Vol 2 - Continental J. Applied Sciences.

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

©Wilolud Online Journals, 2007.
ASSESSMENT OF ORGANOCHLORINE AND POLYCHLORINATED PESTICIDES RESIDUES IN

SOME WESTERN NIGERIA RIVERS
P.A. Egwaikhidea, S.O. Okeniyib S.O. Ohikhenac and C.E. Gimbad

a
Department of Chemistry and Centre for Biomaterials Research, University of Benin, Nigeria
b
Department of Chemistry, Nigerian Defense Academy, Kaduna.
c
Department of Polymer Technology, Federal Polytechnic, Auchi, Nigeria
d
Department of Chemistry, Ahmadu Bello University, Zaria.
ABSTRACT
The degree of contamination and pollution of some rivers in Western Nigeria by
organochlorine and polychlorinated Pesticide Residues were investigated. The
oirganochlorine and polychlorinated pesticides analysed showed total DDT (Dichloro
diphenyl trichloroethane), ranged from ND – 0.3953 ppb. Aldrin and lindane were
detected in the range of 0.0029 – 0.1904ppb and 0.0067 – 0.6341 ppb respectively.
Total Polychlorinated biphenyl’s (PBB) level; ranged from ND – 8.9916 ppb. Other
Organochlorine Pesticides analysed gave varying levels of heptachlorepoxide, (range
ND – 0.0037 ppb), hexachlorobenzene (range ND – 0.0092 ppb), endosulfan (range
ND – 0.0851 ppb) and dieldrin (range 0.0049 – 0:6590 ppb) respectively. The
organochlorine and polychlorinated pesticide residue peaks were identified by
comparison with some reference mixed standard run under the same conditions.
Statistical analysis of the data revealed significant difference in the organochlorine
concentration between the rivers (P<0.32).
KEYWORDS: Contamination, pollution, organochlorine pesticides, polychlorinated

biphenyl, rivers, grap sampling
INTRODUCTION
Pesticides are manufactured and used as a means of directly or indirectly controlling, preventing,
destroying, mitigating, attracting or repelling any pest or altering the growth, development or
characteristics of any plant that is not a pest (Lee, et al. 1982). Thus pesticides are important by protecting
and increasing the quantity and quality of food, commodities, building materials, clothing and ornaments,
in improving animal health and in combating diseases transmitted by man. They are deliberately designed
to alter the balance of ecology. However, the premature release of medicinal, industrial and agricultural
chemicals has caused numerous environmental problems at all levels of life (Gwenzi, 1986, Barbash and
Resek, 1996).
The toxic effect on man and animals through direct contact during crop spraying and residues on treated
crops represent a risk to consumers, the consequences of the chemical, the length of exposure, the route of
entry and absorption to the body and the dose of the chemical concentration (UNEP 1992). While some
pesticides exhibit their toxicology acutely, others remain chronic as a result of exposure from multiple
sources making their implication in health challenges difficult to assess (Salau, 1993), with increasing use
of pesticides and other chemicals, high agricultural borne diseases, but with attendant degradation of the
environment and harmful effect on human (USEPA, 2006, NPIRS 2006).
Concern about exposure to organochlorine has risen chiefly because of their carcinogenicity in animals and
their ubiquity, bio-concentration persistence in human tissues (Kanja, et al. 1992)
1
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 and
discharge of waste waters from clean up equipment’s used for pesticide formulation and applications (Van
Schoubroeck, 1989).
The purpose of this study is to provide baseline information on the level of organochlorine pesticides in
Western Nigeria water bodies using some rivers to indicate the degree of environmental pollution.
MATERIALS AND METHODS

Materials
Sample Collection and Preservation
The water samples analyzed were collected from different rivers in Osun, Oyo, Ogun, Ondo and Lagos
States of Western Nigeria. 8 samples each of the various rivers were collected using Grab sampling
technique and analysed for organochlorine pesticides residues. Sampling was done upstream. Water
samples were kept in plastic bottles and preserved in a refrigerator maintained at 4oC prior to analysis. The
pH 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 model
equipped with 441 model UV detector filled with 25nm filter, model UGK septum less injector and SE 120
model recorder was used to analyse the clean-up hexane extracts.
Samples collection and Preservation

The samples were collected from different rivers in Osun, Oyo, Ogun, Lagos and Ondo States. Grap
sampling technique (Schofield, 1980) was employed for the samples collection. Sampling was carried out
upstream. Eight samples were collected from each sampling point. The PH of the water samples were
immediately taken on collection of the samples. The water samples were placed in rubber bottles and
immediately preserved after collection in ice in order to minimize degradation of the pesticides on the field
and these samples were transferred to the Freezer as soon as they were brought to the laboratory.
Methods
The analytical procedure (APHA – AWWA – WPCF) Standard Methods for the examination of water and
wastewaters (1980), with slight modification was used. The water analysis involved the extraction using
15% diethyl either in hexane. The extracts were cleaned using tetraoxosulphate (vi) acid clean up and
ethanolic 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 detector
fitted with 254nm filter.
Extraction and clean – up procedures

A 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 layer
was 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 above
repeated.
A third extraction was carried out using 60ml of hexane only and the process repeated. The separating
funnel 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 ethanolic
potassium hydroxide clean – up methods thus.
2
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 inversion
for two minutes and then centrifuged for 10 minutes. The top hexane layer was removed gently with an
eppendof 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 centrifuge
tube was capped and put into a water bath for 30 minutes at 50oC. 4ml of 2% sodium chloride in 0.1m
orthophosphoric acid was added and the centrifuge tube shaken by inversion gently for 2 minutes and then
centrifuged for 10 minutes. The upper hexane layers was removed gently and injected into the HPLC.
Quantification of Pesticides Residue

The chromatograms of the samples were compared with those of the mixed standards, which were injected
into HPLC. These were used to estimate the concentration of the Organochlorine and Polychlorinated
pesticide 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 i
W Phstd Vs F
Where
V = Total Volume of the solvent used for extraction
W = Sample Weight taken in grams
Crms = Concentration of reference mixed standard
Phs and Phstd = Peak height of sample and standard respectively
Vs and Vstd = Volume of sample and standard injected in ml respectively
F = Concentration factor.
RESULTS AND DISCUSSION

