1

Haemo-immune System Remediation Potential of

Phyllanthus muellerianus Root Extract in Clarias gariepinus

exposed to 2, 4 Dimethyl Amine

Fidelis Bekeh Ada; Ezekiel Olatunji Ayotunde & Kenneth Igbang Sunday

Department of Fisheries and Aquatic Science,

Cross River University of Technology, Obubra Campus, Nigeria.

For correspondence: fbekehada@yahoo.com; fidekisa@gmail.com; skenneth022@gmail.com;

eoayotunde@ygmail.com; +22348068546835

ABSTRACT Chemical weed application in farms is increasing due to its advantages over other methods of weed

control. This is not without price. These herbicides attack other organisms that were not targeted. Equally

affected are the fishes. Some plant products have been identified as having ability to promote the health of

organisms. Phyllanthus muellarianus was used here to remedy changes that occurred in the blood of

Clarias gariepinus after damage by a herbicide 2, 4-Dimethyl Amine. The water qualities such as

dissolved oxygen concentration, pH, conductivity and temperature, were observed in the tanks where

these fish were exposed to these parameters did not change significantly from the control (α = 0.05).

Haematological parameters like lymphocyte count, Erythrocyte sedimentation rate, protein concentration

and haemoglobin were not significantly different between treatments. Other parameters like total white

blood cells count, granulocyte counts, red blood cells counts, Mean cell volume, mean cell haemoglobin,

mean cell haemoglobin concentration, platelet count, albumen and mean platelet volume were

significantly different among treatment (α = 0.05). The Control group are those that were not treated with

2, 4-Dimethyl Amine. These have same haematological parameters with those that were exposed to 2, 4-

Dimethyl Amine but were later treated with Phllanthus muellarianus. Phyllanthus muellarianus is

therefore a remedy for blood damage in this fish.

Keywords Haematology, immune system, 2, 4-Dimethyl Amine, Clarias gariepinus, Phyllamthus

muellarianus, aqueous root extract

INTRODUCTION

Weeds have been the major hindrance to increased food production worldwide. Their effects have been

emphasized by various authors (Akobundu, 1987; De Datta and Baltazar, 1996; Labrada, 1996). Though

chemical weed control started developing since the Second World War particularly in the mid-1940s

(Squibb, 2010) it has increased significantly over the past decades (Labrada, 1996; Aydin et al., 2005;

Ada et al., 2013). This is due to their economic advantage over traditional human labour of hand pulling

of weeds; or sometimes using hoe and cutlass. Because of advantages of chemical method of weed

control, it has led to increases in herbicide use (Labrada, 1996). Although herbicide use has increased

productivity, there are several problems associated with the use of the herbicides including outright

poisoning and modifications of non-target organisms (Svoboda et al, 2009, Ada et al, 2012).

Agrochemicals applied to fields do harm to non-target organisms like fish and food organism of fish, for

instance; paraquat caused hypoglycemia (reduced sugar concentration in the blood) of Clarias gariepinus

(Kori-siakpere et al, 2007). Up to 10 mg/L glyphosate lead to hyperglycemia (higher than normal sugar

concentration in the blood) in neutropical fish, Prochilodus lineaus (Langiano and Martinez, 2007) and in

International Journal of Innovative Environmental Studies Research 6(2):1-16, April-June, 2018

© SEAHI PUBLICATIONS, 2018 www.seahipaj.org ISSN: 2354-2918

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Channa strata, endosulfan equally raised Channa strata blood sugar (Deshmukh, 2016). 2, 4-D DMA

(Dimethyl Amine) imposed hypoglycemia on mice (Mikov et al., 2010). These cases of hyperglycemia

were due to oxidative stress in the fish. Some of these pesticides remain stored in food organisms (Ahmed

et al., 2014.). The organochlorines have good qualities as herbicides but are not environment friendly.

