Vol 2- Continental J. Applied Sciences.

8/9/2019 Vol 2- Continental J. Applied Sciences.

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Continental J. Applied Sciences 2:1 - 6, 2007Wilolud Online Journals, 2007.

ASSESSMENT OF ORGANOCHLORINE AND POLYCHLORINATED PESTICIDES RESIDUES INSOME WESTERN NIGERIA RIVERS

P.A. Egwaikhide a, S.O. Okeniyi b S.O. Ohikhena c and C.E. Gimba daDepartment of Chemistry and Centre for Biomaterials Research, University of Benin, Nigeria

bDepartment of Chemistry, Nigerian Defense Academy, Kaduna.cDepartment of Polymer Technology, Federal Polytechnic, Auchi, Nigeria

dDepartment of Chemistry, Ahmadu Bello University, Zaria.

ABSTRACTThe degree of contamination and pollution of some rivers in Western Nigeria byorganochlorine and polychlorinated Pesticide Residues were investigated. Theoirganochlorine and polychlorinated pesticides analysed showed total DDT (Dichlorodiphenyl trichloroethane), ranged from ND 0.3953 ppb. Aldrin and lindane weredetected in the range of 0.0029 0.1904ppb and 0.0067 0.6341 ppb respectively.Total Polychlorinated biphenyls (PBB) level; ranged from ND 8.9916 ppb. OtherOrganochlorine Pesticides analysed gave varying levels of heptachlorepoxide, (rangeND 0.0037 ppb), hexachlorobenzene (range ND 0.0092 ppb), endosulfan (rangeND 0.0851 ppb) and dieldrin (range 0.0049 0:6590 ppb) respectively. Theorganochlorine and polychlorinated pesticide residue peaks were identified bycomparison with some reference mixed standard run under the same conditions.Statistical analysis of the data revealed significant difference in the organochlorineconcentration between the rivers (P


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P.A. Egwaikhide et al : Continental J. Applied Sciences 2:1 - 6, 2007

Organic pesticides enter natural water from direct application for control of aquatic weeds, trash fish,aquatic insects, percolation and run off from agricultural lands, drift from industrial waste water anddischarge of waste waters from clean up equipments used for pesticide formulation and applications (VanSchoubroeck, 1989).

The purpose of this study is to provide baseline information on the level of organochlorine pesticides inWestern Nigeria water bodies using some rivers to indicate the degree of environmental pollution.

MATERIALS AND METHODSMaterialsSample Collection and PreservationThe water samples analyzed were collected from different rivers in Osun, Oyo, Ogun, Ondo and LagosStates of Western Nigeria. 8 samples each of the various rivers were collected using Grab samplingtechnique and analysed for organochlorine pesticides residues. Sampling was done upstream. Watersamples were kept in plastic bottles and preserved in a refrigerator maintained at 4 oC prior to analysis. ThepH of the water samples was also taken immediately on collection of the water samples.

All chemicals used, unless otherwise stated were of analytical grade. Glass wool, Waters 204 HPLC modelequipped with 441 model UV detector filled with 25nm filter, model UGK septum less injector and SE 120model recorder was used to analyse the clean-up hexane extracts.

Samples collection and PreservationThe samples were collected from different rivers in Osun, Oyo, Ogun, Lagos and Ondo States. Grapsampling technique (Schofield, 1980) was employed for the samples collection. Sampling was carried outupstream. Eight samples were collected from each sampling point. The P H of the water samples wereimmediately taken on collection of the samples. The water samples were placed in rubber bottles andimmediately preserved after collection in ice in order to minimize degradation of the pesticides on the fieldand these samples were transferred to the Freezer as soon as they were brought to the laboratory.

MethodsThe analytical procedure (APHA AWWA WPCF) Standard Methods for the examination of water andwastewaters (1980), with slight modification was used. The water analysis involved the extraction using15% diethyl either in hexane. The extracts were cleaned using tetraoxosulphate (vi) acid clean up andethanolic potassium hydride base clean up instead of the column clean up described in APHA AWWA WPCF. The cleaned extracts were later injected into the HPLC equipped with 441 model UV detectorfitted with 254nm filter.

Extraction and clean up proceduresA litre of the water sample was measured and transferred into a 2 litre separating funnel. 60ml of 15%

diethyl either in hexane was added and the separating funnel was shaken vigorously for 2 minutes and left

for at least 20 minutes for complete separation of the phases. After separation, the bottom aqueous layerwas drained into a 250ml conical flask through a funnel containing sodium sulphate, which has been pre-washed with 15% diethyl either in hexane.

A second extraction was carried out with another 60ml 15% diethyl either in hexane and the process aboverepeated.

A third extraction was carried out using 60ml of hexane only and the process repeated. The separatingfunnel was rinsed with 50ml of hexane.

The extracts were concentrated to 15ml with a rotary evaporator and to 5ml with nitrogen gas.Concentration factor is 45. The water extracts were cleaned by tetraoxosulphate (vi) acid and ethanolicpotassium hydroxide clean up methods thus.


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2mls of the hexane extract was transferred into a centrifuge tube and 2ml of concentrated tetraoxosulphate(vi) acid was added using eppendof pipette. The centrifuge tube was capped and shaken gently by inversionfor two minutes and then centrifuged for 10 minutes. The top hexane layer was removed gently with aneppendof pipette avoiding the aqueous layer and then injected into the HPLC.

Another 2ml of the water extract was put into a centrifuge tube with eppendof pipette and 2 pellets of potassium hydroxide were added. 2ml of ethanol was added and 0.1ml of distilled water. The centrifugetube was capped and put into a water bath for 30 minutes at 50 oC. 4ml of 2% sodium chloride in 0.1morthophosphoric acid was added and the centrifuge tube shaken by inversion gently for 2 minutes and thencentrifuged for 10 minutes. The upper hexane layers was removed gently and injected into the HPLC.

Quantification of Pesticides ResidueThe chromatograms of the samples were compared with those of the mixed standards, which were injectedinto HPLC. These were used to estimate the concentration of the Organochlorine and Polychlorinatedpesticide residues. 1ml of the standards and samples were injected throughout.The concentration, C of the residue in ppm was calculated using.

C = V x Phs x Vstd x Crms x iW Phstd Vs F

WhereV = Total Volume of the solvent used for extractionW = Sample Weight taken in gramsCrms = Concentration of reference mixed standardPhs and Phstd = Peak height of sample and standard respectivelyVs and Vstd = Volume of sample and standard injected in ml respectivelyF = Concentration factor.

RESULTS AND DISCUSSIONThe multiple regressions analysis of the organochlorine and polychlorinated pesticide residuesconcentrations in Western Nigerian Rivers and water bodies revealed that

P > 0.32

Analysis of variance shows that the compound does not influence one another. There may be a relationshipbetween each of the rivers with the compound especially lindare (p=0.72) diedrine(p=0.35) aldrine(p=0.91) and PCB (p=0.92) op DDT, pp DDT and eldrin were ignored because there was no variance.

The most frequently occurring residues are lindane, hexachlorobenzene, heptachlor, endosulfan and aldrin,while the levels of endrin, op DDT and ppDDT were non detectable. Few of the samples analysed were

found to contain PCBs, which range from 0.4348 8.9916 ppb and aldrin 0.0029 0.1904 ppb. Thehighest lindane concentration of 0.6341 ppb was obtained for sample WW 2. This high contamination couldbe due to the extensive use of lindane, which is marketed as Gammalin 20 and used by farmers forcombating black pod disease in cocoa or most probably be due to it being misused by some Fishermen forkilling fishes. In the later case, it is sprayed directly into the water and there were fishing activities in-placein most places sampled (Agunloye, 1984). Samples WW 2 showed the highest concentration of aldrin(0.1904 ppb). This could be due to run off from agricultural land, because aldrin is a major component of aldrex 40, which is used for crop protection. The levels of organochlorine residues exhibited in samplescollected from the same point at different times are different. That is why the analysis of the samples wasdone in triplicates and the mean recorded. This difference in concentration could be due to the fact thatwhen the water is highly turbulent, there is mixing tendency for a lot of things, which can be carried by thewater compared with when it has low tide. This difference in concentration of residues exhibited at


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different times is comparable with studies reported by other workers (FEPA, 1991, Adjert, 1986). There isno noticeable effect on pH and temperature of the samples.

Lagos state has the highest contamination of lindane, endosulfan and DDT and this could probably be dueto discharge of effluents from the chemical industries and agricultural land run off.Ondo state exhibited the highest level of aldrin and this may be due to aldrin being used for the treatment of cocoa plant, which is a major crops produce grown in Ondo state.

The perusal of mean values of the pesticides in samples analysed demonstrates that on the whole,contamination is at a low level. These are comparable with those reported in surface waters of NorthAmerica (Staare, et al. 2000). It can be seen that some residues are at higher concentrations compared withothers. This may probably be due to the extensive usage of pesticides in some areas (Ecobichon, 1986).

CONCLUSIONSince, population growth, increasing urbanization and industrialization and rising standard of living haveall contributed to an increase in both the amount and the variety of organochlorines and polychlorinatedpesticide residue generated in rivers and water bodies in Nigeria, Frequent studies should be carried out onthese rivers and water bodies to give a baseline information on the level of organochlorine pesticidespresent to ascertain the degree of environmental pollution from time to time.

REFERENCE:Adjerl T.O (1986). Study on the long term effect insects associated with cocoa. Cocoa Research instituteInfo. 99 120

Agunloye T.O. (1984): A survey of chlorinated hydrocarbons in river of southern Nigeria. M.Sc project,department of chemistry, university of Ibandan, Nigeria.

APHA AWWA and WPCF (1980). Standard methods for the examination of water and waste-waters, 15 th edition published by American Public Health Association. Washington D.C.