The multiple regressions analysis of the organochlorine and polychlorinated pesticide residues
concentrations 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 relationship
between 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 pp’DDT were non detectable. Few of the samples analysed were
found to contain PCB’s, which range from 0.4348 – 8.9916 ppb and aldrin 0.0029 – 0.1904 ppb. The
highest lindane concentration of 0.6341 ppb was obtained for sample WW2. This high contamination could
be due to the extensive use of lindane, which is marketed as Gammalin 20 and used by farmers for
combating black pod disease in cocoa or most probably be due to it being misused by some Fishermen for
killing fishes. In the later case, it is sprayed directly into the water and there were fishing activities in-place
in most places sampled (Agunloye, 1984). Samples WW2 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 samples
collected from the same point at different times are different. That is why the analysis of the samples was
done in triplicates and the mean recorded. This difference in concentration could be due to the fact that
when the water is highly turbulent, there is mixing tendency for a lot of things, which can be carried by the
water compared with when it has low tide. This difference in concentration of residues exhibited at
3
different times is comparable with studies reported by other workers (FEPA, 1991, Adjert, 1986). There is
no 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 due
to 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 North
America (Staare, et al. 2000). It can be seen that some residues are at higher concentrations compared with
others. This may probably be due to the extensive usage of pesticides in some areas (Ecobichon, 1986).
CONCLUSION
Since, population growth, increasing urbanization and industrialization and rising standard of living have
all contributed to an increase in both the amount and the variety of organochlorines and polychlorinated
pesticide residue generated in rivers and water bodies in Nigeria, Frequent studies should be carried out on
these rivers and water bodies to give a baseline information on the level of organochlorine pesticides
present 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 institute
Info. 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, 15th
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 Governing
factors. 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 science
of Poisons. New York: McGraw Hill 643 – 689
Federal Environmental Protection Agency now Federal Ministry of Environment (1991). Guidelines and
Standards for Environmental Pollution control in Nigeria.
Gwenzi, W.D. (1986) Pesticides in soil and water Soil. Science society of America. Inc. Publisher Madison
Wisconsin, U.S.A.
Kanja LW, Skaare J.U, Ojwang S.B.O., maitail C.K. (1992). A comparison of organochlorine pesticide
residues 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 water
vol. 2 by Chau, A.S.Y, Afgan B.K. C.R.C. Press Inc. Boca Raton 14,71
4
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. Water
pollution 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. Academy
Press Ltd. 101 – 105
Received for Publication: 11/10/2007

Accepted for Publication: 20/10/2007
Corresponding Author:
P.A. Egwaikhide
Department of Chemistry and Centre for Biomaterials Research, University of Benin, Nigeria
E-mail: - pegwaikhide@yahoo.com
5
The results of the concentration (ppb) levels of Organochlorine and Polychlorinated Pesticide Residues in Western Nigeria rivers are shown in Table 1.
Rivers/Water Pesticides residue concentrations (ppb)
bodies
sampled
sCode PH Temp. Heptachlorioe Heptachor HCP opDDT Pp DDT Dieldrin Endosulfa Aldrin Lindanes E PCB Endrin
o
C p n
oxide
Oshun River ORO 6.91 26.3 ND 0.0010 0.002 ND ND 0.0141 0.0100 0.0037 0.0126 ND ND
Oshogbo 8
Ogun River LLR 6.68 27.0 ND 0.0037 0.002 ND ND 0.0074 0.430 0.0051 0.1852 ND ND
Lagos 1
River Aye RAO 6.80 26.1 ND 0.0008 0.000 ND ND 0.0074 0.0430 0.0132 0.0270 ND ND
Ogun 7
Badagry / BNR 0.0008 ND ND ND 0.0145 0.0057 0.510 0.0419 0.4348 ND
Noque River
Ogbese River ORO 7.05 26.2 ND 0.0008 ND ND ND 0.0107 0.0110 0.1904 0.0280 ND ND
Ondo D
Owena River OWO 7.15 26.5 ND ND 0.003 ND ND 0.0087 0.0029 0.1888 0.0163 8.9916 ND
Ondo D 9
Lagos Lagoon WW1 6.73 26.5 ND ND 0.000 ND ND - 0.0143 0.0960 0.0424 ND ND
8
Lagos Lagoon WW2 6.75 26.8 0.0001 ND 0.004 ND ND 0.0235 0.0857 0.1904 0.6341 8.9916 ND
1
Lagos Lagoon WW3 7.12 26.7 ND 0.0037 0.001 ND ND 0.0049 0.0143 0.0153 0.0164 0.6721 ND
7
Lagos Lagoon WW4 6.09 26.6 ND 0.0008 0.002 ND ND 0.0659 0.0851 0.0051 0.6341 ND ND
2
ND = Not Detected
6
Continental J. Applied Sciences 2: 7 - 11, 2007
© Wilolud Online Journals, 2007.
KINETICS OF THE ADSORPTION OF MUCIN ONTO COMMERCIALLY PURE TITANIUM

SURFACE
OMONIYI K. ISRAEL1*. LORI A. JOSEPH2 AND OGBODOBRI O. JUDE2

1*
Ahmadu Bello University, School of Basic and Remedial
Studies, P.M.B. 6007, Funtua-Nigeria
2
Chemistry Department, Ahmadu Bello University, Zaria-Nigeria.
ABSTRACT
The study investigated the kinetics of adsorption of the salivary protein, mucin on to
titanium, since the adsorption of proteins onto a biomaterial stand as the most
important issue in determining their biocompatibility. The time profiles for the
adsorption 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 higher
than that by the use of 10mg/ml mucin solution. Two kinetic models were applied for
determining the rate and mechanism of adsorption of mucin onto commercially pure
Ti powder. The present study showed that the adsorption rate constant is increased as
adsorbate concentration is increased. Also, at low concentration a thin boundary layer
is formed.
KEY WORDS: mucin, titanium, kinetic model, rate constant
INTRODUCTION
The adsorption of proteins on solid surfaces is an important phenomenon taking place immediately a
foreign material contacts a biological system. It is thus, involved in situations of bio- and blood
compatibility and in fouling in the process industry (Bornzin and Miller, 1982). Protein adsorption appears
to 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 spontaneous
desorption (Lundstrom, 1985). Mucin is a glycoprotein that covers the surface of the buccal cavity and
epithelial organs. Titanium as being reckoned with as an excellent material for dental and orthpaedic
applications, a thorough elucidation of the kinetics of mucin adsorption onto its surface is of great
significance in the bioengineering of titanium implants, towards the fabrication of the perfectly bio-friendly
Ti implants. This study applied two kinetic models for determining the specific rate constant of adsorption
of mucin onto Ti implant in vitro, in what will be called the short time range.

Materials
Mucin was obtained from Nacatai, Tesque Inc., Kyoto, Japan (Batch No. MIP960). It is a well-
characterised acidic protein with a molecular weight of 4.0x105. Commercially pure titanium (Ti) powder
of particle size 200mµM 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 (TiO2) as shown by X-ray photo-electron
spectroscopy (XPS) data (Sutherland et al., 1993).
Protein Adsorption Study

Samples of titanium (50mg or 100mg) were weighed into polypropylene (PP) centrifugation tubes and
10ml of mucin solution (10mg/ml or 50mg/ml) was added to each experimental group, (in triplicate).
7
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 Ti
Weight of Lagergren Kad (Min-1) Bhattacharya and
Ti/mucin Conc. Venkobacharya Kad
(mg/ml) (Min-1)
50mgTi(10mg/ml) 8.06 x 10-2 7.97 x 10-2
100mgTi 7.81 x 10-2 7.25 x 10-2