They are characterized by low volatility, chemical stability, lipid solubility and slow biotransformation

and degradation (Squibb, 2010). Atrazine has been suspected to cause endocrine system disruption,

carcinogenicity, epidemiological disorder and low sperm count in man. It has been implicated for low

birth weight, birth defect, menstrual problems, retinal and muscular degeneration and mammary tumors

(Ackermann, 2007). Some of these herbicides such as organochlorines that have soil activities persist in

the soil for long time where they have more chances of attacking non target organisms. Chinalia et al.

(2007) reviewed toxicity of 2, 4-dichlorophynoxyacetic acid and stated that classifying toxins based on

range of concentration which they kill either target or non-target organisms can be confusing. According

these authors, similar organisms may have distinct toxicological range of a particular toxin. Some toxins

too cause different responses in organisms.

2, 4-Dimethyl Amine, is an herbicide introduced into aquatic environment to control weeds in rice farms.

Rice and fish survive in similar habitats although they occupy different niches. They both have affinity

for riparian environment (Ada et al., 2013). Therefore, chemicals used in weed control in rice farm may

not only annihilate weed plants but also non-target organisms. 2, 4 Dimethyl Amine is easily degradable.

Yet it is found in natural water bodies because of its high water solubility (Qurratu, and Reehan, 2016.). It

has been detected in the sperm and urine of rats (Aydin et al., 2005). It is used for the control of weeds in

rice fields as well as wheat, maize and other type of crops where it can find its way ultimately to water

bodies (Farah et al., 2004). At concentrations of less than 0.1 mg/L, it serves as growth hormones but

becomes toxic to plant at elevated concentrations (Chinalia et al., 2007). Qurratu and Reehan (2016)

noted that phenoxyacetic acid herbicides showed hepatotoxic and nephrotoxic effects in animal when they

were exposed to high level of these herbicides. It also showed genotoxicity and cytotoxicity effect in

human red blood cells (Solonski et al., 2007). Other workers have conflicting reports that 2, 4-Dimethyl

has low toxicity, and lacks geno-toxicity, cytotoxicity, carcinogenicity and neurotoxicity and

carcinogenicity (Charles et al., 2001).

Despite that this herbicide is frequently used in aquatic environment, earlier work on this herbicide is

mostly carried out in mammals found in terrestrial habitats (Berkley et al., 1963; Charles, 2001;

Bakowski, 2003; Aydin et al., 2005; Chinalia et al., 2007; Qurratu and Reehan, 2016).

It is not enough to realize that herbicides are harmful to target or non-target organisms. Just as a farmer

will not stop to cultivate corn in the field due to fear of wild animals; it may also be unfair to recommend

that the use of herbicide should stop because they are dangerous to non-target organisms. It may be more

logical to think of ways of remedying or preventing attack of the herbicide on non-target organisms. This

is because the aims of Eco toxicologist include the task of remedying of harmful effects of the

environmental toxins. Rasool et al. (2010) remedied the destructive effect of paracetamol in the liver of

rats using Galic acid. Ajiboye et al. (2011) had used aqueous extract of Annona senegalensis leaves to

remedy carbon tetrachloride-induced hepatocellular damage in rats. Others include the work of Aly et al.

(2008) and Siyavash et al. (2016). One plant that has been shown to have human health benefits is

Phyllanthus muellarianus. It has been observed to possess haemopoeitic ability in man ((Burkill, 1994)

and in fish (Ada et al., 2016). This plant is able to cause proliferation of the keratinous cells and

formation of collagen in man (Agyare et al., 2011). This is the reason why it is used for wound healing

traditionally in Africa (Burkill, 1994; Agarwal et al., 2007). In analyzing the composition of Phyllanthus

muelarianus, Agyare et al. (2011) pointed out that it contains the following: ellagitannins geraniin ,

corilagin, furosin, the flavonoids quercetin-3-O-[beta]-D-glucoside (isoquercitrin), kaempferol-3-O-

