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Second Quarter E-Book Volume 2: April-June 2017

ISSN: 2536-7064

Journal of Bioscience and Biotechnology Discovery

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Journal of Bioscience and Biotechnology Discovery (JBBD) (ISSN: 2536-7064) is an Open Access, Peer-Review Journal that publishes original and high-quality research articles in all areas of the subject such as Biotechnology, Microbiology, Bioinformatics, Genetics, Biochemistry, Botany, Molecular Biology, Enzyme Engineering, Ecology, Virology, Zoology, Pathology, Physiology etc. The Journals is committed to advancing and sharing creative, innovative and emerging ideas that are beneficial to the field of Biosciences and Biotechnology. Journal of Bioscience and Biotechnology Discovery publishes per article as soon as the manuscript is accepted and e-book every quarter. All published articles and E-Books are freely accessible on our website. Hard copies of the E-book will be made available to the interested authors on request. Editorial Office: [email protected] Customer Care: [email protected] Submit Articles: [email protected] Website: http://www.integrityresjournals.org

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Dr. Awatief Fahmey Hifney Department of Botany and Microbiology Faculty of Science, Assiut University Assiut 71516, Egypt Dr. Abd El-karim Mohammed Abd El-Latif Department of Zoology Faculty of Science, Fayoum University P O Box 63514, Egypt Dr. Muhammad Irfan Department of Zoology Faculty of Life-Sciences University of the Punjab Lahore, Pakistan Prof. Sergio Ruffo Roberto Agricultural Science Center Department of Agronomy Londrina State Univeristy, CCA - Campus Londrina, PR, BRAZIL Assist. Prof. Mustafa SAKALDAŞ Department of Horticulture and Postharvest Physiology Faculty of Agriculture Çanakkale Onsekiz Mart University Lapseki- Çanakkale, Turkey Assoc. Prof. Manal Eid Department of Botany Faculty of Agriculture Suez Canal University P.O. Box: 41522-Ismailia, Egypt Prof. Keerikkattil Paily Joy INSA Senior Scientist Department of Biotechnology Cochin University of Science and Technology Kerala, India

Editors

Table of Content: Volume 2: April – June 2017

Articles Pages

Biological control of pathogenic and secondary (non pathogenic) fungi associated with Barley (Hordeum vulgare) seeds Ihsan Flayyih Hasan AI-Jawhari

10-14

Chemical composition and medicinal potentials of the bark of Erythrophleum ivorense A. Chev R. O. Ogboru, L. O. Akideno and E. A. Owoeye

15-20

Biochemical and haematological safety evaluation of the ethanol extract of the stem bark of Blighia sapida using Swiss albino mice Chinweizu Ejikeme Udobi, Betseabasi Edward Umoh and Ememobong Gideon Asuquo

21-28

Physicochemical properties of a flowing stream in Jabi, Abuja, Nigeria Solomon, R. J. and Kehinde, O. A.

29-35

Assessment of heavy metal concentration in vegetables grown within fungicide treated Cocoa plantations in Akamkpa Local Government Area, Cross River State, Nigeria and implications to human health F. E. Ekpo.

36-41

The resultant physiochemical effect of organic, artificial and mixed diets of Clarias gariepinus to enhance growth Solomon, R. J. and Zainab, A. U.

42-48

Table of Content: Volume 2: April – June 2017

Articles Pages

Growth respond and nutrient utilization of Clarias gariepinus fed bean cake Chiokwe, G. I. and Solomon, R. J.

49-62

The effects of dietary, energy and feed utilization of compounded feed (vital chicken feed and fish meal) on the growth rate of Clarias gariepinus Solomon, R. J. and Adeola, A. A.

63-73

Modulatory properties of Telfairia occidentalis leaf extract on pancytopenia, electrolyte imbalance and renal oxidative damage in rats Johnson Olaleye Oladele, Olu Israel Oyewole, Olamilekan Kabir Bello And Oluwaseun Titilope Oladele

74-78

Journal of Bioscience and Biotechnology Discovery Volume 2. Page 10-14. Published 4th April, 2017

ISSN: 2536-7064: Article Number: JBBD-06.03.17-028 www.integrityresjournals.org/jbbd/index.html

Full Length Research

Biological control of pathogenic and secondary (non pathogenic) fungi associated with Barley (Hordeum

vulgare) seeds

Ihsan Flayyih Hasan AI-Jawhari

Department of Environment and Pollution, Marshes Research Centre, Thi-qar University, Iraq. Email: [email protected].

Copyright © 2017 AI-Jawhari. This article remains permanently open access under the terms of the Creative Commons Attribution License 4.0, which

permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received 6th March, 2017; Accepted 29th March, 2017

ABSTRACT: Plant pathogenic fungi are a major problem in agriculture with effects on yield and quality of agricultural product. In this study, antagonistic effects of five fungi biocontrol agent Aspergillus niger, Aspergillus ostianus, Aspergillus versicolor, Penicillium sp., Trichoderma harzianum and one species of bacterium Pseudomonas aeruginosa isolated from the seeds of barley were evaluated against plant pathogenic fungi Rhizoctonia solani and secondary (non-pathogenic) fungi Aspergillus flavus. The ability of isolated microorganisms in antagonizing or inhibiting the growth of phytopathogenic fungi was tested by measuring the growth inhibition percentage over control. P. aeruginosa obtained higher inhibition with R. solani and A. flavus (88%, 76%) respectively, but A. ostianus obtained the lower inhibition with R. solani and A. flavus (59%, 53%) respectively. The results also showed that significant differences were recorded in mycelial growth of pathogens in presence of biocontrol agent when compared with control. Also, the results showed that non-significant differences were recorded between R. solani and A. flavus when treated with different antagonisms. Key words: Bacteria, phytopathogenic, antagonism, competition, pollution, fungicides. INTRODUCTION Fungal plant pathogens are the most important factors that cause serious losses to agricultural products annually. Fungicides are commonly used to control the diseases in plants. Modern agriculture heavily depends on the application of agrochemicals for fertilizing soils and diseases control. However, the over use of chemical compounds poses potential risk to human health and the environment and can also lead to resistance in causal agents (Akintokun and Taiwo, 2016). It is always better to adopt biological method as an alternative disease control method in order to reduce the hazards, which is also ecology conscious and eco-friendly. Antifungal compounds produced by microorganisms may be used as biocontrol agents. Some soil borne fungi, bacteria and actinomycetes have been identified and used as antagonistic microbes. A number of bacterial species have been tested as biocontrol agents. Antifungal metabolites produced by bacteria like Pseudomonas

spp., Bacillus spp. (Moita et al., 2005; Siddiqui et al., 2005; Nourozian et al., 2006; ChristyJeyaseelan et al., 2012). For instance Pseudomonas fluorescens used against Rhizoctonia solani and Pythium damping off of cotton and Bacillus used for seeds treatment (Agrios, 2005). The mechanisms underlying these bacterial antagonisms for plant pathogens involve antibiotics, competition for nutrients or space, enhancement for root and plant development, induction of plant resistance and or inactivation of the pathogen's enzymes (Harman, 2000). Cereals can be very susceptible to toxigenic fungal growth in the field, during storage or during processing. Their presence in stored products can significantly decrease quality and economic value of the harvested grain (La Penna et al., 2004) . The purpose of this paper was to study the activity of A. niger, A. ostianus, A. versicolor, Pencillium sp, T. harzianum and P. aeuginosa isolated from barley seeds against R. solani

http://creativecommons.org/licenses/by/4.0/

and A.f lavus.

MATERIAL AND METHODS

Isolation and Identification of Fungal isolates

The fungi strains used in this study were isolated from barley seeds. Barley seeds were collected from local market in AI-Nasiriya city (South of Iraq). Potato Dextrose Agar (PDA) supplemented with 250 mg/L Chloramphenicol to suppress bacterial growth was used to isolate fungi from the seeds. Plates and media were incubated at 25

oC in the dark. Single colonies were

picked from the plates under a dissecting microscope and transferred to PDA medium to allow fungal development. Stock cultures were maintained on the potato dextrose agar slant, subcultured periodically until pure cultures were gotten and stored at 4

oC. Fungal isolates were

examined under light microscope and morphologically identified using Pictorial atlas of soil and seed fungi provided by Watanabe (2000).

Isolation and Identification of Bacterial isolates

The bacterium used in this study was isolated from barley seeds. Nutrient agar supplemented with 30 µg/L Nystatin to suppress fungi growth was used to isolated bacteria from the seeds. Plates and media were incubated at 37

oC

in the dark. Stock cultures were maintained on nutrient agar, subculture periodically until pure cultures were gotten and then stored at 4

oC. Bacterial isolates were

examined under light microscope and identified using morphological characters, gram stain, biochemical tests and API20E (Holt et al.,1994) and taxonomical keys provided in the bacteriological keys (Reddy, 2010; Bergy's and Holt, 1994).

Antagonistic assay (dual culture method)

The isolated bacterium was streaked as a thick from edge to center of PDA plates. Then a 4 mm diameter disc of pathogenic fungus R. solani was cut from an actively growing culture by a sterile cork borer and placed onto the center of PDA plates. The petri dishes were incubated at 25

oC in incubator in dark till the complete

growth was observed in control plates. The same procedure also was carried out for A. flavus. The antagonistic effect of five fungi A. niger, A. ostianus, A. versicolor, Penicillium sp and T. harzianum with R. solani and A. flavus separately were recorded and the percentage growth inhibition compared with control were calculated according to the formula given by Vincent (1927). The experiments were carried out in three replicates.

I =𝑅1 − 𝑅2

𝑅1 𝑋 100

AI-Jawhari 11 Where: I = percentage inhibition of mycelia growth, R1 = Mycelial growth in control and R2 = Mycelial growth in treatment. Preparation of filter of antagonistic fungi and bacteria 50 mL of Potato Dextrose Broth (PDB) was transferred to 250 mL conical flasks and sterilized with 121

oC

, 15

' Ib

' at

15 mins by using an autoclave. This was followed by the addition of a 4 mm diameter disc of antagonistic fungi from 7 days old culture separately. Also, the procedure was carried out with antagonistic bacteria of 2 day old culture, but control treatment flasks were contained (PDB) without any microorganisms. All flasks were placed in an incubator in the dark at 25

oC

for two weeks.

At the end of incubation, the extracts were filtered through sterilized Buchner funnel under aseptic conditions and the filtrate was further filter through 0.45 µm sterilized Millipore filter paper. Effect of antagonistic filter of fungi and bacteria on a diameter of mycelia growth of R. solani and A. flavus The filtrate of antagonistic fungi and bacteria of about 10% was added to PDA medium. After mixing the filtrate and medium, the mixture was poured in sterilized petri dishes, while the control treatment only 10% from PDB was added to PDA . Then a 4 mm diameter of each mycelia disc was cut using a sterile cork borer and placed in the center of the PDA plates separately under aseptic conditions. Mycelia disc from R. solani and A. flavus on PDA medium without antagonistic fungi and bacteria were used as control. The culture were incubated in incubator in the dark for 7 days and diameter of the fungal mycelia growth was measured (Matar et al., 2009). The experiments were carried out in three replicates. Statistical analysis The results of the antagonistic activity were subjected to statistical analysis using analysis of variance ANOVA test with the aid of software SPSS windows version 10.0.

RESULTS AND DISCUSSION Effect of antagonistic organisms on mycelia growth of pathogenic and secondary fungi The antagonistic effect of fungi and bacteria isolates was carried out by using five isolates including A. niger, A. ostianus, A. versicolor, Penicillium sp and T. harzianum and one isolate of P. aeruginosa. They were all tested against R. solani and A. flavus. After a week of incuba-tion, the growth of targeted fungal pathogens towards and

12 J. Biosci. Biotechnol. Discov.

Table1. Effect of antagonistic organisms on mycelia growth of pathogenic and secondary fungi after 7days incubation.

Biocontrol agent Pathogens Mycelia growth in control (cm)

Mycelial growth of

pathogen in presence of

biocontrol agent (cm)

% inhibition of mycelia growth over

control

Aspergillus niger R. solani 8.5 2.5* 71

A. flavus 8.5 3.0* 65

A. ostianus R. solani 8.5 3.5* 59

A. flavus 8.5 4.0* 53

A. versicolor R. solani 8.5 3.0* 65

A. flavus 8.5 2.5* 71

Penicillium sp R. solani 8.5 2.0* 76

A. flavus 8.5 3.0* 65

Trichoderma harzianum R. solani 8.5 3.5* 59

A. flavus 8.5 2.0* 76

Pseudomonas aeruginosa R. solani 8.5 1.0* 88

A. flavus 8.5 2.0* 76

*Significance P< 0.05.

away from the antagonistic fungi isolates were recorded. The results showed that significant differences were recorded in mycelial growth of pathogens in presence of biocontrol agent when compared with control (Table 1).

The percentage inhibition of mycelia growth compared with control was tabulated. All antagonistic fungal isolates and P. aeruginosa significantly inhibited the mycelia growth of pathogens. The highest inhibition of mycelia growth was recorded for P. aruginosa against R. solani (Table.1). These results were in agreement with the findings of (Shaikh Farah and Sahera, 2016). They showed that both bacterial isolates of P. aeruginosa and Bacillus subtilis could inhibit the mycelia growth of R. solani and Fusarium oxysporum (Tharmila et al., 2013). Also, Tharmila et al. (2013) showed that Pseudomonas maltophila had the highest antifungal activity against all tested fungi on PDA medium. This antagonistic ability was due to the production and secretion of antifungal compounds that was able to reduce the growth of fungi. Also, P. aeruginosa can produces antibiotics such as HCN, phycocyanin, pyrolnitrin and pseudomonic acid. The results (Table 2) showed that the filtrate of T. harzianum decreased the growth of R .solanion solid medium (PDA) to 4.03 cm and the inhibition percent reached to 59% (Table.1). The results also showed that non-significant differences were recorded between R. solani and A. flavus when treatment with different antagonisms (Table 2). These results were in line with the findings of (Harman et al., 1989) which showed that the only T. harzianum isolate tested was able to exhibit the wide spectrum of inhibition. However T. harzianum and T. koningii have also been applied in soil, and on cowpea leaves, as a biocontrol agent against Rhizoctonia

solani on cotton in a greenhouse environment (Lartey et al.,1994; Latunde,1991), and against wood degrading fungi (Canessa and Morrell, 1996). Both T. viride and T. harzianum are recognized biopesticides mainly against Rhizoctonia, Sclerotinia and Botrytis (Lewis and Papavizas, 1991). Under in vitro studies R. solani, Pythium ultimum and Chalara alegans were strongly inhibited by T. viride, T. harzianum, T. pseudokoningii and T. koningii. These results indicate that the biocontrol efficacy of Trichoderm aseems to perform not only in the medium, but also in the field level (Marchetti et al., 1992). Mycoparasitism involving lytic enzymes have been already described as the mechanism of action of Trichoderma isolates in the biological control of commercial important plant pathogens (Bruce et al., 1995). Trichoderma employs a variety of antagonistic mechanisms for combating other fungi. The simplest one is probably competition for non - structurally - bound nutrients, however, volatiles and soluble antifungal metabolites are also involved (Horvath et al., 1995). These metabolites are composed of harzianic acid, alamthicins, tricholin, peptaboles, antibiotics, 6-phenyl-alpha-pyron, massoilactone, viridian, glioviridin, glisoprenins, heptelidic acid, and other suppressive compounds (Vey et al., 2001; Kucuk and Kivanc, 2004). Also, T. virens strain, that produced high amount of glioviridin antibiotics, protected cotton seedlings from seedling blight caused by P. ultimum (Chetet al., 1997). The most effective T. harzianum isolates against take-all agent produced pyron antibiotic (Monte, 2001). In the same time, general PDB was a more suitable medium for the production of antibiotic compounds (Santamarina et al., 2002).

Table 2. Effect of filter of antagonistic organisms on mycelia growth of Rhizoctonia solani and A.niger on solid medium.

Biocontrol agent R.solani A.flavus

Aspergillus niger 3.50* 4.2

A .ostianus 3.5 ** 3.24

A .versicolor 4.17 ** 5.5

Penicillium sp 2.5** 2.9

Trichoderma harzianum 4.03** 2.5

Pseudomonas aeruginosa 3.17** 1.2

Control 8.5 8.0

*(cm), ** Non significance P< 0.05.

Effect of filter of antagonistic organisms on mycelia growth of R.solani and A.flavus

Table 2 showed that the filtrate of Penicillium sp decreased the mycelia growth of R. solani and A. flavus on PDA plates. This result was due to the release of antibiotic by fungus. These results were in agreement with the findings of Jijakli and Lepoivre (1999) which showed that Penicillium guillermondii was able to degrade fungal cell walls by producing enzyme β- 1,3 glucanase .

It also shown from Table 2 that A. niger, A. ostianus and A. versicolor decreased the mycelia growth of R. solani and A. flavus on solid medium (PDA). This was due to the ability of these fungi to have higher growth and to compete with other fungi on PDA. These results were similar to the findings of Ikotum and Agboola (1992) which showed that A. niger had ability of high growth and compete with Curvularia lunata. However these ability might also be due to the possibility that these fungi produced toxin materials, and these toxins were able to inhibit pathogenic fungi. Also, the present study was similar to the findings of Egorov (1985) which showed that the genus of Aspergillus produces many antibiotics such as fumagillin and griseofulavin. These antibiotics have a wide spectrum of action against microorganisms.

Conclusion All fungi isolated in this study had antagonistic ability against pathogenic fungal strains and secondary fungi. P. aeruginosa showed the highest inhibitory effect on mycelia growth and with an inhibition percentage up to 88%. According to our results, it is concluded that P. aeruginosa is the best biocontrol agent against fungal pathogens associated with barley seeds. This is due to it possibility to reduce and control the environmental pollution by reduction of chemical treatments. It should persuade the farmers to choose biological control.

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Chemical composition and medicinal potentials of the bark of Erythrophleum ivorense A. Chev

R. O. Ogboru*, L. O. Akideno and E. A. Owoeye

Forestry Research Institute of Nigeria, Moist Forest Zonal Research Station, P. M. B 2444, Benin-city, Edo- State, Nigeria.

*Corresponding author. Email: [email protected], [email protected]

Copyright © 2017 Ogboru et al. This article remains permanently open access under the terms of the Creative Commons Attribution License 4.0, which


Received 3rd February, 2017; Accepted 20th March, 2017

ABSTRACT: Determination of the chemical composition of the bark of Erythrophleum ivorense A.chev showed some proximate compounds such carbohydrates, protein, lipids and vitamin C, and not absent in essential food fibre. Essential minerals of sodium, phosphorus, iron, copper and zinc in contents levels needed for a healthy life, except sodium, with a very high content level hence should be diluted properly when administered as a laxative. Some phytochemicals such as phenols, steroids, tannins, flavanoids, alkaloids, saponin and cadiac glycosides were present which gives credence to the medicinal benefits of the bark of the wood despite it wide timber used for construction and toxic nature. Key words: Erythrophleum ivorense bark, phytochemical screening, proximate content, mineral elements, total antioxidant capacity. INTRODUCTION The role of forest in our atmosphere cannot be over emphasized. The necessity to study forest plant species according to WHO (2001) was based on extensive use of plant portions in folk medicine and offered a basis for the daily living of the people. A large number of African indigenous trees species have been recognized to have health defensive properties and uses (Okeno et al., 2003; Einosho and Ayorinde, 2008). Erythrophlem ivorense belongs to the family Caesalpiniacae (Leguminosae- Caesalpinioideae) and is found majorly in the Gambia to the Central African Republic and Garbon. The timber of Erythrophleum Ivorense in Nigeria, is marketed as ‘Iyin’ in Edo, ‘erun’ in yoruba and ‘ihi’ in Igbo (Aigbokhan, 2014). It is called ‘Ordeal tree’, ‘missanda’, ‘alui’, ‘bolondo’, ‘Sasswood tree’(English). Lim du Gabon, Tali (France) and Mancone (Italian) aroundparts of Africa where it is found (Burkill, 1985). Some species of plant in the Erythrophleum genus are also identified to be venomous and toxic to some livestock. The bark of Erythrophleum ivorense traded as ‘sassy-bark’, ‘man cona bark’, ‘casca bark’ or ‘ecorce de tali’ and has several medicinal uses (ITTO, 2004). Bark extracts of Erythrophleum ivorense are taken orally in Sierra Leone as an emetic and purgative and is applied externally to relieve pain (Richter

and Dallwitz, 2000). According to Betti (2004), it is taken orally as a laxative and outwardly to relieve pains. In Ivory- Coast, extract from young branches of crushed Erythrophleum ivorense is rubbed on skin to treat chicken pox (PROTA1, 2008). The bark and occasionally the seeds are widely used as hunting and ordeal poison (Barkarr and Janos, 1996). In Liberia and Garbon, the bark of Erythrophleum ivorense is preferred to that of Erythrophleum suaveolens (Bosch et al., 1996). Erythrophleum ivorense occurs scattered in evergreen primary and secondary forest and moist semi-deciduous forest (Diabate et al. 2005). It has been classified as a non-pioneer light demander. Seedlings are often found in smaller forest gaps. Erythrophleum ivorense can be propagated in the nursery as its seeds take three weeks to germinate. The bark is also used as fish poison in Sierra Leone (PROTA, 2008). The wood is quite hard and hefty and suitable for woodwork, flooring, rail way sleepers, harbor and dock work, turnery, bridges and constructions. It is also used for boat building and wheel hubs. It makes excellent charcoal and good firewood. In Seirra-Leone and Ivory Coast the bark is used for tanning (PROTA, 2008). A bark decoction added to fermenting palm wine would make it a more potent drink (Voorhoeve, 1979). In trade


16 J. Biosci. Biotechnol. Discov. statistics, Erythrophleum ivorense is regarded as the fourth most important timber of Cameroon and was imported to China in 2005 (PROTA, 2008). It is an evergreen tree that can grow up to 40 metres tall, bole cylindrical but sometimes fluted at base with or without buttresses. Its leaves are alternate and flowers are bisexual. The toxicological nature of the specie has been reported by Amoah et al. (2014). They worked on methanol extract on the bark and were administered to rats which caused an increase inflammatory in the liver and kidney of the rats. The phytochemical screening of the plant also revealed the presence of alkaloids, tannins, flavonoids, steroids, cardiac glycosides and terpenoids but they were not quantified. Also Sima et al. (2016) worked on phytochemical screening identifying the phytochemicals, antioxidant and antimicrobial properties of the bark of Erthrophleum ivorense A. Chev. Hence, this research work quantified the exact amount of the phytochemicals present contained in the bark Erythrophleum ivorense and the proximate analysis to show if it is fit for consumption. This study therefore attempts to identify the chemical compositions contained in the bark of Erythrophleum ivorense through proximate analysis, phytochemical screening, mineral analysis and total antioxidant capacity.

MATERIALS AND METHODS Collection of plant samples Bark samples of Erythrophleum ivorense A. Chev were obtained from the Federal Department of Forestry, Benin City, Edo State, Nigeria in October, 2016. It was authenticated by Mr Emmanuel Isebemhe, the taxonomist of the moist Forest Research station. The Bark sample was air-dried for one month at the research station, Benin City and pulverized to powder and sent to the Delta State University Chemistry Laboratory in Abraka for the chemical analysis. Preparation of aqueous extract of plant samples The aqueous extract of bark of Erythrophleum ivorense sample was primed by soaking 10 g of powdered samples in 200 ml of distilled water for 12 hours. The extracts were tried for carotenoids, terpenoids, alkaloids, tannins, flavanoids, saponin, cardiac glycoside, anthraquinone, phenols, polyphenols and steroids Phytochemical analysis Biochemical tests were conducted on the aqueous extract of the bark sample by using standard methods adopted by Sofowora (1993), Harbone (1973), Oyoyede (2005), Ogboru et al. (2015) and Owoeye et al. (2016).

Qualitative analysis on phytochemical constituents Test for tannins 0.5 g of powdered sample was boiled in 20 ml of distilled water in a test tube and then filtered. The filtration method used here is the normal method, which includes a conical flask and filter paper. 0.1% of FeCl2 was added to the filtrate and observed for brownish green or a blue black coloration, which shows the presence of tannins.

Test for saponins 2 g of powdered bark sample was boiled together with 20 ml of distilled water in a water bath and filtered. 10 ml of the filtrate was mixed with 5 ml of distilled water in a test tube and shaken vigorously to obtain a stable persistent froth. The frothing was then mixed with 3 drops of olive oil and observed for the formation of emulsion, which indicates the presence of saponins.

Test for flavonoids A few drops of 1% NH3 solution was added to the aqueous extract of the bark sample in a test tube. A yellow coloration was seen indicating flavonoids compounds was present.

Test for cardiac glycosides 1 ml of concentrated H2SO4 was prepared in a test tube. 5 ml of aqueous extract from the bark sample was mixed with 2 ml of glacial CH3CO2H containing 1 drop of FeCl3. The above mixture was carefully added to the 1 ml of concentrated H2SO4 so that the concentrated H2S04 was underneath the mixture. A brown ring appeared, indicating the presence of the cardiac constituent.