(10mg/ml)
50mgTi(50mg/ml) 8.52x10-2 9.24x10-2
100mgTi 7.25 x 10-2 7.07 x 10-2

(50mg/ml)
The mixture were continuously shaken for incubation times of 10,15,30,45,60 and 70 minutes in an
incubator (1H 150, Gallenkamp Co., England) at 370C.
Subsequently, the Ti particles were collected after centrifugation, and mucin which did not adsorb strongly
to the Ti particles were removed by rinsing with 10ml of double distilled water. The supernatant were
separated and removed again. The Ti particles with the adsorbed mucin of an experimental group were then
heated at 600C 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 6000C for 30 minutes in a furnace in order
to desorp the mucin.. The weight of mucin adsorbed was calculated by comparing the weight after drying at
500C and that after heating at 6000C.
RESULTS AND DISCUSSION

The 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 Ti
particles 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 100mg
group of Ti particles as the incubation time extended, using 50mg/ml mucin solution. Statistical analysis by
one-way ANOVA showed that, there was a significant difference in the amount of mucin adsorbed onto the
50mg Ti particles group compared to that onto the 100mg group, using 10mg/ml mucin solution. There was
no significant difference between the two experimental groups by the use of 50mg/ml mucin solution
(P<0.01). At the end of the 60 minute experiment, the average amount of mucin adsorbed by using
50mg/ml mucin concentration was 2.4 + 0.11 times higher than that by the use of 10mg/ml mucin solution.
Kinetics of adsorption of mucin onto Ti

Two kinetic models were applied for determining the rate and mechanism of adsorption of mucin onto
commercially pure Ti powder. These are based on the assumption that the adsorption follows the first order
rate equation.
The two models are the Lagergren and BhattaCharya/Venkobacharya.
The Lagergren equation is given as Log (qe – qt) = Log qe – (Kad/2.303)t
Where qe and qt are the amounts of mucin adsorbed at equilibrium and time, t respectively, t is time, Kad is
the specific rate constants (min-1)
8
The Second kinetic model is the Bhattacharya and Venkobacharya first order equation. The equation is
given 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 is
the concentration at equilibrium.
As presented in Figures 2 and 3, a straight line with high correlation coefficient, R2 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 with
correlation coefficient in the range of 0.9847 – 0.999. This further reaffirms the fit of the experimental data
to 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 50mg
of Ti particles.
Mode of Particle diffusion

The kinetic data was also analysed by a diffusion-controlled adsorption model. The intra-particle
diffusion is model by the equation.
qt = Kpt ½ + C
Where Kp is the intra-particle diffusion rate constant (in mg min-1), C is the boundary layer thickness and qt
is 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 <R2<
0.9912 confirmed the presence of intra-particle diffusion process as the rate-determining step. Many
experimental confirmed diffusion-controlled adsorption from nonflowing solutions at low concentration
(Bornzin and Miller, 1982).
As displayed in Figures 6 and 7, the low values of intra-particle diffusion rate constants observed,
compared to that for lysozyme (molecular weight, 14,000 Da) reported by Kandori et al. 2004 may be due
to high molecular weight of mucin.
Figure 2: Lagergren plots for mucin (10mg/ml concentration) adsorption onto Ti Figure 3: Lagergren plots for mucin (50mg/ml concentration) adsorption onto Ti
0.8 1
y = -0.037x + 1.1509
0.6 0.8 2
R = 0.9912
0.4 0.6
0.2 0.4
y = -0.0315x + 0.8747
2
R = 0.9963
L o g (q e - q t)
L o g (q e - q t)
0 Series1 0.2 Series1

0 5 10 15 20 25 30 35 40 45 50 Series2 Series2
100 mg Ti 100 mg Ti
-0.2 0
50 mg Ti 50 mg Ti
y = -0.0339x + 0.9189 0 5 10 15 20 25 30 35 40 45 50
2
R = 0.98
-0.4 -0.2
y = -0.035x + 0.835
2
R = 0.9873
-0.6 -0.4
-0.8 -0.6
-1 -0.8
Time (mins) Time (mins)
9
Figure 5: Bhattacharya and Venkobacharya plots for mucin (50mg/ml concentration)

Figure 4: Bhattacharya and Venkobacharya plots for mucin (10mg/ml concentration)
adsorption onto Ti
adsorption onto Ti
0
0
0 5 10 15 20 25 30 35 40 45 50
0 5 10 15 20 25 30 35 40 45 50
y = -0.0346x + 0.1961 -0.2

-0.2 2 y = -0.0401x + 0.0334
R = 0.9949 2
R = 0.999
-0.4
-0.4
-0.6
y = -0.0315x + 0.0455
2
-0.6 R = 0.9847
-0.8
Lo g [1 - U (T)]
y = -0.0307x - 0.222
Log (qe - qt)
Series1 2
Series1
R = 0.9904
Series2 -1 Series2
-0.8
100 mg Ti 100 mg Ti
50 mg Ti 50 mg Ti
-1.2
-1
-1.4
-1.2
-1.6
-1.4
-1.8
-1.6 -2
Time (mins) Time (mins)
Figure 6: Variation of the amount of mucin (10mg/ml concentration) transported into Ti

oxide film with time
Figure 7: Variation of the amount of mucin (50mg/ml concentration) transported into Ti
8 oxide film with time
14
7
y = 0.9011x + 0.7699
2
y = 0.9159x + 6.3491
R = 0.9912 12 2
R = 0.9412
6
10 y = 1.5612x + 1.7732
2
R = 0.9171
5
qt (mg)
qt Series1
(mg) 8
4 Series2 Series1
100 mg Ti Series2
50 mg Ti 100 mg Ti
y = 0.7888x - 0.8404 6 50 mg Ti
3 R2 = 0.949
4
2
1 2
0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
1/2 1/2
t (min-1) t (min-1)
CONCLUSION
Adsorbed mucin molecules become more tighly bound as the contact time with the adsorbent surface
increases (Lori and Ogbodobri, 2005). The present study showed that the adsorption rate constant is
increased as adsorbate concentration is increased. Also, at low concentration a thin boundary layer is
formed. At high concentration, the surface is covered by a thicker boundary layer since there is very little
time 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 great
significance in the fabrication of the ever-expected perfectly bio-friendly implants and prostheses.
ACKNOWLEDGEMENTS
We 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.
REFERENCES
Bornzin, G.A. and Miller, I.F. (1982). In: Kandori, K., Miyagawa, K. and Ishikawa, T. (2004). “Adsorption
Of 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 Onto
Various Synthetic Calcium Hydroxyapatite Particles.” Journal of Colloid and Interface Science, 273:406-
413.
10
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 to
Titanium.” Proceedings of the 28th 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.