[beta]-D-glucoside (astragalin), quercetin-3-O-D-rutinoside (rutin), gallic acid, methyl gallate, caffeic

acid, chlorogenic acid. 3.5-dicaffeoylquinic acid and caffeoylmalic acid (phaselic acid). In this

experiment, the plant Phyllanthus muellarianus extract is used to elucidate its ability to repair

damages/alterations done on blood of Clarias gariepinus exposed to 2, 4 Dimethyl Amine. Induced

charges done on health of organisms include the works of Pandey and Pandey (2001) that used fungicides

Ada et al. ….. Int. J. Innovative Environ. Studies Res. 6(2):1-16, 2018

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to induce alterations on some haematological parameters of freshwater catfish, Heteropneustes fossilis

and Okpashi et al. (2014) has induced diabetes in rats using different plant extracts. Catap et al. (2015)

challenged the immune system of Oreochromis niloticus with Aeromonas hydrophila and remedied it

with boiled corn (Zea mays) silk.

Blood components have been used to determine the effects of toxicants on fish. This is because blood is

extremely vulnerable to environmental flux (Pandey and Pandey, 2001). Blood is an important diagnosing

component of organisms to determine stress and disease (Seth and Saxena, 2003). Blood has ability to

supply efficient correlation between the organism and environment. Physiological adaptations that

culminate in blood picture changes are usually in response to: temperature, salinity, nativity, pH, oxygen

tension, respiratory metabolism, age, sex, blood constituents, seasonal variations and weight (Randall,

1970). The haematology of fish is very important to biologists as it is a sensitive marker of physiological

and biochemical functions, nutrition, health, stress and diseases in reaction to varying environmental

conditions.

MATERIALS AND METHODS

The tanks used in this experiment were thoroughly washed with soap and disinfectant and allowed to dry

for one week. 20 liters of water from CRUTECH fish farm was put in the different aquaria, containing

various concentrations of Phyllanthus muellerianus powder, measured according to the required

concentrations. Ten fish each that were exposed to sub-lethal concentration of 2, 4-Dimethyl Amine (10

mg/L) were randomly selected and stocked in these aquaria. Fish in one treatment was not subjected to 2,

4, Dimethyl Amine, and was regarded as the control. The treatments were replicated in triplicates (APHA,

1995).

Roots of Phyllanthus muellarianus were collected from Obudu Local Government Area of Cross River

State Nigeria. They were collected by excavating the soil around the tree using a spade. The roots were

cut off with the use of a stainless steel cutlass. They were transported to Obubra fresh within two days.

They were put inside perforated sack bags to avoid possible fermentation.

Roots of Phyllanthus muellarianus were cleaned, sun-dried for 8 days and grated in a stainless steel grater

and further pounded using pistil and mortar. The grinded roots were filtered through 100 micron sieve to

obtain fine powder from the roots. The powder was stored in desiccators waiting for the experiment

(Olatayo, 2004; Olufayo, 2009; Adewoye, 2010; Ajiboye et al., 2017).

The weight of the powder was measured directly in milligrams using a high precision digital weighing

balance (model no: KD-CN) to constitute the various concentrations. Each volume of water for treatment

was 20 L. The quantity of powder to constitute the concentration in a particular tank was W x 20, where

W = weight in mg/L.

The concentrations used were 0.0, 10, 20, 30, 40, 50 and 60mg/L. Ten fish that were exposed to sub-lethal

concentration of 2, 4-Dimethyl Amine were randomly selected and stocked in each aquarium for

monitoring. Administration was therefore by absorption through the skin, gills and other absorptive

surfaces

Mercury in glass thermometer was used to measure temperature. Dissolved Oxygen concentration was

measured electronically using 970 JENWAY DO2 meter to the nearest 0.1 mg/L. The pH meter model

3505 JENWAY was used to determine the pH. A heparinised needle and syringe was inserted mid

ventrally to remove blood from the dorsal blood vessel lying below the vertebral column of the fish

(Lewbert, 2001). 3 – 5ml of blood was taken from each replicate for haematological analysis. The

samples was put in EDTA containers and transported immediately to the laboratory for analysis

(Akinwande et al., 2004; Adewoye, 2010).