Test for phenols

To 3 ml of extract, 3 ml of lead acetate solution was added. The occurence of white precipitates indicates the presence of phenols.

Test for alkaloids

To 3 ml of filterate, 1 ml of Hagers reagent (Saturated picric acid solution) was added. The appearance of yellow precipitate shows alkaloids present.

Test for steroids

2 ml of acetic anhydride was added to 0.5 g extract with 2 ml of sulphuric acid and observes for the colour change

from violet to blue or green in samples indicating the presence of steroids.

Test for terpenes

0.5 g of plant extract in a test tube was added with 2 ml of chloroform and 5 ml of concentrated sulphuric acid carefully. A reddish brown interface to show positive result for terpenes.

Test for anthraquinones

0.5 g of plant extract is shaken with 10 ml of benzene and filtered. 5 ml of 10% ammonia is added to the filterate. The mixture is shaken and the presence of pink, red or violet colour indicates the presence of anthraquinones.

Quantitative analysis on phytochemical constituents Phenols

The quantity of phenols was determined using the spectrophotometer method. The fat free plant sample was boiled with 50 ml of Ether (CH3CH2)2O) for 15 mins. 5 ml of the boiled sample was then pipette into 50 ml flask, and 10 ml of distilled water was added. After the addition of distilled water, 2 ml of NH4OH solution and 5 ml of concentrated CH3(CH2)CH2OH was added to the mixture. The sample was made up to the mark and left for 30 mins to react for colour development and measured at 505 nm wavelength using a spectrophotometer.

Cardiac glycosides

The Baljets reagent method was used (95 ml of 1% picric acid + 5ml of 10% NaOH). 1 g of powdered sample was soaked overnight with 70% ethanol and filtered. The extract was purified by lead acetate and Na2HPO4 and 1 ml of freshly prepared Baljets reagent was added. The solution was put in the curvette and read at 495 nm in the UV spectrophotometer.

Alkaloids

5 g of plant sample was prepared in a beaker and 200 ml of 10% CH3CO2H in C2H5OH was added to the powdered plant sample. The mixture was covered and allowed to stand for four hours. The mixture was then filtered and the extract was allowed to become concentrated in a water bath until it reaches ¼ of the original volume. Concentrated NH4OH is added until the precipitation is complete. The whole solution is allowed to settle and the precipitate was collected and washed with dilute NH4OH and then filtered. The residue is alkaloid which is then dried and weighed.

Ogboru et al. 17 Tannins Quantity of tannins was determined by using the spectrophotometer method. 0.5g of plant sample is weighed into a 50 ml plastic bottle. 50 ml of distilled water is added and stirred for 1hour. The sample is filtered into a 50 ml volumentric flask and made up to mark. 5ml of the filtered sample is then pipette out into test tube and mixed with 2 ml of 0.1M FeCL3 in 0.1M HCl and 0.008MK4Fe(CN)6.3H2O. The absorbance is measured with a spectrophotometer at 395nm wavelength within 10mins. Saponins 20 g of powdered back of plant sample was put in a cornical flask and 100 ml of 20% C2H5OH was added to the plant sample. The sample was heated over a hot bath for 4 hours with continous stiring at about 55

0C. The

mixture was then filtered and the residue re-extracted with another 200 ml of 20% C2H5OH. The combined extracts were reduced to 40 ml over a water bath at about 90

0C.

The concentrated solution was then transferred into a 250 ml separator funnel and 20 ml of (CH3CH2)2O was added to the extract and shaken vigorously. The aqueous layer was recovered while the (CH3CH2)2O layer was discarded and the purification process was repeated twice. 60 ml of n-C4H9OH was added and the combined n-C4H9OH extracts was washed twice with 10 ml of 5% Nacl. The remaining solution was then heated in a water bath and allowed to evaporation. After the evaporation, the samples were dried in the oven to a constant weight.

Flavonoids 10 g of the plant sample was repeatedly extracted with 100 ml of 80% aqueous methanol at room temperature. The entire solution was then filtered through filter paper and the filterate was transferred into a water bath and the solution was evaporated into dryness. The sample was then weighed until a constant weight was obtained.

Proximate composition determinations The proximate composition of the bark of Erythrophleum ivorense A.Chev was determined using the methods of the AOAC (1990). These analysis included carbohydrate, crude protein, moisture content, ash, crude fibre, fat and vitamin C.

Antioxidant analysis Total Antioxidant capacity was determined by the Phospho-molybdenum method according to the procedure


Table 1. Phytochemistry.

Phytochemicals Qualitative Remarks Quantitative remarks(mg/kg)

Tannin + 31.17+0.04

Flavanoid + 33.76+0.25

Alkaloid + 58.89+0.20

Saponin + 41.63+0.11

Cardiac glycoside + 37.45+0.16

Anthraquinon - -

Phenolic + 10.08+0.38

Terpenes - -

Carotenoid - -

Steriods + 06.03+0.02

(+) Present (-) Absent.

described by Prieto et al. (1991). This was also adopted by Vernon et al. (1999), Zongo et al. (2010) and Ogboru et al. (2016). Total Phenols were determined using the method described by Obadoni and Ochuko (2001). For the extraction of phenolic content, the fat free sample was boiled with 50 ml of ether for 15 mins. 5 ml of the extract was pipette into a 50 ml volumetric flask and then 10 ml of distilled water was added. The samples were made up to the mark and left to react for 30 mins for colour development. The absorbance at 505 nm was read at a UV-spectrophotometer.

Mineral element analysis

Milled samples (5 g) were burnt to ash in a furnace at 550

0C for 24 hours. The resulting ash was cooled in a

desiccator and weighed. 2 ml of Concentrated HCl was added to melt the ash and a few drops of HNO3 were added (AOAC, 1990). The solution was placed in a steaming water bath and evaporated almost to dryness. The contents were transferred to 100 ml volumetric flask and diluted to volume with distilled water and appropriate dilutions were made for each element before analysis.

Determination of Sodium (Na) and Phosphorus (P) by flame photometer Principle

The flame photometer emits radiant energy when atoms of an element arrival to their ground state after their excitation by the high temperature of the flame. The degree of emission is related to the concentration of the element in the solution. Na and K analysis of the sample were done by the method of flame photometry. Determination of Iron (Fe), Zinc (Zn) and Copper (Cu) The same wet sample solutions of the bark of Erthrophleum ivorense was analyzed for the determination of Iron content, Zinc and Copper using the Atomic

absorption spectrophotometer with Model Agilent technologies 55A. Different electrode lamps were used for each mineral. The equipment was run for standard solutions of each mineral before and during determination to check that it is working properly. The dilution factor was 100.

RESULTS AND DISCUSSION Phytochemistry The phytochemical screening of the bark of Erythrophleum ivorense which was sourced from Federal Department of Forestry, Benin City, revealed that alkaloids (53.89 ± 0.20 mg/kg), Phenolic compounds (10.08 ± 0.38mg/kg), Flavanoids (33.76 ± 0.25 mg/kg), Tannins (31.17 ± 0.04 mg/kg), Saponins (41.63 ± 0.11 mg/kg), Cardiac glycosides (37.45 ± 0.16 mg/kg) and steroids (06.03 ± 0.02 mg/kg) were significantly present in the sample (Table 1).

Erythrophleum alkaloids have similar pharmacological activities as digitoxine and ouabain which have shown to have anti-hypersentive effects, antiarrhythmic effect, antimalarial activity and anticancer actions (PROTA, 2008).

Anthraquinones and steroids constituents promote the plant in the treatment and therapeutic applications as arrow poisons or cardiac drugs as laxatives. The presence of anthraquinones was reported to have anti-oxidant, antimicrobial, anti-viral, anti-malaria and anti-tumor activities. The presence of alkaloids also indicates that the bark of Erythrophleum ivorense can be use as muscle relaxant in clinics. The presence of flavonoids in a plant indicates its anti-allergic, anti-inflammatory, anti-cancer, anti-oxidantand hypo-lipidemic effects (Skene and Sutton, 2006). Flavanoids have also been reported to be potent antioxidant and free radicals scavengers capable of protecting cell membranes from damage (Noda et al., 2000)

Tannins are used in the dyestuff productions as caustics for cationic dyes (tannin dyes) and also inks (iron gallate

Table 2. Proximate Analysis.

Parameters Mg/kg

Carbohydrates 57.61+0.08

Protein 201.47+0.02

Moisture content 4.38+0.16

Ash 63.11+0.38

Fibre 130.84+0.04

Fat 0.02+1.16

Vitamin C 8.06+0.20

Table 3. Mineral Analysis.

Mineral elements (ppm)

Zinc 21.66+0.02

Copper 3.77+0.05

Sodium 214.53+0.04

Iron 42.24+0.14

Phosphorus 2.143+0.01

Table 4. Antioxidant Analysis.

Parameters Values

Total Phenolic (GAE100-1) 10.08+0.03

Vitamin C (mg/kg) 8.06+0.09

Total Antioxidant Capacity mg/Vce 63.38+0.10

ink). In the food industries tannins are used to clarify wines, beer and fruit juices. It can also be used as coagulants in rubber production (Gyamfi and Aniya, 2002). Lately, tannins have enticed scientific attention, especially due to amplified incidence of deadly illnesses such as various cancer and AIDS. Tannin rich medicinal plants are used to heal a lot of illnesses; such as leucorrhoea, rhinorrhea and diarrhea. The presence of a few phytochemicals gives belief to the medicinal benefits that the bark of this plant encompasses. Proximate analysis Proximate study revealed that the bark contains little nutrients and minerals. Macro nutrient like protein and Carbohydrates were determined with value of 201.47 ± 0.02 and 57.61 ± 0.08 mg/kg respectively. Other macro nutrients were also relatively high in content level. Crude Lipid had a small content level of 0.02 ± 01.16 mg/kg. Proximate analysis for crude Fibre, Vitamin C, Ash, and Moisture also revealed their contents levels to be 130.84 ± 0.04, 8.06 ± 0.02, 52.37 ± 0.38 and 4.38 ± 0.16 mg/kg respectively (Table 2).

Ogboru et al. 19 Mineral analysis The existence of essential minerals and their contents levels analyzed showed that Erythrophleum ivorense contains sodium (Na; 214.53 ± 0.04) mg/kg, phosphorus (P; 2.143 ± 0.01) mg/kg, Iron (Fe; 17.42.24 ± 0.02) mg/kg, Zinc(Zn; 21.66 ± 0.02) mg/kg and Copper (Cu; 3.77 ± 0.05) mg/kg respectively (Table 3). The values ranges are non-lethal, therapeutic and idea for the maintenance of good health except for sodium that was on the very high side. Total antioxidant analysis Erythrophleum ivorense bark was found to be very opulent in anti-oxidants and phenols with total anti-oxidant capacity of 63.38.02 ± 0.12 mgVce and phenol total content of 10.08 ± 0.03 GAE100

-1 (Table 4). Total

Anti-oxidant Capacity in Erythrophleum ivorense was moderate. The vital role of anti-oxidants is to neutralise the effect of free radicals which damage health cells. It also strengthens the immune system and prevent diseases (e.g. cardiovascular disease and cancer), thus imparting good health and longevity. Phenols help in providing the body defence against oxidative stress or damage arising from oxidising agents and free radicals particularly when the internal enzymatic mechanisms fail or become inadequate. Besides, they are known for their excellent ability to prevent fatty acids from oxidative decay. Conclusion It can be inferred from the phytochemical analysis that the bark of Erythrophleum ivorense can be said to have some medicinal potentials due to some phytochemicals present. But because high doses of the bark extract is an enormously strong, rapid acting cardiac poison, in warm blooded animals causing shortness of breath, seizures and cardiac arrest in a few minutes. It is advised that the bark be administered with caution despite it alkaloid content and high sodium level.

ACKNOWLEDGEMENT We want to appreciate the Laboratory Technologist of chemistry department, Mr. Aghogho, Delta State University for his assistance in the laboratory techniques for this work.

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Biochemical and haematological safety evaluation of the ethanol extract of the stem bark of Blighia sapida

using Swiss albino mice

Chinweizu Ejikeme Udobi1*, Betseabasi Edward Umoh2 and Ememobong Gideon Asuquo1

1Pharmaceutical Microbiology Unit, Department of Pharmaceutics and Pharmaceutical Technology, Faculty of

Pharmacy, University of Uyo, Nigeria. 2Centre for Wetlands and Waste Management Studies, Faculty of Agriculture, University of Uyo, Nigeria.

*Corresponding author. Email: [email protected]

Copyright © 2017 Udobi et al. This article remains permanently open access under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received 16th February, 2017; Accepted 28th March, 2017

ABSTRACT: The ethanol extract of the stem bark of Blighia sapida was studied for its toxic effects on certain hematological and biochemical indices of the swiss albino mice. Three different concentrations of the extract were administered to different groups of the mice through their drinking water for 14 days in the sub-acute test and 28 days in the case of the chronic test. Preliminary phytochemical screening showed that the plant contains bioactive compounds such as saponins, polyphenols, flavonoids, terpenes, resins, alkaloids, carbohydrates, cardiac glycosides, and tannins. The LD50 of the extract was determined to be 500 mg/kg body weight using the Lorke method. Other results obtained showed a significant decrease in some haematological indices such as PCV, HB, RBC, MCV, WBC, lymphocytes and platelet levels as the doses administered increased from low to high. There was also, a significant decrease in the levels of certain biochemical indices such as total protein, albumin, globulin, urea and AST, while the levels of ALT and ALP were significantly increased. The ethanol extract of the stem bark of Blighia sapida showed a slight toxicity to the swiss albino mice. It also showed ability to precipitate some level of anaemia and hepatic damage. Result obtained also confirms that the extract has anticoagulant properties. The safety implications of these effects are highlighted. Key words: Ethanol extract, stem bark, swiss albino mice, biochemical, haematological. INTRODUCTION Medicinal plants play a vital role in the management of various diseases (Olatunji and Atolani, 2009). Scientific investigations of medicinal plants have been initiated in many countries because of their contributions to health care. The continual search for and the interest in natural plant products for use as medicines has continued to act as the catalyst for exploring methodologies involved in obtaining the required plant materials and probing their constituents for safety for human use (Okoli, 2004).

The amount of pharmacological substances and chemicals being used in the human community today has increased tremendously. These are today in the form of or as constituents of food substances, medicines, and beverages, other industrial and household products.

However, pharmacological substances may result in chronic toxicity in the living system when used over a long period of time or acute toxicity may result when large quantities capable of eliciting immediate toxic effect are used. These effects may be mild or severe, depending on the nature of the substance.

Preparations from plants known to have medicinal values have been used in Africa by the locals mostly in their crude form for a very long time. In most of these places, these preparations are the only medications known to serve the populace. They meet the medicinal needs of the people by curing a wide range of diseases. This is because plants are known to have the ability to produce and store a wide range of chemical substances.


22 J. Biosci. Biotechnol. Discov. Some of these substances have the ability to defend the plants against microbial attack while others confer some other abilities on the plants. The importance of these plants is however undermined by the fact that most of them exhibit different levels of toxicity (Veeramuthu et al., 2006).

Toxicity study is therefore necessary before these plants can be used as medications by humans. Toxicity study is the determination of potential hazards a test substance may likely produce and the characterization of its action. Most of the toxicity testing is carried out on experimental animals (Craig, 1999). The advantages of using animal models in toxicity testing are enormous. They include the possibility of clearly defined genetic constitution and their amenity to controlled exposure, controlled duration of exposure, and the possibility of detailed examination of all tissues (Arome, 2013).

Blighia sapida belongs to the family Sapindaceae. In Nigeria, it is known as akee and ishin. It has many other local names in other western Africa countries (Micheal et al., 1998). The tree is usually densely branched and symmetrical with smooth gray bark. It is about 40ft (12m) tall and possesses evergreen (rarely deciduous) alternate leaves.

All parts of the plant have been used in traditional practice for the treatment of several ailments. For example, the aqueous extract of the seeds of Blighia sapida administered as parasite expellant. The crush new foliage is applied on the forehead as headache relief and the juice from the leaf is applied as eye drops in cases of conjunctivitis. Various preparation and combinations of the extracts have also been made for the treatment of diseases such as dysentery, epilepsy, yellow fever (Kubmarawa et al., 2007) and diabetics (Kokwaro, 2000). The crushed seeds have been used to treat dental decay while the crushed bark are used for wound healing and decoction of the bark is used for constipation (Ekue et al., 2010). The plant has also been reported to be effective against cold and pain when applied; it is also acaricidal and insecticidal (Ashurust, 2001).

The inedible portion of the plant contains the toxins hypoglycin A and hypoglycinB (Blanke et al., 2006). Hypoglycin A is found in both seeds and arils, while hypoglycin B is found only in the seeds. Hypoglycin is converted in the body to methylene cyclopropyl acetic acid (MCPA). Hypoglycin and MCPA are both toxic. MCPA inhibits several enzymes involved in the breakdown of acyl CoA compounds. Hypoglycin combines irreversibly with Coenzymes A, carnitine and carnitine acyltransferases I and II reducing their bioavailability and consequently inhibiting beta oxidation of fatty acids. Beta oxidation normally provides the body with ATP, NADH and acetyl CoA which is used to supplement the energy produced by glycolysis. Glucose stored are consequently depleted leading to hypoglycemia (Olatunji and Atolani, 2009). Blighia sapida has been used extensively in the West African region for

medicinal purposes neglecting the toxicity aspect of the plant. Results obtained from this study will therefore guide in confirming their safety and hence usage in traditional medical practice. MATERIALS AND METHODS Plant collection and authentication The stem bark of B.Sapida was collected from Itak Ikot Akap, Ikono Local Government Area, Akwa Ibom State, Nigeria. The authentication of the plants was done by Prof. Margaret Bassey of the Department of Botany and Ecological studies, Faculty of Science, University of Uyo and a voucher specimen with herbarium No. 69(a) was deposited in the herbarium of the Faculty of Pharmacy, University of Uyo, Nigeria. Preparation and extraction of plant material The stem bark of B.sapida was properly washed, chopped into pieces and air dried for two weeks. They were ground into powder and extracted using 75% ethanol. The extract was obtained by soaking 300 g of the dried powdery samples in 2500 ml of ethanol for 48 hrs during which the mixture was intermittently shaken. It was later filtered through Whatman filter paper. The extracts were evaporated to dryness at 40

oC in a water

bath. This temperature was maintained to avoid the chemical components from being destroyed. Animal treatment The animals used for this study were Swiss albino mice. They were maintained at the Experimental Animal House of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Nigeria. They were kept in rat cages and fed with standard pellet feeds and allowed free access to clean fresh water. All experimental protocols were in compliance with the Faculty of Pharmacy, University of Uyo Ethics on Research in Animals as well as internationally accepted principles for laboratory animal use and care. Phytochemical screening Phytochemical screening was done to determine the chemical constituents of the plant extract. These include test for alkaloids, Cardiac Glycosides, Cyanogenic Glycosides ,flavonoids, resins, Polyphenols (Trease and Evans 2009), saponins, phlobatanins, Anthraquinones, carbohydrates Balsams, (Sofowora, 2008) Terpenes, tannins (Harborne, 1973).

Toxicity studies

Acute toxicity test

Determination of Lethal dose (LD50): The Lorke method was used to determine the concentration of the extracts of the stem bark of Blighia sapida that killed 50% of the test animal population (Lorke, 1983). The animals used for this study were Swiss albino mice (weighing between 22 and 34 g). They were divided into groups, with three animals per group. The test was in two phases. In the first phase, each group of the animals was administered with 3000 mg/kg, 2000 mg/kg and 1500 mg/kg of the extract. The animals were monitored for 24 hrs and mortalities noted. In the second phase, each group was administered with 250 mg/kg, 500 mg/kg and 1000 mg/kg of the extract. They were monitored for 24 hrs and mortalities noted. All extract administrations were done through the intraperitoneal route. The Lethal Dose (LD50) was determined using Highest dose that gave no mortality (Do) and Lowest dose that produced mortality (D100) (Lorke, 2003).

Sub-acute toxicity test

After 14 days of administration of the extracts of the stem bark of Blighia sapida, the mice were randomly selected irrespective of sex and weight and divided into four groups which were the Low Dose (LD), Middle Dose (MD), High Dose (HD) and the Control groups. The LD50 was used as baseline dose (i.e. low dose) while the middle and the high doses were obtained by doubling and tripling the LD50 respectively according to the method of the American Association of Clinical Chemistry, (2014). The animals were fed with standard pellets feeds and clean water. The test groups had their extracts added to their drinking water to avoid injection abscess which may kill the animal if given by injection (Pincus et al., 2011), while the control group was fed with standard pellets feed but given only potable drinking water. Doses of the extract administered were 500 mg/kg, 1000 mg/kg and 1500 mg/kg. The doses were administered daily through the same route for 14 days. Observation was done every 24 hours. After the test period, the animals were suffocated using chloroform and sacrificed. Sterile syringes with needles were used to collect the blood from the heart of the sacrificed animal by process known as Cardiac Puncture (American Association of Clinical Chemistry, 2014). The blood samples were analyzed for the effect of the extracts on some hematological and biochemical indices using the Mindray 5 part Differential Haematological Auto-analyzer (BC-5300)

Hematological safety evaluation: Using the blood sample obtained, laboratory analysis was carried out to determine the effect of the extracts on certain hematological parameters in the blood. They include Pack Cell Volume (PCV), Hemoglobin (HB), Mean

Udobi et al. 23 Corpuscular Hemoglobin Concentration (MCHC), Mean Corpuscular Values (MCV), Mean Corpuscular Hemoglobin (MCH), Red Blood Cell (RBC), White Blood cell (WBC), Lymphocytes, Neutrophils, Monocytes, Eosinophil, and Platelets. The sample specimen bottle was properly labelled, imputed into the computer attached to the auto analyzer and saved accordingly. 3 to 5 ml of blood in a specimen bottle was mixed thoroughly to avoid clusters or larger particles from blocking the tiny tubing in the auto analyzer as this will damage the machine. The auto analyzer was turned on and allowed to self-calibrate (self-check). When it was ready, a beeping noise was heard as prompt sound. The analyzer brought down a prop (a tube-like part of the machine) and sucked the blood sample from the specimen bottle after which the cycle of analysis began. The analyzer automatically separated every parameter and sent the result to the monitor which was later printed. Biochemical safety evaluation: Laboratory analysis was also carried out to determine the effect of the extract on certain biochemical parameters in the system which includes total protein, albumin, urea, globulin, Aspartate aminotransferase (AST), Alkaline Phosphatase (ALP) and Alanine Aminotransferase (ALT). The method was as in heamatological parameters. The analyzer automatically separated the hematological parameters and biochemical parameters and displayed all the result on the monitor. Chronic toxicity: This was determined after 28 days of administration of the extracts of the stem bark of B. sapida. The mice were randomly selected and divided into four groups; the Low Dose, Middle Dose, High Dose and the Control groups. The test mice were fed with standard pellets feed and the extract added to their drinking water while the control was fed with the standard pellets feed and potable drinking water only. The extracts were added to their drinking water to avoid injection abscess which may kill the animal if given by injection (Pincus et al., 2011). The test animals were also divided into three groups with three animals in each group. Three different doses of the extract namely, high dose (HD), middle dose (MD) and low dose (LD) were administered to each group. The LD50 was used as baseline dose (i.e. low dose) while the middle dose and the high dose were obtained by doubling and tripling the LD50 according to the method of the American Association of Clinical Chemistry, (2014). Dose of B. sapida extract administered were 500 mg/kg for Low dose (LD), 1000 mg/kg for Middle dose (MD) and 1500 mg/kg for High dose (HD). After 28 days, the animals were suffocated using chloroform and sacrificed. Sterile syringe with needle was used to collect the blood from the heart of the sacrificed animal by process known as Cardiac Puncture (Pincus et al., 2014). The blood sample was collected and stored in a heparinized specimen bottle with anti-


Table 1. Results of the phytochemical screening of the ethanol extract of the stem bark of Blighia sapida.

Constituents Indications

Saponins +

Polyphenols +

Flavonoids +

Steroid glycosides -

Terpenes +

Phlobatanins -

Aanthraquinone -

Cyanogenic glycoside -

Balsam +

Resins +

Alkaloids +

Carbohydrates +

Cardiac glycosides +

Cardenolides +

Tannins +

Table 2. Result of the acute toxicity test for ethanol extract of the stem bark ofBlighia sapida.

Phases Dose (mg/kg) No. of mice Mortality % Mortality LD50(mg/kg)

1 3000 3 3 100

√D0XD100

=500mg/kg

2000 3 3 100

1500 3 3 100

2 1000 3 3 100

500 3 2 66.67

250 3 0 0

D0, hightest dose that gave no mortality, 250 mg/kg body weight; D100, lowest dose that produced mortality, 1000 mg/kg.

coagulant and the specimen bottle with the blood sample was shaken briskly to prevent the blood from clotting. The blood samples were taken to the laboratory to be analyzed for the effect of the extracts on some hematological and biochemical indices

Statistical analysis

Statistical analysis was done using windows SPSS package (SPSS version 15.0). Data was analyzed using one way ANOVA followed by post hoc t-test least significance difference (LSD). The data were expressed as mean ± standard error and values of p< 0.05 were considered significant.