OMONIYI K. ISRAEL
Ahmadu Bello University, School of Basic and Remedial, Studies, P.M.B. 6007, Funtua-Nigeria
E-mail: israelflourish@yahoo.com
11
© Wilolud Online Journals, 2007.
SPATIAL VARIATION IN HEAVY METAL CONCENTRATION IN SOIL PROFILES IN EMENE

INDUSTRIAL AREA OF ENUGU, SOUTH EASTERN NIGERIA.
1
Anyika, C. C and 2Nnabude, P.C
1
Department of Soil Science, Federal University of Technology, Minna. 2Department of Botany, Nnamdi
Azikiwe University, Awka.
ABSTRACT
An investigation into the extent of metals in soils around the Emenite and the defunct
Niger steel factories, which are closer to human habitation and private farms, was
conducted.Arsenic, chromium and lead were determined by Energy Dispersive X-ray
Fluorescence (EDXRF) with isotopic source (109Cd). In the control soil samples, mean
soil pH was 6.4, while mean soil pH at the study location was 6.9. Arsenic and lead
concentrations decreased though not significantly with increasing distance from the
factory 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. Chromium
concentration increased marginally with a concentration of 1168.20 Mg ha-1. Highest
concentrations of 87.95 Mg ha-1 and 121.90 Mg ha-1 for arsenic and lead respectively
were 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 for
the control locations were 46.79 Mg ha-1, 39.45 Mg ha-1 and 691.20 Mg ha-1 for
arsenic, lead and chromium respectively. While at the study location, average metal
concentrations 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 higher
than the values at the control location and implied that serious soil contamination
resulted from the deposition of dust particles and fume emissions from the factory. It
is recommended that bioremediation should be used to transform the pollutants into
less hazardous forms and that people should not live or carryout agricultural activities
within 300 m from the industrial area.
KEYWORDS: Heavy metals, Spatial variation, Depth variation, Soil profiles,

Southeastern Nigeria, Typic Paleudult.
INTRODUCTION
The level of inorganic contaminants released into the environment from industrial sources every year is
now estimated to exceed that from organic and radioactive
sources combined and a fair share of these inorganic substances ends up contaminating the soils. (Okonkwo
and Eboatu, 1999)
The effects of soil contamination with these heavy metals which are toxic to plants in large amounts can be
much more damaging and permanent than those of organic sources. Metals are strongly absorbed by the
soil clay and humus and so do not leach to any extent. Once metals contaminate a soil, it remains so
indefinitely. They may find their ways into many environments through agricultural crops, soil surface and
ground water’s 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 contamination
include: Hg, Pb, Cd, As and Cr. (Mercury, lead, Cadmium, Arsenic and Chromium) hence the need to
investigate their levels in the soil around the Emenite and the defunct Niger steel factories in the Emene
12
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 and
other animals. Cadmium and arsenic are extremely poisonous; mercury, lead and chromium are moderately
so.
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 other
processing techniques, release into the atmosphere tons of these elements, which can be carried for miles
and later deposited on the vegetation and soil. Lead is a gasoline additive that is released into the
atmosphere and carried to the soil through rain and wind. (Nriagu and Paycna, 1988). Arsenic was for
many 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 metals
are found as constituents in specific organic pesticides and in domestic and industrial sewage sludge, where
they pollute agricultural land, metals in sewage become adsorbed by the solid matter and so are retained in
the sludge. The liquid effluent, which is eventually discharged to the rivers, has very low metal content. So
the 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 (most
notably lead, because of the changes in gasoline formulation) are decreasing. All are daily ingested by
humans 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 have
been carried out. A survey of heavy metal concentration in pastoral soils was conducted in the early 1990’s,
in New Zealand, and only one of the five heavy metals i.e. Cadmium was present at elevated levels, though
the 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 most
industrial 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 metres
respectively may be ignorant of the level or environmental pollution that may result from the factory which
uses some raw materials like cement from limestone, which has cadmium contents as well as fumes
released 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 steel
factories, obtained from both the spatial distance and depths.

Environment of Study/ Location
Emene is the study area. Emene is located in Enugu east local government area of Enugu state, southeastern
Nigeria. Emene is situated 2 km North of Enugu town. Emene is a host to numerous industries, including
the 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 70

10’ E and 60 15’E of the Greenwich
meridian and latitudinally, it stretches
from 60 30’ N and 70 30’ N of equator.
13
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: Forest
Climate: very humid
Mean annual rainfall: >1650mm
Length of raining season: >250 days
Land 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 site
Drainage: 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 109Cd annular source and a [Si (Li)]
detector (lithium – drifted silicon detector), which has a resolution of 170eV for the 5.90keV line. The
detector and the source are placed on the Dewar, which contained liquid nitrogen for the purpose of cooling
the detector (Fig. 1).
109
Sample Target
Cd Si(Li) X-Ray
Excitation source Detector OSCILLO
SCOPE
Integral 572 7100/7150 Read out

Dewar Preamp AMPLIFIER 44 of 7450 Analogue to digital
GATED MCA converter
BIASED
Amplifiers
456 480
HV POWER PULSER
SUPPLY
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 cadmium
source acts as an excitation source that emits Ag-K X-rays (22.1keV) in which all elements having
characteristic energies lower than that quoted above were accessible for detection in the samples. There is
also the molybdenum (Mo) target, which serves as a source of monochromatic x-rays. The x-rays are
excited 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 – digital
converter (ADC) card. The molybdenum target is used for absorption correction. The absorption factor thus
determined is then used by the software package (AXIL-QXAS) to quantify the concentration of the
elements in the samples. The software is also used to correct for the contribution of the Zr-K to the Mo – K
peak by subtraction. The spectra for the samples were collected for 3000 seconds and subsequently
evaluated. (Idris et al., 2000). The data was finally analyzed statistically, using statistical package for
Social Science Windows. (SPSS).
14
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 from
depths 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 the
cassava farm up to the rail road, totaling 21m.
(b) Control Sample Collection
The control sample, B, (B1, B2, and B3) was collected at the interior of Oye Emene1km from
the study area. Three replicates of the soil samples were collected in the same way as the
study sample.
PROCEDURE:
Disturbed soil samples were collected using the core sampler. The sampler was pushed into the soil and the
samples were collected at a depth of 0-15cm for the surface samples. The auger was used to dig deeper
into the soil and collection made with the core sampler, up to a depth of 75cm. A grid of 20cm by 20cm
was 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 location
while 15 samples were collected for control studies. All samples were collected in 3 replicates to give a
total of 105 samples. The samples (20g) each were then filled in clean cellophane bags, which had already
been 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 wire
surrounding the factory was between 5m and 6m respectively. The sampling started at the boundary
between the factory and the cassava farm and ended 3m before the railroad. Similarly, opposite the factory
about 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 pestle
to grain size of less than 125µm. This was done for the purpose of homogenization. Pellets of 19mm
diameter were then prepared from 0.5g powder mixed with 3 drops of organic liquid binder (PVC dissolved
in toluene) and pressed afterwards at 10 tons with a hydraulic press. (SPECAL P/N 15 011), the pellets
were finally weighed and then taken for Analysis.
Soil pH Determination Studies.

• 10g of <2mm sieved soil from 20g sample was weighed into a 50ml disposable cup.
• 20ml of 0.01M CaCl2 solution was added and the suspension stirred and allowed to stand for
30 minutes with occasional stirring. This is to allow most of the sediments to settle.
• The pH was determined by immersing the glass electrode into the partially settled suspension.
Make sure the electrode does not reach the bottom of the container.
• Record the pH to one decimal place.
• The pH meter was checked against two buffers usually at pH 4.0 and 7.0 for non-calcareous
soils.
• The pH reading was taken at 25o
RESULTS AND DISCUSSION.