Behavioural charateristics that were watched out for included; erratic swimming, air gulping, rate of

operculation, death, Loss of balance, Excessive mucus secretion, Tail movement, Molting and Barbell

deformation (Ayoola, 2008; Ada et al., 2017; Ayotunde et al., 2015)

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Haematological analysis was carried out by the use of computerized, automated haematology analyzer

(sysmex kx-21N™). This method was selected because of its speed, which is fast and could avoid the

problem of delay, as in manual methods.

Blood analysis was repeated using blood samples collected after exposing the fish to sub-lethal

concentrations of Phyllanthus muellarianus extracts. Parameters that were investigated included:

erythrocyte count, erythrocyte sedimentation rate (ESR), red blood cell distribution width (RDWc),

haemoglobin (Hb), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC),

packed cell volume (PCV), leukocyte count (WBC), differential white blood cells count, mean cell

volume (MCV), Platelet, mean platelet volume (MPV) and platelet distribution width (PDWc) (Okomoda

et al., 2013).

Figure 1; White blood cells counts in Clarias gariepinus sub- adults, in remediation attempt

using Phyllanthus muellarianus root extract after exposure to 2,4 Di methyl Amine. There were

significant differences between the means (α = 0.05). means with different letters are statistically

different while those with similar letters are the same.

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Table 1: the physicochemical parameters of water exposed to 2, 4-Dimethyl Amine (24 and 96 hours bioassay) and Phyllanthus

muellarianus root extract. Only conductivity varied among treatments when exposed to 2,4-Dimethyl Amine

Range finding test 2,4 D Definitive test 2,4 D Remediation with Phyllanthus muellarianus root

extract

Conc.

(mg/L)

Temp

. oC

pH Conductivity

(µm/cm)

DO

(mg/L)

Conc.

(mg/

L)

Temp. oC pH conductivity

(µm/cm)

DO

(mg/L)

Conc.

(mg/L)

Temp

. oC

pH Conductivity

(µm/cm)

DO

(mg /L)

0.0 27.00

+ 00

6.93

+0.28

43.33

+2.51c

5.70

+0.36

5.0 27.40

+0.64

8.76

+0.23

42.93

+0.73bc

5.03

+0.05

0.0 26.32

+0.84

7.48

+0.39

44.02

+3.20

4.29

+0.01

15.0 26.25

+ 0.3

6.96

+0.49

39.66

+2.30ab

5.76

+0.40

10.0 27.50

+0.57

7.56

+0.56

41.33

+4.05b

5.26

+0.20

20.0 26.48

+0.24

7.24

+0.42

43.42

+2.92

4.98

+0.28

30.0 27.15

+0. 6

7.26

+0.11

41.38

+3.05b

5.63

+0.57

15.0 27.60

+0.52

7.76

+0.25

33.33

+3.78a

5.63

+0.20

30.0 25.74

+0.63

6.93

+0.22

43.88

+3.33

4.68

+0.33

45.0 27.00

+ 0.0

7.13

+0.32

44.66

+5.86c

6.63

+0.28

20.0 27.23

+0.23

7.60

+1.38

42.33

+2.76b

5.63

+0.60

40.0 26.64

+0.73

7.29

+0.13

44.14

+0.73

4.24

+0.92

60.0 26.90

+ 1.1

7.30

+0.42

38.00

+11.87a

5.86

+0.40

25.0 27.23

+0.28

8.16

+0.05

44.33

+0.56cd

5.76

+0.60

50.0 25.32

+0.22

6.98

+0.20

44.28

+3.20

4.49

+0.73

75.0 27.10

+ 0.2

6.96

+2.09

48..22

+5.29c

5.93

+0.59

30.0 27.40

+2.46

8.36

+0.72

46.02

+0.00d

5.03

+0.89

60.0 25.42

+0.32

7.58

+0.62

44.31

+4.82

4.44

+0.37

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Figure 2; Lymphocytes counts in Clarias gariepinus sub-adults treated with Phyllanthus

muellarianus root extract after exposure to 2, 4Dimethyl Amine. There were no significant

differences between the means of lymphocytes counts (α = 0.05)