RESULTS

Phytochemical screening

The result of the phytochemical screening of the ethanol

extract of the bark of B.sapida revealed the presence of saponins, tannins, terpenes, cardenolide, Balsam, alkaloid, carbohydrate, flavonoids and cardiac glycosides. However phlobatanins, cyanogenic glycoside, Steroid glycoside and anthraquinones were absent (Table 1).

Acute toxicity Results of the acute toxicity test of the ethanol extract of the stem bark of B.sapida are presented in Table 2. On administration of the extract, physical signs like dullness, rubbing of nose and mouth on the floor of the cage along with restlessness were shown by the animals. When the LD50 was computed, it was found to be 500 mg/kg.

Sub-acute toxicity Hematological safety evaluation

The ethanol extract of the stem bark of B. sapida was

Udobi et al. 25

Table 3a. Result showing the sub-acute effect of the administration of the ethanol extract of the stem bark of Blighia sapida on Red blood cell indices of Swiss albino rats.

Groups PCV (%) HB (g/dL) RBC (x106/µL) MCV (µm

3) MCH (pg) MCHC (g/dL)

Low dose 46.9±1.3** 14.4±1.2** 7.57±1.2** 52.8±2.3** 15.6±1.5 28.8±2.9**

Middle dose 46.1±5.6** 13.1±3.5* 7.29±1.9** 51.7±3.4** 15.1±2.5 30.1±2.0

High dose 42.8±2.7* 12.1±2.4** 6.90±1.5* 50.3±2.4 13.8±3.0** 30.7±1.3

Control 48.2±1.3 15.7±1.4 9.32±1.1 49.4±1.0 15.8±0.9 31.2±1.2

Values presented as mean ± SEM, Significance relative to control: **p<0.01, *p<0.05.

Table 3b. Result showing the sub-acute effect of the administration of the ethanol extract of the stem bark of Blighia sapida on Total WBC, differential and platelet count of Swiss albino rats.

Groups WBC (x103/µL) LYM (x10

3/µL) NEU (x10

3/µL) MON (x10

3/µL) EOS (x10

3/µL) Platelet (x10

3/µL)

Low dose 13.5±1.5* 10.5±0.8* 2.1±0.3* 0.23±0.2 0.03±0.1 6255±17.6*

Middle dose 11.3±4* 8.4±2.8* 1.5±0.6* 0.3±0.1 0.0-0.0 567±19.2*

High dose 11.0±2.4* 7.8±2.1** 1.2±0.9** 0.7±0.2* 0.0-0.0 555±30.3*

Control 7.9±1.2 6.3±0.8 0.7±0.1 0.4±0.1 0.03±0.1 538±22.1


Table 3c. The sub-acute effect of the ethanol extract of the stem bark ofBlighia sapida on biochemical indices of Swiss albino rat.

Groups Total protein (g/L) Albumin (g/L) Globulin (g/L) Urea (mmol/L) AST (iu/L) ALT (iu/L) ALP (iu/L)

Low dose 82.7±2.3* 39.3±1.5* 43.7±1.5* 6.4±0.1** 105.3±2.1* 33.3±2.1 243±17.8*

Middle dose 75.3±1.5* 35.3±1.5 39.3±2.1** 6.1±2.2** 100.7±2.5* 41.3±1.2* 317±5.3*

High dose 64.3±1.5** 35.3±3.2 27.3±1.5* 4.7±0.5 97.7±6.4** 41.3±1.5* 319.3±9.5*

Control 71.3±0.6 37±2.0 35.7±1.2 5.7±0.1 94.7±1.5 34.3±2.3 335±1.0

iu/L (international unit/litre), Values presented as mean ± SEM, Significance relative to control: **p<0.01, *p<0.05.

observed to cause decrease in the PCV, HB, RBC, MCV, WBC, lymphocytes, neutrophils and platelet levels as the doses administered increased from low dose to high dose while the MCHC level increased as shown in Table 3a and 3b.

Biochemical safety evaluation

The ethanol extract of the stem bark of Blighia sapida was observed to cause decrease in total protein, albumin, globulin, urea and aspartate aminotransferase (AST) levels as the doses administered increased from low dose to high dose while alanine aminotransferase (ALT) and alkaline phosphatase (ALP) level increased (Table 4c).

Chronic toxicity test

Hematological safety evaluation

The ethanol extract of the stem bark of Blighia sapida was observed to cause decrease in the packed cell volume, hemoglobin, red blood cell, mean corpuscular

volume, white blood cell, lymphocytes, neutrophils and platelet levels as the doses administered increased from low dose to high dose while the mean corpuscular hemoglobin concentration level increased (Table 4a and 4b).

Biochemical safety evaluation The ethanol extract of the stem bark of Blighia sapida was observed to cause decrease in total protein, albumin, globulin, urea and aspartate aminotransferase (AST) levels as the doses administered increased from low dose to high dose while alanine aminotransferase (ALT) and alkaline phosphatase (ALP) level increased. DISCUSSION In developing countries, herbal products from medicinal plants found everywhere within the environment have become famous in healthcare and some have been falsely considered as safe because they are obtained from natural sources. Bioactive compounds from


Table 4a. Result showing the chronic effect of the administration of the ethanol extract of the stem bark of Blighiasapida on Red blood cell indices of Swiss albino rats.

Groups PCV (%) HB (g/dL) RBC (x106/µL) MCV (µm3) MCH (pg) MCHC (g/dL)

Low dose 43.3±0.6** 11.1±1.3* 4.6±2.1* 49.4±2.2 12.5±1.2** 32.8±2.9**

Middle dose 38.3±1.5* 10.2±2.6* 3.97±1.1* 47.5±1.7** 10.8±2.2** 34.1±4.3**

High dose 36.3±3.2* 8.7±1.7* 3.3±1.0* 45.9±2.0** 9.6±2.2* 36.6±5.3*

Control 48.2±1.3 15.7±1.4 9.32±1.1 49.4±1.0 15.8±0.9 31.2±1.2


Table 4b. Result showing the chronic effect of the administration of the ethanol extract of the stem bark of Blighia sapida on Total WBC, differential and platelet count of Swiss albino mice.

Groups WBC (x103/µ/L) LYM (x10

3/µ/L) NEU (x10

3/µ/L) MON (x10

3/µ/L) EOS (x10

3/µ/L) Platelet (x10

3/µ/L)

Low dose 10.8±0.95* 8.5±0.7* 1.1±0.2* 0.3±0.3 0.0-0.0 642±12.5*

Middle dose 8.1±3.0* 5.3±0.9 0.8±0.4 0.5±0.1 0.0-0.0 559±22.0*

High dose 6.8±2.2** 5.1±0.9 0.5±0.4 0.8±0.2 0.0-0.0 548±25.8*

Control 7.9±1.2 6.3±0.8 0.7±0.1 0.4±0.1 0.03±0.1 538±22.1


Table 4c. Result showing the chronic effect of the ethanol extract of the stem bark ofBlighia sapida on biochemical indices of Swiss albino rat.

Groups Total protein (g/L) Albumin (g/L) Globulin (g/L) Urea (mmol/L) AST (iu/L) ALT (iu/L) ALP (iu/L)

Low dose 78.7±2.5* 37.6±2.5 39±1.7** 3.6±0.7** 98±2.1* 39.3±4.5 249±18.7*

Middle dose 72.3±2.0** 32.3±1.2** 34.3±4.0 3.1±1.4** 96±2.0** 44.3±1.5* 322.7±5.5*

High dose 61±1.0* 32±2.6** 23±1.0* 2.1±0.6* 92.6±3.3** 45.7±2.1* 326.7±2.9*

Control 71.3±0.6 37±2.0 35.7±1.2 5.7±0.1 94.7±1.5 34.3±2.3 335±1.0


medicinal plants are concluded to be safe without understanding the possible health effects and thus commonly used in self-medication (Vaghasiya et al., 2011). Plants have produced these chemicals to protect themselves but research has shown that these chemicals in plant can also protect human against diseases (Falodun et al., 2006). These phytochemicals are also known to perform other functions. Some work as antioxidants, some exhibit hormonal actions, some stimulate enzymes, some interfere with DNA replication, and others have anti-bacterial effect while some possess other physical effect. Some of these plants chemicals though not distinguishable from the other metabolites which have therapeutic effect and defend the plants are toxic to humans. This explains why a large number of plants that produces them cannot be eventually useful in the formulation of drugs even though their parts may be biologically active.

The ethanol extracts of the stem bark of Blighia sapida was analyzed for the presence of phytochemical compounds which could be responsible for their medi-cinal use in traditional medicine (Tona 2008; Sofowora,

1999 and Olusegun, 2013). These chemicals could also include toxic component if there are any. Result obtained showed that the stem bark of B. sapida contained Saponin, Alkaloids, terpenes, cardiac glycosides Tannin and Balsam (Table 1). This result agrees with that of Olusegun (2013) and Ubulom et al. (2013). These phytochemical compounds have pharmacological effects and have been the basis of chemical synthesis of drugs used in modern medicine. They are also responsible for their use in traditional medicine (Sofowora, 2008).

The acute toxicity evaluation revealed that the lethal dose (LD50) of the ethanol stem bark extract of Blighia sapida was 500 mg/kg (Table 2). According to Homburger (1989), this extract can be said to be slightly toxic (500 to 5000 mg/kg). The behavioral changes observed in the mice on administration were dullness, rubbing of nose and mouth on the floor of the cage along with restlessness.

The ethanol extract of the stem bark of B. sapida, both at sub-acute and chronic administrations (Tables 3a and 4a) caused a significant reduction in the PCV which is evident in the reduction in RBC and HB. This suggests

that the plant could precipitate some level of anaemia if the animals are exposed to it for a long period of time (Fahey, 2005). Meanwhile, reductions in the numbers of RBC (anaemia) may be associated with bone marrow suppression, as observed in cattle grazing on small quantities of bracken fern over several months (Knight and Walter, 2001).

The white blood cell (WBC) count for both the sub-acute and chronic toxicity testing (Table 3b and 4b) were also decreased significantly. This decrease suggests that the extracts did not exhibit toxic effect even at high doses to trigger the release of the white blood cells. The lymphocyte, the main effector cells of the immune system (Mc Knight et al., 1999; Tédong et al., 2007) was also observed to decrease appreciably indicating that the extracts did not exert challenge on the immune system of the treated animals. The extract was also observed to have caused a significant decrease in the Platelets count. The reduction in platelet count suggests that blood clotting is impacted (American Association for Clinical Chemistry, 2014). Platelets also known as thrombocytes help to mediate blood clotting, which is a meshwork of fibrin fibres. The fibres also adhere to damaged blood vessels; therefore, the blood clot becomes adherent to any vascular opening and thus prevents further blood clot (Wu and Thyagarajan, 1996; Andrews et al., 1997; Cox and Cox, 2000). The extract could thus precipitate thrombocytopaenia which is the presence of low level platelets in the circulatory system as well as slow clotting of blood. This observation of decreased platelet level in the circulatory system by the extract also means that it may have anticoagulant property.

The ethanol extract of the stem bark of Blighia sapida caused a significant decrease in the AST level in the blood at both sub-acute and chronic toxicity test (Table 3c and 4c). ALT and AST are produced in the liver and are good markers of damage to liver cells but not necessarily the severity of the damage (Rej, 1989). They are normally present at low levels in the blood so if the liver cells are damaged, it would be expected that some of the enzymes leak into the blood and increase in levels. The decrease in level of AST observed in this study may reflect no damage of liver cells. However, an increase in ALT level was observed. ALT is known to increase in liver disease and it has been used as a tool for measuring hepatic necrosis (Bush, 1991). The Increase in ALP may be considered as a sensitive indicator of cholestasis in early stages or mild circumstances preceding other indicators such as hyperbilirubinemia (Bush, 1991). The extract showed a decrease in urea level at both sub-acute and chronic toxicity test; low blood urea levels are not common and are not usually a cause for concern. This result may be seen when the system is overhydrated (too much fluid volume) or in cold weather when the body does not excrete much fluid from the system (Vasodilation) (Arise et al., 2012). The ethanol extract of the stem bark of Blighia sapida at sub-acute and chronic

Udobi et al. 27 toxicity test result showed a decrease in the Albumin level. Albumin, apart from being a useful indicator of the integrity of glomerular membrane, is also important in determining the severity of disease (Adedapo et al., 2005). Decrease in albumin level may be due primarily to reduction in synthesis by the liver and secondary to reduced protein intake which further confirms a possible hepatic damage (Luskova et al., 2002; Jyotsna et al., 2003).

Conclusion Findings from this study confirm that ethanol extract of the stem bark of Blighia sapida showed a slight toxicity to the Swiss albino mice. It also showed ability to precipitate some level of anaemia. Results obtained also confirmed that the extract had anticoagulant properties as well as the ability to cause hepatic damage.

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Journal of Bioscience and Biotechnology Discovery Volume 2. Page 29-35. Published 3rdth May, 2017



Physicochemical properties of a flowing stream in Jabi, Abuja, Nigeria

Solomon, R. J.* and Kehinde, O. A.

Department of Biological Sciences, Faculty of Science, University of Abuja, Abuja, Nigeria.

*Corresponding author E-mail: [email protected]

Copyright © 2017 Solomon and Kehinde. This article remains permanently open access under the terms of the Creative Commons Attribution License

4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received 5th April, 2017; Accepted 28th April, 2017

ABSTRACT: The physicochemical qualities of flowing stream located in Jabi, Abuja, Nigeria was assessed over the duration of 3 months. Parameters measured include pH, temperature, electrical conductivity, turbidity, biological oxygen demand (BOD), dissolved oxygen, chemical oxygen demand, nitrate, sulphate, acidity, alkalinity, and carbon levels and these were simultaneously monitored in the flowing stream using standard methods. Unacceptably, high levels of the assayed parameters were observed in Nitrate (1.39 to 1.44 mg/L), while dissolved oxygen (3.2 to 11.6 mg/L) and turbidity (8.6 to 9.1 NTU) were totally outside the compliance levels of the Federal Environmental Protection Agency (FEPA) Guidelines and World Health Organization (WHO) tolerance limits for domestic uses. The study has revealed that there was an adverse impact on the physiochemical characteristics of the flowing stream as a result of discharge of inadequately treated waste products from surrounding communities. This poses a health risk to several rural communities which rely on flowing stream primarily as their sources of domestic water and also harmful to aquatic life. There is need for the intervention of appropriate regulatory agencies to ensure flowing stream is hygienic for consumption and aquatic life through proper channelling. Kew words: Biological oxygen demand, chemical oxygen demand, Chloride, Fluoride, dissolve oxygen, Jabi, Magnesium, Nitrate, pH, physicochemical, temperature, total hardness, turbidity. INTRODUCTION Water is the most abundant compound on the Earth surface, covering about 70 percent of the planet. In nature, water exists in liquid, solid and gaseous states. It is in dynamic equilibrium between the liquid and gas state at standard temperature and pressure. At room temperature, it is a tasteless and odorless liquid, nearly colorless with a hint of blue. Many substances dissolves in water and it is commonly referred to as the universal solvent. Because of this, water in nature and in use is rarely pure. However, there are also many compounds that are essentially, if not completely, insoluble in water. Water is the only common substance found naturally in all three common states of matter and it is essential for all life on earth. Water makes of 55 percent to 78percent of the human body.

Fresh water has become a scarce commodity due to over exploitation and pollution (Ghose and Basu 1968,

Gupta and Shukle, 2006; Patil and Tijare, 2001). Pollution is caused when a change in physical, chemical or biological condition in the environment harmfully affects quality of human life, animals and plant (Thompson et al., 2004; Okoye et al., 2002). Industrial, sewage and municipal are been continuously added to water bodies hence affects the physiochemical quality of water making them unfit for use on livestock and other organisms (Dwivedi and Pandey, 2002).

Uncontrolled domestic water discharged into pond has resulted in eutrophication of ponds as evidence by substantial algal bloom, dissolved oxygen depletion in the subsurface water leads to the death of large fish and other oxygen dependent organisms (Rast and Thornton, 1996; WRC, 2000).

Effluent discharged into environment with enhanced concentration of nutrient, sediments and toxic substances



30 J. Biosci. Biotechnol. Discov. may have a serious negative impact in quality of the receiving water body when discharged untreated or partially treated (Forenshell, 2001; Schulz and Howe, 2003). Water pollution by effluent has become a question of considerable pubic and scientific concern in the light of the evidence of their extreme toxicity to human health and to biological ecosystem (Katsuro et al., 2004). The occurrence of heavy metals in industrial and municipal sewage effluence constitute a major source of heavy metal entering aquatic media. Hence, there should be regular assessment of this sewage effluence to ensure that adequate measures are taken to reduce pollution level to the minimal.

Worldwide, water bodies are primarily means for disposal of waste, especially the effluence from industrial, municipal sewage and agricultural practices that are closed to them This effluence can alter the physical, chemical and biological nature of receiving water body (Sandoyin, 1991). The initial effect of waste is to degrade physical quality of the water. Later biological degradation becomes evident in terms of number, variety and organization of the living organisms in the water (Gray, 1989). Often, the water bodies readily assimilate waste materials they receive without significant deterioration of some quality criteria; the extent of this referred to as its assimilative capacity (Fair, 1971). However the water quality is deteriorating day by day due to anthropogenic impute of dissolve nutrient and organic matter and industrial effluent which is built up on its bank. So, it is of vital importance to monitor and simulate the water quality parameters to ascertain whether the water is still suitable for various uses. Water contaminated by effluent from various sources is associated with heavy disease burden (Okoh, 2007) and this could influence the current shorter life expectancy in the developing countries compared with developed nations (WHO, 2002). Due to population explosion and moderate rapid urbanization, people rely heavily on water sources of doubtful quality in the absence of better alternatives, or due to economic and technological constraint to adequately treat the available water before use (Aina and Adedipe, 1996, Calamari and Naeve 1994). The scarcity of clean water and the pollution of fresh water have therefore led to a situation in which one-fifth of the urban dwellers in developing countries and three-quarter of their rural dwelling population do not have access to reasonably safe water supplies (Lloyd and Helmer, 1992)

Effluent are composed mainly of either organic, inorganic matter or both and toxic substances depending on its source. Inorganic matter in effluent are formulated using various chemical containing nitrogen, phosphorous and potassium. These elements especially phosphorous stimulate the growth of microscopic plant while nitrogen promotes overgrowth of aquatic vegetation which degrades water quality. Potassium promotes productivity of aquatic animals such as fish (Wurts 2000). Organic

matter in effluent are formulated using various chemicals containing carbon, nitrogen and phosphorus. Organic matter promotes the growth of zooplanktons as well as macro benthic invertebrates (Adigun, 2005). Organic matter also stimulates the growth of decomposers such as bacteria and fungi.

Bacteria and fungi are very critical to the breakdown of the toxic components of the effluent. It has been observed that dissolved oxygen in water is required during the decaying of the organic matter, which may lead to the depletion of oxygen in the water body and cause harmful substance to accumulate (Watson and Cichra, 2006). Organic matter contains high concentration of ammonia, which may occur as bubbles attached to the block solid materials known as benthic deposits.

Contamination of the environment by effluent viewed as an international problem because of the effect on the ecosystem in most countries. In Nigeria, the situation is no better by the activities of most industries and populace towards waste disposal and management which usually leads to the increasing level of pollution of the environment. Sewage discharge is a major component of water pollution which is compounded in areas where waste water treatments are inefficient. Such is the case of the Jabi stream which is a running stream.

This receiving stream serves as a source of water to some communities downstream which are used for a variety of purposes like irrigation, drinking, aquatic life and other domestic uses without prior treatment. The present study on the physiochemical parameters is significant in that some of these parameters may prove lethal to aquatic flora, fauna and ultimately humans who are usually at the top of the food chain. The receiving stream serves as a convenient means of cleaning the highly loaded sewage and carries waste away from its discharge point. The need to know the quality of the water from the receiving stream has informed this study. The study will also provide information on the performance efficiency of the sewage lagoon.

MATERIALS AND METHOD Sampling point were the river is situated is at Utako with Latitude 008

0 7.44 E, Longitude 005

0 9.08 N with

elevation 285 m above sea level using global position system (GPS) to determine the coordinate and elevation. Sampling Samples were collected in 250 ml glass bottle for dissolved oxygen and biological oxygen demand. Also samples were collected in plastic bottle for other physiological parameters, pre cleaned by washing with

non-ionic detergents and rinsed in tap water. Before sampling, the bottles were rinsed three times with sample water before being filled with the sample. The actual samplings were done midstream by dipping each sample bottle at approximately 20 to 30 cm below the water surface, projecting the mouth of the container against the flow direction. The samples were then transported in cooler boxes.

Physicochemical analysis All field meters and equipment were checked and calibrated according to the manufacturer’s specifications. pH, temperature, electrical conductivity, transparency, dissolved oxygen, organic matter, organic carbon and chemical oxygen demand (COD) of the samples were determined by titration while biological oxygen demand (BOD) was determined five days after sampling. Samples were kept in BOD bottles in cool cupboard. Acidity and alkalinity were determined in the laboratory on the day of sampling. The concentrations of sulphate and nitrate were determined in the laboratory by using standard colorimeter.

Statistical analysis The obtained data were subjected to descriptive statistical analysis (95% confidence limit). The computation were achieved with the use of statistical package for social science (SPSS) to determine the mean, standard deviation, threshold and coefficient of variation and range values.

RESULTS AND DISCUSSION The water samples were collected for over a period of three months. One sample was collected from each sampling point. The results were presented in Tables 1, 2 and 3. The physiochemical characteristic of water is important determinant of aquatic system. Their characteristic is greatly influenced by climate, vegetation and general composition of water.

pH Water in a pure state has a neutral pH. As a result, water is neither acidic nor basic. The pH values vary significantly (p>0.05) in the water samples collected (from Jabi water flow) at month interval and ranged from 7.25 to7.50. Generally, the obtained pH values fall within the World Health Organization standard of 7.0 to 8.5 and the water quality ranges 6.5 to 8.5 for drinking water and meant for full contact recreation (DWAF, 1996; WHO, 1984; 1989). The European Union also set pH protection

Solomon and Kehinde 31 limits of 6.0 to 9.0 for fisheries and aquatic life (Chapman, 1996).

Temperature The temperature profile of the flowing stream at Jabi varies significantly (P>0.05) and ranged 19 to 23

0C. 25

0C

is the recommended limit for no risk according to the FEPA water quality guidelines for domestics use (DWAF, 1995) while 40

0C is recommended limit according to

WHO. Based on these guidelines, the temperature of the stream did not pose any threat to the homeostatic balance of the receiving water bodies, in conformity with the report of Jaji et al. 2007. Turbidity The turbidity profile varies significantly (p>0.05) amongst the water samples collected from the water body and ranged from 8.62 to 9.11 NTU. The turbidity values obtained from the sampling points was higher than WHO standard of 5 NTU (WHO, 2004). None of the sample collected met the requirement of FEPA guidelines of 0 to 1 NTU for turbidities in water for domestic use (DWAF, 1998). These values are grossly exceeded in the water sample and disqualify the receiving water body for direct domestic use. Also the excessive turbidity in water can cause problem with water purification processes such as flocculation and filtration, which may increase treatment cost (DWAF, 1998). High turbid waters are often associated with the possibility of microbiological contamination, as high turbidity makes it difficult to disinfect water properly (DWAF, 1998).

Dissolve Oxygen The dissolved oxygen profile through the period varied significantly (p>0.05) and ranged from 3.2 to 11.6 mg/l .Dissolved oxygen is an important factor used for water quality control. The effect of waste discharge on a surface water is largely determined by the oxygen balance of the system and its presence is essential in maintaining biological life within a system (DFID, 1999). Dissolved oxygen concentrations in unpolluted water normally range between 8 and 10 mg/l and concentration below 5 mg/L adversely affect aquatic life (DFID, 1999; Rao, 2005). Dissolve oxygen for standard water for drinking purpose is 6 mg/L whereas for sustaining fish and aquatic life is 4 to 5 mg/L (Rao, 2005). The dissolve oxygen value from this study fell short of the recommended standard (3.2 to 11.6 mg/l). For water quality variable such as dissolved oxygen, water quality criteria are set at the minimum acceptable concentration to ensure the maintenance of biological function.


Table 1. Physicochemical properties of the flowing stream in Jabi.

Parameter Unit Jun 2014 Jul 2014 Aug 2014

DO Mg/l 3.2 8.4 11.6

BOD Mg/l 1.6 0.8 6.0

Temperature oC 23 19 23

Conductivity Us/cm 289 284 295

TDS Ppm 142 141 146

pH

----- 7.45 7.25 7.50

Total hardness Mg/l 257 260 262

Calcium hardness Mg/l 115 113 110

Magnesiun hardness Mg/l 155 157 152

Turbidity Mg/l 8.92 9.11 8.62

Chloride Mg/l 43.4 44.3 48.7

Alkalinity Mg/l 110 118 113

Iron Mg/l 0.56 0.59 0.6

Flouride Mg/l 0.3 0.2 0.2

Sodium Mg/l 24.7 22.4 26.6

Phosphate Mg/l 0.87 0.89 0.91

Sulphate Mg/l 15.7 16.2 15.7

COD Mg/l 2.0 4.0 4.0

DO, Dissolve oxygen, BOD, Biological oxygen demand, COD, Chemical oxygen demand and TDS,Total dissolve solid.