Spatial Variation in Chromium, Arsenic and Lead Concentrations:
15
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, well
above the control value of 691.2 Mg ha-1. The analysis of variance showed a significant variation in
concentration, between 12m and 3m. The results generally showed an increase in concentration away from
the factory. The control value of 691.2 Mg ha-1 obtained under the control was significantly lower than the
values obtained within the vicinity of the factory. This implies that the soil at the factory location is
polluted with chromium.
The results (figure 2.) showed that arsenic concentration at the middle distance (6 – 12 m) differed
significantly from the extreme points (3 and 18 m). The results showed no consistent trend in metal
distribution. At the surface, the major increase to 86.68 Mg ha-1 was obtained at a spatial distance of 12m
and still remained elevated at 74.73Mg ha-1 at 18m, well above the control value of 46.79 Mg ha-1. The
analysis of variance showed a general lack of significant variation in concentration with the spatial
distance. However, they all varied significantly from the control. This implies that the soils are polluted
with arsenic. The trend generally followed a gentle decrease in concentration away from the factory with
spatial distance.
The results for lead (figure 2.) showed no consistent pattern of metal distribution with respect to distance
away from the factory. At the surface, there was a major increase to 110.10Mg ha-1 at 9m and 103.76Mg
ha-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 study
location. This implies that the soils are polluted. The trend followed a decrease in concentration away from
the factory site.
)
a
h1500
/
g
M
(
n1000
o
it
ra
t
500
n
e Cr
c 0
n
o
C 3 6 9 12 15 18 As
Distance(m)
Pb
Fig. 2. Showed the spatial variation in chromium, arsenic and lead concentrations in soil with respect to
distance away from the factory location.
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, well
above the control value of 691.2 Mg ha-1. The analysis of variance showed a significant variation in
concentration, between 12m and 3m. The results generally showed an increase in concentration away from
the factory. The control value of 691.2 Mg ha-1 obtained under the control was significantly lower than the
values obtained within the vicinity of the factory. This implies that the soil at the factory location is
polluted with chromium.
The results (figure 2.) showed that arsenic concentration at the middle distance (6 – 12 m) differed
significantly from the extreme points (3 and 18 m). The results showed no consistent trend in metal
distribution. At the surface, the major increase to 86.68 Mg ha-1 was obtained at a spatial distance of 12m
and still remained elevated at 74.73Mg ha-1 at 18m, well above the control value of 46.79 Mg ha-1. The
analysis of variance showed a general lack of significant variation in concentration with the spatial
distance. However, they all varied significantly from the control. This implies that the soils are polluted
16
with arsenic. The trend generally followed a gentle decrease in concentration away from the factory with
spatial distance.
The results for lead (figure 2.) showed no consistent pattern of metal distribution with respect to distance
away from the factory. At the surface, there was a major increase to 110.10Mg ha-1 at 9m and 103.76Mg
ha-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 study
location. This implies that the soils are polluted. The trend followed a decrease in concentration away from
the factory site.
PERCENTAGE INCREASE IN HEAVY METAL CONCENTRATION OVER CONTROL:

Percentage increase over
200
150
control
100
50
0
Cr
3 6 9 12 15 18
Distance(m)
As
Pb
Figure 3. Component bar chart showing percentage increase of 3 heavy metal concentrations over control
concentration
From the data presented in the barchart, (figure 3.), the percentage increase in chromium at the study
location over the mean control level was highest of 69 % for chromium at a spatial distance of 18m while
the 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 lowest
increase 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 highest
percentage increase of 180% at the 9m spatial distances and the lowest increase of 163 % over the control
at the 18m spatial distances.
DEPTH VARIATION IN CHROMIUM, ARSENIC AND LEAD CONCENTRATIONS: (Mg ha – 1)

)
a
h1200
/
g
M 1000
(
n 800
io
t 600
a
rt 400
n 200
e Cr
c
n
o
0
C
0-15 As
15-30 30-45 45-60 60-75
Depth (cm)
Pb
Figure 4. Graph of Depth variation against the concentration of 3 heavy metals (Distribution of heavy
metals in subsoil)
17
Mobility: The results showed the following sequence of mobility:

LEAD > ARSENIC > CHROMIUM. This sequence is similar to that observed in other environments by
other authors, for example, at a copper smelter (Elizabeth et. al., 2000) a Zinc factory, a lead - zinc
production site and mining, sites (Alloway, 1990). Differences in the mobility sequence of metals might be
due 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 a
significant role in fixing metals and preventing them from being leached out of the soil profile. There is
however 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 the
45-60cm and 60-75cm, which were not significant. The trend showed a pattern of decreasing values in the
profiles.
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 no
significant difference in Arsenic concentration with depth, with the exception of the 15 - 30 cm and 30 - 45
cm, which varied significantly. The trend generally showed a marginal decrease in concentration with
depth.
STATISTICAL ANALYSIS/ COMPARISON OF pH AT THE VARIOUS SPATIAL DISTANCES AND

DEPTHS USING THE UNPAIRED T-TEST PROCEDURE:
Table 1: Table of unpaired t - test for soil pH

Variate Mean S. D t-Cal. P- Value Remark
3m 7.0 0.295 6.399 < 0.0001 Sig
1km 6.4 0.198
6m 7.0 0.342 5.820 <0.0001

1km 6.4 0.198 Sig
3m 6.9 0.404 4.759 0.0001 Sig

1km 6.4 0.198
12m 7.0 0.233 2.405 0.0230 Sig

15m 6.7 0.348
12m 7.0 0.233 8.113 <0.0001 Sig

18m 6.8 0.331
15m 6.8 0.348 3.674 0.0010 Sig

1km 6.4 0.198
18m 6.8 0.331 3.8912 0.007 Sig

1km 6.4 0.198
18
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 – 75
cm 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 not
statistically significant and were not presented. This was in agreement with the results of Kabala. and
Singh, (2000) working on mobility and fractionation of heavy metals in soil profiles within the vicinity of a
copper smelter in Poland, despite the fact that they used a depth of 120cm and samples collected from four
locations within a Copper smelter. This is to be expected as all samples were taken within the same area
and therefore, soil was subjected to the same physico - chemical processes, with the exception of the
control. This implies that the soils at the factory location differ significantly from the control, meaning that
the soils near the Emenite factory are polluted.
TEST OF HYPOTHESIS ON SOIL pH AND ARSENIC, CHROMIUM AND LEAD

