbomeh (2016) had a similar report when Clarias gariepinus were exposed to different concentrations of Cypermethrin. According to their report, reductions in platelet numbers with time of exposure suggest interference between the chemical and thrombocytopoiesis in the bone marrow.


Figure 13 Albumin level in blood of Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There were no significant differences between the means (α = 0.05)


Figure 14 Mean Platelet Volume (ft) of Clarias gariepinus sub-adult exposed to 2, 4-Dimethyl Amine

Note: There were no significant difference between means (α = 0.05)


Authors’ contributions

Associate professor Fidelis Bekeh Ada served as a team leader in this work. He supervised, and at the end wrote the report of this research work. Mr. Kenneth Igbang Sunday is an MSc candidate. He carried out all the practical aspects and provided fund for this research work. This work is for His award of the degree of masters of Science in Fisheries Management. All authors read and approved the final manuscript.



We are thankful for the contribution of Mr. Igri of the central laboratory, Faculty of Agriculture, University of Calabar, Nigeria for keeping to schedule of carrying out haematological analysis for this work.



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