Barbash, J.E. and Resek, E.A. (1996). Pesticides in Ground water: Distribution, Trends and Governingfactors. Ann Arbor Press Chelsea Vol(2)

Burse, VW, Head, S.L, Korver M.P Mcclure, P.C, Donahue, J.F, Needham, L.L, (1990): Determination of selected Organochlorine pesticides and Polychlorinated by Phenyl in Human Serum. J. Anal.Toxicol.14:137-142)

Ecobichon D.J. (1986). Toxic effects of pesticides of Caserrett and Doulls. Toxicology. The basic scienceof Poisons. New York: McGraw Hill 643 689

Federal Environmental Protection Agency now Federal Ministry of Environment (1991). Guidelines andStandards for Environmental Pollution control in Nigeria.

Gwenzi, W.D. (1986) Pesticides in soil and water Soil. Science society of America. Inc. Publisher MadisonWisconsin, U.S.A.

Kanja LW, Skaare J.U, Ojwang S.B.O., maitail C.K. (1992). A comparison of organochlorine pesticideresidues in material adipose tissue, maternal blook cord blood and human milk from mother infant pairs.Springer New York vol. 22(1)

Lee, H, Chau, A.S.Y, Kawahara, F (1982). Organochlorine Pesticides: In analysis of pesticides in watervol. 2 by Chau, A.S.Y, Afgan B.K. C.R.C. Press Inc. Boca Raton 14,71


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National Pesticide Information Retrieval System, Purdue, University (2006).

Salau A.A. (1993): Environmental Crisis and Development in Nigeria. An inaugural lecture: serves No.13:Port Harcourt, University of Port Harcourt Publishing House 24 26

Schofield, T. (1980): Sampling of water and waste water. Practical aspect of sample collection. Waterpollution control 79: 468

Staare, J.U, Bernhoft, A, Aderocher A, Gabrielsen G.W, Gokbyr G.W and Henriksen (2000).Organochlorine in top predator at svalbard: Occurrence, levels and effects. Toxicol Lett. 112/113: 103 109

United Nations Environment Programme (1992). Waste: Environmental Data Report 1991/92. third Ed.Prepared for UNEP by the Grains Monitoring and Assessment Research Centre. London Uk 333 359

United State Environmental Protection Agency (2006). Drinking water standards and health Adviisories(EPA 822 R 06 013). Washington D.C.

Van Schoubroeck F.H.J(1989): Managing pest and pesticides in small scale Agriculture. London. AcademyPress Ltd. 101 105

Received for Publication: 11/10/2007Accepted for Publication: 20/10/2007

Corresponding Author:P.A. Egwaikhide

Department of Chemistry and Centre for Biomaterials Research, University of Benin, NigeriaE-mail: - [email protected]


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The results of the concentration (ppb) levels of Organochlorine and Polychlorinated Pesticide Residues in Western Nigeria rivers are shownRivers/Water

bodiessampled

Pesticides residue concentrations (ppb)

sCode P H Temp.oC

Heptachlorioep

oxide

Heptachor HCP opDDT Pp DDT Dieldrin Endosulfan

Oshun RiverOshogbo

ORO 6.91 26.3 ND 0.0010 0.0028

ND ND 0.0141 0.0100

Ogun RiverLagos

LLR 6.68 27.0 ND 0.0037 0.0021

ND ND 0.0074 0.430

River AyeOgun

RAO 6.80 26.1 ND 0.0008 0.0007

ND ND 0.0074 0.0430

Badagry / Noque River

BNR 0.0008 ND ND ND 0.0145 0.0057

Ogbese RiverOndo

OROD

7.05 26.2 ND 0.0008 ND ND ND 0.0107 0.0110

Owena RiverOndo

OWOD

7.15 26.5 ND ND 0.0039

ND ND 0.0087 0.0029

Lagos Lagoon WW 1 6.73 26.5 ND ND 0.0008

ND ND - 0.0143

Lagos Lagoon WW 2 6.75 26.8 0.0001 ND 0.0041

ND ND 0.0235 0.0857

Lagos Lagoon WW 3 7.12 26.7 ND 0.0037 0.0017

ND ND 0.0049 0.0143

Lagos Lagoon WW 4 6.09 26.6 ND 0.0008 0.0022

ND ND 0.0659 0.0851

ND = Not Detected


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Continental J. Applied Sciences 2: 7 - 11, 2007 Wilolud Online Journals, 2007.

KINETICS OF THE ADSORPTION OF MUCIN ONTO COMMERCIALLY PURE TITANIUMSURFACE

OMONIYI K. ISRAEL 1*. LORI A. JOSEPH 2 AND OGBODOBRI O. JUDE 2 1*Ahmadu Bello University, School of Basic and Remedial

Studies, P.M.B. 6007, Funtua-Nigeria2Chemistry Department, Ahmadu Bello University, Zaria-Nigeria.

ABSTRACTThe study investigated the kinetics of adsorption of the salivary protein, mucin on totitanium, since the adsorption of proteins onto a biomaterial stand as the mostimportant issue in determining their biocompatibility. The time profiles for theadsorption of mucin onto titanium, Ti particles showed that the average amount of mucin adsorbed by using 50mg/ml mucin concentration was 2.4 + 0.11 times higherthan that by the use of 10mg/ml mucin solution. Two kinetic models were applied fordetermining the rate and mechanism of adsorption of mucin onto commercially pureTi powder. The present study showed that the adsorption rate constant is increased asadsorbate concentration is increased. Also, at low concentration a thin boundary layeris formed.

KEY WORDS: mucin, titanium, kinetic model, rate constant

INTRODUCTIONThe adsorption of proteins on solid surfaces is an important phenomenon taking place immediately aforeign material contacts a biological system. It is thus, involved in situations of bio- and bloodcompatibility and in fouling in the process industry (Bornzin and Miller, 1982). Protein adsorption appearsto be mainly irreversible in many cases (Kim and Lee, 1975), conformation during or after the adsorption.Model calculations indicate that the kinetics of exchange reactions can be faster than spontaneousdesorption (Lundstrom, 1985). Mucin is a glycoprotein that covers the surface of the buccal cavity andepithelial organs. Titanium as being reckoned with as an excellent material for dental and orthpaedicapplications, a thorough elucidation of the kinetics of mucin adsorption onto its surface is of greatsignificance in the bioengineering of titanium implants, towards the fabrication of the perfectly bio-friendlyTi implants. This study applied two kinetic models for determining the specific rate constant of adsorptionof mucin onto Ti implant in vitro , in what will be called the short time range.

MATERIALS AND METHODSMaterials

Mucin was obtained from Nacatai, Tesque Inc., Kyoto, Japan (Batch No. MIP960). It is a well-characterised acidic protein with a molecular weight of 4.0x10 5. Commercially pure titanium (Ti) powderof particle size 200m M was obtained from BDH Chemical Ltd. (Poole, England). It has a purity of 99.7%+ by analysis with atomic absorption spectrophotometry (Buck Scientific 200A).

Ti powder surface consist mainly of titanium dioxide (TiO 2) as shown by X-ray photo-electronspectroscopy (XPS) data (Sutherland et al., 1993).

Protein Adsorption StudySamples of titanium (50mg or 100mg) were weighed into polypropylene (PP) centrifugation tubes and10ml of mucin solution (10mg/ml or 50mg/ml) was added to each experimental group, (in triplicate).


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OMONIYI K. ISRAEL et al : Continental J. Applied Sciences 2: 7 - 11, 2007

:

Table 1: The rate constants, Kad for the adsorption of mucin onto TiWeight of Ti/mucin Conc.(mg/ml)

Lagergren Kad (Min -1) Bhattacharya andVenkobacharya Kad(Min -1)

50mgTi(10mg/ml) 8.06 x 10 -2 7.97 x 10 -2

100mgTi(10mg/ml)

7.81 x 10 -2 7.25 x 10 -2

50mgTi(50mg/ml) 8.52x10 -2 9.24x10 -2

100mgTi

(50mg/ml)

7.25 x 10 -2 7.07 x 10 -2

The mixture were continuously shaken for incubation times of 10,15,30,45,60 and 70 minutes in anincubator (1H 150, Gallenkamp Co., England) at 37 0C.

Subsequently, the Ti particles were collected after centrifugation, and mucin which did not adsorb stronglyto the Ti particles were removed by rinsing with 10ml of double distilled water. The supernatant wereseparated and removed again. The Ti particles with the adsorbed mucin of an experimental group were thenheated at 60 0C for 5 hours in an oven and then weighed by using an analytical balance (H15, E. mettler Co.,Switzerland, accuracy 10 -4g). The samples were then heated to 600 0C for 30 minutes in a furnace in orderto desorp the mucin.. The weight of mucin adsorbed was calculated by comparing the weight after drying at50 0C and that after heating at 600 0C.

RESULTS AND DISCUSSIONThe time profiles for the adsorption of mucin are shown in Figure 1, the amounts of adsorbed mucin (mg)onto Ti increased steadily from 1.6 to 4.4 in the 50mg group; and from 3.6 to 6.9 in the 100mg group of Tiparticles as the incubation time extended, using 10mg/ml mucin solution. The amounts of adsorbed mucin(mg) onto Ti increased steadily from 5.9 to 12.2 in the 50mg group; and from 8.9 to 12.8 in the 100mggroup of Ti particles as the incubation time extended, using 50mg/ml mucin solution. Statistical analysis byone-way ANOVA showed that, there was a significant difference in the amount of mucin adsorbed onto the50mg Ti particles group compared to that onto the 100mg group, using 10mg/ml mucin solution. There wasno significant difference between the two experimental groups by the use of 50mg/ml mucin solution(P


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The Second kinetic model is the Bhattacharya and Venkobacharya first order equation. The equation isgiven as:

Log [1 (U)T] = ( Kad / 2.303 )t

Where U (T) = Ci Ct/Ci-Ce

Ci, Ct are initial concentration and concentration at time t respectively, Kad is the rate constant and Ce isthe concentration at equilibrium.

As presented in Figures 2 and 3, a straight line with high correlation coefficient, R 2 in the range of 0.9800 0.9963 indicates acceptability of the model.