CONCENTRATION:
The results showed that P>0.05, therefore, we accept H0 and conclude that there is no significant correlation
between soil pH and Arsenic concentration.
The results showed that P>0.05, therefore, we accept the H0 and thus conclude that there is no significant
correlation between soil pH and chromium concentration.
The results showed that P>0.05, therefore, we accept H0 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 only
difference been that they worked on iron and manganese at a depth of 216 cm. They thus concluded that
this 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 metal
concentration, they pointed out other factors like: plant specie, organic matter content, clay content and
moisture content.
NORMALITY TEST:
The Anderson-Darling Normality test was used. Both the analysis of variance and correlation techniques
are based on the assumption that the data is normally distributed. However, the assumption for this test is
also carried out to access the relative or unique contributions of soil pH in predicting metal distribution in
soils. 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 that
the data is normally distributed. This is in agreement with the results of (Tazisong, et. al., 2004): although
exchangeable iron in their results did not strictly meet the assumption, but the deviation or skewness was
not 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 Agency
has established permissible limits of these metals in soils. The table below (table 2). compared these limits
with what was obtained at the control and factory locations.
19
Table 2. Heavy metals (Mg ha-1 ) in the study area in relation to the permissible limits.
Heavy Permissible Permissible Factory Control

metal limit limit Location Location
UK1 USEPA2 Enugu Enugu
Arsenic 75 75 75.80 46.79

Chromium 3000 3000 1038.3 691.2
Lead 500 420 97.25 39.45
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 Mg
ha -1 ) dry weight is above the permissible limit of 75 Mg ha-1 dry weight for sludge application in soil set
up by the USEPA, and this could lead to deterioration in soil quality and possible contamination of the food
chain. 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 the
factory and depth variation is also concentration dependent and that these 3 metals have similar distribution
characteristics in the area studied. It has been established that the levels of arsenic, chromium and lead in
the soil around the Emenite factory is as a result of emission of fumes and dusts from the factory. The
general trend in this work agrees with previous works despite the fact that they were mainly in temperate
locations. 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 and
meteorological 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 critical
level. There is therefore a danger of build up of larger doses either through inhalation, absorption through
the 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 it
degenerates dangerously.
ACKNOWLEDGEMENT: The author’s wishes to acknowledge Prof. L. A. Dim of the Centre for Energy
Research and Training A.B.U. Zaria, Prof.L.N. Moghalu of Department of Geography, Meteorology and
Environmental 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 is
hereby recommended:
1) People should not leave or carry out agricultural activities within a distance of at least 300m from
the 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
20
surroundings. The law further stipulates that factory, which emits dust, fumes or other impurities, which are
injurious or offensive to the employees, must take practicable measures to protect the employees and to
provide 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 the
state attorney general to sue the Emenite factory which is the only factory close to human habitation and
private farms around the industrial estate or they can also rely on the doctrine of strict liability (Rylands Vs
Fletcher) 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 our
agricultural land, the following is recommended:
FOR ARSENIC: Transformation of arsenic 3 to arsenic 2, which can be taken up by plants can be achieved
by redox reaction. Also applications of sulphates of zinc, iron and aluminum, which tie up the arsenic in
insoluble forms.
Finally, this study should be taken up at a regular interval of about 3 years since pollution is going on in the
area on daily bases.
REFERENCES
Alloway, B.J. (1990b). ‘’The origin of heavy metals in soils’’. In Steinborn, M. and Breen, J. (eds.), Heavy
Metals 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 profiles
in 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 in
bauxites from Mambilla Plateau, Northern Nigeria Using Isotopic X- Ray Fluorescence Spectroscopy”.
Proceedings. Of the 23rd Annual Conference, of the Nigeria Institute of Physics. “The Millenium
Conference.” pp 190-192.
Kabala C, and Singh, BR, (2000). “Fractionation and Mobility of Copper, Lead, and Zinc in Soil Profiles
in 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 Florida
soils. Journal Environmental Quality, 26:765 – 775.
21
National Population Commission (NPC). (1991). Emene census final results of 1991 population census
of Nigeria. NPC. Enugu.
Nriagu, E.M. and Pacyna, J.M. (1988). “The role of sorbed humic substances on the distribution of organic
inorganic 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 from
aqueous 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. 1st Edition. Onis
Excel Publishing. Zaria, p 8.
Oviasogie, P.O. and Ukpebor, E. E. (2003). “Assessment of some forms of Pb, Mn and Cu in
petrochemicals 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, Food

and 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).

Anyika C.C.
Department of Soil Science, Federal University of Technology, Minna.
Email: anyika 3C @ yahoo.com
22
INFLUENCE OF MATERNAL PARITY ON TRANSPLACENTAL TRANSMISSION OF MALARIA

AND THE CONSEQUENT EFFECT ON NEONATAL BIRTH WEIGHT IN ABRAKA, DELTA
STATE, NIGERIA
a
Pender, K.E., bIgweh, J.C and cOnyesom, I*.
a.
Department of Physiology, College of Health Sciences,
Delta State University, Abraka.
b
Department of Physiology, College of Medicine,
University of Nigeria, Enugu Campus.
c
Department of Medical Biochemistry, College of Health Sciences,
Delta State University, Abraka.
ABSTRACT
This study investigates the effect of maternal parity on the pattern of transplacental
transmission of malaria and the consequent influence on neonatal birth weight. Forty-
five cord blood samples were collected from the neonates of consenting and randomly
selected women who were delivered of babies in General Hospital, Abraka, Delta
State, Nigeria. The cord blood samples were prepared for malarial parasite count
using standard procedure. Results show that 42.2% of the samples were positive for
malarial parasite, out of which 52.6% and 47.4% were from neonates of multiparous
and primiparous mothers, respectively. The mean birth weight of malarial infected
neonates from multiparous and primiparous mothers, respectively. The mean birth
weight of malarial infected neonates from multiparous and primiparous mothers were
3.17±0.53 and 3.09±0.54; P>0.05, respectively. The birth weights for uninfected
neonates from multiparous and primiparous mothers were 3.43±0.63 and
3.22±0.43;P>0.05, respectively. 94.7% of the infected cases were caused by
Plasmodium falciparum and the remaining 5.3% by P. ovale strategies for reducing
fetomaternal transmission should be sought.
KEYWORDS: Abraka, maternal parity, malaria, Plasmodium falciparum, placenta.
INTRODUCTION
Malaria is the commonest cause of fever in the tropics. In malaria endemic areas, gravid women often
present 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 predilection
of malarial parasite for placenta is due to avid binding to surface chondroiton sulphate on the
synicitiotrophoblast (Beesen and Reeder, 2001).
Placental parasitisation interferes with placental function and transplacental transmission may lead to birth
weight reduction, which is a poor prognostic factor (Coller and Scaly, 2005).
Malaria related growth retardation predominantly affects the primiparious and is most severe with
Plasmodium falciparum (McGready and Billie, 2004; Mac-Gregor and Avery, 1974).
Reported cases of transplacental transmission of malaria and effects on neonatal birth weights has been
reported 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 weights
have been made in the Gambia (MacGregor and Wilson, 1983), Cote de Ivoire (Larkin and Thuma, 2003),
Cameroon (Akum and Kwoh, 2005).
23
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 history
associated with fetal growth restriction. The history were obtained from the booking information. The
study was approved by our Faculty’s Research and Bioethics Committee.
Specimen Collection: Forty-five cord blood samples were obtained from the selected women who also
gave their informed consent. At the end of the second stage of labour, the cord was clamped at two points
and divided. The distal cord was elevated while attached to the placenta, then 5 ml syringe was used to
collect about 2 ml of umbilical arterial blood and placed in a heparinised bottle and analyzed within the
hour 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 hepatitis
B 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 spread
out untidily to cover approximately one square centimeter for the thick film; it was allowed to dry. For the
thin film, a drop of blood was placed near one end of the slide; another slide angled at 45 degrees to the
drop of blood was used to make a thin smear on the first. This was labeled and allowed to dry. After
drying, smears were fixed in methanol for five seconds, stained with Giemsa; then 20 drops of distilled
water were dropped on the smears and allowed to dry. Thick and thin films were examined using 1000
magnification with oil immersion lens. Parasite density was calculated as the number of infected RBCs per
1000 RBCs. Statistics: The data obtained were summarized by way of mean and standard deviation and
the means were compared by the use of Students t-test. Level of significance was set at P<0.05.
RESULTS
Data obtained from the study are presented on Table 1. Table 1, shows the parity related prevalence and
mean birth weights.
Out of the 19 samples of cord blood positive for malaria parasites, 18 were P. falciparum with a prevalence
of 94.7%, 1 was P. ovale with a prevalence of 5.26%. P. vivax and P. Malariae were not identified. The
mean birth weight for the primigravid positive for malarial parasite was 3.090±0.54 kg and similar
multigravid group was 3.170±0.525 kg showing a difference of 180 g. ( P>0.05).
DISCUSSION
Data (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 fetal
growth restriction due to malaria. Similar to earlier studies in areas of high transmission (Larkin and
Thuma, 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 be
initiated in order to encourage especially primigravid women to utilize antenatal services. The need for
vector 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 and
the risk of low birth weight infants.
24
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 the
birth 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 sero
evaluation 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 University
Press, London.
Lamikanra, O.T. (1986). Study of malaria parasitaemia in pregnant women, placenta, blood and new borns
in 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 Epidemiol 146 (10): 826-
831.
McGready, R. and Billie, B. (2004). The effect of Plasmodium histopathology in an area of low malaria
transmission. 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:
it’s 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 double
blind placebo controlled study. J Inf Dis 169: 593-603.
Table 1: Effect of maternal parity on cord blood parasitaemia and birth weight
Maternal parity
Multigravidae Primigravidae
Cord blood Malarial Parasitemia
N, no of subjects Positive Negative Positive Negative
Infectious malarial parasite 10 19 9 7
10 0 8 0
Plasmodium falciparum
Plasmodium vivax 0 0 0 0
Plasmodium ovale 0 0 1 0
Plasmodium malariae 0 0 0 0
Birth weight (kg) 3.17±0.53 3.43±0.63 3.09±0.54 3.22±0.43
Dr I. Onyesom
Postal: P.O. Box 144, Abraka, Delta State, Nigeria. E-mail: onyesominno@yahoo.co.uk
25
TOPICAL LIPOSOMAL DIBUCAINE DELIVERY SYSTEM: DEVELOPMENT AND