Table 2. Summary of basic statistics for flowing stream in Jabi.

Physsiochemical

parameter Range Mean

Standard deviation

Threshold

X + 2S

Coefficient of Variation

DO( mg/L) 3.2-11.6 7.73 4.24 16.2 5.49

BOD (mg/L) 0.8-6.0 2.80 2.80 8.4 1.00

Temperature 19-23 21.7 2.31 26.3 1.06

Conductivity 284-295 289 5.51 300 1.91

TDS 141-146 143 2.65 148 1.85

PH 7.25-7.50 7.4 0.13 7.66 1.76

Total hardness 257-260 260 2.52 265 9.69

Calcium 110-115 113 2.52 118 2.23

Magnesium 152-157 155 2.52 160 1.63

Turbidity 8.62-9.11 8.88 0.25 9.38 2.82

Chloride 43.4-48.7 45.5 2.84 129 6.24

Alkalinity 110-118 114 4.04 122 3.54

Iron 0.56-0.6 0.58 0.02 0.62 3.45

Fluoride 0.2-0.3 0.23 0.06 0.35 2.61

Nitrate 1.39-1.44 1.43 0.02 1.47 1.39

Sodium 22.4-26.3 24.5 1.06 26.6 0.08

Phosphate 0.87-091 0.89 0.02 0.93 2.25

Sulphate 15.7-16.2 15.9 0.29 16.5 1.82

COD 2.0-4.0 3.33 1.15 5.63 3.45

DO, Dissolve oxygen, BOD, Biological oxygen demand, COD, Chemical oxygen demand.

Biological/chemical oxygen demand Both the BOD and COD test are measure of the relative

oxygen – depletion effect of a waste contaminant. Both have been widely adopted as measure of pollution effect. The BOD test measures the oxygen demand of biodegra-

Solomon and Kehinde 33

Table 3. The mean values of physiochemical parameter of flowing stream in Jabi compare with standard limit.

Parameter Stream FEPA WHO

DO mg/L 7.73 NOT < 2 6 BOD mg/L 2.80 10 50 Temperature 21.7 26 40 Conductivity 289 70 5

Turbidity 8.88 < 1 5

Alkalinity 114 ND 250 pH 7.4 6-9 7.0-8.5 Nitrate 1.43 20 <45 COD mg/L 3.33 80 1000 Chloride 45.5 240 250 Fluoride 0.23 1.2 1.5 TDS mg/L 260 148 159

DO, Dissolve oxygen, BOD, Biological oxygen demand, COD, Chemical oxygen demand, FEPA, Federal environmental protection agency, ND,Not determined, and WHO, World Health Organization.

dable pollutants whereas the COD test measures the oxygen demand of oxidizable pollutants. The COD is a determinant of the level organic matter and carbon.

The chemical oxygen demand (COD) of the water sample generally varied from 2.0 to 4.0 mg/L. At the month of July and August there was increase in the rate of COD. These could be due domestic activities and industrial activity which had a negative impact on the quality of fresh water and subsequently cause harm to aquatic animals (Morrison et al., 2001). The biological oxygen demand varies significantly from the water sample collected (0.8 to 6.0 mg/L) (Table 1). When this present result was compared with result of COD and BOD of the treated water bodies from developed countries, it was observed that the concentration of COD and BOD differ as reported by UNEP (1993) (Table 3). These results disqualify the use of the water for both domestics and drinking purpose (Fatoki et al., 2003).

Nitrate

The most highly oxidized form of nitrogen compounds is commonly present in surface and ground water because it is the end product of aerobic decomposition of organic nitrogenous matter. Unpolluted natural water usually contains only minute amounts of nitrate (Jaji et al., 2007). In this study, the nitrate (N) concentrations ranged between 1.39 to 1.44 mg/L (Table 2) which vary significantly (p> 0.05). It is important to note that nitrate level in the water body could be a source of eutrophication as the obtained values exceeded the recommended limit for FEPA.

Total hardness

The total hardness of the sample collected varies

significantly (p>0.05). The total hardness values obtained from samples varied from 257 to 262 (Tables 1 and 2). The results obtained were higher than that of WHO standard for drinking water. The standard for the total hardness for drinking water by the WHO is 150 mg/L (WHO, 2004).

Chloride

The total chloride of the water samples obtained varies significantly (p>0.05). The values for the total chloride obtained were 43.4 to 48.7 which was less than the WHO standards for drinking water (WHO, 2004). The standard for total chloride is 250 mg/L. These imply that this water can be used for drinking and domestic purpose (Tables 1 and 3).

Fluoride and Magnesium

The total fluoride of the water sample varied significantly (p>0.05). The values obtained varied from 0.2 to 0.3, which is lesser than the standard for total fluoride for drinking water (Tables 1, 2 and 3). The standard for total fluoride for drinking water and domestic use is 1.5 mg/L. These results corresponded with that of Ogunfowokan et al. (2005). Magnesium also test result also varied significantly (p>0.05). The values obtained ranged from 152 to 157 which is higher than that of the WHO standard for drinking water (Table 3). The allowable value for magnesium concentration is 0.20 (WHO, 2004).

Conclusion The study revealed that there was an adverse impact on the physiochemical parameter on the steam by discharge

34 J. Biosci. Biotechnol. Discov. of sewages and industrial waste in to the stream and contaminates the stream. Waste from house hold may also be dump into the stream or carried in to the stream through drainage systems. This may pose a health risk to rural communities which rely on the water as a drinking source since the physiochemical parameter varied significantly from the standard set by the WHO and FEPA. Recommendation There is need for the intervention of appropriate regulatory agencies to ensure production of high quality water. Appropriate drainage system should constructed and channel away from water bodies that are used for domestic and industrial purpose. Proper publications and awareness to individuals and industries to avoid dumping of refuse or waste in water bodies meant for drinking and other domestic use. Good sewage drainage system should be constructed; and not channeled to water bodies. Proper recycling of domestic and industrial waste should be encouraged especially in the developing countries. REFERENCES Adigun, B. A. (2005). Water quality management in Aquaculture

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drinking-water quality, Draft for review and comments. Nitrates and Nitrites in drinking-water, World Health Organization. (WHO/SDE/WSH/04.08/56).

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Journal of Bioscience and Biotechnology Discovery Volume 2. Page 36-41. Published 18th May, 2017



Assessment of heavy metal concentration in vegetables grown within fungicide treated Cocoa plantations in

Akamkpa Local Government Area, Cross River State, Nigeria and implications to human health

F. E. Ekpo

Department of Biological Sciences, Akwa Ibom State University, Ikot Akpaden, Mkpat Enin, P. M. B. 1167, Uyo, Akwa Ibom State, Nigeria. Email: [email protected].

Copyright © 2017 Ekpo. This article remains permanently open access under the terms of the Creative Commons Attribution License 4.0, which


Received 6th April, 2017; Accepted 1st May, 2017

ABSTRACT: The study was carried out to investigate the concentrations of heavy metals in soil samples and edible vegetables (Heinsia crinita and Gnetum africana) grown within fungicide treated cocoa plantation in Akamkpa communities of Cross River State Nigeria. In each sampling community, the edible vegetables and soil samples were collected for the period of three months (March to May) and were replicated three times. The levels of heavy metals in soil and edible vegetable samples collected were determined using Atomic Absorption Spectrophotometer. The concentrations of heavy metals in soil samples collected from fungicide treated coca plantations were higher than those recorded in the control location in all the sample locations. The concentrations of heavy metals in edible vegetables (Heinsia crinita and Gnetum africana) collected from fungicide treated cocoa plantations exceeded the maximum permissible limits values of heavy metals for edible vegetables. The soils and edible vegetables (Heinsia crinita and Gnetum africana) in the fungicide treated cocoa plantations in Akamkpa communities are contaminated by heavy metals and may pose serious adverse health effect to the consumers and the ecosystem. Key words: Akamkpa communities, edible vegetables, fungicide treated Cocoa plantation, heavy metals. INTRODUCTION The use of chemical pesticides in cocoa plantations in Nigeria has seen a sharp increase in recent years. Farmers in Akamkpa Local Government Area of Cross River State use chemical pesticides as a preventive and control measures against the possibility of devastating crops loss from pests and diseases and also to increase its yield. Chemical pesticides have been employed in cocoa plantations to control and eradicate pests and diseases and also to improve the yield of the crops in the area for several decades. However, there has been a rapid increase in the quantity and use of pesticides in cocoa plantations over the past two decades (Hodgson, 2003). The use of chemicals pesticides to control fungal diseases of cocoa has already caused grave damage to the soil, vegetables, water resources and the entire ecosystem, resulting in the health risk (Alloway, 1995).

Pesticides are widely used in cocoa plantations in Akamkpa Local Government Area probably due to the farmers’ perception of insects and diseases control. This is because it is profitable, and no better alternatives measures are available, then spraying of pesticide is a good investment in the area (George, 2006). Pesticides are synthetic chemicals that are purposely applied to the environment with aim to suppress diseases and pests of plant and animal and also to improve their output.

Although, there is an increasing demand for cocoa beans (Taylor, 2000), pests and diseases pose a major challenge to its production (Bowers et al., 2001). Phytophthora pod rot (black pod) is the most prevalent disease of cocoa in Akamkpa Local Government Area (Isong, 2007). The disease is caused by Phytophthora palmivora and Phytophthora megakarya. The control of


black pod disease is a major challenge for world cocoa cultivation. According to Tan and Tan (1990), several methods have been adopted by researchers and farmers to control Phytophthora pod rot of cocoa. The most common is the routine use of fungicides to supplement good farm management practices. Among the different types pesticides known, coper based fungicides are extensively used by farmers, because of their cost effectiveness and their broad spectrum activity. According to Bempah et al., (2011), copper based fungicide is toxic, persistent nature, and the residual effect in the crops grown is detrimental to health. Repetitive applications (6 to 9 sprays/year) of copper-based fungicides in cocoa plantation bring serious risks to human health and an adverse effect on the sustainability of the agro-ecosystem.

Juraske et al. (2001) observed that exposure to pesticide residues through the diet is assumed to be five times higher than exposure routes of air and water in magnitude. According to the World Health Organization (WHO, 2001), food consumption consists an average of 30% (Based on mass) of fruit and vegetables, and vegetables are the most frequently consumed. Since vegetables are mainly consumed raw or semi-processed, it is expected that they contain higher pesticides residue levels. Given the potential risk of pesticides for public health, the use of pesticides to control cocoa diseases in cocoa plantations in farming communities of Akamkpa Local Government Area is of great concern to the consumers because of its health implication. Although, pesticide residues in foodstuffs have been carried out for decades in most developed countries (Claeys et al., 2011). However, edible vegetables grown within fungicide treated cocoa plantations in Akamkpa Local Government Area are not much investigated from pesticide contamination point of view. Everyday, people are being encouraged by physicians to consume more vegetables for their health benefit. Therefore, the objective of this study was to assess the concentration of heavy metals in edible vegetables grown within fungicide treated cocoa plantations in two communities of Akamkpa Local Government Area of Cross River State and to create awareness on the effects of heavy metals on human beings and the environment and the potential health risks associated with the pesticide residue with regard to consumers. MATERIALS AND METHODS Study area The study was conducted in two cocoa plantations in Akamkpa Local Government Area of Cross River State Nigeria. The cocoa plantations were located at Old Ndepiji community and Owon community. In each of this community, there is a cocoa plantation that was

Ekpo 37 established in early 1980s by the community. Cocoa (Theobroma cacao L.) is a major cash crop cultivated in these communities and it is a source of livelihood to the farmers. The dominant cultural practice of cocoa plantation in these communities involves planting of the cocoa trees on forestlands with various indigenous food crops, vegetables, fruit trees and medicinal species that has economic importance to the farmers and also provide shade for the cocoa plants. In these communities, the popularly eaten food is mainly carbohydrate. Indigenous vegetables play useful role in producing food quality like proteins, minerals, vitamins and fats. Farmers in these communities depend on indigenous vegetables grown in the cocoa plantations. Cocoa play a significant role in the lives of the rural economy in the area, as the industry is dominated by large numbers of smallholder peasant farmers who depend on the cocoa for their sustenance. Cocoa plant, like all living organisms are attacked by a wide range of pests and diseases. When this happens expected production targets by the farmers are not met, their economies and livelihood are adversely affected. A preventive and curative measure which is the application of chemical pesticides, mostly coper based fungicides are therefore necessary in the cocoa plantations to control pests and diseases and to increase the yield (Adeyeye and Balah, 2007). Indigenous vegetable and fruit crops such as Heinsia crinita, Gnetum Africana, Lasienthera Africana, Mangifera indica are grown within cocoa plantation for consumption and to provide income to the farmers. These vegetables are consumed on daily basis. They are cooked in almost all the hotels, restaurants and bars in these communities. These indigenous vegetables are also importance dishes in all traditional feasts or parties (naming ceremony, birth day party, traditional marriage, wedding ceremony and burial ceremony) in the communities. Each year, chemical pesticides mostly copper based fungicides are sprayed in cocoa plantations where these indigenous vegetables and fruit crops are grown together with the cocoa trees. According to Ukpong (2010) chemical pesticides are sprayed in cocoa plantations for about 6 to 9 times of high doses in a year depending on the variation of rainfall pattern to control or reduce the impact of pests and diseases in their farms. Realizing the wide usage of these indigenous vegetables (Heinsia crinita, and Gnetum africana) in the studied communities, it become necessary to ascertain the concentrations of heavy metals in these vegetables and the effect to the well-being of the consumers. Materials Samples of soil and leaves of Heinsia crinita, (Atama) and Gnetum Africana (Afang) were collected from treated cocoa plantations in the two different communities (Old Ndepiji and Owon) in Akamkpa Local Government Area of Cross River State and the control location was a bush


Table 1. The mean Concentrations (mgkg-1

) of heavy metals in soils from Fungicide treated cocoa plantations in Communities of Akamkpa L. G Area, Cross River, Nigeria State.

Heavy metals Old Ndepiji Community Owon Community Mfanosing Community

Fe (mgkg-1

) 5.17±0.28 5.22±0.05 3.05±0.01

Zn (mgkg-1

) 4.30±0.15 4.60±0.02 2.45±0.10

Cu (mgkg-1) 7.28±0.15 7.22±0.20 2.01±0.05

Pb (mgkg-1

) 2.95±0.02 2.68±0.05 0.05±0.10

Cr (mgkg-1

) 3.35±0.05 3.45±0.20 1.02±0.01

Cd (mgkg-1) 2.01±0.01 2.10±0.05 0.01±0.02

Ni (mgkg-1

) 4.25±0.15 3.75±0.25 0.10±0.30

fallow at Mfanosing where there is no cocoa plantation for the period of three months (March to May). In each of the community, samples of soil and leaf of (Heinsia crinite and Gnetum africana) were collected from fungicide cocoa plantations and untreated pesticides bush fallow at Mfamosing (control) location. Samples of soils and leaves were taken from three different places in each of the studied community and this was done by a random process from the field. The collected samples were sealed and labeled with a unique sample identity and taken to Central Research Laboratory, Akwa Ibom State Ministry of Science and Technology for heavy metals analysis. Methods Preparation and preservation Samples of leaf Heinsia crinita (Atama) and Gnetum africana (Afang) were washed in fresh running water to eliminate dust, dirt, possible parasites and then were again washed with deionized water. The clean leaves samples were air-dried and placed in an electric oven at 65°C for 48 hours. The dry leaves samples were homogenized by grinding using a ceramic coated grinder and used for metal analysis. All soil samples were spread on plastic trays and allowed to dry at ambient temperature for 8 days. The soil samples after drying were ground with a ceramic coated grinder and sieved through a nylon sieve. The final samples were kept in labeled polyethene containers at ambient temperature before analysis. Digestion and determination Five (5) grams of leaves samples were weighed into 50ml beakers, followed by the addition of 20 ml mixture of analytical grade acids HNO

3:HCIO

4 in the ratio 5:1. The

digestion was performed at a temperature of about 150°C for 2 hours. After cooling, the solution was made up to a final volume (50 ml) with distilled water. The metal

concentrations were determined by atomic absorption spectrometry using a Unicam Model 299 Atomic Absorption Spectrophotometer (AAS). Analysis of each sample was carried out three times to obtain representative results and the data reported in mg/kg (on a dry weight basis). Data analysis Data were generated in triplicates and expressed as mean (±) standard deviation and was determined according to the method of Steel and Torrie (1980). Significant differences were determined by the LSD (probability level of 0.05 was considered statistically significant).

RESULTS The mean values of heavy metals Copper (Cu), Lead (Pb), Zinc (Zn),Iron (Fe), Cadmium (Cd), Chromium (Cr) and Nickle ( Ni) contents of soil samples obtained from two cocoa plantations (Old Ndepiji and Owon) and the control at Mfanosing is shown in Table 1. The level of heavy metals contents observed from the soil samples in Old Ndepiji and Owon cocoa plantations were significantly higher than those recorded from the control location. The mean values of heavy metals contents obtained from plant samples (Heinsia crinite and Gnetum africana) in two cocoa plantations and the control location is shown in Table 2. The mean values of heavy metals contents obtained from the plants samples in Old Ndepiji and Owon cocoa plantations were significantly higher than those obtained from the control location. Several studies have shown that plants grown in heavy metals polluted soils have higher concentrations of heavy metals than those grown in unpolluted soils (Gutormsen et al., 1995; Kabata-Pendias and Pendias, 1992).

The mean values of Cu obtained from soil samples in two fungicide treated cocoa plantations communities (Old Ndipiji and Owon) were 7.28±0.28 mg/kg and 7.22±0.05 mg/kg respectively. While the mean values Cu recorded

Ekpo 39

Table 2. The mean Concentrations (mgkg

-1) of heavy metals in edible vegetables from Fungicide cocoa plantations in

Communities of Akamkpa L. G Area, Cross River State, Nigeria.

Heavy metals Vegetables Old Ndepiji Community Owon Community Mfanosing Community

Fe Heinsia crinita 13.02±0.02 12.19±0.15 5.25±0.10

Gnetum africana 11.05±0.10 12.05±0.00 4.90±0.05

Zn Heinsia crinita 11.54±0.05 13.15±0.12 5.66±0.10

Gnetum africana 12.01±0.01 12.98±0.10 5.00±0.20

Cu Heinsia crinita 12.05±0.01 12.15±0.01 2.14±0.11

Gnetum africana 12.15±0.02 12.50±0.15 2.09±0.05

Pb Heinsia crinita 3.75±0.05 5.78±0.20 1.04±0.25

Gnetum africana 3.01±0.10 6.25±0.01 0.98±0.15

Cr Heinsia crinita 4.75±0.10 5.16±0.10 0.55±0.10

Gnetum africana 5.50±0.05 5.35±0.01 0.75±0.30

Cd Heinsia crinita 2.95±0.15 4.35±0.70 1.05±0.10

Gnetum africana 2.55±0.11 4.65±0.50 1.01±0.05

Ni Heinsia crinita 7.11±0.02 7.21±0.02 0.95±0.00

Gnetum africana 8.05±0.10 7.03±0.10 1.05±0.10

at control location (Mfamosing) was 2.05±0.01 mg/kg. The mean values of Fe and Zn contents obtained from soil samples in Old Ndepiji were 5.17±0.10 mg/kg and 4.30±0.15 mg/kg respectively. The mean values of Fe and Zn recorded in soil samples obtained from Owon cocoa plantations were 5.20±0.25 mg/kg and 4.25±0.01 mg/kg respectively. While in Mfamosing (control location) the mean values of Fe and Zn in the soil samples were 3.12±0.01 mg/kg and 2.45±0.05 mg/kg respectively. The concentrations of Pb and Cd recorded from soil obtained in Old Ndipiji cocoa plantation were 2.99±0.05 mg/kg and 2.01±0.01 mg/kg respectively. In Owon cocoa plantation the concentrations of Pb and Cd were 2.68±0.02 and 2.10±0.10 respectively. While in Mfamosing (Control location) the mean values of Pb and Cd were 0.05±0.01 and 0.01±0.01 respectively. Similar trend of Ni and Cr contents were recorded in soil samples obtained from the two different cocoa plantations and the control location at Mfamosing. The higher concentrations of heavy metals recorded in the soil samples obtained from two fungicide treated cocoa plantations in the study communities could be attributed to chemical pesticides sprayed to control the black pods disease of cocoa in the plantations which the chemical pesticides contained a high level of these metals. Soil samples obtained from control location (Mfamosing) had reduced heavy metals contents because the area is not affected with these pesticides.

The two indigenous vegetables (Heinsia crinite and Gnetum africana) collected from two fungicide treated cocoa plantations, regardless of locations recorded a higher concentrations of heavy metals in the plant samples. The mean values of Cu contents in plants samples (Heinsia crinite and Gnetum africana) obtained from Old Ndepiji were 12.05±0.01 mg/kg and 12.15±0.02 mg/kg respectively. In Owon cocoa plantation, the

concentrations of Cu in plant samples (Heinsia crinite and Gnetum africana) were 12.15±0.01 mg/kg, 12.00±0.00 mg/kg respectively. While at the Mfamosing, control location the concentration of Cu in the indigenous vegetables (Heinsia crinite and Gnetum africana) were 2.14±0.11 mg/kg, 2.09±0.05 mg/kg respectively. The mean values of Fe contents obtained from the indigenous vegetables (Heinsia crinite and Gnetum africana) in Old Ndepiji cocoa plantations were 13.02±0.02 mg/kg and 11.05±0.10 mg/kg respectively. In Owon community, the mean values of Fe contents recorded from (Heinsia crinite and Gnetum africana) were 12.19±0.15 mg/kg and 12.02±0.00 mg/kg respectively. While at Mfamosing, control location the concentration of Fe obtained from the plant samples (Heinsia crinite and Gnetum africana) were 5.25±0.10 mg/kg and 4.95±0.05 mg/kg respectively. The Concentrations of Pb and Cd contents observed in plant samples (Heinsia crinite and Gnetum africana) collected from Old Ndepiji cocoa plantations were (3.75 ±0.05 and 3.01±0.01 ), (2.90±0.15 and 2.15±0.02) mg/kg respectively. Similar trend of Pb and Cd contents were recorded in plant samples (Heinsia crinite and Gnetum africana) obtained from Owon cocoa plantations. While at Mfamosing (control location) the mean values of Pb and Cd observed in the plant samples (Heinsia crinite and Gnetum africana) were (1.04 ±0.05 and 0.51±0.15), (1.02±0.03 and 0.98±0.10) mg/kg respectively. The low values of heavy metals observed in plant samples collected at Mfamosing bush fallow (control site) may be attributed to the fact that the area is free from chemical pesticides. The mean values of Ni contents recorded in indigenous vegetables (Heinsia crinite and Gnetum africana) obtained from Old Ndepiji and Owon community cocoa plantations were (7.11±0.02, 8.05±0.10) and (7.21±0.02, 7.05±0.10) mg/kg respectively. In Mfamosing,

40 J. Biosci. Biotechnol. Discov. the mean values of Ni recorded in (Heinsia crinite and Gnetum africana) were (1.75±0.25 and 1.20±0.10) mg/kg respectively. However, the high concentrations of heavy metals recorded in the indigenous vegetables collected from the two cocoa plantations may be due to bioaccumulation of these metals in the plant tissues. DISCUSSION The concentrations of the heavy metals observed in soils obtained from fungicide treated cocoa plantations in the communities of Akamkpa Local government Area of Cross River state, Nigeria were higher than the concentrations of heavy metals observed in soil samples from Mfanosing bush fallow (control location). The higher concentrations of heavy metals recorded in the soil samples collected from fungicide treated cocoa plantations may be due to the frequent applications of chemical pesticides to control the diseases of cocoa and to increase its yield in the plantations. The result was in line with the work of (Nzegbule, 2007) who reported the higher concentration of heavy metals in soil obtained from pesticides treated cocoa plantations in Umuahia, Abia State of Nigeria. The soil samples obtained from fungicide treated cocoa plantations in the two communities (Old Ndepiji and Owon) showed a significant increased (p<0.05) for all the metals analyzed. The result obtained from the analysis showed that the soil and vegetables obtained from the fungicide treated cocoa plantation were higher than those obtained from the control location. The lower values of heavy metals observed at the control site (Mfamosing bush fallow ) in the soil samples may also be attributed to the fact that the area is free from chemical pesticides application. Since the area does not engage in cocoa plantation practice. The concentrations of the heavy metals in the soil samples obtained from the fungicide treated cocoa plantations communities were higher than the FEPA (1991) and WHO (2001) permissible limits. While the concentrations of heavy metals recorded in the soil samples obtained from the Mfamosing bush fallow (control location) were within the permissible limits. This implies that the soil samples obtained from fungicide treated cocoa plantations in Akamkpa Local Government Area of Cross River State are contaminated with heavy metals and pose serious environmental and health risk to the rural farmers.