The Bhattacharya and Venkobacharya plots (Figures 4 and 5) also gave a linear relationship withcorrelation coefficient in the range of 0.9847 0.999. This further reaffirms the fit of the experimental datato a first order kinetic as suggested by the Largergren model.

From Table 1, the adsorption rate constants is higher as adsorbate concentration is increased by using 50mgof Ti particles.

Mode of Particle diffusionThe kinetic data was also analysed by a diffusion-controlled adsorption model. The intra-particlediffusion is model by the equation.

qt = K pt + C

Where K p is the intra-particle diffusion rate constant (in mg min-1

), C is the boundary layer thickness and qtis the amount of mucin adsorbed (mg) at time t.

As presented in Figures 6 and 7, a linear relationship with correlation coefficient values 0.9171


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Figure 4: Bhattacharya and Venkobacharya plots for mucin (10mg/ml concentration)adsorption onto Ti

y = -0.0315x + 0.0455R2 = 0.9847

y = -0.0346x + 0.1961

R2 = 0.9949

-1.6

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

00 5 10 15 20 25 30 35 40 45 50

Time (mins)

L o g

( q e

- q t )

Series1

Series2100 mgTi

50mg Ti

Figure 5: Bhattacharya and Venkobacharya plots for mucin (50mg/ml concentration)adsorption onto Ti

y= -0.0307x - 0.222R

2= 0.9904

y= -0.0401x+ 0.0334R

2= 0.999

-2

-1.8

-1.6

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

00 5 10 15 20 25 30 35 40 45 50

Time (mins)

L o g [ 1 -

U (

T ) ]

Series1Series2100mg Ti50 mgTi

Figure 6: Variation of the amount of mucin (10mg/ml concentration) transported into Tifilm with time

y= 0.9011x+ 0.7699 R 2 =0.9912

y= 0.7888x- 0.8404 R 2 =0.949

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7 8 t1/2 (min-1)

qt(mg) Series1 Series2

100mgTi 50mgTi

Figure 7: Variation of the amount of mucin (50mg/ml concentration) transported into Tifilm with time

y= 0.9159x+ 6.3491 R 2 =0.9412

y = 1.5612x+ 1.7732 R 2 = 0.9171

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6 7 8 t1/2 (min-1)

qt (mg)

Series1 Series2 100mgTi 50mgTi

CONCLUSIONAdsorbed mucin molecules become more tighly bound as the contact time with the adsorbent surfaceincreases (Lori and Ogbodobri, 2005). The present study showed that the adsorption rate constant isincreased as adsorbate concentration is increased. Also, at low concentration a thin boundary layer isformed. At high concentration, the surface is covered by a thicker boundary layer since there is very littletime for the molecules to change conformation before the surface is covered.

Kinetic study of the rate constant of adsorption of proteinic systems onto solid surfaces is of greatsignificance in the fabrication of the ever-expected perfectly bio-friendly implants and prostheses.

ACKNOWLEDGEMENTSWe are grateful to Prof. Lori A. Joseph, Department of Chemistry, Ahmadu Bello University, Zaria -Nigeria, for stimulating research work on the present topic, and for supporting the work.

REFERENCESBornzin, G.A. and Miller, I.F. (1982). In: Kandori, K., Miyagawa, K. and Ishikawa, T. (2004). AdsorptionOf Immunogamma Globulin Onto Various Synthetic Calcium Hydroxyapatite Particles. Journal of Colloid and Interface Science, 273:406-413.

Kandori, K., Miyagawa, K. and Ishikawa, T. (2004). Adsorption Of Immunogamma Globulin OntoVarious Synthetic Calcium Hydroxyapatite Particles. Journal of Colloid and Interface Science, 273:406-413.


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Kim, S.W. and Lee, R.G. (1975). In: Baier, R.E. (Edition). Applied Chemistry at Protein Interfaces,Advanced Chemical Series, 145:218.

Lori, J.A. and Ogbodobri, J.O. (2005). Determination of the Adsorptive Properties Of Mucin toTitanium. Proceedings of the 28 th Annual International Conference of the Chemical Society of Nigeria,Maiduguri, Nigeria, p. 56-59.

Lundstrom, I. (2005). Models of Protein Adsorption on Solid surfaces. Progress in Colloids Polymer Science , 70:76-82.

Sutherland, D.S. Forshaw, P.D., Allen, G.C. Brown, I.T and Williams, K.R. (1993). Surface Analysis of Titanium Implants. Biomaterials, 14:893-899.


Corresponding Author:OMONIYI K. ISRAELAhmadu Bello University, School of Basic and Remedial, Studies, P.M.B. 6007, Funtua-NigeriaE-mail: [email protected]


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Continental J. Applied Sciences 2: 12 - 22, 2007 Wilolud Online Journals, 2007.

SPATIAL VARIATION IN HEAVY METAL CONCENTRATION IN SOIL PROFILES IN EMENEINDUSTRIAL AREA OF ENUGU, SOUTH EASTERN NIGERIA.

1Anyika, C. C and 2Nnabude, P.C1Department of Soil Science, Federal University of Technology, Minna. 2Department of Botany, Nnamdi

Azikiwe University, Awka.

ABSTRACTAn investigation into the extent of metals in soils around the Emenite and the defunctNiger steel factories, which are closer to human habitation and private farms, wasconducted.Arsenic, chromium and lead were determined by Energy Dispersive X-rayFluorescence (EDXRF) with isotopic source ( 109Cd). In the control soil samples, meansoil pH was 6.4, while mean soil pH at the study location was 6.9. Arsenic and leadconcentrations decreased though not significantly with increasing distance from thefactory site. Numerically, concentration peaked at 12 m (86.68 Mg ha -1) and 9 m(110.10 Mg ha -1) away from the factory for arsenic and lead respectively. Chromiumconcentration increased marginally with a concentration of 1168.20 Mg ha -1. Highestconcentrations of 87.95 Mg ha -1 and 121.90 Mg ha -1 for arsenic and lead respectivelywere obtained at 15-30cm depth. On the other hand, the chromium concentration of 1135.10 Mg ha -1 was obtained at 0 15 cm depth. Average metal concentrations forthe control locations were 46.79 Mg ha -1, 39.45 Mg ha -1 and 691.20 Mg ha -1 forarsenic, lead and chromium respectively. While at the study location, average metalconcentrations were: Arsenic; 75.80 Mg ha -1 dry weight, Chromium; 1038.5 Mg ha -1 dry weight, Lead; 97.25 Mg ha -1 dry weight. These were very significantly higherthan the values at the control location and implied that serious soil contaminationresulted from the deposition of dust particles and fume emissions from the factory. Itis recommended that bioremediation should be used to transform the pollutants intoless hazardous forms and that people should not live or carryout agricultural activitieswithin 300 m from the industrial area.

KEYWORDS: Heavy metals, Spatial variation, Depth variation, Soil profiles,Southeastern Nigeria, Typic Paleudult.

INTRODUCTIONThe level of inorganic contaminants released into the environment from industrial sources every year is

now estimated to exceed that from organic and radioactivesources combined and a fair share of these inorganic substances ends up contaminating the soils. (Okonkwoand Eboatu, 1999)

The effects of soil contamination with these heavy metals which are toxic to plants in large amounts can bemuch more damaging and permanent than those of organic sources. Metals are strongly absorbed by thesoil clay and humus and so do not leach to any extent. Once metals contaminate a soil, it remains soindefinitely. They may find their ways into many environments through agricultural crops, soil surface andground waters where they undergo a process of redistribution and are now detected at different levels of concentration in the food chain. (Oviasogie and Ukpebor, 2003). Most frequent metal contaminationinclude: Hg, Pb, Cd, As and Cr. (Mercury, lead, Cadmium, Arsenic and Chromium) hence the need toinvestigate their levels in the soil around the Emenite and the defunct Niger steel factories in the Emene


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Anyika, C. C and Nnabude, P.C: Continental J. Applied Sciences 2: 12 - 22, 2007

industrial estate, Enugu state. To a greater or lesser degree, all of these elements are toxic to humans andother animals. Cadmium and arsenic are extremely poisonous; mercury, lead and chromium are moderatelyso.

There are many sources of inorganic chemical (Heavy metals) contaminants that can accumulate in soils.The burning of fossil fuels, emission from chimney of industries and metal refineries, smelting, and otherprocessing techniques, release into the atmosphere tons of these elements, which can be carried for milesand later deposited on the vegetation and soil. Lead is a gasoline additive that is released into theatmosphere and carried to the soil through rain and wind. (Nriagu and Paycna, 1988). Arsenic was formany years used as an insecticide on cotton, tobacco, fruit crops, lawns, and as a defoliant or vine killer.

The area of land likely to be affected from industrial sites is not very extensive. Some of these heavy metalsare found as constituents in specific organic pesticides and in domestic and industrial sewage sludge, wherethey pollute agricultural land, metals in sewage become adsorbed by the solid matter and so are retained inthe sludge. The liquid effluent, which is eventually discharged to the rivers, has very low metal content. Sothe solid sludge, which is of potential value to the farmer as organic manure rich in nitrogen and phosphate,is also a potential hazard when metals are present in large amounts. Additional localized contamination of soils with metals results from ore smelting fumes, industrial wastes, and air pollution. ( Okieimen et al.,1986 ).

Some of the toxic metals are being released to the environment in increasing amounts, while others (mostnotably lead, because of the changes in gasoline formulation) are decreasing. All are daily ingested byhumans either through the air or through food, water and soil.

To evaluate the distribution of Heavy metals in the environment due to mans activities various works havebeen carried out. A survey of heavy metal concentration in pastoral soils was conducted in the early 1990s,in New Zealand, and only one of the five heavy metals i.e. Cadmium was present at elevated levels, thoughthe concentrations were well below the level identified as warranting further investigations(ANZECC/NHMRC, 1992). Similar studies have also been carried out around textile factory and mostindustrial sites. But very little work has been reported in Nigeria and indeed Africa at large.