CHARACTERIZATION
Nounou, M.M.*, El-Khordagui, L.K., Khalafallah, N.A. and Khalil, S.A.

Department of Pharmaceutics, Faculty of Pharmacy, University of Alexandria, Egypt
ABSTRACT
Formulation of local anesthetics in controlled delivery systems provides safer and
more effective anesthesia. The aim of this study was to develop a liposomal dibucaine
base (DB) local anesthetic delivery system. DB-loaded multilamellar vesicles (MLVs)
with different characteristics were obtained by varying lipid composition, drug
loading, induced charge and pH of the hydration buffer. Liposomes were
characterized for morphology, size, entrapment efficiency (EE) and stability including
leakage stability. Results indicate that amongst formulations tested, negatively
charged liposomes with the lipid composition phosphatidyl choline: cholesterol:
dicetyl phosphate 7:6:1 and drug: lipid ratio 90mg: 300mg prepared with pH 9
hydration medium sowed good in vitro characteristics in terms of EE (≈ 90%),
sustained drug release (≈ 20% in 12 hrs) and low burst effect. However, they
exhibited relatively poor leakage stability. A delivery system was prepared by
incorporating negatively charged DB-loaded liposomes in a 2% HPMC gel base. Gel
formulations were assessed in vitro for drug leakage stability and in vivo using the pin
prick test in Guinea pigs. Incorporation of liposomes in the gel enhanced their leakage
stability. Release characteristics of the liposomal gel could be modulated by
combining different proportions of free and liposomal DB. The in vivo performance
of a combination gel provided a superior local anesthetic profile (fast onset and
prolonged duration of action) compared to a non-liposomal DB gel and a liposomal
gel formulation with no free drug added. The DB liposomal gel developed offers great
potentials as a local anesthetic delivery system.
KEYWORDS
Dibucaine base, dicetylphosphate, in-vitro release, release stability, multilamellar
vesicles, liposomal gel, local anaesthesia, pin prick test, sustained release.
INTRODUCTION
Topical anesthetics were introduced in the latter half of the 19th century, starting with a description of the
topical uses for cocaine (1). The need for the development of an effective topical anesthetic preparation
prompted 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 insufficient
amount 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 topical
anesthetic effect. Several studies indicated that upon application of a "thick layer" of EMLA cream for at
least 2 hours, a reasonable effect develops (0.5 mm deep anesthesia) in adults and in children (3). It could be
concluded that the achievement of significant rapid, deep, and long lasting skin anesthesia is a challenging
problem. However, to achieve sufficient anesthetic effect on intact skin, prolonged application and high
concentration of drug (10-30%) are required (4).
Among the most effective topical anesthetics and of particular interest is Dibucaine, a potent long acting
local anesthetic.
26
Nounou, M.M et al: Continental J. Applied Sciences 2: 26 - 37, 2007
Table 1: Dibucaine gel formulations