Figure 3; Granulocyte counts in Clarias gariepinus sub adult subjected to different levels of

Phyllanthus muellarianus root extract after exposure to 2, 4-Di-methyl Amine. There were

significant differences between means (α = 0.05)

b

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Figure 4; Erythrocyte sedimentation rate in in blood Clarias gariepinus sub-adults treated with

Phyllanthus muellarianus root extract after exposure to 2, 4-Dimethyl Amine. There were no

significant difference between the means (α = 0.05)

Figure 5; Plasma protein (g/dl) concentrations in Clarias gariepinus sub-adult subjected to different

levels of Phyllanthus muellarianus root extract, after exposure to 2,4-Dimethyl Amine. There were

no significant differences between means (α = 0.05)

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Figure 6; Changes in the red blood cells counts of Clarias gariepinus sub-adults to remediation attempt

using Phyllanthus muellarianus root extract after exposure to 2,4-Dimethyl Amine. There were

significant differences between the means (α = 0.05)

Figure 7; Haemoglobin (g/dl) in Clarias gariepinus sub-adult subjected to different levels of

Phyllanthus muellarianus root extract, after exposure to 2,4-Dimethyl Amine. There were no

significant differences between means (α = 0.05)

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Figure 8; Changes in mean cell volume (fl) of Clarias gariepinus sub- adults, to remediation attempt

using Phyllanthus muellarianus root extract after exposure to 2, 4-Dimethyl Amine. There were

significant differences between the means (α = 0.05)

Figure 9; Mean cell haemoglobin (pg) in Clarias gariepinus sub adult subjected to different

concentrations of Phyllanthus muellarianus root extract after exposure to 2,4 Di- Methyl Amine.

There were significant differences between means (α = 0.05)

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Figure 10; Mean cell haemoglobin concentration (g/dl) in Clarias gariepinus sub adult subjected to

different levels of Phyllanthus muellarianus root extract, after exposure to 2, 4 Di-methyl Amine.

There were significant differences between means (α = 0.05)

Figure 11; Platelet counts in Clarias gariepinus sub adult subjected to different levels of Phyllanthus

muellarianus root extract after exposure to 2,4 Di- Methyl Amine. There were significant

differences between means (α = 0.05)

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Figure 12; Albumin level in Clarias gariepinus sub- adults, to remediation attempt using

Phyllanthus muellarianus root extract after exposure to 2,4 Di methyl Amine. There were

significant differences between the means (α = 0.05)

Figure 13; Mean Platelet Volume (fl) of Clarias gariepinus sub- adults, in remediation attempt using

Phyllanthus muellarianus root extract after exposure to 2,4 Di methyl Amine. There were

significant differences between the means (α = 0.05).

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Figure 14 Heamatocrit or packed cell volume in different levels of treatment and that when exposed

to 2, 4-Dimethyl Amine. No significant difference were observed between the means of different

treatments

DISCUSSION

According to Fondriest Environmental (2018), „Conductivity measures the water‟s ability to conduct

electricity‟. She further explained that pure water is a poor conductor of electricity. Impurities like salts

and other inorganic chemicals dissolve in water form electrically charged particles called ions. These

increase the water‟s ability to conduct electricity. Dissolved salts and other inorganic chemicals increases

conductivity as salinity increases. But organic compounds, such as sugars, oils, and alcohols, do not form

ions that conduct electricity. In this our experiment, 2, 4-Dimethyl was observed to cause change in

conductivity of the water. During treatment where Phyllanthus muellarianus was used, the conductivity

did not show any variation, suggesting that the different components which caused erythropoiesis were

mostly organic in nature. Other water quality parameters such as dissolved oxygen concentration, pH and

water temperature did change with treatments.