Host communities surrounding the Emenite factory within a distance of 6 metres and 12 metresrespectively may be ignorant of the level or environmental pollution that may result from the factory whichuses some raw materials like cement from limestone, which has cadmium contents as well as fumesreleased from chimneys of the factory.

The aim of this study is to investigate the heavy metal levels; lead, chromium, arsenic (Pb, Cr and As)emitted as fumes and dust particles in soil profiles sampled near the Emenite and the defunct Niger steelfactories, obtained from both the spatial distance and depths.

MATERIALS AND METHODSEnvironment of Study/ LocationEmene is the study area. Emene is located in Enugu east local government area of Enugu state, southeasternNigeria. Emene is situated 2 km North of Enugu town. Emene is a host to numerous industries, includingthe Emenite factory, the defunct Niger steel factory, gas plant, pharmaceutical industry, amongst others.The 1991 National Population Commission (NPC) gave the population as: Males; 21,927. Females; 22,604.Total; 44,531. Emene shares the same physical characteristics with the Enugu area.

Location: Emene is in Enugu East. (Longitude 7 0

10 E and 6 0 15E of the Greenwichmeridian and latitudinally, it stretchesfrom 6 0 30 N and 7 0 30 N of equator.


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Soil Taxonomic classification: Typic Paleudult (FDALR, 1985).Haplic Acrisol (FAO/UNESCO, 2003)Soil parent material: Saprolite from sedimentary basin.Geology: Sedimentary basin.Geomorphology: Nearly level plain.Topography: Crest, 0 1 % slope.Ecological zone: ForestClimate: very humidMean annual rainfall: >1650mmLength of raining season: >250 daysLand use: Cassava, ( Manihot spp .), banana, coconut.Main tree species: Iroko, mahogany, obeche.Main grass species: Andropogon tactorum and Imperata cylindrica.Soil erosion hazard: None at profile siteDrainage: Well drained.

Source: Anyika,C.C. and Nnabude,P.C., 2005.

MATERIALS:The X-ray spectrometer used is a system with an in-built 25mCi 109 Cd annular source and a [Si (Li)]detector (lithium drifted silicon detector), which has a resolution of 170eV for the 5.90keV line. Thedetector and the source are placed on the Dewar, which contained liquid nitrogen for the purpose of coolingthe detector (Fig. 1).

Fig 1. Equipment Arrangement for the Experiment.

INSTRUMENTATION:

An X-ray spectrometer requires an X-ray source and a means of dispersing the X-rays. The cadmiumsource acts as an excitation source that emits Ag-K X-rays (22.1keV) in which all elements havingcharacteristic energies lower than that quoted above were accessible for detection in the samples. There isalso the molybdenum (Mo) target, which serves as a source of monochromatic x-rays. The x-rays areexcited through the sample by primary radiation and they penetrate the sample on their way to the detector.The detector is in turn coupled to a multi channel analyser and a computer controlled analog digitalconverter (ADC) card. The molybdenum target is used for absorption correction. The absorption factor thusdetermined is then used by the software package (AXIL-QXAS) to quantify the concentration of theelements in the samples. The software is also used to correct for the contribution of the Zr-K to the Mo Kpeak by subtraction. The spectra for the samples were collected for 3000 seconds and subsequentlyevaluated. ( Idris et al., 2000 ). The data was finally analyzed statistically, using statistical package forSocial Science Windows. (SPSS).

Sample TargetSi(Li) X-RayDetector

Dewar

109 CdExcitation source

Analogue to digitalconverter

IntegralPreamp

80PULSER

56HV POWERSUPPLY

7100/7150of 7450MCA

OSCILLOSCOPE

4GATEDBIASEDAmplifiers

572AMPLIFIER

Read out


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SAMPLE COLLECTION:(a) Soil Samples; at each of the 4 sampling sites (site E1 E2 and E3 and B), a depth profile (to

study the possibility of leaching into ground water) of the soil comprising of samples fromdepths of 0-15cm, 15-30cm, 30 45cm, 45-60cm, 60 75cm, was carried out at an interval of 3m, 6m, 9m, 12m, 15m and 18m respectively from the boundary of the factory with thecassava farm up to the rail road, totaling 21m.

(b) Control Sample CollectionThe control sample, B , (B1, B2, and B3) was collected at the interior of Oye Emene1km fromthe study area. Three replicates of the soil samples were collected in the same way as thestudy sample.

PROCEDURE:Disturbed soil samples were collected using the core sampler. The sampler was pushed into the soil and thesamples were collected at a depth of 0-15cm for the surface samples. The auger was used to dig deeperinto the soil and collection made with the core sampler, up to a depth of 75cm. A grid of 20cm by 20cmwas used to demarcate the distance between the 3 replicates while the collection was done at intervals of 3m, 6m, 9m, 12m, 15m and 18m respectively. A total of 90 samples were collected from the study locationwhile 15 samples were collected for control studies. All samples were collected in 3 replicates to give atotal of 105 samples. The samples (20g) each were then filled in clean cellophane bags, which had alreadybeen labeled. They were then air dried for five days under room temperature to ensure constant weight.The locations of the samples were at the back of the cement discharge bay, substation and production plant.All of which emits fumes and dust, respectively. The distance from these locations to the barbed wiresurrounding the factory was between 5m and 6m respectively. The sampling started at the boundarybetween the factory and the cassava farm and ended 3m before the railroad. Similarly, opposite the factoryabout 7m away is the Niger Steel factory, which ceased operations over 20 years ago.

SAMPLE PREPARATION: The samples were ground manually to powder with an agate mortar and pestleto grain size of less than 125 m. This was done for the purpose of homogenization. Pellets of 19mmdiameter were then prepared from 0.5g powder mixed with 3 drops of organic liquid binder (PVC dissolvedin toluene) and pressed afterwards at 10 tons with a hydraulic press. (SPECAL P/N 15 011), the pelletswere finally weighed and then taken for Analysis.

Soil pH Determination Studies. 10g of


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The results (figure. 2) showed no consistent trend of metal distribution in Chromium concentration with

respect to distance from contamination source. At the surface, there was a major increase to 1168.2 Mg ha-1

at 18m spatial distances and still remained elevated at a high surface value of 1033.3 Mg ha -1 at 9m, wellabove the control value of 691.2 Mg ha -1. The analysis of variance showed a significant variation inconcentration, between 12m and 3m. The results generally showed an increase in concentration away fromthe factory. The control value of 691.2 Mg ha -1 obtained under the control was significantly lower than thevalues obtained within the vicinity of the factory. This implies that the soil at the factory location ispolluted with chromium.

The results (figure 2.) showed that arsenic concentration at the middle distance (6 12 m) differedsignificantly from the extreme points (3 and 18 m). The results showed no consistent trend in metaldistribution. At the surface, the major increase to 86.68 Mg ha -1 was obtained at a spatial distance of 12mand still remained elevated at 74.73Mg ha -1 at 18m, well above the control value of 46.79 Mg ha -1. Theanalysis of variance showed a general lack of significant variation in concentration with the spatialdistance. However, they all varied significantly from the control. This implies that the soils are pollutedwith arsenic. The trend generally followed a gentle decrease in concentration away from the factory withspatial distance.

The results for lead (figure 2.) showed no consistent pattern of metal distribution with respect to distanceaway from the factory. At the surface, there was a major increase to 110.10Mg ha -1 at 9m and 103.76Mgha -1 at 18m, well above the control value of 39.45Mg ha -1. The analysis of variance showed lack of significant variation with the spatial distances with the exception of 3m and 18m, 1km and the studylocation. This implies that the soils are polluted. The trend followed a decrease in concentration away fromthe factory site.

0

500

1000

1500

3 6 9 12 15 18Concentration(Mg/ha)

Distance(m)

Cr

As

Pb

Fig. 2. Showed the spatial variation in chromium, arsenic and lead concentrations in soil with respect todistance away from the factory location.

The results (figure. 2). showed no consistent trend of metal distribution in Chromium concentration withrespect to distance from contamination source. At the surface, there was a major increase to 1168.2 Mg ha -1 at 18m spatial distances and still remained elevated at a high surface value of 1033.3 Mg ha -1 at 9m, wellabove the control value of 691.2 Mg ha -1. The analysis of variance showed a significant variation inconcentration, between 12m and 3m. The results generally showed an increase in concentration away fromthe factory. The control value of 691.2 Mg ha -1 obtained under the control was significantly lower than thevalues obtained within the vicinity of the factory. This implies that the soil at the factory location ispolluted with chromium.

The results (figure 2.) showed that arsenic concentration at the middle distance (6 12 m) differedsignificantly from the extreme points (3 and 18 m). The results showed no consistent trend in metaldistribution. At the surface, the major increase to 86.68 Mg ha -1 was obtained at a spatial distance of 12mand still remained elevated at 74.73Mg ha -1 at 18m, well above the control value of 46.79 Mg ha -1. Theanalysis of variance showed a general lack of significant variation in concentration with the spatialdistance. However, they all varied significantly from the control. This implies that the soils are polluted


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with arsenic. The trend generally followed a gentle decrease in concentration away from the factory withspatial distance.

The results for lead (figure 2.) showed no consistent pattern of metal distribution with respect to distanceaway from the factory. At the surface, there was a major increase to 110.10Mg ha -1 at 9m and 103.76Mgha -1 at 18m, well above the control value of 39.45Mg ha -1. The analysis of variance showed lack of significant variation with the spatial distances with the exception of 3m and 18m, 1km and the studylocation. This implies that the soils are polluted. The trend followed a decrease in concentration away fromthe factory site.

PERCENTAGE INCREASE IN HEAVY METAL CONCENTRATION OVER CONTROL:

0

50

100

150

200

3 6 9 12 15 18

Distance(m)

P e r c e n t a g e

i n c r e a s e

o v e r

c o n t r o l

CrAsPb

Figure 3. Component bar chart showing percentage increase of 3 heavy metal concentrations over controlconcentration

From the data presented in the barchart, (figure 3.), the percentage increase in chromium at the studylocation over the mean control level was highest of 69 % for chromium at a spatial distance of 18m whilethe lowest increase of 50 % was observed at the 9m spatial distances.