The edible vegetables (Heinsia crinite and Gnetum africana) obtained from fungicide treated cocoa plantations communities of Akamkpa Local Government Area recorded the highest concentrations of heavy metals. The concentrations of the metals in edible vegetables obtained from fungicide treated cocoa plantations were higher than those obtained from the control location (Mfamosing bush fallow). The result was in line with the work of Ekpo et al. (2014) who reported

the bioavailability of heavy metals in chemical polluted environment. The bioavailability of heavy metals recorded in edible vegetables obtained from fungicide treated cocoa plantation may be attributed to the frequent applications of this pesticide to increase the yield of cocoa and also to control the black pods of cocoa caused by a fungus (Phytophthora palmivora) in the cocoa plantations.

The situation is increasingly becoming a concern to the rural farmers in cocoa producing communities in Akamkpa Local Government Area of Cross River State as the business expanding and pesticides applications are carried out without any control measures.

The results obtained from laboratory analysis indicate that soils and edible vegetables obtained from fungicide treated cocoa plantations communities in Akamkpa Local Government Area are contaminated with heavy metals.

Scientists have reported that heavy metals encouraged tumor and mutations at greater amounts in animals and they have capacity of producing genetic harm to germ cells of both male and female animals (Hayes, 2004). Wagner (2003) reported that heavy metals are growing toxins which through biomagnifications and bioaccumulations in plants affect the health of humans. Heavy metals are extremely poisonous at comparatively smaller amount in animal including human beings. Ingesting food or drinking water with very greater degree of heavy metals relentlessly infuriates the stomach results in diarrhea and vomiting (Hayes, 2004). Dudka and Miller (2009) reported ways for the connection of heavy metals to the human body comprises direct breathing of polluted air, drinking of polluted water and direct contact with soil and ingestion of food comprising of plants grown in metal-polluted soil.

Heavy metal-polluted food can severely reduce some vital nutrients in the body that are accountable for declining immunological defenses, growth delay, reduced psychosocial abilities, and increases greater occurrence of upper gastrointestinal cancer degrees (Arora and Musa, 2008).

More et al. (2003) reported that heavy metals are non-biodegradables and they are recognized as environmental contaminants causing cytotoxic, mutagenic and cancerous (carcinogenic) effects in animals. The biotic half-lives of these heavy metals are lengthy and furthermore they have capacity to store in various organs of the body and therefore causing health problems in human. Continuous exposure of heavy metals in soil and edible vegetables may lead to the accumulation of these heavy metals in kidneys, causes kidney diseases, lung disorders like bronchiolitis, emphysema and alveolitis (Smith, 2010). Heavy metals are also known for its toxicity and negative impacts on human health. Absorption of swallowed heavy metals may have a severe danger to public health. Some long lasting negative impacts of Lead toxicity includes colic, constipation and anemia (blood related disorder) (Bolger

et al., 2000).

Although, a number of heavy metals are essential for biological systems, it’s become toxic when their concentration level exceeds those required for correct nutrition. According to Walter et al. (2005), metals toxicity occurs when an organisms is unable to cope with additional metal concentration by direct usage, storage and excretion. Conclusion The concentrations of heavy metals recorded in edible vegetable obtained from fungicide treated cocoa plantations in Akamkpa Local Government Area of Cross River State, Nigeria were higher than the FEPA and WHO permissible limits. Therefore, edible vegetables and fruits grown in fungicide treated cocoa plantations communities pose health hazards for consumers. REFERENCES Adeyeye, F., & Balah, H. (2007). Fungicide-Derived Copper

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Journal of Bioscience and Biotechnology Discovery Volume 2. Page 42-48. Published 23rd May, 2017



The resultant physiochemical effect of organic, artificial and mixed diets of Clarias gariepinus to enhance

growth

Solomon, R. J.* and Zainab, A. U.

Department of Biological Sciences, Faculty of Science, University of Abuja, Abuja, Nigeria.


Copyright © 2017 Solomon and Zainab. This article remains permanently open access under the terms of the Creative Commons Attribution License


Received 30th April, 2017, Accepted 20th May, 2017

ABSTRACT: This study was carried out at the University of Abuja permanent site Gwagwalada, Abuja to determine the water quality parameters of Clarias gariepinus fed with organic, artificial and mixture of organic and artificial diets. The materials and the fingerlings were all purchased from Ajima farms, Kuje Area Council. After seven days of acclimatization, a group of ten (10) fishes were stocked in three treatments of plastic rubber with carrying capacity of 60 litres. Treatment A were fed with artificial (Coppens), treatment B with organic (chicken droppings) while treatment C with mixture of artificial and organic (both Coppens and chicken droppings) and the experiment ran for a period of 90 days. They were fed 7 days a week (twice daily) between 8 and 6 pm by hand casting at the rate of 4% body weight. The water quality parameters were taken once a week. Nitrate, pH and ammonia were calculated using combii 11 strip while temperature was taken using mercury in glass thermometer. The temperature was between (26 to 27°C), pH (5 to 8 mg/l), nitrate (0.01 to 60 mg/l), ammonia (0.01 to 2.15 mg/l) and dissolved oxygen (5 to 6.6) respectively. All quality water parameters taken were found to be within the recommended range for optimum growth performance of Clarias gariepinus. Key words: Artificial, organic (chicken droppings), combii 11 strip. INTRODUCTION Water is vital to the existence of all living organisms, but this value resources is increasingly being threatened as human population grow and demand more water of high quality for domestic purposes and economic activities. It is now generally accepted that aquatic as holding tanks that supply water for human activities. Rather these environments are complex materials that require careful use to sustainable ecosystem functioning well into the future.

Rivers are the most important freshwater resources for man, unfortunately disposal of sewerage, industrial waste and plethora of human activities which affects their physio-chemical characteristics and micro biological quality (koshy and Naya, 1999). Pollution of the aquatic environment is a serious and growing problem. Increasing numbers and amount of industrial agricultural and commercial chemicals discharged into the aquatic

environment have led to various deleterious effects on aquatic organisms. Aquatic organisms including fish accumulate pollutants directly from contaminated water indirectly via the food chain (Hammer, 2004).

Owing to the large quantity of effluence discharged to the receiving waters, the natural processes of pathogen reduction are inadequate for protection of public health. In addition industries wastes that alter the water PH and provide excessive bacterial nutrients often compromise the ability of natural process to inactivate and destroy pathogens (Gerardi and Zimmerman, 2005). The extent of discharge of domestic and industrial effluents is such that rivers receiving untreated effluent cannot provide the dilution necessary for their survival as good quality water sources. The transfer of unfavorable release from industries is detrimental to human and animal health safety (Adekunle and Eniola, 2008). Disposal of sewage



waste into a large volume of water could increase the biological oxygen demand (BOD) to such a high level that all the available oxygen may be removed, consequently causing the death of all aerobic species (Maduka, 2004). Prevention of river pollution requires effective monitoring of physics, chemical and micro-biological parameters (Chandra et al., 2006).

African Catfish

The African catfish Clarias gariepinus belongs to the family clariidae (Air-breathing Catfishes), order siluriformes (Catfishes), class actinopterygii (ray-finned fishes), sub-phylum vertebrata, phylum chordate and kingdom Animalia. The family clariidae is divided in to two genera viz: Clarias and Heterobrachus. There are over hundred species in this family occurring naturally throughput species in this family occurring naturally throughout most of Africa and the southern half the Java and the Philippines (Little et al., 1999). Clarias gariepinus is generally considered to be one is generally considered to be one of the most important tropical catfish species for aquaculture. It has an almost pan-African distribution, ranging from the mile to West Africa and from Algeria to southern Africa. They also occur in Asia minor (Israel, Syria and South of Turkey) (C. gariepinus) at various geographical locations bears different names. It is called C. lazera in northern and central Africa, (C. gariepinus) in South Africa (Vivean et, al. 1985). C. gariepinus is characterized with naked skin and elongate with fairly long dorsal and anal fins. The dorsal fin has 61-80 soft rays and the anal fin has 45-65 soft rays. They have strong pectoral fin with spines that are serrated on the outer side (Taugels, 1986). In C. gariepinus, exchange of respiratory gas (i.e oxygen and carbon dioxide) takes place through the gills. Like any other mudfish, it has accessory breathing (arbores cent) organ which enables the fish not only to like in stagnant pools but to travel over damp ground (C. gariepinus differ from other catfishes in having an auxiliary breathing organ in this special pocket attached to the second and fourth gill arches and are responsible for the ability of (c. gariepinus) to live out of water much longer than other catfishes.

Feed and feeding development

Feed and feeding of catfishes in grow out ponds are perhaps the most documented in literature. Various efforts have been made to establish the crude protein and amino acid requirement of C. gariepinus (Ayinla and Akande,1988) recommended 35% and 40% crude protein (CP) for raising table size and brood acid (EAA) required by warm water fish species only 3 EAAS studied have been documented and these are arginine, methionine and lysine. In order to formulate and compound aqua feeds that will meet the nutrient requirement of the catfish at affordable cost, several conventional and non-

Solomon and Zainab 43 conventional animal by-product and plant residues have been tested to substitute or replace fish meal. Feeding development has moved from the use of single ingredient, broadcasting un-pelleted meal to pelleting and in fact the use of pelleted floating fish which has made a big difference to aquaculture development in Nigeria as C. gariepinus is being raised to maturity within 6 months (Ayinla and Akande,1988).

Conventional feed sources

These are feed stud that are regularly used in the formulation of fish feed. Their usage is standardized and widely acceptable. Many of these are cheap and readily available in very large quantity. They are usually agro-industrial by products. Example include wheat bran, groundnut cake and rice bran, some are animal based (fish meal, blood meals, shrimp meal). Whereas others are plant based for example maize, soya bean meal, cotton seed meal. Generally these materials are relatively cheap and available throughout the year. However, Otubusin, (1992) pointed out that the effectiveness of a feed preferred is a determinant rather than cheapness. Hence balance is therefore needed, if aquaculture is profitable.

Non-conventional feed stuff

These are locally available feed stuff that is not standardized. The usage is not widely-spared and they are not consumed by man in most cases. Utilization in aqua-feed is very common especially in rural area of sub-Saharan African among low income group that are normally come from three sources. They include, kitchen wastes plant sources of feed and animal sources.

Animal sources

The non conventional feed stuff of animal origin are high quality feed ingredients that could compare to some extent with the conventional types. These are cheaper by virtue of the fact that there is no competition for human consumption. However, the only problem with these feed stuffs is their unavailability in large commercial quantities for the sustenance of aquaculture industries. In most part of Africa, these are available in small quantities and their production is inconsistent and sporadic in nature. Examples include tadpole meal, maggots earthworm meal, housefly larvae among others etc (Mohammed, 2009; Ugwumba, 2003; Ibiyo and Olowesegun, 2004).

Plant source of feed These are generally known as non-conventional plant feed stuff (NCPF). These are in abundant, almost in every locality in Africa their potential and utilization in


Table 1. Proximate composition analysis of feed ingredients.

Ingredients MO CP CF CA P LS MT MR (Kcalg-1

)

Fish meal 12.57 66.23 1.08 5.14 2.89 4.85 2.62 2861

Groundnut Cake 19.10 41.40 7.74 0.19 0.63 1.59 0.57 2892

Blood meal 15.12 76.20 1.46 0.29 0.09 6.90 1.00 3080

Dried brewers grains 12.89 18.60 20.00 0.20 0.16 0.81 0.98 1980

White maize meal 13.09 9.38 2.31 0.032 0.12 0.07 0.24 3434

MO, moisture, CP, crude protein, CF, crude fibre, P, phosphorus, LS, lysine, MT, methionine + cysteine,

ME, Metabolizable energy.

aqua culture fed have been reviewed (Ugwumb, 2003). Their levels of inclusion in aqua feed varies and largely depends on their availability nutrients level, processing technique, species of fish and cultural farming pattern prevalent in the locality (Mohammed, 2009). The recommended level of inclusion of NCPF so many factors which limit higher level of incorporation and 305 of the diet. There are such as low protein content, amino acid imbalance and presence of ant nutritional factors (Alegbeleye et al., 2001).

Kitchen wastes

This is being used at household level of aqua culture especially in backyard fish farming where ruminants from house fold wastes are used to feed the fish, category are cassava peel was fed to tilapia. Recommended additional of 20% methonine with 205 inclusion of cassava peel also kitchen remnants like bread, cooked rice and yam are commonly used in the culture of fish (Alegbeleye et al., 2001).

Aim of the study

Fish farming is expanding rapidly throughout the world and has a high potential for the provision of valuable protein in less developed countries (Little and Edwards, 2003). Due to global population expansion, demand for high quality animal protein, especially from aquatic sources is rising. Increasing the aquaculture production is clearly needed to meet this demand in the third millennium, especially as the capture fisheries resources are declining due to over fishing, habitat destruction and pollution (Dunham et al., 2000). Hence, the study aim to analyze the physiochemical properties of the environmental conditions of the University of Abuja for Clarias gariepinus survival and growth.

MATREIALS AND METHOD

Study area

The study was carried out at the University of Abuja located at the Federal Capital Territory, Abuja. It is

located along the Kaduna-Lokoja express road covering about 11,824 hectares. Abuja has an area of 1,043 km² and a population of 157770 at the census of 2006.

Aquarium and treatment Thirty fingerlings of Clarias gariepinus were procured from Ajima farm in Kuje Area Council. These fingerlings were collected in an open plastic rubber with oxygenated water and were transported to the study area by car. After seven days of acclimation, a group of 10 fishes were stocked in three treatments of plastic rubber with carrying capacity of 60 L. Treatment A were fed with inorganic diet (Coppens), treatment B with organic (chicken droppings) while treatment C with mixture of inorganic and organic (both Coppen and chicken droppings). The experiment ran for a period of 90 days. They were fed seven days per week (twice daily at 8.00 am and 6 pm) by hand casting at the rate of 4% of their body weight.

Biochemical composition of feed ingredients used in basal diet formulation Ingredients used in the formulation of diets (B) include: fishmeal, groundnut cake, blood meal, dried brewers grains and white maize meal (Table 1). The results of the analysis show that groundnuts cake had the highest (19.10%) moisture content, while fish meal had the lowest (12.57%). In terms of crude protein, blood meal had the highest (76.20%) value while maize meal is recorded the lowest (9.38%). The least of crude fibre is dried brewers grain with the value of (20.00%) while the lowest (1.08%) was recorded in fish meal. Fish meal had the highest (5.14%) calcium contents while maize meal had the lowest (0.03%).

Nutrients compositions Nutrients composition of food ingredients was obtained from proximate ingredient as shown in (Table 1). The organic fertilizer was analyzed. Nutrients analyzed include: moisture (MO), Crude protein (CP), crude fibre

(CF), calcium (CA), phosphorous (P), lysine (LS) and methionine + cystcine (MT). This was done according to the method of (AOAC 1999). Estimation of metabolizable energy (ME) of ingredient for the feed was calculated by converting the gross energy using the following equation as described by (Miller and Payne, 1959). Evaluation of fish performance Fish in each experimental tank were weighed every week to know the growth and health status of the fish and to adjust the feeding rate. Data obtained were for estimation of average weight gained (final weight-initial weight), mean length increment (final length-initial length), specific growth rate (100 x Ln final average weight - Ln initial average weight/days), Length-weight relationship was calculated using the convectional formulae for calculating the length-weight relationship.

W = aLb ------------------(1)

The equation (1) and data were transformed into logarithm before the calculations were made therefore equation (1) becomes:

Log W = log a+b log l ------------- (2)

Where, W = weight of the fish (g), L = standard length of the fish (cm), a = constant and b = an exponent

Condition factor

The condition factor “k” was also calculated for individual fish for each month using the conventional formulae (Worthington and Richard, 1930).

K = w × 100/L3 -------------(3)

Where, K = condition factor, W = weight (g) and L = standard length (cm)

Water quality analysis

Water quality parameters of the experimental tanks was analyzed at every 7 days during the period of study for the minimum and maximum temperature, Ammonia, pH, dissolved oxygen and Nitrate thermometer, ammonia, pH, Nitrate and dissolve oxygen were determined using combi II (test strip). Data generated from the experimental analyze variance (ANOVA) which was carried out to test the variation of the water quality parameters. These were found to be within acceptable limit for fish growth and health. Phosphorus was very high (2.89%) in fish meal and very low (0.09%) in blood meal. The highest (4.85 and 2.62%) lysine and methionine values were observed in fishmeal while lowest (0.27 and 0.24%) were seen in white maize feed

Solomon and Zainab 45 Table 2. Composition of organic fertilizer and formulated feed used in 90 days in tank a, b, and c.

Table 3. Nutrient composition, organic, formulated feeds, and coppens used in the culture of Clarias gariepinus for 90days.

Nutrients Organic fertilizer Basal diet Coppens

Dry matter 92.30 90.88 90.092

ME 3126.00 3102.16 3045.30

Methionine 1.66 0.70 0.893

Lysine 2.83 1.93 1.866

Phosphorus 0.80 0.47 0.827

Calcium 0.74 0.52 1.110

Lipids 15.10 7.01 12.50

Crude fibre 2.35 6.30 2.18

Crude Protein 39.00 35.00 44.00

respectively. Metabolizable energy was very high (3434 Kcal kg

-1) in white maize meal and lowest (1980 kcal Kg

-

1) values was recorded in dried breweries grains (Tables

2 and 3).

Production parameters The list weight gained and the highest from week 0 to 12 in Tank A, B and C were 0 to 14.02, the least and the highest length gained in all the Tanks A, B and C from weeks 0 to 12 were 0 to 6.8 and the survival rate were 80 to 100 respectively which shows that not only the feed but the water parameters were within the acceptable rate for the growth and survival rate of the fish.

RESULTS AND DISCUSSION Water quality parameters are one of the major importance factors in aquaculture production the water quality parameters such as temperature, pH, Nitrate, Ammonia and dissolved oxygen were determined during the study period. The abnormal concentration of any of these water quality parameters may have been the cause of the fish death. However numeration and density stress are additional parameters for fish death. The temperature

Ingredients Tank A Tank B Tank C

Organic fertilizer - 50.00 50

Coppens 50.00 - 50

Salt 0.25 0.25 0.25

Fish meal 4.00 4.00 4.00

Groundnut cake 12.90 12.90 12.90

Blood meal 6.00 6.00 6.00

Dried brewers grain 12.95 12.95 12.95

White maize meal 14.90 14.90 14.90


Table 4. Summary of water quality parameters for Tank A.

Parameters Sum Average Range

Temp (°C) 345 26.53846 1.00

pH (mg/l) 79.8 6.13846 5.00

Ammonia (mg/l) 17.11 1.31615 1.82

Nitrate (mg/l) 150.14 11.5923 5.90

Dissolved acid 79.8 6.13846 5.00

Table 5. Summary of water quality parameters for Tank B

Parameter Sum Average Range

Temp °C 344 26.4615 1.00

pH (mg/l) 103.60 7.92308 1.10

Ammonia (mg/l) 6.05 0.46538 1.57

Nitrate (mg/l) 150.12 11.54769 59.90

Dissolved acid 79.9 6.14615 5.0

Table 6. Summary of water quality parameters for Tank C.

Parameter Sum Average Range

Temp °C 346 26.61538 1.00

pH (mg/l) 104 8 0.80

Ammonia (mg/l) 16.63 1.279231 1.82

Nitrate (mg/l) 150.13 11.54846 59.90

Dissolved acid 76.6 6.046154 1.00

recorded during the study period ranged between 26°c to 27

0c respectively. The temperature reading in all the

experiment (Tank A, B and C) were within a permissible range. These shows that the readings are within a required or tolerable range for the culture of fish for optimum fish performance. The mean hydrogen 10 m (pH) concentration recorded for the treatments, ranged from between 7.0mg/L to 8.5mg/L. This shows that the acidity of the experimental water were in the range that supported the survival of the fishes into the experimental tanks and fall within the recommended pH range of 6.5 mg/L to 85 mg/L for good performance. The concentration of Ammonia in the experimental water was low. Tank A has a mean ammonia level of 1.36 mg/L, Tank B 0.45 mg/l and Tank C 1.20 mg/L respectively. The low level of Ammonia concentration in the experimental water could be due to the low level of biomas. The low level of oxygen may be due to metabolic activities of the fishes and of bacteria decaying organic material such as underutilized feel. The mean value of dissolved oxygen of the experimental water was found to be between 5.5 mg to 6.6 mg/L. The ranges of the dissolved oxygen of the experimental water remain within

acceptable limits (Huet, 1972). In the early week of the study, Nitrate was high but gradually lowed as the growths of the fishes were achieved in all the experiment. The average values of Nitrate in the experimental water, 11.55 mg/L for tank A, 11.54 mg/L for tank B and 11.54 mg/L for tank C respectively may have adequately supported the growth of the fishes (Tables 4, 5 and 6 and Figures 1, 2 and 3). Conclusion The result of this study shows that Tank A and C has moderate values of all the physiochemical parameters studied compared to Tank B perhaps this could be the reason why the fishes in Tank A and C did well than those in than B. Generally, all water quality parameters studied were found to be within the recommended ranges for optimum performances of Clarias gariepinus. This indicates that the environmental conditions of the permanent site of the University of Abuja may offer condusive conditions for not only Clarias gariepinus survival and growth but for other fishes.

Solomon and Zainab 47

Figure 1. Chart for Water Quality parameters for Tank A (Inorganic diet).

Figure 2. Chart for Water quality parameters for Tank B (organic diet).

Figure 3. Chart for water quality parameters for Tank C (organic and inorganic diet).

0

10

20

30

40

50

60

70

Water quality parameter measurements (Tank A

inoganic diet coppeans)

Temp (◦c)

PH (MG/L)

AMMONIA (MG/L)

NITRATE (MG/L)

DISSOLVED OXYGEN

0

10

20

30

40

50

60

70

Water quality parameter measurements Tank B {organic diet (chicken dropping)}

Temp (◦c)

PH (MG/L)

AMMONIA (MG/L)

NITRATE (MG/L)

DISSOLVED OXYGEN

0

10

20

30

40

50

60

70

Water quality parameter measurements (Tank C

mixture of organic and inoganic diet )

Temp (◦c)

PH (MG/L)

AMMONIA (MG/L)

NITRATE (MG/L)

DISSOLVED OXYGEN

48 J. Biosci. Biotechnol. Discov. REFERENCES Adekunle, A. S., & Eniola, I. T. K. (2008). Impact of industrial

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Ayinla, O. A., & Akande, G. R. (1988). Growth response of Clarias gariepinus on silage based diets. Nigeria Institute of Oceanography and Marine Research. Technical Paper, 62, 15.

Chandra, R., Singh, S., & Raj, A. (2006). Seasonal bacteriological analysis of Gola river water contaminated with pulp paper mill waste in Uttaranchal, India. Environmental monitoring and assessment, 118(1), 393-406.

Dunham, R. A., Majumdar, K., Hallerman, E., Bartley, D., Mair, G., Hulata, G., Pongthana, N, Bakos, J, Penman, D., & Gupta, M. (2000). Review of the status of aquaculture genetics. In Aquaculture in the Third Millennium. Technical Proceedings of the Conference on Aquaculture in the Third Millennium, Bangkok, Thailand Pp. 137-166.

Gerardi, M. H, & Zimmerman M.C (2005) Waste Water Pathogens. John Wiley and sons Inc; Hobaken NJ, USA: Pp. 3-4.

Hammer, M. J. (2004). Water and waste water Technology. 5th

Edition Practice Hall Inc; Upper Saddle River, NJ USA, Pp. 139-141.

Huet, M. (1972). Textbook of fish culture, breeding and cultivation of fish. Fishing News (books) Ltd. England. p. 33.

Ibiyo, L. M. O., & Olowosegun, T. (2004). The potential for improving profitability in aquaculture pp. 45-53. In processing of the 19

th Annual conference of the fisheries society of

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farming systems. Inland water resources and aquaculture service. Animal production service. FAO, Rome.

Little, D. C., & Muir, J. F., & Price, R. (1999). Fish farming in tropical fresh water. Stoas Box 78m 6700 AB Wageningen, Netherlands. p. 64.

Maduka, H. C. C. (2004). Water pollution and Man’s health in environment Degradation, Reclamation conservation and pollution control for the rural women and the youths. Green-line publisher, Ado Ekiti, Nigeria, Pp. 198-203

Miller, D. S., & Payne, P. R. (1959). Aballistic bomb calorinmeter. British Journal of Nutritious, B501-508.

Mohammed, F. A. S. (2009). Histopathological studies on Tilapia zilli and Solea vulgaris from lake Quran, Egypt. World J. Fish Mar. Sci, 1, 29-39.

Otubusin S. O. (1992). Aquaculture food security and environment invented. Lecture presented at the formal launching at the National Association of fisheries students (NAFIS). University of Agriculture, Abeokuta (UNAAB).