The total white blood cells count increased significantly when the fish was exposed to the herbicide, 2, 4-

Dimethyl amine. Upon treating the fish with Phyllanthus muellarianus aqueous root extract, the total

white blood cell count reduced to equate with the natural population. Increase in white blood cells is a

usual response in organisms to toxins, however. As shown in Figure 1, white blood cells population of

fish from the natural environment was 3.19 million per litre. Upon exposure to 2, 4-Dimethyl Amine, the

blood population increased to 15.91 million cells per litre. The increase in the white blood cells

population is due to the introduction of the herbicide (poison). This is always a response of white blood

cells to fight, detoxify toxic proteins and to develop immunity in the body of an organism (Ahmed et al.,

2014).

Lymphocytes function in producing antibodies, and mediate cellular and hormonal immune responses

Abbas et al. (2010). Though there were no significant increase in lymphocyte numbers (Fig 2) across

treatments in the present study, the observed insignificant changes could be attributed to

immunostimulatory role of Phyllanthus muellarianus root extracts. This phenomenon was observed in the

works of Aly et al. (2008) and Siyavash et al. (2016) .

The red blood cells kept increasing with increase in concentration of root extract. Though red blood cells

participate in conferring immunity to organisms, they are not directly involved in fighting. The red blood

cells involvement in the fight is that of transporting oxygen, nutrients and wastes for the fish‟s optimal

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physiological functions (Taylor et al. 1097; Ochei and Kolhatkar, 2000). The elevation of white blood

cells population is attributable to two factors namely; due to replication (Ochei and Kolhatkar, 2000) and

the release of marginal cells into the blood stream during toxin attack (Barker et al. 2001). As Ajiboye et

al. (2017) stated, „dead cells release chemicals which initiate inflammatory response leading to the

activation of Kupffer cells‟ in the liver and recruitment of neutrophils and monocytes.

Mean cell volume, mean cell haemoglobin and mean cell haemoglobin concentration (Figures 8, 9 and

10) were lower when exposed to high concentrations of Phyllanthus muellarianus. These parameters are

derivatives of red blood cells count (Figure 6). The red blood cells population was higher at this higher

concentration of Phylanthus muellarianus extract. That shows that the extract was able to cause

multiplication of red blood cells (erythropoiesis). With higher numbers of cells in the volume invariably

means that individual cell volume is reduced and carry less amount of haemoglobin per cell. It is seen that

the haemoglobin in g/dl (Figure 7) was not significantly different among treatments. The less quantity of

haemoglobin per cell was compensated for by the higher cell numbers carrying haemoglobin. Similar

trend was observed in platelet count (Figure 11) and individual size (Figure 13). This dynamics of

younger cells with smaller size having being a result of rapid replication of the older and larger cells was

explained by Baker et al. (2001). Ada et al. (2015) had observed the haemopoietic potential of this plant,

Phyllanthus muellarianus in Clarias gariepinus. Ajiboye et al. (2017) observed that the plant leaf

aqueous extract do attenuate or retard antioxidant enzymes activities in this fish.

It is known that Erythrocyte Sedimentation increases during inflammation and increase or abnormal

immunoglobulin. But plasma albumen concentration shows the Erythrocyte Sedimentation Rate (Baker,

et al, 2001). In our experiment, Figures 4 and 5 show that protein and erythrocyte sedimentation rate

followed similar pattern of decrease or increase which were however not significant among treatments.

However, physical observation shows that they were both decreasing with concentration. Baker et al.

(2001) explained that albumen concentration has inverse relationship with erythrocyte sedimentation rate

as was observed in this experiment. Albumin level in this treatment is relatively higher than that of the

control. Similar report was reported by Bello et al. (2014) when Clarias gariepinus were fed Walnut

leaves Tetracarpidium conophorum and Onion bulbs Allium cepa Linn. They reported the increase in

Albumin level to have been an immune response to certain constituents of the extracts.

Phyllanthus muellarians has been used extensively for human medicine. It is haematopoietic. This study

has revealed that it could be used to cure some blood disorders in fish also. For efficient growth and

metabolism especially during therapy, root extract of this plant could be used for recovery of damaged

blood tissue in fish.

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