For arsenic, the highest percentage increase of 85% was at the 12m spatial distances, while the lowestincrease of 60% was observed at the 18m spatial distances.

For lead, which had the highest increase over control amongst the three heavy metals had the highestpercentage increase of 180% at the 9m spatial distances and the lowest increase of 163 % over the controlat the 18m spatial distances.

DEPTH VARIATION IN CHROMIUM, ARSENIC AND LEAD CONCENTRATIONS: (Mg ha 1)

Figure 4. Graph of Depth variation against the concentration of 3 heavy metals (Distribution of heavymetals in subsoil)

0200400600800

10001200

0-15 15-30 30-45 45-60 60-75Concentration(Mg/ha)

Depth (cm)

Cr

As

Pb


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Mobility: The results showed the following sequence of mobility:LEAD > ARSENIC > CHROMIUM. This sequence is similar to that observed in other environments byother authors, for example, at a copper smelter ( Elizabeth et. al., 2000 ) a Zinc factory, a lead - zincproduction site and mining, sites (Alloway, 1990). Differences in the mobility sequence of metals might bedue to differences in the methods used, soil types including soil pH, degree of contamination and sources of contamination. In this study, the relatively high pH and clay content present at the profiles might play asignificant role in fixing metals and preventing them from being leached out of the soil profile. There ishowever a possibility of leaching at shallow depths as obtained in these results.

The results (figure 4.) showed that chromium concentration decreased significantly in the order: 0-15cm>15-30cm>30-45cm>45-60cm>60 - 75cm.The analysis of variance showed that chromium varied significantly with depth, with the exception of the45-60cm and 60-75cm, which were not significant. The trend showed a pattern of decreasing values in theprofiles.

The results for arsenic (figure 4.) showed that arsenic concentration decreased in the order: 15 30 cm > 30- 45 cm > 0 15 cm > 60 75 cm > 45 60 cm. The analysis of variance showed that there was nosignificant difference in Arsenic concentration with depth, with the exception of the 15 - 30 cm and 30 - 45cm, which varied significantly. The trend generally showed a marginal decrease in concentration withdepth.

STATISTICAL ANALYSIS/ COMPARISON OF pH AT THE VARIOUS SPATIAL DISTANCES ANDDEPTHS USING THE UNPAIRED T-TEST PROCEDURE:

Table 1: Table of unpaired t - test for soil pHVariate Mean S. D t-Cal. P- Value Remark

3m1km

7.06.4

0.2950.198

6.399 < 0.0001 Sig

6m

1km

7.0

6.4

0.342

0.198

5.820


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The result for lead (figure 4.) showed that lead concentration decreased in the order:

15 30 cm > 30 45 cm > 60 -75 cm > 45 - 60 cm > 0 -15 cm.

The analysis of variance showed that lead varied significantly with depth with the exception of the 60 75cm and 45 60 cm depths. The results presented graphically in figure 4 shows that the concentration of lead was decreasing with increasing depth. The trend follows a decrease in concentration with depth.

The results, with the exception of those presented in table 1. showed that most relationships were notstatistically significant and were not presented. This was in agreement with the results of Kabala. andSingh, (2000) working on mobility and fractionation of heavy metals in soil profiles within the vicinity of acopper smelter in Poland, despite the fact that they used a depth of 120cm and samples collected from fourlocations within a Copper smelter. This is to be expected as all samples were taken within the same areaand therefore, soil was subjected to the same physico - chemical processes, with the exception of thecontrol. This implies that the soils at the factory location differ significantly from the control, meaning thatthe soils near the Emenite factory are polluted.

TEST OF HYPOTHESIS ON SOIL pH AND ARSENIC, CHROMIUM AND LEADCONCENTRATION:The results showed that P>0.05, therefore, we accept H 0 and conclude that there is no significant correlationbetween soil pH and Arsenic concentration.

The results showed that P>0.05, therefore, we accept the H 0 and thus conclude that there is no significantcorrelation between soil pH and chromium concentration.The results showed that P>0.05, therefore, we accept H 0 that there is no significant correlation between

soil pH and lead concentration.This general lack of significant correlation between soil pH and arsenic, chromium and lead concentrations,is in agreement with the results of ( Tazisong, et. al.,2004 ), in their study in Alabama ultisols, the onlydifference been that they worked on iron and manganese at a depth of 216 cm. They thus concluded thatthis general lack of correlation might be due to narrow pH ranges within the soils ( Ma et. al., 1997 ).Similarly, Fleming and Parle, (1977) concluded that pH is not the only factor influencing metalconcentration, they pointed out other factors like: plant specie, organic matter content, clay content andmoisture content.

NORMALITY TEST:The Anderson-Darling Normality test was used. Both the analysis of variance and correlation techniquesare based on the assumption that the data is normally distributed. However, the assumption for this test isalso carried out to access the relative or unique contributions of soil pH in predicting metal distribution insoils. Normality was tested for arsenic chromium and lead. The results represented graphically in figures

4.4 4.6, shows that arsenic (P=0.017), chromium, (P=0.000), lead (P=0.000), respectively, meaning thatthe data is normally distributed. This is in agreement with the results of (Tazisong, et. al. , 2004): althoughexchangeable iron in their results did not strictly meet the assumption, but the deviation or skewness wasnot great and therefore were not transformed. However, their exchangeable manganese met the assumption.

CONCENTRATION OF ARSENIC, CHROMIUM AND LEAD AT THE STUDY LOCATION:HEAVY METAL CONCENTRATION IN RELATION TO LANDUSE IN THE PROJECT AREA:The United Kingdom Department of Environment and the United States Environmental Protection Agencyhas established permissible limits of these metals in soils. The table below (table 2). compared these limitswith what was obtained at the control and factory locations.


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Table 2. Heavy metals (Mg ha-1

) in the study area in relation to the permissible limits.

1. United Kingdom Department of Environment, (1990), 2. U.S. Environmental Protection Agency, (1993)

From the foregoing, it could be observed from the results of our study that the level of arsenic (75.80 Mgha -1 ) dry weight is above the permissible limit of 75 Mg ha -1 dry weight for sludge application in soil setup by the USEPA, and this could lead to deterioration in soil quality and possible contamination of the foodchain. The levels of lead and chromium are still below the permissible limits.

CONCLUSIONS:We thus conclude that the spatial variability in metal concentration depends on distance away from thefactory and depth variation is also concentration dependent and that these 3 metals have similar distributioncharacteristics in the area studied. It has been established that the levels of arsenic, chromium and lead inthe soil around the Emenite factory is as a result of emission of fumes and dusts from the factory. Thegeneral trend in this work agrees with previous works despite the fact that they were mainly in temperatelocations. The few areas of differences is attributable to sampling techniques, like depth of investigation,nature of soil, nature and volume of metals emitted by the industry, weather, topography, climatic andmeteorological conditions, nature of instrument used for analysis.

Finally, the levels of heavy metal in the soil around the Emenite factory are gradually rising to a criticallevel. There is therefore a danger of build up of larger doses either through inhalation, absorption throughthe skin, bioaccumulation or consumption of plants or ingestion of soil by children. The consequences of which have been cited in the literature review. Hence there is a need to address the problem before itdegenerates dangerously.

ACKNOWLEDGEMENT: The authors wishes to acknowledge Prof. L. A. Dim of the Centre for EnergyResearch and Training A.B.U. Zaria, Prof.L.N. Moghalu of Department of Geography, Meteorology andEnvironmental Management Nnamdi Azikiwe University, Awka and Prof. James C. Nwafor of Department

of Geography University of Nigeria Nsukka.

RECOMMENDATIONS:Based on the inferences, findings and experiences in the course of this research work, the following ishereby recommended:

1) People should not leave or carry out agricultural activities within a distance of at least 300m fromthe industrial estate.

2) Strict enforcement of the urban and regional planning laws should be ensured.3) The Emenite Ltd must comply with the following statutory laws:

a. Sections 247 of Nigerian criminal code which makes it an offence for anybody to impair the quality of the atmosphere anywhere thereby making it noxious to human health.b. They must comply with the factories decree 1987 CAP 126 laws of the Federation of Nigeria 1990,which makes provision for ensuring a pollution free environment at the work place and adjoining

Heavymetal

PermissiblelimitUK 1

PermissiblelimitUSEPA 2

FactoryLocationEnugu

ControlLocationEnugu

ArsenicChromiumLead

753000500

753000420

75.801038.397.25

46.79691.239.45


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surroundings. The law further stipulates that factory, which emits dust, fumes or other impurities, which areinjurious or offensive to the employees, must take practicable measures to protect the employees and toprovide exhaust appliances.C. They must also comply with sections 13 (s) and (d) of the environmental impact assessment decree No.86 of 1992.(Official gazette).

The members of the Emene community can also rely on the doctrine of statutory nuisance and petition thestate attorney general to sue the Emenite factory which is the only factory close to human habitation andprivate farms around the industrial estate or they can also rely on the doctrine of strict liability (Rylands VsFletcher) to safeguard their health and environment, by borrowing a leaf from the Wazirpur area of northern Delhi region. In that case the factory has to be substantially compensated.4. BIO REMEDIATION OF SOIL: To avoid contamination of food chain and further degeneration of ouragricultural land, the following is recommended:

FOR ARSENIC: Transformation of arsenic 3 to arsenic 2, which can be taken up by plants can be achievedby redox reaction. Also applications of sulphates of zinc, iron and aluminum, which tie up the arsenic ininsoluble forms.Finally, this study should be taken up at a regular interval of about 3 years since pollution is going on in thearea on daily bases.