Taugels, G. (1986). A Systematic revision of the Africa species of the genus clarias (pisces cllaridae). Annual Muse Royal del’ Afrique centrate, Pp. 247-199.

Ugwumba, A. O. (2003). Aquaculture options and the future of fish supply in Nigeria. The zoologist, 2, 96-122.

Viveen, W. J. A. R., Richter, C. J. J., Van Oordt, P. G. W. J., Janssen, J. A. L., & Huisman, E. A. (2013). Practical manual for the culture of the African catfish (Clarias gariepinus).




Growth respond and nutrient utilization of Clarias gariepinus fed bean cake

Chiokwe, G. I. and Solomon, R. J.*

Department of Biological Sciences, Faculty of Science, University of Abuja, Abuja - Nigeria.


Copyright © 2017 Chiokwe and Solomon. This article remains permanently open access under the terms of the Creative Commons Attribution License


Received 1st May, 2017; Accepted 19th May, 2017

ABSTRACT: The study was conducted to assess the effect of bean cake on the growth of Clarias gariepinus. The feed was formulated and fed to Clarias gariepinus for a period of 8 weeks while using Coppens and Multifeed as the controls. Fishes were fed twice daily at 4% body weight/day. There were 12 fishes per experimental tank. The result obtained from the study indicated that the highest growth rate occurred in the fishes fed with coppens and multifeed and the lowest being Bean cake. The analysis of the result done using ANOVA however showed that there was no significant difference (p>0.05) in the mean weight, mean length, food conversion efficiency (FCE) and the survival rates (SR), but showed a significant difference (p<0.05) in the weight gain, length gain, specific growth rate (SGR) and mean growth rate (MGR). Bean cake could be used for the sustenance of Clarias gariepinus but not for optimum growth. Key words: Bean cake, Clarias gariepinus, coppens, growth rate, multifeed. INTRODUCTION Fish is an important source of both food and income to many people in developing countries like Nigeria. Fish is known to be the one of the main source of protein to man providing about 17 to 38% of total of total protein intake in various countries and about 40% of normal protein intake of the average Nigeria (Dada and Gnanados, 1983). It also provides fat, minerals, oil and vitamins which are accessory food factors whose deficiency leads to malnutrition and in man. In Africa, as much as 5% of the population, some 35million people depends wholly or partly on the fisheries sector for their livelihood (FAO, 1996a). It is estimated that by 2050, when world population is projected to be over 9 billion, Africa will have to increase food production by over 300%, Latin America by 80% and Asia 70% to provide minimally adequate diets for the projected population of 2 billion, 810 million and 54billion people in the respective regions (Anon, 1997).

The consumption and demand for fish as a cheap source of protein on the increase in Africa, because of the level of poverty in the land. The vast majority of the fish supply in most cases comes from the rivers in the continent. While capture fisheries based on species that

are presently exploited seem to have reached their natural limit (FAO, 1996b), there is considerable potential to expand aquaculture in Africa in order to improve food security (Kapetsy, 1994; Engel, 1997; Jamiu and Ayinla, 2003). Although potentials abound in the continent for the development of viable fish farming, one of the major hindrances to the development of aquaculture industry in Africa is the lack of locally produced fish feed. Fish requires high quality feed for growth and attainment of market size within the shortest possible time. Therefore local production of fish feed is very crucial to the development and sustainability of aquaculture in Africa.

Feed accounts for over two third of the variable cost of fish culture operation in an intensive management system (Akiyama, 1988; Eyo, 1990).

The African catfish (Clarias gariepinus) is choice fish species in Nigeria. It commands high demands from consumers and is mostly preferred by aquaculture. This is due to the ideal characteristics of this species (Eding and Kamstra, 2001), which includes high growth rate at high stocking densities, a high food conversion, good food quality and smoking characteristics as well as year round production (Ita, 1985). Fish culture production in



50 J. Biosci. Biotechnol. Discov. Nigeria includes stocking of lakes and production in ponds, cages and tanks (Ita, 1985). Pond culture is the most prevalent (Akinwole and Faturoti, 2006). Virtually all aspects of ponds culture of African catfish (Clarias gariepinus) in Nigeria has been developed and documented to ensure profitable production of the species. The appreciable quality of water and large expanse of land required for pond culture abound in Nigeria, but however fish feed has been a limitation for its expansion.Beans is one of the most important food legume crops in the semi-arid tropics covering Africa, Asia, Southern Europe and Central and South America. Beans are drought resistance tolerant and warm weather crop. They are well adapted to the drier region of the tropics, where other legumes do not perform well. Beans are high in protein and are readily available in the market. Beans are used for various things for example; Beans could be boiled and eaten with other food like rice, bread. They are also boiled with sauce and eaten alone; beans are the favorite meal of school children because of their sustainability and high protein content, they are also eaten as cake, popularly called akara. Beans are also fed to livestock such Poultry birds and so on (Singh et al., 2003).

Despite the fact that beans is rich in protein which is what fish need for optimum growth, beans can be used as alternative supplement feed. However, no attention has been drawn to the use of beans as an alternative supplement. More so, researchers seemed to have neglected the potentials that beans hold for fish growth. Consequently, this research work is undertaken with a view to establishing the effects of beans on the growth of Clarias gariepinus.

Aquaculture development and growth in Africa have been on low ebb despite the vast aquatic resources that abounds in the continent. Since the introduction of aquaculture to Africa, some decades ago, there have been a lot of innovations, technological advancement and progress in the areas of genetics, seed propagation, pond construction and farm management in general. Despite breakthroughs recorded in these areas, most farmers in Africa still rely heavily on imported feed ingredients and fish feeds from European countries, which makes fish farming expensive as fish feed accounts for at least 60% of the total cost of production (Jamiu and Ayinla, 2003). This has contributed in no small measure to the slow pace at which aquaculture is advancing in Africa. This project, however reviewed critically the potentials of locally manufactured fish feed in enhancing, improving and sustaining aquaculture development in Nigeria and Africa at large. Aquaculture development in Africa Aquaculture development in Africa is insignificant compared to the rest of the world (Changadeya et al.,

2003). According to Hetch (2000) the entire continent contributed only 0.4% to the total world aquaculture production for the period 1994 to 1995. In the year 2000 it contributed a mere 0.97% of the total global aquaculture (FAO, 2003).

Although, the history of aquaculture is relatively recent in sub-saharan Africa compared to Asia and some other parts of the world. Most known aquaculture systems have been introduced over the last 35years (FAO, 1996a, 1996c). The growth, expansion and production of aquaculture in northern part of Africa especially Egypt is more advanced in techniques and technicalities in comparison to the sub- Saharan regions (FAO, 2003). In sub-Saharan regions, aquaculture in most places is still essentially rural, secondary and part time activity taking place in small farms with small fresh water ponds (FAO, 1996a). The systems that are generally practiced range from extensive to semi intensive cultural systems with limited fish yield, which are mostly consumed directly or sold locally (CIFA, 1998). Almost all fishing is carried out by rural small scale operators in small fresh water ponds as a secondary activity to agriculture. Although there is abundant potential for the development and expansion of aquaculture in this region, factors such as the novelty of aquaculture, the general poor economic conditions in most countries and the relative paucity of entrepreneurial skills and credit facilities hamper its development (FAO, 1997). Aquaculture development in most African countries is primarily focused on socio-economic objectives such as nutritional improvement in rural areas, income generation, diversification of farm activities and creation of employment especially in rural communities where opportunities for aquaculture in northern part of Africa are limited (CIFA, 1998). This approach over the years has resulted in sustained aquaculture growth in some African countries such as Ivory Coast, Egypt, Ghana, Malawi, Nigeria, and Zambia (Jamiu and Ayinla, 2003). While there is still room for enhancing aquaculture production in Africa through improved production systems, genetics and general farm management principles, the desired and expected growth of aquaculture to meet the ever increasing demand for fish and satisfy its socio-economic functions is only achievable through cost effective and high quality fish feed. Aquaculture development in Nigeria Despite the fact that fish farming started in Nigeria over fifty years ago, it is not until very recently that aquaculture made substantial contribution to domestic fish supply. After many years of dormancy, the fisheries and aquaculture sector in Nigeria has been brought to the fore front of the national development agenda. Apart from ongoing activities of the presidential initiative on fisheries and aquaculture development, Nigeria also hosted the

NEPAD “Fish for All summit” in Abuja in August 2005. This summit was a major success with the participation of 30countries and international organizations. It is worthwhile to note the active participation and exhibition mounted by CAFAN at the summit which concluded by adopting the “Abuja Declaration” on sustainable fisheries and aquaculture development for Africa and the NEPAD plan of action.

The role of fishing in realizing food security in Nigeria cannot be overstated. It accounts for a major source of food protein. For example, fishery products domestic consumption provides approximately 22% of the protein requirement in Nigeria. It also generates employment for 36 million people directly through aquaculture. This means that any attempt to neglect fishing by the government maybe to our own peril (FAO, 2003). Interestingly, demand for fish has continued to increase, not only in Nigeria but also worldwide. Despite that, marine capture historically still accounts for over 80% of the world fish supply, however the capture fisheries have not been able to meet up with the growing demand due to increased fishing pressure and the resultant over-fishing syndrome (FAO, 2003). It has been postulated by FAO, that if Nigeria is to bridge the serious fish supply gap, the country must invest heavily in modern systems of aquaculture as well as poly-culture for rural communities, while providing enhanced capacities for capture fisheries development. There is no doubt that Nigeria possesses a good environment rich enough to stimulate growth in aquaculture given the right attitude by the government. The underdeveloped aquaculture sector has the potential of 0.65 to 1.2 million tonnes of fish production annually in Nigeria. But it is presently producing 16.619 to 25.264 tonnes annually and it is produced mainly from outdoor, dugout extensive fish ponds, and it is basically 3% of the country’s fish production potentials. Outdoor dugout pond farming has always been misconstrued as cheap and easy to operate due to lack of solid infrastructural requirements, low maintenance cost and low feeding costs.

According to the Nigerian institute of oceanography and Marine Research, the country has 1,000,000 hectares in fresh water of swamp suitable for aquaculture. This sounds as good news for fish farmers, however, the exploitation of this potentials remains minimal. For instance, an estimated area dedicated to fish farming in 1998 was given as about 5,000 hectares in fresh water and 230 hectares in brackish water. Production estimate was put at 1 tonne per hectare per year for small scale ponds and 2 to 4 tonnes per hectares per year for commercial farmers. This production was however achieved by using low semi-intensive fish farming culture level. The development of semi-intensive fish farming in Nigeria lies in the establishment of fish culture projects; such projects would increase fish production by 656:815 metric tonnes yearly. Due to the location of the fresh water swamps and the mangrove swamp in Niger delta,

Chiokwe and Solomon 51 the rudimentary level of aquaculture development in Nigeria and particularly in Niger delta region is of great concern, especially when the potential for its development remains attractive. Aquaculture and food security in Africa Nutritionally, fish is one of the cheapest and direct sources of protein and micronutrient for millions of people in Africa (Ben et al., 2005), with steady decline in capture fisheries, aquaculture is a readily, veritable tool in the provision of fish eaten all over the continent. Unlike some other animal products, fish is widely acceptable. Its acceptability cuts across social, cultural and religious background. To maintain food fish consumption at the present level of 5 to 45 kg per person per year depending on the country, supplies of fish must increase tremendously. However, with the current supply trends combined with ever increasing population, the per capita consumption of fish in Africa is stagnating and in sub- saharan Africa has fallen drastically (Muir et. al., 2005). To arrest this deplorable condition and boost production of fish, aquaculture remains the only feasible option that can sustain adequate fish supply in Africa. Fish feed development Fish feed technology is one of the least developed sector of aquaculture particularly in Africa and other developing countries of the world (FAO, 2003). Feed is one of the major input in aquaculture production, it is one of the fundamental challenges facing the development and growth of aquaculture in the African continent. Fish feed development in Sub-Saharan Africa has not made a significant progress in aquaculture as expected.

According to Hetch (2002), it is observed that the research on inexpensive feed ingredients has not contributed greatly to aquaculture development in Africa and suggested that more research on how best plant protein can be used as fish feed, play very vital role in aquaculture growth and expansion. In fact, it is a major factor that determines the profitability of aquaculture venture. Jamiu and Ayinla, (2003) reported that feed accounts for at least 60% of the total cost of fish production in Africa, which to a large extent determines the viability and profitability of fish farming enterprise. As aquaculture becomes intensive, most farmers in Africa depend largely on imported fish feed from European countries for the productivity and sustainability of the industry. For example, in Nigeria an estimated 4,000 tonnes of quality fish feed are imported into the country each year (AFP, 2007). This has contributed in no small way in increasing the total cost of production which will ultimately translate to high cost of fish, thereby making it expensive for the teeming population of the poor people

52 J. Biosci. Biotechnol. Discov. living in Sub-Saharan Africa. In some countries like Kenya, Namibia, Malawi, Nigeria, Uganda, Madagascar, Ghana and Cote D’ivoire, where little quality of fish feeds are produced locally, the quality is very poor and production rate inconsistent. This corroborated the submission of Jamiu and Ayinla, (2003) that the low quality of fish feed and its attendant high cost are the major factor limiting the development of aquaculture in Africa. Hence, research in fish nutrition that will utilize locally available ingredients and fabricated equipment without reducing the quality of the feed is urgent and crucial to the overall success of aquaculture development, growth and expansion in Africa. For many aquaculture ventures to be viable and profitable, it must have a regular and adequate supply of balanced artificial diet for the cultured fishes. This is so because the dissolved nutrient environment are seasonal and might be insufficient or may not occur in required proportion to meet the nutritional demand of cultured fishes (Ugwumba and Ugwumba, 2003). There is therefore the need to develop and encourage fish farmers to make use of ideal pond fertilization programs, non-conventional feed resources, feed stuff processing, refinement and formulation that take cognizance of the requirement of the various species and their stages. In comparison to livestock feeds, fish feeds are unique in that they are pelleted and the size of the pellet depends to a large extent on size and age of the fish involved. Fish feed is very important in the efficiency and overall performance of fish in the pond and least lost feed production which will reduce the cost of production of fish. This is why any attention towards the production of effective and cheap feed will benefit fish farmers in Nigeria and Africa at large. The feed produced and used widely in Africa are categorized into conventional and non-conventional feed stuff. The categorization is based on the availability and acceptability of the feed stuff involved.

Literature on the use of beans as fish feed has been scarce and very difficult to come by. However, beans has been used to feed livestock, example; poultry birds, in some part of the country. The reported proximate analysis of beans (cowpea) showed that it has good potential as a source of protein for livestock feeding (Aremu, 1990). This study is therefore geared at exploring the possibility of using bean cake as fish feed, which if successful will cut down the cost of fish feed by replacing some of the expensive conventional fish feeds. MATERIALS AND METHODS Experimental design Three rectangular transparent plastic tanks of 50 × 26cm × 26 cm with a capacity of 30 litres each were used for the experiment, with water level maintained at 35cm (25 litres). There were three treatments designated as Tank

A, Tank B, and Tank C. The species of fish used was Clarias gariepinus fingerlings of length 0 to 16 cm and weight 0 to 20 g. The fishes were obtained from Mallam Dankishiya’s fish farm and transported to the biological garden of University of Abuja between 6 to 8am to reduce mortality due to increased temperature. The fishes were acclimated for seven days in the biological garden. At the end of the acclimation period, the fishes were randomly selected and assigned to different tanks at a stocking rate of 12 fingerlings per tank. The fishes were starved for 24hrs to empty their gut content and prepare them for the experimental feed. This practice also helps to make the fish hungry and thus more responsive to the new diet. The fishes were fed 4% of their gross body weight per tank. Tank A was fed Coppens fish feed, Tank B was fed the formulated Bean Cake while Tank C was fed Multifeed feed. The initial individual weight, length, mean length and mean weight were recorded. The aquaria were covered with mosquito net to prevent the fingerlings from jumping out, intrusion of insects and other foraging bodies (Lizard, geckos, spiders etc), fresh water was used throughout the experiment. Depleted water was replaced with fresh water of 25 litres after each cleaning. During cleaning, which involved scrubbing of the tanks with sponge, disinfection of the tanks using potassium permanganate to prevent contamination caused by the leftover food particles and washing with clean water? Weighing of fish The weight of the fishes were determined with the aid of a E-Zurich (Swiss made) weighing balance. Each fish was weighed individually thereafter the process was carried out weekly with the final weighing done at the end of the 8 weeks period of the experiment. The weighing of the fingerlings was done in order to get weight gain by the fingerlings weekly, after they have been subjected to feeding with the various feeds assigned to the various tanks at 4% gross body weight of the fishes per tank. Length of fish The lengths of the fishes were determined using a measuring board and a meter rule. This involved measuring the total length of individual fishes to the nearest centimeter. In measuring the length the fishes was placed on a board with its snout closed and touching the edge of the board, and the caudal fin pressed together and the reading taken from the graduated measuring board and recorded. This was done weekly to get the length gain per week. Feed Formulation The following materials were used to formulate the

experimental bean cake feed; Ife brown beans, vitamin premix, methionine, lysine, bone meal and starch. The materials were procured from gwagwalada market and Agro Vet Stores. The beans and the bone meal were grinded individually in a grinding machine to obtain a fine powder.

The ingredients were mixed together thoroughly and formed into a paste using starch as the binding agent. The paste was pelleted using a locally made pelleter. The resultant pellets were sun dried for 48 h. the dried feed was then taken for proximate analysis to the percentage compositions of crude protein, crude fat, crude fibre and ash. The remaining feed was then stored in an air tight container to prevent moisture from penetrating into it and also for subsequent feeding of the fingerlings.

Feeding and measurement

Three feeds were used for the experiment, they includes; coppens feed for aquaculture, multi feed for aquaculture, and the formulated experimental bean cake respectively. The feeds were assigned to various tanks; Tanks A was fed coppens, Tank B was fed the experimental feed and Tank C was fed multifeed. The fishes were fed 4% of their gross body weight per tank per day. The process involved weighing all the fishes in each tank using a weighing balance and calculating 4% of the gross body weight of the fishes per tank. This amount was measurement out from the bulk of the feed and fed to the fingerlings. The fingerlings were fed twice daily, half of the measured feed in the morning (7.00 to 9.00am) and the remaining half in the evening (5.00 to 7.00 pm). At the beginning of every new week, the process was repeated and thus the new gross weights were used. The duration for the experiment was 8weeks.

Physiochemical Parameters

During the first week of the experiment, the physiochemical parameters of the water were carried out daily. Parameters such as temperature, pH, ammonia and dissolved oxygen were measured. This was to enable me know the time of the week to change the water (i.e when the water starts getting to toxic for the fingerlings survival). However, parameters such as the temperature were measured daily. Both the water temperature and the atmospheric temperature were read to the nearest 0°C with the aid of mercury in glass thermometer. The dissolved oxygen was determined once a week after the first week of daily measurements by titration with 0.1NaOH and the azide modification of the Winkler method (America Public Health Association, 1976), pH was determined with the aid of a digital pH meter. Ammonia was determined by a spectropho-tometer, using the Phenolhydrochloride method, nitrite was measured using urinalysis test strip kit (Sterling, 1985).

Chiokwe and Solomon 53 Nutrient utilization parameters

Mean weight gain (%): This was calculated as

Mean Length Gain (%): This was calculated as

Specific growth rate (SGR)

Where, WT = Final Weight, Wt= Initial weight, T = Time (Days) and Ln = Natural logarithm

Food conversion efficiency (FCE)

Mean Growth Rate (MGR)

Where W2= Final Weight, W1 = Initial Weight, t = Period of the experiment in days and 0.5 = Constant.

Weight Gain: This was calculated as W2 - W1 Where, W2 = final mean weight value and W1 = Initial mean weight value

Survival rate (SR)

Statistical analysis Analysis of growth data using analysis of variance (One-way, ANOVA) was used for this study.

RESULTS Results of production parameters for the three treatments are presented in (Tables 1, 3, and 5). While the physio-


Table 1. Production Parameter For Treatment A (Coppens).

Production Parameters Weeks

0 1 2 3 4 5 6 7 8

Gross weight (g) 110.42 141.78 176.196 217.92 268.304 275.231 307.373 333.861 386.210

Mean weight (g) 9.201 11.815 14.683 18.160 21.942 25.021 27.943 30.351 35.110

Weight gain (g) 0.00 2.614 2.823 3.477 3.782 3.079 2.922 2.408 4.759

Total length (cm) 114.24 138.108 152.196 176.508 195.348 207.90 232.562 255.431 278.564

Mean length (cm) 9.520 11.509 12.683 14.709 16.279 18.900 21.142 23.221 25.324

Length gain (cm) 0.00 1.989 1.174 2.026 1.58 2.621 2.242 2.079 2.103

Specific growth rate (%) 0.00 3.527 3.339 3.538 3.104 2.858 2.645 2.436 2.392

Mean growth rate (%) 0.00 0.687 0.580 0.511 0.451 0.393 0.347 0.310 0.286

Gross weight (g) 0.00 59.187 49.777 49.33 43.387 29.234 26.541 19.585 35.636

Survival rate (%) 100 100 100 100 100 91.67 91.67 91.67 91.67

Figure 2. Physiochemical parameter for treatment A (Coppens) (Weekly Mean Values).

Physiochemical parameter Weeks

1 2 3 4 5 6 7 8

Atmospheric temperature (°C) 26.6 27.9 27.5 28.3 29.5 27.8 26.6 27.5

Water temperature (°C) 24.9 25.3 26.1 26.4 26.8 24.7 23.8 24.1

pH 7.4 7.6 7.8 7.9 8.1 8.3 8.5 8.6

Dissolved Oxygen (mg/l) 6.52 6.19 6.03 5.81 4.96 4.47 4.19 3.98

Ammonia (mg/l) 0.01 0.12 0.19 0.26 0.34 0.41 0.44 0.45

Nitrite (mg/l) 0.01 0.01 0.02 0.02 0.03 0.04 0.05 0.05

chemical parameters for the treatments are given in (Tables 2, 4 and 6) respectively. Tables 1 and 2 show the production of and physiochemical parameters for treatment A (coppens), Tables 3 and 4 show the production and physiochemical parameters for treatment B (Bean Cake), and Tables 5 and 6 show the production and physiochemical parameters for treatment C (Multifeed) respectively. Also, Figures 1 to 7 show the graphical representations of the production

and physiochemical parameters for treatment A; Figures 2 and 8 show the graphical representations for the production and physiochemical parameters for treatment B; and Figures 3 and 9 show the graphical representations of the production and physiochemical parameters for treatment C. However, (Figures 4, 5 and6) show the graphical representations of the survival rates, Specific Growth Rates (SGR) and Mean Growth Rates (MGR) for the three treatments respectively.

From (Figure 4), it can be observed that the fingerings survived at the same rate for the first two weeks, but treatment B recorded mortalities on the 3rd and the 6th weeks, treatment A also recorded a mortality on the 5th week and treatment C recorded a mortality on the 6th week. Figure 5 compare the Specific Growth Rates (SGR) of the three treatments. Treatments A and C showed a progressive decline on the SGR while treatment B showed an increase on the SGR from

Chiokwe and Solomon 55

Table 3. Production Parameters For Treatment B (Bean Cake).

Production Parameters Weeks

0 1 2 3 4 5 6 7 8

Gross weight (g) 118.452 126.684 140.916 146.52 158.532 174.988 172.21 184.31 199.80

Mean weight (g) 9.871 10.557 11.743 13.320 14.412 15.908 17.221 18.431 19.980

Weight gain (g) 0.00 0.686 1.182 1.577 1.092 1.496 1.313 1.210 1.549

Total length (cm) 125.40 132.12 142.692 138.831 147.752 155.232 149.81 157.30 168.21

Mean length (cm) 10.450 11.01 11.891 12.621 13.432 14.112 14.981 15.730 16.821

Length gain (cm) 0.00 0.54 0.881 0.730 0.811 0.680 0.869 0.749 1.091


Mean growth rate (%) 0.00 0.188 0.231 0.250 0.228 0.220 0.206 0.192 0.183

Food conversion efficiency (%)

0.00 14.478 23.326 27.978 19.472 23.591 19.197 17.566 21.011

Survival rate (%) 100 100 100 91.67 91.67 91.67 83.33 83.33 83.33

Table 4. Physiochemical parameter for treatment B (Bean Cake) weekly mean values).


1 2 3 4 5 6 7 8



pH 7.4 7.6 7.8 8.1 8.4 8.6 8.7 8.9


Ammonia (mg/l) 0.01 0.16 0.23 0.31 0.35 0.41 0.45 0.5

Nitrite (mg/l) 0.01 0.02 0.02 0.03 0.05 0.07 0.09 0.11

week 1 to week 3 and declined through to week 8. Physiochemical parameters The atmospheric temperature throughout the study period varied between 26°C and 30°C, while the water temperature varied between 23°C and 27°C (Tables 2, 4 and 6).The concentration of

dissolved oxygen was between 6.73 mg/l and 3.49 mg/l, which is within the permissible limit standard of dissolved oxygen for aquatic life recommended by the Federal ministry of Environment (2006). Also, Eding and Kamstra, (2001) reported that the standard value of dissolved oxygen for African catfish (Clarias gariepinus) is between 2.9 to 6.8 mg/l. Treatment B recorded the lowest amount of dissolved of

oxygen (3.4 mg/l). The concentration of the dissolved oxygen declined weekly (Tables 2, 4 and 6) due to the growth of the fishes and therefore increases the demand for dissolved oxygen, was as the same volume of water is being used. Ammonia concentration in the three treatments throughout the experiment ranged between 0.01 mg/l and 0.5 mg/l. Nitrite concen-tration ranged between 0.01 and 0.11. Treatment B


Table 5. Production parameter for treatment C (Multi feed)

Production parameters Weeks

0 1 2 3 4 5 6 7 8

Gross weight (g) 122.16 147.792 173.976 204.12 242.412 271.932 272.041 296.791 326.282

Mean weight (g) 10.180 12.316 14.498 17.010 20.201 22.661 24.731 26.981 29.662

Weight gain (g) 0.00 2.136 2.182 2.512 3.191 3.460 3.07 3.25 2.681

Total length (cm) 111.852 142.596 161.184 186.252 206.652 231.612 231.121 243.221 258.137

Mean length (cm) 9.321 11.883 13.432 15.521 17.221 19.301 21.011 22.111 23.467

Length gain (cm) 0.00 2.562 1.549 2.089 1.70 2.080 1.71 1.10 1.356


Mean growth rate (%) 0.00 0.487 0.418 0.376 0.340 0.347 0.275 0.250 0.230

Food conversion

efficiency (%) 0.00 43.713 36.910 36.097 39.082 35.683 28.224 29.867 22.583

Survival rate (%) 100 100 100 100 100 100 91.67 91.67 91.67

Table 6. Physiochemical parameter for treatment C (Multi feed) weekly mean values.


1 2 3 4 5 6 7 8



pH 7.4 7.6 7.8 8.1 8.3 8.4 8.6 8.7


Ammonia (mg/l) 0.01 0.13 0.18 0.27 0.35 0.42 0.46 0.47

Nitrite (mg/l) 0.001 0.01 0.01 0.02 0.02 0.03 0.04 0.05

recorded the highest value of nitrite concentration (0.11 mg/l). DISCUSSION The growth pattern in the three treatments revealed that the highest growth occurred in treatment A and C respectively. The reported

crude protein value of fish in literature is 65% (Annune, 1990), while that of beans (vigna unguiculata) is 25 % (Henshaw, 2008). Thus, from the above values, the feeds fed to treatment A and C has higher protein content than bean cake and therefore a higher biological value than bean cake, fact which is confirmed by fishes fed on coppen and multifeed which showed the highest weight gain. However, coppens had the highest

weight gain (4.759g), followed by Multi-feed (3.460 g) and then bean cake (1.577 g); length gain (2.621 cm), (2.562 cm) and (1.091cm); specific growth rate (3.572%), (2.721%), and (1.427%); and mean growth rate (0.687%), (0.487%) and (0.250%) (Tables 1, 3, and 5) respectively, this could be attributed to their difference in percentage of crude protein contents of the feeds, coppens (45%), Multi-feeds (42%)


Figure 1. Production parameters for treatment A.

Figure 2. Production parameters for treatment B.

450

400 gross weight

350

mean weght

300

weight gain

250

total length 200

150 mean length

100 length gain

50 food conversion efficiency

0

Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8

250

gross weight

200

mean weght

150 weight gain

total length

100

mean length

50 length gain

food conversion efficiency

0



Figure 3. Production parameters for treatment C.

Figure 4. Survival Rates (%) of the Treatments A, B and C.

350

300 gross weight

250 mean weght

weight gain 200

total length

150

mean length

100

length gain

50

food conversion efficiency

0


120

100

80

Treatment A

60

Treatment B

40

Treatment C

20

0

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8


Figure 5. Specific Growth Rates (%) of the three treatments.

Figure 6. Mean Growth Rates (%) of the three treatments.

4

3.5

3

2.5

Treatment A

2

Treatment B

1.5

Treatment C

1

0.5

0


0.8

0.7

0.6

0.5

Treatment A

0.4

atment B

0.3

Treatment C

0.2

0.1

0



Figure 7. Physiochemical Parameters for treatments A.

Figure 8. Physiochemical parameters for treatments B.

35

30

25

atmospheric temp. (oC)

20

water temp. (oC)

pH

15

dissolved oxygen

10 ammonia

nitrite

5

0


Figure 8: Physiochemical Parameters for treatments B

30

25

20

atmospheric temp. (oC)

water temp. (oC)

15

pH

dissolved oxygen

10

ammonia

nitrite

5

0



Figure 9. Physiochemical Parameters for treatments C.

and bean cake (28.6%). More so, another reason for the low performance of the

fishes fed bean cake could be the lack of exudation of fish flavour from the diet (Chow, 1981), this then explains the low consumption rate of baen cake observed during the experiment and further explains why there was left over of feed in the aquarium with bean cake compared to others 15-20 min after they were fed.

The low consumption rate of bean cake could also be implicated in the low weight of observed and also the highest mortality rate recorded in Treatment B from the inception of the experiment to its termination. The effect of bean cake and the other feeds on the growth and productivity of Clarias gariepinus was statistically analysed using the one-way ANOVA. The analysis showed that their was a significant difference in weight gain (F = 7.596155; df = 24; P-value = 0.00278; P<0.05), length gain (F = 6.9350852; df =24; P-value = 0.004209; P<0.05), Specific growth rates (F = 5.310648; df = 24; P- value = 0.012314; P<0.05), Mean growth rate (F = 4.535696; df = 24; P-value = 0.021336; P<0.05).

However, the analysis showed that there was no significant difference in mean weight (F = 2.611572; df = 24; P-value = 0.094148; P>0.05), mean length (F = 2.022346; df = 24; P-value = 0.154286; P>0.05), food conversion efficiency (F = 3.370884; df = 24; P-value = 0.051261; P>0.05), survival rate ( F = 2.697197, df = 24; P-value = 0.08773; P>0.05).

Conclusion It is however concluded from the experiment that bean cake feed does not contain the nutrient necessary for the optimum growth and development of Clarias gariepinus, but contains nutrients for the maintenance and the sustenance of Clarias gariepinus and is therefore recommended to be used for the maintenance of Clarias gariepinus, because it is less expensive and readily available. REFERENCES AFP (2004). Inventory of feed producers in Nigeria. Published

by aquaculture and inland fisheries project. Annex of the National special programme for food security with the Agriculture development programme in all states and FCT Abuja, Nigeria. p. 118.

Akinwole, A. O., & Faturoti, E. O. (2006). Biological performance of African Catfish (Clarias gariepinus) cultural in recirculating system in Ibadan. Aquacultural Engineering, 36, 18-23.

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Annune, P. A. (1990). Preliminary Investigation on the suitability

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atmospheric temp. (°C)

20

water temp. (°C)

pH

15

dissolved oxygen

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nitrite

5

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http://www.fao.org/




The effects of dietary, energy and feed utilization of compounded feed (vital chicken feed and fish meal) on

the growth rate of Clarias gariepinus

Solomon, R. J.* and Adeola, A. A.

Department of Biological Sciences, Faculty of Science, University of Abuja, Abuja - Nigeria.


Copyright © 2017 Solomon and Adeola. This article remains permanently open access under the terms of the Creative Commons Attribution License


Received 3rd May, 2017; Accepted 22nd May, 2017.

ABSTRACT: The experiment was conducted at the research unit of the Biological science Department, University of Abuja for a period of Twelve weeks. The experiment was set up to determine the effects of chicken feed (vital chicken feed) in the diet of Clarias gariepinus. The effects variables and nutrient utilization were determined from the practical diets using varying levels of 100% coppens in tank A (42% protein,13% crude fibre,6.7% ash),50% of vital chicken feed with 50% of fishmeal in tank B (20% crude protein,10% ash,9%crude fibre) and a combination of 30% vital chicken feed plus 70% fish meal in tank C (25% crude protein,12% fats,13% crude fibre,8% ash). The feeds were formulated and fed to Clarias gariepinus fingerlings with lengths (0 to10 cm) and weights (0 to 20 g). They were fed at 4% body weight twice daily. The optimum growth and survival rate were calculated. Key words: Chicken feed, fish meal, growth rate. INTRODUCTION Over the past decades aquaculture has grown in leaps and bounds in response to an increasing demand for fish as a source of protein globally (Akinrotimi et al., 2007; Bard et al., 1976; Halver, 1978). According to FAO (2006), fish supplies from capture fisheries will therefore not be able to meet the growing global demand for aquatic food. Hence, there is need for a viable alternative fish production system that can sufficiently meet the demand and aquaculture fits exactly into the role (De Silva and Davy,1992; De Silva and Anderson,1995).

As aquaculture production becomes more and more intense in Nigeria, fish feed will be a significant factor in increasing the production and profitability of aquaculture (Akinrotimi et al. 2007; Anyanwu and Clifford, 2007). There is an increasing demand for fish and fish protein which resulted in widespread over fishing in wild fisheries (Fagbenro, 1997). The global returns for fish farming recorded by the FAO in 2008 totalled 33.8 million tones worth about US 60 billion (Fishery and Aquaculture statistics: Aquaculture Production, 2008, FAO Year book Rome).

There are two major categories of aquaculture; extensive aquaculture based on local photosynthetical production and intensive aquaculture, limiting growth here is the available food supply by natural sources, commonly zooplankton feeding on pelagic algae or benthic animals such as crustaceans and mollusces. In order to tap all available food sources in the pond, the aquaculturist will choose fish species which occupy different [places in the pond ecosystem e.g a filter algae feeder such as tilapia, a benthic feeder such as carp or catfish (De Silva and Davy,1992; De Silva and Anderson,1995). In intensive aquaculture, the optimal water parameters for cold and warm water fish includes; Acidity; pH 6 – 9, Alkalinity > 20mglL, ammonia,<0.02 mglL; calcium: >5 mglL, carbondioxide <5-mmglL; chloride >4.0mglL, chloride: 0.003 mgIL etc. In these kinds of systems, fish production per unit of surface can be increased at all will as long as sufficient oxygen, fresh water and food are provided. The cost of fish feed which must contain a much higher level of protein (up to 60%) than cattle food and a balanced amino acid composition



64 J. Biosci. Biotechnol. Discov. as well. However, these higher protein level requirements are a consequence of the higher food conversion efficiency (FCE – kg of feed per kg of animal product) of aquatics animals; fish like salmon have FCE’s in the range of 1.1 kg of feed per kg of salmon, whereas, chickens are in the 2.5 kg of feed per kg chicken range.

Essential here is aeration of the water; as fish need a sufficient oxygen level for growth. This is achieved by bubbling, cascade flow or aqueous oxygen. Catfish, Clarias spp can breathe atmospheric air and can tolerate much higher levels of pollutants than trout or salmon which makes aeration and water purification less necessary and makes Clarias species especially suited for intensive fish production. In most Clarias farms about 10% of the water volume can consist of fish biomass. Within intensive and extensive aquaculture methods there are numerous specific types of fish farms each has benefits and applications unique to its design; Some of includes: the cage system, Irrigation ditch or pond systems, indoor fish farming etc. The indoor fish farming is an alternative to outdoor open ocean cage aquaculture, is through the use of a recirculation aquaculture system (RAS). A RAS is a series of culture tanks and filters where water is continuously recycled and monitored to keep optimal condition of water quality. The water is treated mechanically through the removal of particulate matter and biologically through the conversion of harmful accumulated chemicals into non-toxic ones. Through this system, many of the environmental drawbacks of aquaculture are minimized including escaped fish, water usage and the introduction of pollutants. The practices also increased feed use efficiency growth by providing water quality (Adeniji and Ovie, 1990; Timmon, 2002). Others include classic farming, Animal slaughter etc. When fish are reared in high density indoor systems or confined in cages and cannot forage freely on natural feeds, they must be provided a complete diet. In contrast, supplemental incomplete, partial diets are intended only to help support the natural food (insects, algae, small fish). Supplemental diets do not contain a full complement of vitamins or minerals, but are used to help fortify the naturally available diet with extra protein, carbohydrate and / or lipid. In an attempt to the objective of culturing fish, two main sources of fish feed have been identified for meeting nutritional requirement of fish. These include the fish on farm feed and the commercial feed. As a result, on farm feeds are mostly used in Nigeria this according to Fagbenro, (1997) amount to 67% of estimated total of 35,570 tonnes of feed produced in the year 2000. Due to the increase in fresh water farming in Nigeria and globally, there could be intense competition for fish meal and fish oil (FAO, 2006), hence the need for alternative source of protein to replace fish meal without reducing the quality of the feed.

Poor feed leads to slow growth, high feed conversion ratio, low survival, disease and poor harvest (Eyo, 2003).

Good quality conditions that are adequate leads to profitability in fish culture managements. In Nigeria today, aquaculture seeks to improve fish yield and fish productivity. Its benefits range from rural development income generation, farm sustainability as well as reduction in vulnerability. This practice also makes use of land which is considered unsuitable for agriculture such as swamps or salure. (Ayinla and Akande, 1988) stated that protein consumed in Nigeria comes from the wild (Eyo, 1996) reported that since aquatic resources are finite although renewable, every effort should be made towards increased fish production through improved resources management and conservation and also intensive aquaculture practices.

Currently, aquaculture production in Nigeria has witnessed slow pace of development aquaculture contributes only about 2500MT of fish annually which is about 69% of domestic fish production, whereas, the projected requirement for fish products by the year 200 was 4 million MT (Ita,1998). Nigeria has high potential in aquaculture which is hardly tapped (Ayinla and Akande, 1988) stated that aquaculture provided food of high animal protein generated income and employment thereby promoting the socio-economic development of Nigerians.

Fish production when combined with improved inland fisheries management to eliminate fish importation and earn substantial foreign exchange. Feeding of catfish in grow out are perhaps the most documented in literature, various efforts have been made to establish the crude protein and amino acid requirement of Clarias gariepinus (Ayinla and Akande, 1988). It was observed that hybrids exhibited a high degree of cannibalism and resulting in high individual growth rate with a corresponding low production (yield) due to high mortality rate. Solomon, (2006) stated that there is a wide range of food and feed stuff that are suitable for fish feeding and their quality is primarily assessed on their nutrient composition such as protein levels. According to Hepher and Pruginin,(1981) protein requirements for optimal growth and feeding of juvenile fishes usually range from 35-55% depending on species stated that typical herbivores such as tilapia require dietary protein. Shephard, 1988) stated that difference in growth increment between monoculture of one specie and Polyculture of several species within the same period. However, one specie might affect the environment to improve the growth condition for the other species therefore. Diet supplementation is an important aspect aquaculture management especially in intensive fish culture end is promising for fish production (Abdelghamy and Ahmad, 2002) in aquaculture, diet is often the single largest operating cost item and can represent sent over 50% of the operating cost in intensive aquaculture (Dolgado et al., 2003). The cost depends on many factors such as protein level, the source and type of ingredients that could be derived from plants and animal resources and manufacture practices.

Apart from developing low cost diet, different feeding management strategies such as on demand feeding regimes and good pond management and husbandry (Abdelghamy and Ahmed, 2002; Bascinar et al., 2007) could improve fish growth. The optimum feeding regime of culture fish is an important aspect of achieving efficient production and also could lead to insignificant saving diet cost. Fish is one of the major inputs in aquaculture production. It is one of the fundamental challenges facing the development and growth of aquaculture. According to Huet (1970); it is observed that the research on inexpensive feed ingredients has not contributed greatly to aquaculture development and suggested that more research on how best plant protein can be used as fish feed is required.

Development and management of fish feed play very vital role in aquaculture growth and expansion, it is a major factor that determines the profitability of aquaculture venture. Jamiu and Ayinla, (2003) reported that feed count for at least 60% of the total cost of fish production which at a large extent determines the viability and profitability of fish farming enterprise. Fish production involves both the intensive and semi – intensive system of production. For any aquaculture venture to be viable and profitable, it must have a regular and adequate supply of balanced artificial diet for the cultured fishes. This is so because the dissolved nutrients that promote primary and secondary production in the natural environment are seasonal and might be insufficient or may not occur in required proportion to meet the nutritional demand of cultured fishes. There is therefore the need to develop and encourage fish farmers to make use of ideal pond fertilization, non-conventional feed resources, feed stuff processing, refinement and formulations that take cognizance of the requirement of the various species and their stages (Ibiyo and Oluwasegun, 2004). Fish feed is important in the efficiency and overall performance of fish in the pond and which will reduce cost of production of fish. This is why any attention toward the production of effective and cheap feed will benefit farmers (Gabriel et al., 2007).

Clarias gariepinus (Burchell, 1822) farming clandare is generally considered to be one of the most important tropical catfish species for aquaculture in West Africa other species includes Heterobrachus and their hybrids. The reasons for their culture are based on their fast growth rate, disease resistance, high stocking growth, high stocking density, aerial respiration, high speed conversion efficiency among others (Oyelese, 2007). Streams, rivers, swamps to flood plains, many of which are subjected to seasoned drying catfishes are cultured conveniently under monoculture and Polyculture system. The monoculture is the culture of the same fish species while Polyculture is the culture of two or more fish species of different habits and ecological niches. This type of culture is favored in pond system (Maar et al., 1966; Wu et al., 2004). There are therefore, the culture of

Solomon and Adeola 65 two species of fish; a system that could be referred to as duo culture. Also, there is the culture of three closely related species of the same family and the same feeding habit, this type of culture could be referred to as trio culture system. Most culturists in Africa especially Nigeria have practiced any of this culturing different species of catfish together or separately have little or no effect on their growth performance (Houlihan et al., 2001). The work of Ogunsanmi and Yunusa, (2008) show that clariid catfish culture under the monoculture system gave weight gain followed by duo culture and least in the tri culture. This result also show that hybrid had the best weight gain in all the three culture systems followed by Clarias gariepinus and least with Heterobrachus longifillis (Faturoti et al., 2002).

MATERIALS AND METHODS

Feed formulation

Vital chicken feed was bought from Agro-kings vet, Gwagwalada, Abuja and fish meal from ideal fish farm in Kado fish market in Abuja. The experimental feed was formulated by binding the vital chicken feed and fish meal with pap in the ratio 1:1 and 3:7, it was then pelleted locally using a tin with holes and then kept under the sun to dry.

Experimental fingerlings

The experimental fish (fingerlings) Clarias gariepinus of about 0 – 10 cm and 0 – 20 g were transported from Agricultural Development Project (ADP), Gwagwalada in Abuja in plastic bowls with well oxygenated water at the early hour of the morning to avoid mortality due to high temperature. A total number of 39 fingerlings of catfish (Clarias gariepinus) of size 0 – 10 cm and 0 -20 g were randomly distributed into 3 circular tanks (13 fish per circular tank). The fingerlings of nearly the same size were acclimatized for seven days and fed with coppens at 4% body weight. The circular tanks were well aerated. At the end of acclimatization period, the fishes were starved for 24hours to empty their content and prepare them for experimental feed. This also makes the fish hungry and thus receptive to the new diet before randomly stocking the fish. The initial total length (cm) of individual fish and mean weight of the fish was recorded before placing them in the rearing containers. The fingerlings were fed 4% body weight daily (6:00am and 6:00pm) respectively.

Proximate analysis of formulated feed

Proximate analysis of the formulated feed was carried out at the Institute of Agricultural Research, Zaria. The proximate analysis also known as Weende analysis or

66 J. Biosci. Biotechnol. Discov. nutritive value is a quantitative method to determine different macronutrients in a feed. They are categorized into moisture (crude water), crude ash (CA),crude protein (CP),fats and lipids and crude fiber. The feed sample was initially dried at 103°C for four hours, the weight loss of the sample was determined and the moisture fraction is calculated. Ashing the sample at 550°C for four hours removes the carbon from the sample, all organic compounds are removed. Also calculating the weight loss of the feed sample (Vital chicken feed mixed with fishmeal) from the dry matter to crude ash content mathematically determines the organic matter fraction. The Nitrogen content of the food is the basis for calculating the crude protein content of the feed. The method established by Kjeidahi converts the nitrogen present in the sample to ammonia which is determined by titration. The carbohydrate in the feed sample was retrieved in two fractions; Crude fibre and nitrogen-free extractives of the proximate analysis. The fraction which is not soluble in a defined concentration if alkalis and acids is the crude fibre (CF), the fraction which contained cellulose, lignin, sugars, pectin and hemicelluloses are the Nitrogen-free extractives(NFE). This fraction was therefore calculated by subtracting crude protein, crude ash and crude fibre from the organic matter.

Feeding and measurement

The proximate analysis of the formulated feeds Coppens feed used in aquaculture (floating diet) contains 42% protein, 13% crude fibre, 6.7% ash, 0.9% phosphorus, 1.0% calcium, 0.2% sodium was used as the control feed for the first treatment tank (tank A) which serves as the control treatment (Lovell, 1998). Vital chicken feed starter mixed with fish meal containing crude protein of 20%, 10% fat, 9% crude fibre, 1.0% calcium, 0.45% phosphorous, 2800kcal metabolized energy for the second treatment (Tank B), vital chicken feed starter mixed with fish meal in 3:7 for the third treatment (Tank C) containing crude protein of 25%, 12% of fats,13% of crude fibre,2.0% calcium,8% of ash,2850 kcal metabolized energy. The fingerlings were fed at 4% body weight twice daily, morning (6am – 8am) and evening (6pm – 8pm). Samplings of fish for weight and length measurement were done by reducing the water volume with a rubber siphone before the fish are collected with a scoop net. Fish weight was taken using a top loading balance (pocket scale), the fingerlings were weighed in groups once a week. The standard length of the fish was taken to the nearest cm with the aid of a measuring board; this was done once a week depleted water was replaced with fresh water to an effective depth of 50 cm after each cleaning.

Physiochemical parameters

Both surface water and atmospheric temperature were

read daily (between 6-8am) to the nearest degree centigrade with the aid of mecury-in-glass thermometer. Dissolved oxygen was determined once a week by titration with 0.1 NaOH the azide modification of the Winkler method (American Public Health Association; 1990). Using the phenylydrochlorite method (Stirling, 1985). pH and Nitrite were determined using the combi-II Urinalysis strip. Food utilization parameters Specific Growth Rate (SGR) This was calculated from data on changes of the body weight over the given time intervals according to the method of Brown, (1957) as follows;

SGR% =LnW2 − LnW1

T − t𝑥100

Where; W1 is the initial weight (gram at t) and W2 is the final weight (gram at T). Food conversion efficiency

FCR =Weight of food consumed per fortnight (g)

Weight gamed by fish per fortnight (g

Weight gain (g) Weight gain (g) is calculated as the difference between the initial and final mean weight gain values of the fish in the aquarium.

Weight gain % =Final weight

Initial weight𝑥100

Survival rate (SR)

The survival rate was calculated as the total number of fish harvested /total number of fish stocked expressed in percentage.

Survival % =Number of fish harvested

Number of fish stocked𝑥100

Relative Weight Gain

Relative Weight Gain (RWG) =W2 – W1

W1𝑥100

Mean Growth Rate (MGR)

This was computed using the standard equation.

MGR =W2 – W1

0.5 (W1. W2)𝑥100

Solomon and Adeola 67

Table 1. Production parameters for treatment A

Parameters Week0 Wk1 Wk2 Wk3 Wk4 Wk5 Wk6 Wk7 Wk8 Wk9 Wk10 Wk11 Wk12 total mean

total weight (g) 106.48 112.67 122.15 129.12 137.74 147.77 149.15 153.2 164.21 175.02 180.6 188.39 201.99 1968.5 151.4223

mean weight (g) 8.19 8.66 9.39 9.93 10.59 11.36 12.42 13.92 14.93 15.91 16.41 17.12 18.36 167.19 12.86077

weight gain (g) 0 0.47 0.77 0.54 0.66 0.77 1.06 1.5 1.01 0.98 0.5 0.77 1.24 10.27 0.79

total length (cm) 99.75 104.5 110 115.61 123.29 128.1 132.5 134.59 138.5 146.55 153.1 160.48 173.7 1720.7 132.3592

mean length (cm) 7.67 8.03 8.46 8.89 9.48 10.19 10.67 12.14 12.59 13.32 13.91 14.58 15.79 145.72 11.20923

length gain (cm) 0 0.36 0.43 0.43 0.59 0.71 0.48 1.47 0.45 0.73 0.59 0.67 1.27 8.18 0.629231

feeding rate (g) 6.92 7.32 7.93 8.39 8.95 9.6 8.94 8.42 9.02 7 7.22 7.53 8.07 105.31 8.100769

specific growth rate (cm) 0 1.49 0.25 0.11 0.1 0.09 0.4 0.03 0.05 0.04 0.04 0.03 0.03 2.66 0.204615

mean growth rate (cm) 6 6.61 9.84 4.2 3.46 10.03 2.98 7.23 1.56 1.07 5.02 5.94 8.5 72.44 5.572308

food conv. Efficiency (%) 0 6.42 9.7 6.43 7.37 8.02 11.85 17.81 11.19 14 6.92 9.42 15.36 124.49 9.576154

survival rate (%) 100 100 100 100 100 100 90 80 80 80 80 80 80 1170 90

Table 2. Physiochemical parameters for treatment A

Parameters WK 0 WK 1 WK 2 WK 3 WK 4 WK 5 WK 6 WK 7 WK 8 WK9 WK10 WK11 WK12

Temperature (°C) 26 26 27.5 27 28 27 28 29 27 28 27 28 27.5

Dissolved O2 (mg/l) 6.42 6.3 6.1 6 5.93 6 6.2 5.9 5.85 6.1 5.8 5.9 6

pH 6 6 7 7.5 6.5 7 8 7 7 7.5 6.5 6 6

Ammonia (mg/l) 0.01 0.25 0.38 0.41 0.58 0.47 0.54 0.58 0.52 0.5 0.48 0.54 0.52

Nitrite (mg/l) 0.01 0.01 0.02 0.02 0.01 0.01 0.02 0.01 0.02 0.03 0.02 0.01 0.02

Where, W1 = initial weight, W2 = final weight, t = period of experiment in days and 0.5 = constant

Percentage Weight Gain %

This is expressed by the equation.