REFERENCESAlloway, B.J. (1990b). The origin of heavy metals in soils. In Steinborn, M. and Breen, J. (eds.), HeavyMetals in Soil and Vegetation at Shallee Mine Silvermines, Co. Tipperary, Limerick. Ireland, 99:1 6.

ANZECC/NHMRC,(1992). http://www.publish.csiro . au/nid/88.htm. Heavy metals in Glebe - Australia.

Elizabeth, M., Brian G., Honway L., Michael W. and Ping D. (2000). Metal partitioning in soil profilesin the vicinity of an exploration environment; http://geea.geoscienceworld.org/cgi/content/full/4/2/171

Federal Department of Land Agricultural Resources (FDLAR). (1985). Reconnaissance Soil Survey of Anambra State of Nigeria.Soil Report, 1985. Federal Department of Agriculture and Land Resources(FDLAR), Lagos: Nigeria.

FAO/ UNESCO. (2003). Soil map of the world legend 1990. FAO: Rome. The Soils of Eastern Nigeria.University of Amsterdam, 4: 186-197.

Federal Military Government of Nigeria. (1992). Environmental Impact Assessment Decree (1992),Decree No. 86. Sections 13 (s) and (d).

Fleming, G. and Parle, P. (1977). Plant herbage and Vegetables from West of Dublin city Irish

Research, 16: 35 48.

Idris, Y., Funtua, I.I. and Umar, I.M. (2000). Measurement of the levels of impure elements inbauxites from Mambilla Plateau, Northern Nigeria Using Isotopic X- Ray Fluorescence Spectroscopy.Proceedings. Of the 23 rd Annual Conference, of the Nigeria Institute of Physics. The MilleniumConference. pp 190-192.

Kabala C, and Singh, BR, (2000). Fractionation and Mobility of Copper, Lead, and Zinc in Soil Profilesin the Vicinity of a Copper Smelter. http//www.singh.ijvf.nlh.no.

Ma, L. Q., Tan, F. and Harris, W. G.(1997). Concentrations and distributions of eleven metals in Floridasoils. Journal Environmental Quality, 26:765 775.


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National Population Commission (NPC). (1991). Emene census final results of 1991 population censusof Nigeria. NPC. Enugu.

Nriagu, E.M. and Pacyna, J.M. (1988). The role of sorbed humic substances on the distribution of organicinorganic contaminants in groundwater. Geoderma. 67: 103-124.

Okieimen, F.E., Osuide, M. O. and Oriakhi, C.O.(1986) Sorption of cadmium, lead and zinc ions fromaqueous solutions by maize (Zea mays) cobs , Nigerian Journal Applied Science, Vol. 3, and 1: 121-126.

Okonkwo, E. M. and Eboatu, A.N. (1999): Environmental Pollution and Degradation. 1 st Edition. OnisExcel Publishing. Zaria, p 8.

Oviasogie, P.O. and Ukpebor, E. E. (2003). Assessment of some forms of Pb, Mn and Cu inpetrochemicals contaminated soil using different extractants. International Journal Chemistry, 13 (3):119 126.

Tazisong, I. A., Senwo, Z. N., Mbila, M. O. and Wang, Y. (2004), Concentration and distribution of iron and manganese fractions in Alabama ultisols. Soil science, 169: (7) 489-497.

UK Department of Environment (1990) http:// www.defra.gov.uk. Department for environment, Foodand Rural Affairs.

United States Environmental Protection Agency (USEPA), ( 1993). Clean Water Act, sec. 503, vol.58, No. 32 (Washington, D. C: U.S. Environmental Protection Agency).


Corresponding Author:Anyika C.C.Department of Soil Science, Federal University of Technology, Minna.Email: anyika 3C @ yahoo.com


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Continental J. Applied Sciences 2: 23 - 25, 2007Wilolud Online Journals, 2007.

INFLUENCE OF MATERNAL PARITY ON TRANSPLACENTAL TRANSMISSION OF MALARIAAND THE CONSEQUENT EFFECT ON NEONATAL BIRTH WEIGHT IN ABRAKA, DELTA

STATE, NIGERIA

aPender, K.E., bIgweh, J.C and cOnyesom, I*.a.Department of Physiology, College of Health Sciences,

Delta State University, Abraka.bDepartment of Physiology, College of Medicine,

University of Nigeria, Enugu Campus.cDepartment of Medical Biochemistry, College of Health Sciences,

Delta State University, Abraka.

ABSTRACTThis study investigates the effect of maternal parity on the pattern of transplacentaltransmission of malaria and the consequent influence on neonatal birth weight. Forty-five cord blood samples were collected from the neonates of consenting and randomlyselected women who were delivered of babies in General Hospital, Abraka, DeltaState, Nigeria. The cord blood samples were prepared for malarial parasite countusing standard procedure. Results show that 42.2% of the samples were positive formalarial parasite, out of which 52.6% and 47.4% were from neonates of multiparousand primiparous mothers, respectively. The mean birth weight of malarial infectedneonates from multiparous and primiparous mothers, respectively. The mean birthweight of malarial infected neonates from multiparous and primiparous mothers were3.17 0.53 and 3.09 0.54; P>0.05, respectively. The birth weights for uninfectedneonates from multiparous and primiparous mothers were 3.43 0.63 and3.22 0.43;P>0.05, respectively. 94.7% of the infected cases were caused byPlasmodium falciparum and the remaining 5.3% by P. ovale strategies for reducingfetomaternal transmission should be sought.

KEYWORDS: Abraka, maternal parity, malaria, Plasmodium falciparum , placenta.

INTRODUCTIONMalaria is the commonest cause of fever in the tropics. In malaria endemic areas, gravid women oftenpresent with placenta parasitized by Plasmodium falciparum Malaria is associated with abortion, still birth,prematurity and low birth weight (Le Hesran, et al ., 1997; Judith and Longmore, 2005). The predilectionof malarial parasite for placenta is due to avid binding to surface chondroiton sulphate on thesynicitiotrophoblast (Beesen and Reeder, 2001).

Placental parasitisation interferes with placental function and transplacental transmission may lead to birthweight reduction, which is a poor prognostic factor (Coller and Scaly, 2005).

Malaria related growth retardation predominantly affects the primiparious and is most severe withPlasmodium falciparum (McGready and Billie, 2004; Mac-Gregor and Avery, 1974).

Reported cases of transplacental transmission of malaria and effects on neonatal birth weights has beenreported in Jos (Egwuyenga and Ajayi, 1995) and Lagos (Lamikanra, 1986) areas of Nigeria.

Similar reports in the West African subregion on transplacental malaria and depression of birth weightshave been made in the Gambia (MacGregor and Wilson, 1983), Cote de Ivoire (Larkin and Thuma, 2003),Cameroon (Akum and Kwoh, 2005).


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Pender, K.E et al : Continental J. Applied Sciences 2: 23 - 25, 2007

Transplacental malaria was more likely to induce intrauterine growth retardation than preterm delivery(Norston and Ter Kulie, 1995). This study determines the influence of maternal parity on the pattern of transplacental malaria transmission and the attendant effects on neonatal birth weights at term in Abraka.

MATERIALS AND METHODS:Patients: Forty-five pregnant women for antenatal care were randomly selected from the General Hospital,Abraka, in Delta state, Nigeria, and standardized by excluding those with medial and obstetric historyassociated with fetal growth restriction. The history were obtained from the booking information. Thestudy was approved by our Facultys Research and Bioethics Committee.

Specimen Collection: Forty-five cord blood samples were obtained from the selected women who alsogave their informed consent. At the end of the second stage of labour, the cord was clamped at two pointsand divided. The distal cord was elevated while attached to the placenta, then 5 ml syringe was used tocollect about 2 ml of umbilical arterial blood and placed in a heparinised bottle and analyzed within thehour of collection. Neonates were weighted within one hour on a Salter scale to the nearest 0.05 kg.

Universal precautions for handling blood were adhered to, in order to avoid the risk of especially hepatitisB surface antigen (Hb Sag) or the Human Immunodeficiency Virus (HIV).

Analysis of Specimens: Two film; thick and thin were made from each sample. A small blob was spreadout untidily to cover approximately one square centimeter for the thick film; it was allowed to dry. For thethin film, a drop of blood was placed near one end of the slide; another slide angled at 45 degrees to thedrop of blood was used to make a thin smear on the first. This was labeled and allowed to dry. Afterdrying, smears were fixed in methanol for five seconds, stained with Giemsa; then 20 drops of distilledwater were dropped on the smears and allowed to dry. Thick and thin films were examined using 1000magnification with oil immersion lens. Parasite density was calculated as the number of infected RBCs per1000 RBCs. Statistics: The data obtained were summarized by way of mean and standard deviation andthe means were compared by the use of Students t-test. Level of significance was set at P0.05).

DISCUSSIONData (Table 1) suggest that transplacental transmission of malaria may be associated with fetal growth

restriction and birth weight reduction and that primigravid may be a risk factor for the severity of fetalgrowth restriction due to malaria. Similar to earlier studies in areas of high transmission (Larkin andThuma, 2003), Plasmodium falciparum was the dominant parasite transmitted, showing a prevalence of 97.4%.

Since falciparum malaria during pregnancy could cause low birth weight, vigorous and effective means of reducing fetomaternal infection should be emphasized. Community enlightenment programmes should beinitiated in order to encourage especially primigravid women to utilize antenatal services. The need forvector control and effective chemo-prophylaxis should be integrated into the antenatal programmes and

services. It is hoped that these measures will reduce the risk of transplacental transmission of malaria andthe risk of low birth weight infants.


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Pender, K.E et al : Continental J. Applied Sciences 2: 23 - 25, 2007

REFERENCES

Akum, A.E, and Kwoh, A.J. (2005). The effect of umbilical cord and placental malaria parasitaemia on thebirth weights of new borns from south west Cameroon. Acta paedietrica . 94 (7): 917-923.

Beesen, J. G. and Reeder, J.C. (2001). Parasite adhesion and immune invasion in placental malaria.Trends parasitol 17: 331-337.