% WG =Wt – W0

W0𝑥100

Where, Wt = Weight at time t and Wo = initial weight Length – Weight relationship

W= aLb -----------------(1)

Log W= Log a + b Log L --------(2) Where W = Weight of the fish (g), L= Standard length of the fish (cm), a= constant and b= Exponent RESULTS AND DISCUSSION Results of physiochemical parameters for treatments A, B and C are represented in (Tables 2, 4 and 6) while production parameters were

given in (Tables 1, 3 and 5). The atmospheric temperature throughout the study period varied between 26 to 29°C but the highest temperature occurred in the seventh week of the experiment in treatment A. Treatments A and B recorded the highest concentration of dissolved oxygen at 6.42 mg/l and 6.84 mg/l respectively while the lowest was found in treatment C at 5.3 mg/l. Ammonia concentration in treatments A, B and C ranges between 0.01 and 1.0 mg/l. The various production parameters in the three treatments show that treatment A has the highest mean weight at 18.36 g. The highest mean length was observed in treatment C (16.26 cm). The results


Table 3. Parameters for treatment B.

Parameters Wk0 Wk1 wk 2 wk 3 Wk4 wk 5 wk 6 Wk7 wk 8 wk 9 wk10 wk 11 wk 12 total mean

total weight (g) 108 117.17 130.1 139.37 147.3 154.15 158.5 162.2 171.2 180.7 188.3 195.7 208.1 2060.82 158.5246

mean weight (g) 8.3 9.01 10.01 10.72 11.33 11.85 13.21 13.51 14.25 15.05 15.69 16.31 17.33 166.57 12.81308

weight gain (g) 0 0.71 1 0.71 0.61 0.52 1.36 0.3 0.75 0.79 0.64 0.62 1.02 9.03 0.694615

total length (cm) 100.8 107 115 121.66 128.1 137.9 139.9 142 152.2 161.4 167.4 175 185.1 1833.39 141.03

mean length (cm) 7.75 8.23 8.84 9.35 9.85 10.6 11.65 11.82 12.68 13.44 13.95 14.58 15.42 148.16 11.39692

length gain (cm) 0 0.48 0.61 0.51 0.5 0.75 1.05 0.17 0.86 0.76 0.51 0.63 0.84 7.67 0.59

feeding rate (g) 7.02 7.61 8.45 9.05 9.57 10.01 9.51 9.73 10.27 7.22 7.53 7.82 8.32 112.11 8.623846



Food conv. Efficiency (%) 0 9.32 11.83 7.85 6.37 5.19 14.3 3.08 7.3 10.94 8.49 7.92 12.25 104.84 8.064615

survival rate (%) 100 100 100 100 100 90 90 90 90 90 90 90 90 1220 93.84615

Table 4. Physiochemical parameters for treatment B.

Parameters WK 0 WK 1 WK 2 WK 3 WK 4 WK 5 WK 6 WK 7 WK 8 WK9 WK10 WK11 WK12

Temprature (°C) 26 27 28 27.5 26 27 28 26 26 27 26.5 28 27 Dissolved O2 (mg/l) 5.96 6.12 6.83 6.24 6 5.9 6.3 6.12 5.8 6.13 5.9 6 6.12 pH 6 7 7.5 6.5 7.5 7 8 7 7 7.5 7.5 6.5 7 Ammonia (mg/l) 0.01 0.34 0.4 0.38 0.5 0.62 0.65 0.68 1 0.7 0.6 0.62 0.58 Nitrite (mg/l) 0.01 0.01 0.02 0.01 0.03 0.03 0.02 0.01 0.03 0.02 0.01 0.02 0.02

Table 5. Production parameters for treatment C.

Parameters Wk0 Wk1 wk2 Wk3 wk4 Wk5 Wk6 wk 7 wk 8 wk 9 wk10 Wk11 wk 12 total mean

total weight (g) 106 111.3 117.21 122.92 129.76 137.8 144.87 149.32 156.37 164.4 171.5 186.59 196.32 1894.3 145.717

mean weight (g) 8.15 8.56 9.01 9.45 9.98 10.6 12.07 13.57 14.21 14.94 15.59 16.96 17.84 160.93 12.3792

weight gain (g) 0 0.41 0.45 0.44 0.53 0.62 1.47 1.5 0.64 0.73 0.65 1.37 0.88 9.69 0.74538

total length (cm) 98.25 103.3 109.42 115.97 122.95 130.9 135.5 139.65 146.1 152.25 158.8 169.5 178.9 1761.4 135.493

mean length (cm) 7.55 7.94 8.41 8.92 9.45 10.06 11.29 12.69 13.28 13.86 14.43 15.4 16.26 149.54 11.5031

length gain (cm) 0 0.39 0.47 0.51 0.53 0.61 1.23 1.4 0.59 0.58 0.57 0.97 0.86 8.71 0.67

feeding rate (g) 6.89 7.23 7.61 7.99 8.43 8.95 8.64 8.21 8.6 6.57 6.86 7.46 7.86 101.3 7.79231



Food conv. Efficiency (%) 0 5.67 5.91 5.5 6.28 6.92 17.01 18.27 7.44 11.11 9.47 18.36 11.19 123.13 9.47154

survival rate (%) 100 100 100 100 100 100 90 80 80 80 80 80 80 1170 90


Table 6. Physiochemical parameters for treatment C.

Parameters WK 0 WK 1 WK 2 WK 3 WK4 WK 5 WK 6 WK 7 WK8 WK 9 WK 10 WK11 WK12

Temprature (°C) 26 27 28 27 26 26.5 27.5 27 28 27 27 28 27

Dissolved O2 (mg/l) 5.5 5.8 6 6.2 5.3 5.5 5.8 6 5.9

6 6.1 5.7

pH 6 6.5 7.5 7 7 8 7 7.5 7 6.5 7 7.5 7

Ammonia (mg/l) 0.01 0.02 0.38 0.42 0.62 0.72 0.92 1 0.95

0.9 1 0.5

Nitrite (mg/l) 0.01 0.02 0.02 0.01 0.03 0.04 0.02 0.02 0.03 0.02 0.01 0.04 0.02

Figure 1.Production parameters for treatment A

from the study agree with Boyd and Lichtokoppler, (1979). The highest survival rate was found in treatment B at 93.8% followed closely by treatment A and C at 90% (Figures 1 to

6). The result showed that the highest mean weight was observed in tank A (18.33 g) which surpassed that of tank B and C (De Silva and

Davy,1992; De Silva and Anderson,1995). The highest mean length (16.26 cm) and the highest specific growth (3.75 cm) were observed in tank C. It could therefore be concluded that a mixture

Fig 1: PRODUCTION PARAMETERS FOR TREATMENT A

0

50

100

150

200

250

week zero

wk one

wk two

wk three

wk four

wk five

wk six wk seven

wk eight

wk nine

wk ten

wk elev.

wk twe.

PR

OD

UC

TIO

N P

AR

AM

ETER

S (W

EIG

HT

AN

D L

ENG

HT)

total weight (g)

mean weight (g)

weight gain (g)

total length (cm)

mean length (cm)

length gain (cm)

feeding rate (g)

specific growth rate (cm)

mean growth rate (cm)

food conv. Efficiency (%)

survival rate (%)


Figure 2. Physiochemical parameters for treatment A.

Figure 3. Production parameters for treatment B.

of vital chicken feed and fishmeal can replace Coppens. Fish farmers can therefore explore the use of vital chicken feed mixed with fishmeal as an alternative to

Coppens (commercial fish feed) in the diet of Clarias gariepinus to reduce cost. The result of the study is in agreement with Bascinar et al., 2007.

0

5

10

15

20

25

30

35

WK 0 WK 1 WK 2 WK 3 WK 4 WK 5 WK 6 WK 7 WK 8 WK9 WK10 WK11 WK12

PH

YSI

OC

HEM

ICA

L P

AR

AM

ETER

S

TEMPRATURE (OC)

DISSOLVED O2 (mg/l)

PH

AMMONIA (mg/l)

NITRITE (mg/l)


Figure 4. Physiochemical parameters for treatment B.

Figure 5. Production parameters for treatment C.


Figure 6. Physiochemical parameters for treatment C.

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Figure 6. Physiochemical parameters for treatment C

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Modulatory properties of Telfairia occidentalis leaf extract on pancytopenia, electrolyte imbalance and

renal oxidative damage in rats

Johnson Olaleye OLADELE1, Olu Israel OYEWOLE1*, Olamilekan Kabir BELLO2 and Oluwaseun Titilope OLADELE1

1Phytomedicine and Toxicology Laboratories, Department of Biochemistry, Faculty of Basic and Applied Sciences, Osun

State University, Osogbo, Nigeria. 2Department of Biochemistry, Faculty of Life Sciences, University of Ilorin, Nigeria.


Copyright © 2017 Oladele et al. This article remains permanently open access under the terms of the Creative Commons Attribution License 4.0,

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received 30th April, 2017; Accepted 25th May, 2017

ABSTRACT: Telfairia occidentalis is a medicinal plant acclaimed to possess many therapeutic potential. The study was carried out to investigate the possible modulatory effects of aqueous leaf extract of T. occidentalis on chemical-induced oxidative stress, renal dysfunction and pancytopenia in rats. Twenty-four (24) adult male rats were divided into 4 groups of 6 rats each. Group I received distilled water, group II, III and IV were administered 5 mg/kg body weight of cadmium chloride to induce oxidative stress. Group III and IV were treated with 200 and 400 mg/kg body weight respectively of aqueous leaf extract of T. occidentalis for 14 days while rats in group II were left untreated. Results obtained showed that administration of cadmium caused significant suppression of haematopoiesis, depleted endogenous antioxidants and increased lipid peroxidation in the kidney as well as alteration of serum electrolytes. Treatment with graded doses of T. occidentalis leaf extract significantly reversed the cadmium induced-biochemical insults as the extract restored the normal renal integrity and boosted renal antioxidant status in rats. The leaf extract also caused significant boost in haematopoiesis in the rats. This result confirmed that aqueous extract of T. occidentalis effectively maintain electrolyte balance, modulates pancytopenia and oxidative renal damage in rats suggesting its protective potentials on anaemia and renal disorders. Keywords: Electrolyte imbalance, oxidative stress, renal damage, pancytopenia, Telfairia occidentalis, Cadmium chloride. INTRODUCTION Medicinal plants possessing natural antioxidants polyphenolic compounds have been shown to have reactive oxygen species (ROS) scavenging and lipid peroxidation prevention effects. Telfairia occidentalis (fluted pumpkin-common name, ugu-Igbo language), is a tropical vine grown in West and Central Africa as leaf vegetable and for its edible seeds. The leaf is used locally as blood booster due to the abundance of blood enriching minerals such as iron, potassium, sodium, phosphorus, vitamins (thiamine, riboflavin, nicotinamide, ascorbic acid) and phytochemicals in the plant (Kayode

and Kayode, 2011). T. occidentalis leaf is also used in the treatment of infertility (Nwangwa et al., 2007), liver problem and diabetes (Eseyin et al., 2005; Adaramoye et al., 2007). Experimental evidence has claimed that the plant has positive effect on haematopoiesis (Alada, 2000). The hypolipidemic effect and the therapeutic usefulness of the leaf extract in hypercholestolemia has also been documented (Oboh et al., 2006).

Many heavy metals such as cadmium have been considered to have deleterious effects on human health (Chounwou et al., 2012). Human exposure to cadmium



may be via medications, cigarette, diets, water, occupation and environmental pollution (Chater et al., 2008). A number of disorders including Parkinson syndrome, hepatorenal degeneration, neuropathies, pneumoconiosis and cancer have been associated with chronic exposure to cadmium (Oyewole et al., 2016).

Upon exposure, cadmium can accumulate in various organs of the body including liver, kidney, pancreas and testis eliciting adverse effects on these organs and altering their physiological and biochemical functions. The kidney is a major target of cadmium-induced toxicity due to the presence of metallothionein, a sulfhydryl group containing protein (Van Kerkhove et al., 2010). Cadmium induced anaemia has been associated with haemolysis and iron-deficient indices (Wang et al., 2012). The present study investigated the ameliorating effects of aqueous leaf extract of T. occidentalis on cadmium chloride-induced oxidative stress, renal dysfunction and pancytopenia in rats.

MATERIALS AND METHODS Chemicals and reagents Urea, creatinine and electrolytes (K

+, Na

+ and HCO3

-) kits

are products of Randox Chemical Limited, England. Antioxidant (CAT, SOD, GST), GSH, ascorbic acid and MDA kits were obtained from Cayman Chemical Michigan, USA. Cadmium chloride is a product of British Drug House Poole, England. Collection of plant material and aqueous extraction Fresh leaves of T. occidentalis was purchased from vegetable section of Igbona Market in Osogbo, Osun State, Nigeria. The leaves were thoroughly washed and blended in water. The paste was filtered to obtain a clear aqueous extract of the leaves. The filtrate was air dried to obtain a powdery form which was used to prepare the 200 mg/kg and 400 mg/kg body weight of extract used in this experiment. Experimental animals Twenty-four male Wistar albino rats weighing between 130 to140 g were used for this experiment. The rats were obtained from the Central Animal House, Osun State University Osogbo, Nigeria. The rats were kept in ventilated cage at optimum temperature and 12 hrs light/dark cycle and fed with commercial growers smash and water ad libitum. The experiment was carried out in accordance with current rules and guidelines established for the care of laboratory animals (NRC, 2011). The rats were acclimatized for two weeks before treatment commenced.

Oladele et al. 75 Experimental design and dose regimen

The 24 Wistar albino rats were sorted into four (4) different groups containing six (6) rats each. Average body weight of each animal group were taken and recorded daily. Administration of extract and cadmium chloride was done using the gavage method with the aid of oral canula. The animals were treated daily for 14 consecutive days. Group I: received distilled water and serve as the control. Group II: received 5 mg/kg body weight of CdCl2. Group III: received 5 mg/kg body weight of CdCl2 and 200 mg/kg body weight of T. occidentalis. Group IV: received 5 mg/kg body weight of CdCl2 and 400 mg/kg body weight of T. occidentalis.

Collection of blood samples

The rats were weighed and sacrificed after 24 hrs of last dose treatment under the influence of chloroform anesthesia. The jugular vein was cut and whole blood for haematological analysis collected into labeled EDTA bottles to prevent clotting. Serum for biochemical analysis were obtained by collecting blood from the jugular vein into separate plain bottles, allowed to clot and centrifuged at 4000 rpm for 30 mins. The serum obtained was stored in a refrigerator at -4 ºC until it was used for biochemical analysis.

Preparation of kidney homogenates

The rats were quickly dissected and the kidneys harvested. The kidneys were rinsed with KCl and blotted with filter paper and weighed. They were then chopped into bits and homogenized in 4 volumes of the homogenizing buffer (0.1 M Tris-KCl, pH 7.4) using a Teflon homogenizer. The resulting homogenate was centrifuged at 12,500 g for 15 mins in a cold centrifuge (4

0C), to obtain the post mitochondrial fraction. The

supernatant was collected and used for biochemical analyses.

Measurement of haematological parameters

Haematological parameters including packed cell volume (PCV), haemoglogbin concentration (Hb), red blood cell count (RBC), white blood cell count (WBC), lymphocyte, reticulocyte and WBC differential counts were measured using the automated multiparameter blood analyzer SYSMEX KX21 as earlier described by Dacie and Lewis (1991).

Biochemical assays

Catalase activity in the kidney homogenate was determined according to the method of Sinha (1972).


Table 1. Haematological parameters in rats administered cadmium chloride and aqueous leaf extract of T. occidentalis.

Parameters Group I Group II Group III Group IV

PCV (%) 31.87±2.96 17.20±2.53* 26.12±3.08 32.48±3.42

Hb Conc. (g/dl) 19.75±1.54 11.87±1.07* 16.38±1.40 17.09±2.02

RBC (X 106µl) 8.91±0.64 3.85±0.32

* 6.68±0.47 7.44±0.90

WBC (X 103µl) 16.52±1.92 10.27±1.55

* 14.99±1.32 15.01±2.06

Lymphocyte (%) 18.13±3.01 10.07±2.58* 15.47±2.93 16.11±2.24

Reticulocytes (%) 12.82±1.86 8.80±1.21* 11.38±1.31 12.43±2.12

Monocyte (%) 50.70±5.85 38.88±4.92* 46.47±5.20 48.66±5.26

Eosinophil (%) 11.43±1.74 7.21±1.09* 9.96±0.79 10.21±1.25

Neutrophil (%) 34.34±3.94 22.82±4.52* 29.51±3.88 31.32±3.17

Basophil (%) 7.18±0.84 3.22±0.36* 6.33±0.64 6.97±0.51

Data presented as Mean ± SD of 6 animals. *Significantly different from normal control group at P< 0.05.

Table 2. Serum urea, creatinine and electrolytes concentrations in rats administered cadmium chloride and aqueous leaf extract of T. occidentalis.


Urea (mmol/L) 49.68±4.66 76.43±5.71* 50.88±4.53 51.35±5.21

Creatinine (mmol/L) 58.61±5.23 88.53±8.11* 62.59±4.10 60.44±5.21

Na+(mmol/L) 82.44±6.21 62.39±5.43* 75.68±6.46 78.30±5.49

K+(mmol/L) 68.77±5.10 89.24±7.12* 74.33±6.55 73.90±4.71

HCO3-(mmol/L) 48.22±3.78 31.55±2.66* 34.69±3.00 38.41±2.39


Superoxide dismutase (SOD) was assayed by the method of Misra and Fridovich (1972). The method of Habig et al. (1974) was used in the determination of glutathione S-transferase (GST) activity. The level of reduced glutathione (GSH) in the samples was determined by the method described by Jollow et al. (1974). Lipid peroxidation (malondialdehyde) was assessed by using the procedure of Varshney and Kale (1990). The ascorbic acid concentration was determined according to the method of Jagota and Dani (1982). Serum electrolytes (Na

+, K

+, HCO3

-), urea and creatinine

were measured using the appropriate kits and method described by the manufacturer (Randox).

Statistical analysis

Data were expressed as mean ± standard deviation (mean ± SD) and analyzed using one-way analysis of variance (ANOVA) with the aid of SPSS 12.0 computer software package (SPSS Inc; Chicago, U.S.A). Student’s t-test was employed for comparison between two sets of data and differences at P<0.05 were considered significant.

RESULTS

Table 1 show the haematological parameters in rats

administered cadmium chloride and aqueous leaf extract of T. occidentalis. Rats administered cadmium alone (group II) recorded significant reduction in haematological indices (PCV, RBC, WBC, Hb, lymphocytes reticulocytes, monocyte, eosinophil, neutrophil and basophil). This reduction was significantly (P<0.05) reversed following treatment with aqueous leaf extract of T. occidentalis (group III and IV).

The results of serum concentrations of urea, creatinine and electrolytes in control and experimental animals are shown in Table 2. Rats exposed to cadmium chloride (group II) showed a significant increase (P<0.05) in serum concentrations of urea and creatinine as compared to the control (group I). Cadmium also caused significant increase (P<0.05) in K

+ while it reduced Na

+

and HCO3- as compared to the control. The altered

values of kidney function indicators were reverted significantly (P<0.05) toward normal in rats treated with 200 and 400 mg/kg body weight of leaf extract of T. occidentalis (group III and IV).

Table 3 shows the effect of cadmium chloride and aqueous leaf extract of T. occidentalis on enzymatic and non-enzymatic antioxidants as well as lipid peroxidation in the rat’s kidney. Renal SOD, GST, CAT, GSH and ascorbic acid were significantly reduced while lipid peroxidation (MDA concentration) was increased in rats treated with cadmium chloride alone (group II). Adminis-

Oladele et al. 77

Table 3. Some oxidative stress indicators in the kidney of rats administered cadmium chloride and aqueous leaf extract of T. occidentalis.


SOD (U/mg protein) 41.31±3.55 25.65±2.68 35.29±3.24 38.52±2.98

CAT (U/mg protein) 28.16±2.22 18.47±3.11 22.82±2.36 24.43±2.54

GST (U/mg protein) 37.89±3.43 27.56±2.99 32.10±2.89 32.78±3.00

GSH (µg/ml) 8.77±0.88 4.96±0.42 7.35±0.54 7.48±0.47

Ascorbic acid (µg/ml) 5.63±0.57 2.89±0.41 5.10±0.32 5.24±0.51

MDA (units/mg protein) 5.49±0.34 10.21±0.89 6.96±0.63 6.87±0.58


tration of graded doses of T. occidentalis leaf significantly attenuates these antioxidants anomalies in the kidney of experimental rats. DISCUSSION Results in Table 1 indicated a marked general decrease in blood cellular elements in the animals (pancytopenia) following administration of cadmium chloride. Exposure of rats to the metal also resulted in anaemia characterized by significant reduction in PCV and Hb concentration. However, treatment with aqueous leaf extract of T. occidentalis significantly increased (P<0.05) all the heamatological parameters. The haematinic effect of this extract may be due to the phytochemical constituents of the leaves which consist of blood enriching minerals such as iron, potassium, sodium, phosphorus, vitamins (thiamine, riboflavin, nicotinamide, ascorbic acid) and phytochemicals (Kayode and Kayode, 2011).

The observed increased serum K+

level and decreased serum Na

+ and HCO3

- in rats administered cadmium

alone indicates electrolyte imbalance. This result agrees with the previous reports that cadmium intoxication induced abnormal serum electrolytes and hyperkalemia (Tabassum and Bajaj, 2013). This electrolyte imbalance might have resulted due to peroxidation of the polyunsaturated fatty acids in the membrane by cadmium which delocalized Na

+-K

+-ATPase from basolateral to

apical membrane. The mechanism of cadmium-induced organ damage has been elucidated to be through alteration of transport pathways (Patra et al., 2012), epigenetic aberrations in DNA expression, the disruption of the redox balance resulting in oxidative stress and impairment of mitochondrial functions to induce apoptosis (Matovic et al., 2012). Treatment with aqueous leaf extract of T. occidentalis significantly reversed the observed alterations in serum electrolytes suggesting its positive effects on homeostasis.

The observed significant increase in the level of urea and creatinine in the serum of cadmium treated rats (group II) as compared to the normal control (group I) is an indication of renal dysfunction. Urea and creatinine are chiefly excreted from the blood via glomerular

filtrations and are routinely measured to assess the kidney health status (Dioka et al., 2004). Increased serum levels of these metabolites are synonymous with decreased in the rate of their excretion which is a pointer to impairment of kidney function (Patil et al., 2007). Aqueous leaf extract of T. occidentalis administration significantly normalized the serum levels of urea and creatinine.

The increased lipid peroxidation and depletion of antioxidants levels in the kidney of rats intoxicated with cadmium is synonymous with oxidative stress. This observation might be a consequence of increased free radical formation as well as the disruption of antioxidant status. However, treatment with aqueous extract of T. occidentalis significantly reversed these alterations confirming the antioxidant properties of the extract. The polyphenolic content of the leaf extract might have contributed directly to the antioxidant action. It is suggested that polyphenols have inhibitory effects on mutagenesis and carcinogenesis in human (Tsao and Akhtar, 2005). Flavonoids in plants are regarded as antioxidant molecules and could therefore reduce cellular oxidative stress (Oboh et al., 2007).

Lipid peroxidation is known to be one of the principal mechanisms of cell injury in aerobic organisms subjected to oxidative stress (Oboh and Rocha, 2007). Neurodegenerative diseases and aging processes resulting from accumulation of free radicals could be inhibited by antioxidant activities of medicinal plants such as T. occidentalis. Conclusion This study demonstrated that aqueous extract of T. occidentalis effectively boosted haematopoiesis, corrected electrolyte imbalance, reduced oxidative stress and attenuates renal dysfunction induced by cadmium chloride exposure in rats. This study suggests that the extract has protective potential on chemical-induced pancytopenia and renal oxidative dysfunction. Regular consumption of T. occidentalis leaf might therefore be a good remedy for management of anaemia while it also maintains kidney integrity.

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