Egwuyenga, O.A. and Ajayi, J.A. (1995). Transplacental passage of Plasmodium falciparum and seroevaluation of new borns in northern Nigeria. J Comm. Dis , 27 (2): 767-83.

Coller J. and Scaly, L. (2005). Oxford Handbook of Clinical Specialties. 6th (ed.), Oxford UniversityPress, London.

Lamikanra, O.T. (1986). Study of malaria parasitaemia in pregnant women, placenta, blood and new bornsin Lagos, Nigeria. Entrz pub med pmid : 8199063.

Larkin, G.I., and Thuma, P.E. (2003) Congenital malaria in hyper endemic area. Entrz pub med pmid 1931868.

Le Hesran, J.Y., Cot, M. and Personne, P.P. (1997). Maternal placental infection with Plasmodium falciparum and malaria morbidity during the first two years of life. Am J Epidemio l 146 (10): 826-831.

McGready, R. and Billie, B. (2004). The effect of Plasmodium histopathology in an area of low malariatransmission. Am J Trop Med Hyg . 70 (4): 398-407.

Mac-Gregor, L.A. and Avery, J.H. (1974). Malaria transmission and fetal growth. BMS 5: 443-436.

Mac-Gregor, L.A and Wilson, M.E. (1983). Malaria infection of the placenta in the Gambia, West Africa:its incidence and relation to still birth, birth weight and placental weight. Trans R Soc Trop Med

Hyg . 77 (2): 232-244.

Norston, F. and Ter Kulie, (1995). Mefloquine prophylaxis prevents malaria during pregnancy. A doubleblind placebo controlled study. J Inf Dis 169: 593-603.

Table 1: Effect of maternal parity on cord blood parasitaemia and birth weightMaternal parity

Multigravidae PrimigravidaeCord blood Malarial Parasitemia

N, no of subjects Positive Negative Positive NegativeInfectious malarial parasite 10 19 9 7

Plasmodium falciparum10 0 8 0

Plasmodium vivax 0 0 0 0Plasmodium ovale 0 0 1 0Plasmodium malariae 0 0 0 0Birth weight (kg) 3.17 0.53 3.43 0.63 3.09 0.54 3.22 0.43Received for Publication: 29/07/2007Accepted for Publication: 20/09/2007

Corresponding Author:Dr I. OnyesomPostal: P.O. Box 144, Abraka, Delta State, Nigeria. E-mail: [email protected]


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Continental J. Applied Sciences 2: 26 - 37, 2007Wilolud Online Journals, 2007.

TOPICAL LIPOSOMAL DIBUCAINE DELIVERY SYSTEM: DEVELOPMENT ANDCHARACTERIZATION

Nounou, M.M.*, El-Khordagui, L.K., Khalafallah, N.A. and Khalil, S.A.Department of Pharmaceutics, Faculty of Pharmacy, University of Alexandria, Egypt

ABSTRACTFormulation of local anesthetics in controlled delivery systems provides safer andmore effective anesthesia. The aim of this study was to develop a liposomal dibucainebase (DB) local anesthetic delivery system. DB-loaded multilamellar vesicles (MLVs)with different characteristics were obtained by varying lipid composition, drugloading, induced charge and pH of the hydration buffer. Liposomes werecharacterized for morphology, size, entrapment efficiency (EE) and stability includingleakage stability. Results indicate that amongst formulations tested, negativelycharged liposomes with the lipid composition phosphatidyl choline: cholesterol:dicetyl phosphate 7:6:1 and drug: lipid ratio 90mg: 300mg prepared with pH 9hydration medium sowed good in vitro characteristics in terms of EE ( 90%),sustained drug release ( 20% in 12 hrs) and low burst effect. However, theyexhibited relatively poor leakage stability. A delivery system was prepared byincorporating negatively charged DB-loaded liposomes in a 2% HPMC gel base. Gelformulations were assessed in vitro for drug leakage stability and in vivo using the pinprick test in Guinea pigs. Incorporation of liposomes in the gel enhanced their leakagestability. Release characteristics of the liposomal gel could be modulated bycombining different proportions of free and liposomal DB. The in vivo performanceof a combination gel provided a superior local anesthetic profile (fast onset andprolonged duration of action) compared to a non-liposomal DB gel and a liposomalgel formulation with no free drug added. The DB liposomal gel developed offers greatpotentials as a local anesthetic delivery system.

KEYWORDSDibucaine base, dicetylphosphate, in-vitro release, release stability, multilamellarvesicles, liposomal gel, local anaesthesia, pin prick test, sustained release.

INTRODUCTIONTopical anesthetics were introduced in the latter half of the 19th century, starting with a description of thetopical uses for cocaine (1) . The need for the development of an effective topical anesthetic preparationprompted researchers to try various approaches. Earlier experiments showed the lack of efficacy of most

local anesthetic drugs in topical vehicles, because of the lack of penetration and delivery of insufficientamount of drug to the target site i.e. the dermally located sensory nerves (2) . The development of EMLA(Eutectic Mixture of Local Anesthetics) was a step forward in the achievement of a more efficient topicalanesthetic effect. Several studies indicated that upon application of a "thick layer" of EMLA cream for atleast 2 hours, a reasonable effect develops (0.5 mm deep anesthesia) in adults and in children (3) . It could beconcluded that the achievement of significant rapid, deep, and long lasting skin anesthesia is a challengingproblem. However, to achieve sufficient anesthetic effect on intact skin, prolonged application and highconcentration of drug (10-30%) are required (4).

Among the most effective topical anesthetics and of particular interest is Dibucaine, a potent long actinglocal anesthetic.


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Nounou, M.M et al : Continental J. Applied Sciences 2: 26 - 37, 2007

Table 1: Dibucaine gel formulations

Code Description Applicationproperties

State of thedrugincorporatedinside the gel

Concentrationof

dibucaine base

Concentrationof HPMC gel

DG dibucaine baseplain gel

Transparent,smooth, viscous,

non tacky gel

100% Freedibucaine base

1 % w / w

4 % w / v

DLG dibucaine baseliposomal gel Wh i t e , o p a q u

e , s m o o t h ,

vi s c o u s ,n on t a c k y g e l

100%Dibucaine base

encapsulatedinside

liposomes

DCG-87.5

Dibucaine basecombinationgel (Free and

liposomaldrug)

87.5% of the

drug isliposomal

encapsulated,the rest is free

drug

Dibucaine is a topical amide local anesthetic that is commonly used in haemorrhoidal preparations. It hasbeen used as a base or hydrochloride salt in creams or ointments containing up to 1% for the relief of painand itching associated with skin and anorectal conditions (5).

Few studies tackeled the incorporation of this drug in novel topical drug delivery system to target the drugto the dermal region and eliminate its toxic systemic action. Lai et al. (6) investigated the epidermalapplication of dibucaine through iontophoresis, Masters et al. (7) investigated the incorporation of dibucaine

in biodegradable polymer matrices, Thoma et al.(8-11)

investigated the incorporation of dibucaine inbiodegradable microparticles and finally, Mezei et al. (12) investigated the possibility of incorporation of dibucaine in multilamellar liposomal vesicles. No topical liposomal Dibucaine product has yet beenmarketed.

As a valuable, biocompatible drug delivery system, liposomes are advocated for topical treatment of diseases, especially in dermatology, due to their ability to provide prolonged release of incorporatedmaterial (13) . Liposomes also increase the permeability of skin for various entrapped drugs and at the sametime way diminish some side effects of these drugs (14) . Several studies indicated that tetracaine in liposomeencapsulated form provides better topical anesthesia than a conventional form (15, 16) . Liposomes appliedtopically were proposed to penetrate into the skin (dermis) through the lipid channels of the epidermis andlocalize the drug within the skin. Due to multilamellar structure of liposomes, sustained release of theencapsulated drug is possible (16) .

However, the main disadvantage in using liposomes topically is the liquid nature of the preparation.Suitable viscosity and application properties of liposomes can be achieved by their incorporation in anappropriate vehicle. Usually, liposomes are applied to the skin in solution or in hydrogels, since stableliposomal creams are difficult to formulate (17) . For topical application of liposomes, hydrophilic polymersare suitable thickening agents, since they make the formulations convenient for application. It has beenconfirmed that liposomes are fairly compatible with viscosity increasing agents such as methylcellulose,hydroxypropyl methylcellulose (18) , chitosan (19) as well as polymers derived from acrylic acid (carbopolresins) (20) . However, the type and concentration of polymer which forms the hydrogel could influence thestability as well as the rate of penetration of liposome entrapped substances into the skin (21) . Concerningthe assessment of anaesthetic effect, The effectiveness of topical anesthetics has been difficult to assessbecause no wholly satisfactory method of comparison has been available (22, 23) . Therefore a paucity of comparative data is available. The most widely used method for the evaluation of local anesthetics is the


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Table 2: Pin prick test results in guinea pigs following the application of dibucaine topical

formulations.Code Control DLG DCG DG

Time(min.)

1 2 3 4 5 M 1 2 3 4 5 MP

1 2 3 4 5 MP

1 2 3 4 5 MP

Scores Scores Scores Scores

0 10 10 10 10 10 10 6 6 5 5 6 6 s* 1 0 0 0 1 0 s*** 2 0 1 0 0 0 s***

15 8 10 9 10 9 9 7 7 5 5 5 5 s*** 2 1 1 2 1 1 s*** 5 3 2 2 1 2 s***

30 9 10 10 10 10 10 6 7 6 5 5 6 s** 1 1 2 1 0 1 s*** 8 5 4 5 4 5 s**

45 9 9 10 10 10 10 5 4 5 4 4 4 s*** 2 2 1 1 1 1 s*** 9 8 7 8 7 8 s*

60 8 10 10 9 9 9 3 3 4 3 2 3 s*** 2 1 0 0 2 1 s*** 8 9 10 9 9 9 ns

120 10 10 8 9 10 10 2 1 2 2 2 2 s*** 1 2 1 1 1 1 s*** 8 9 9 9 10 9 ns

180 10 9 9 10 10 10 4 2 1 2 2 2 s*** 2 1 2 1 1 1 s*** 9 10 10 10 9 10 ns

240 9 9 10 10 10 10 4 2 2 2 3 2 s*** 3 1 1 2 2 2 s*** 10 9 10 10 10 10 ns300 10 10 10 9 10 10 5 3 2 3 2 3 s*** 2 2 2 2 1 2 s*** 10 10 10 9 10 10 ns

360 10 10 10 10 10 10 4 2 2 2 2 2 s*** 3 2 3 2 2 2 s*** 10 10 10 10 10 10 ns

Notes:M = Medianns=Non Significant Data (P>0.05)s*=Significant Data at P


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Figure 1: Electron microscope photo of dibucaine liposomal dispersion D 6pH9-90(-)(75,000 X)

0

5

10

15

20

25

Zero time One week Two weeks Four weeks

I n t e r c e p t

( % r

e l e a s e

d )

Figure 4: Intercept of Higuschi plots of release data from D 6pH9-90(-) liposomal

dispersion As a function of storage at 4 0C

Figure 2: Release stability profiles at 32 0C of dibucaine liposomal dispersion (D 6pH5-90(-)) asa function of storage time at 4 0C

Figure 3: Release stability profiles at 32 0Cof dibucaine liposomal dispersion (D 6pH9-90(-)) as a function of storage time at 4 0Cover four weeks

0

5

10

15

20

25

Zero time One week Two weeks Four weeks

I n

t e r c e p t ( %

r e l e a s e d )

Figure 5: Intercept of Higuschi plots of release data from D 6pH5-90(-) liposomaldispersion As a function of storage at 4 0Cover four weeks

0

20

40

60

80

100

0 2 4 6 8 10 12Time (hr.)

C u m m u l a t i v e r e l e a s e o f d i b u c a i n e

, %

DG

DCG-87.5

DLG

Figure 6: Release profiles at 32 0C of dibucaine topical formulations.

0 1 0

2 0

3 0

4 0

5 0

0

2

4

6

8

1 0

1 2

1 4

T i m

e ( h

o u r )

Z e r o

D a y s

S e v e n

d a y s

F o u r t e e n

D a y s

2 8 D

a y s

0 5 1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

0

2

4

6

8

1 0

1 2

T i m

e ( h

o u r )

Z e r o

D a y s

S e v e n

d a y s

F o u r t e e n

D a y s

2 8 D

a y s


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-5.00

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

DG DCG-87.5 DLG

I n t e r c e p

t ( %

r e

l e a s e

d )

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

S l o p e

( %

/ S Q R T

t )

Intercept Slope Figure 7: Slope and intercept of Higuschi plots of release data fromdibucaine gels as a function of gelcomposition.

0

5

10

15

20

25

30

0 2 4 6 8 10 12 14Time (hr.)

% c

u m u

l a t i v e r e

l e a s e

o f d i b u c a

i n e

b a s e

f r o m

D i b u c a

i n e

L i p o s o m a

l G e

l ( D L G )

Zero Days

Seven Days

Fourteen Days

28 days

Figure 8 : Release stability profilesat 32 0C of dibucaine liposomal gel(DLG) stored at 4 0C.

80

82

84

86

88

90

92

94

9698

100

0 5 10 15 20 25 30Time (days)

% D

i b u c a i n e r e t a i n e d

D6pH9-90(-)

D6pH5-90(-)

DLG

0

20

40

60

80

100

0 2 4 6 8 10 12Time (hr.)

% C

u m m u l a t i v e r e l e a s e o f d i b u c a i n e b a s e g e l s

DG

DLG

Figure 9: Percent dibucaine retained in Figure 10: Release profiles at 32 0C of dibucaine liposomal gel (DLG) and dibucaine liposomes after one hour release at 32 0Cgel (DG) as control

0

20

40

60

80

100

0 2 4 6 8 10 12Time (hr.)

% C

u m m u l a t i v e r e l e a s e p r o f i l e o f d i b u c a i n e b a s e l i p o s o m a l

d i s p e r s i o n ( D 6 p H 9 - 9

0 ( - ) ) Control

D6pH9-90(-)

Figure 11: Release profiles at 32 0C of dibucaine liposomal dispersion (D6pH9-90(-))and dibucainesolution as control


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015

3045

60120

180240

300360

DG

control

0

1

2

3

4

5

6

7

8

9

10

P a

i n S

c o r e

( o u t o

f 1 0 )

Time (min.)

DG control

015

3045

60120

180240

300360

DLG

control

0

1

2

3

4

5

6

7

8

9

10

P a i n S c o r e ( o u t o f 1 0 )

Time (min.)

DLG control

015

3045

60120

180240

300360

DCG

control

0

1

2

3

4

5

6

7

8

9

10

P a i n S c o r e ( o u t o f 1 0 )

Time (min.)

DCG control Figure 12: Graphical presentation of median (n=5 animals) pain score in guinea pigs.

slowly hydrated using 10 ml of either phosphate buffer pH 5.6 (D 6pH5-90(-)) or bicarbonate buffer pH 9.0(D 6pH9-90(-)).

The process of hydration involved rotation at a low speed at 43C in the rotary evaporator (with novacuum) for 30 minutes followed by hand shaking for 15 minutes at 43C in a thermostatically controlledwater bath. The resulting liposomal dispersion was left to mature over night at 4C. This method wasreported by Bangham, Stansfield and Walkins (30).

Separation of free dibucaineThis was achieved by centrifugation of the prepared dispersions at 16500 rpm (27800 Xg) for 90 minutes at-5C (Beckman model J2-21 centrifuge). The resulting liposomal concentrate was washed twice each with5 ml of either phosphate buffer pH 5.6 or bicarbonate buffer pH 9.0 and recentrifuged for a further 90

minutes. The resulting liposomal concentrates were refrigerated.

Entrapped drug was determined by lysis of liposomes with chloroform/methanol 7/3 V/V. Dibucaineconcentration was determined spectrophotometrically at 241 nm (31) using the lysis mixture as blank asdescribed by Mezei et al. (12) .

Dibucaine liposomal dispersions were examined by transmission electron microscopy using 2% ammoniummolybedate aqueous solution as a negative stain. Liposome vesicle size was determined using the laserdiffraction particle size analyzer.

Preparation of dibucaine liposomal gelThe calculated amount of hydroxypropyl methylcellulose powder was dusted slowly over phosphate buffersolution of pH 5.6 under continuous agitation in an ice bath, and then the mixture was strongly agitated for45 minutes. The prepared dispersion was then left in the refrigerator (4-5 0C) overnight. A clear solutionwas obtained which gelled at room temperature (32, 33) . Gel was degassed by centrifugation if necessary (34) .Dibucaine base liposomal concentrate (D 6pH9-90(-)) was added to the prepared gel by trituration so as toyield finally a 1% w/w drug and a 4% w/v gelling agent concentration(Dibucaine Liposomal Gel [DLG]).

Dibucaine liposomal gel loaded with free and liposomal dibucaine was prepared through mixing dibucaineliposomal gel [DLG] with dibucaine gel [DG] in the proportions shown in Table 1.In vitro drug release from dibucaine liposomal dispersions and gels

Drug release from liposomes was studied using a dialysis method. Dialysis bags were spectra/Por 2 of 12,000-14,000 dalton molecular weight cut off. The bags were soaked before use in distilled water at roomtemperature for 12 hours to remove the preservative followed by rinsing thoroughly in distilled water. In


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case of dibucaine liposomal dispersions, the dialysis bags were fitted on the tablet dissolution tester paddle

(Electrolab tablet dissolution tester USP 24) by attaching a stainless steel part to allow fixing the dialysisbags to it. Dibucaine liposomal concentrate (equivalent to 2 mg dibucaine base) dispersed in one ml of either phosphate buffer pH 5.6 or bicarbonate buffer pH 9 was filled in a 10 cm initial length, 6.4 mmdiameter dialysis bag. The bag was closed at both ends with cotton thread and tested for leakage. The finallength of the bag after tying was 80.2 cm. The dialysis bag was attached horizontally fully stretched to thepaddle which was then immersed in the tablet dissolution tester beaker containing 250 ml of the releasemedium (phosphate buffer pH 5.6 containing 7% propylene glycol and 25 % methanol). The bag was fullyimmersed under the surface. The temperature was set at 320.2 0C and the speed of rotation of the paddlesat 100 rpm.

Control bags were prepared and tested along with the liposomal dispersions. The control bags eachcontained 2 mg Dibucaine base dissolved in one ml of the release medium. A representative liposomaldispersion was examined by electron microscopy before and after the release run.

While in case of dibucaine liposomal gel, the tablet dissolution tester was used in the release studies. Aknown weight (equivalent to 10 mg dibucaine base) of each gel formulation shown in Table 1 was filled instainless steel cups (Radius 1.5 cm, height 0.5 cm). The surface of the gel was made flat; the cups werefitted with the dialysis sheet by a rubber band and immersed in the bottom of the beakers of the tabletdissolution tester filled with 250 ml of release medium.

The paddles were positioned in the beakers of the tablet dissolution tester under the surface of the dialysismedium. The temperature was set to 32 0.2 0C and the speed of rotation of the paddles of the dissolutiontester was set to 100 r.p.m. Aliquots of the release medium were periodically withdrawn for analysis andreplaced with equal volume of fresh release medium (adjusted at 32 0.2 0C). Each release experiment wascontinued for 12 hours.

Aliquots of the release medium were withdrawn for analysis at different time intervals and replaced withfresh medium. Each release run was continued for 12 hours. The absorbance of the collected samples, afterdilution if necessary with release medium, was measured at max 244 nm. The results recorded were themean value of three runs carried out for each liposome concentrate.

Release stability of dibucaine liposomal dispersions and gelsPossible leakage of dibucaine base from mu

Vol 2- Continental J. Applied Sciences.

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