State of the
Concentration
Application drug Concentration
Code Description of
properties incorporated of HPMC gel
dibucaine base
inside the gel
Transparent,
dibucaine base 100% Free
DG smooth, viscous,
plain gel dibucaine base
non tacky gel
100%
Dibucaine base
White, opaque, smooth,
dibucaine base viscous, non tacky gel
4 % w/v
DLG encapsulated
1 % w/w
liposomal gel
inside
liposomes
87.5% of the
Dibucaine base
drug is
combination
liposomal
DCG-87.5 gel (Free and
encapsulated,
liposomal
the rest is free
drug)
drug
Dibucaine is a topical amide local anesthetic that is commonly used in haemorrhoidal preparations. It has
been used as a base or hydrochloride salt in creams or ointments containing up to 1% for the relief of pain
and 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 drug
to the dermal region and eliminate its toxic systemic action. Lai et al.(6) investigated the epidermal
application 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 in
biodegradable 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 been
marketed.
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 incorporated
material (13). Liposomes also increase the permeability of skin for various entrapped drugs and at the same
time way diminish some side effects of these drugs(14). Several studies indicated that tetracaine in liposome
encapsulated form provides better topical anesthesia than a conventional form(15, 16). Liposomes applied
topically were proposed to penetrate into the skin (dermis) through the lipid channels of the epidermis and
localize the drug within the skin. Due to multilamellar structure of liposomes, sustained release of the
encapsulated 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 an
appropriate vehicle. Usually, liposomes are applied to the skin in solution or in hydrogels, since stable
liposomal creams are difficult to formulate(17). For topical application of liposomes, hydrophilic polymers
are suitable thickening agents, since they make the formulations convenient for application. It has been
confirmed 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 (carbopol
resins) (20). However, the type and concentration of polymer which forms the hydrogel could influence the
stability as well as the rate of penetration of liposome entrapped substances into the skin (21). Concerning
the assessment of anaesthetic effect, The effectiveness of topical anesthetics has been difficult to assess
because 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
27
Table 2: Pin prick test results in guinea pigs following the application of dibucaine topical
formulations.
Code Control DLG DCG DG
Time 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5 M
(min.) P P P
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 ns
300 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 = Median
ns=Non Significant Data (P>0.05)
s*=Significant Data at P<0.05
s**= Significant Data at P<0.01
s***=Significant Data at P<0.001
P=Parametric Data Manipulation(one way ANOVA followed by Tukey-Kramer Test)
"Pin prick method" (4, 15, 18, 24-27). In connection with this method, a wide variety of stimuli have been used in
measuring the degree of local anesthesia such as alcohol swabs, very fine needles (gauge 23 or 25), very
large needles (gauge 18 or 19, or an epidural needle), safety pins, screwdrivers, clamps or thorn of thistle
(28)
. Some anesthetists use their fingernails to pinch patients to test the presence of sensory blockage (29).
The main drawback of this technique is the variation of the intensity of the stimulus resulting from the wide
assortment of methods, besides, these stimuli may not be uniformly reproducible.

Chemicals
Dibucaine base was obtained from Ward Blenkinsop Co. (London, England), Cholesterol 99% Extra pure
was purchased S.d.Fine-Chem Ltd. (Mumbai, India). L-α- phosphatidylcholine, type X-E: from dried egg
yolk, Stearylamine and Dicetylphosphate were from Sigma Chemical Co. (St. Louis, USA). Hydroxypropyl
methylcellulose high viscosity grade (4000 c.p.) was a generous gift from Alexandria
Pharmaceuticals(Alexandria, Egypt).
Spectra/Por® dialysis membrane 12,000-14,000 molecular weight cut off was received from Spectrum
Laboratories Inc. (USA).
All other materials and solvents were of analytical grade and double-distilled water was used.
Liposome preparation
The lipid components (300 mg total lipid) consisting of phosphatidylcholine with a lipid: cholesterol:
dicetylphosphate molar ratio of 7:6:1, were weighed into a 250 ml long necked pear shaped quick fit round
bottom flask and dissolved in 5 ml chloroform/methanol 7/3 V/V. Dibucaine base dissolved in 3 ml of the
same solvent mixture were added to the lipid solution. The organic solvent was removed under reduced
pressure at 43ºC using a rotary evaporator (Rotavapor, Type R 110). The resulting thin lipid film was
28
25
20
I n te r c e p t (% r e l e a s e d )
15
10
0
Zero time One week Two weeks Four weeks
Figure 1: Electron microscope photo of
dibucaine liposomal dispersion D6pH9-90(-)
(75,000 X)
Figure 4: Intercept of Higuschi plots of
release data from D6pH9-90(-) liposomal
dispersion As a function of storage at 4 0C
25
14
Fourteen Days
Seven days
Zero Days
28 Days
12
20
In te rc e p t (% r e le a s e d )
10
15
8
Time (hour)
6
10
4
5
2
0
C u m u la t iv e % r e le a s e d
50
40
30
20
10
0
Zero time One week Two weeks Four weeks
Figure 2: Release stability profiles at 32 0C of
dibucaine liposomal dispersion (D6pH5-90(-)) as Figure 5: Intercept of Higuschi plots of
a function of storage time at 4 0C release data from D6pH5-90(-) liposomal
dispersion As a function of storage at 4 0C
over four weeks
12
Fourteen Days
Seven days
Zero Days
28 Days
10
100
DG
6 Time (hour) 8
DCG-87.5
80
DLG
C um m ulativ e r elea se of dibucaine, %
60
4
40
2
20
0
C u m u la tiv e % r e le a s e d
45
40
35
30
25
20
15
10
0
0 2 4 6 Time (hr.) 8 10 12
Figure 3: Release stability profiles at 32 0C Figure 6: Release profiles at 32 0C of

of dibucaine liposomal dispersion (D6pH9-90(- dibucaine topical formulations.
)) as a function of storage time at 4 0C
over four weeks
29
45.00 35.00 30
% c u m u l a t iv e re le a s e o f d i b u c a i n e b a s e f ro m
40.00
30.00
D ib u c a in e L i p o s o m a l G e l ( D L G )
35.00 25
30.00 25.00
In te r c e p t (% r e le a s e d )
20
S lo p e (% / S Q R T t)
25.00
20.00
20.00 Zero Days
15
15.00
15.00 Seven Days
10.00 10.00 10
Fourteen Days
5.00
5.00 28 days
0.00 5
-5.00 DG DCG-87.5 DLG 0.00

Intercept Slope 0
0 2 4 6 Time (hr.) 8 10 12 14
Figure 7: Slope and intercept of
Higuschi plots of release data from Figure 8 : Release stability profiles
dibucaine gels as a function of gel at 32 0C of dibucaine liposomal gel
composition. (DLG) stored at 4 0C.
100 100
98
% D ib u c a in e r e t a in e d
96
% C u m m u la t iv e r e le a s e o f d ib u c a in e b a s e g e ls
80
D6pH9-90(-)
94 DG
D6pH5-90(-)
92
DLG 60 DLG
90
88
86 40
84
82 20
80
0 5 10 15 20 25 30
Time (days) 0
0 2 4 Time (hr.) 6 8 10 12
0
Figure 9: Percent dibucaine retained in Figure 10: Release profiles at 32 C of
dibucaine liposomal gel (DLG) and dibucaine liposomes after one hour release at 32 0C
gel (DG) as control
100
% C u m m u lative re le as e p ro file o f d ib u ca in e b as e lip o s o m al
80
Control
d is p e rsio n (D 6p H 9-9 0(-) )
60
D6pH9-90(-)
40
20
0
0 2 4 Time (hr.) 6 8 10 12
Figure 11: Release profiles at 32 0C of dibucaine liposomal dispersion (D6pH9-90(-))and dibucaine

solution as control
30
10
10 10
9
9 9
8
8 8
7
7 7
P a in S co r e (o u t o f 10 )
P ai n S core(out of 10)
P a i n S c o r e (o u t o f 1 0 )
6
6 6
5
5 5
4
4 4
3
3 3
2
2 2
1
1 1
0
0 0
0
0 15 0
15 30 15
30
control 45 control 30
control
45 60 45
60 120 60
120
DG Time (min.) 180 DLG 120
DCG
Time (min.) 180 240 Time (min.) 180
240 300 240
300 360 300
360 360
DG control DLG control 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 (D6pH5-90(-)) or bicarbonate buffer pH 9.0
(D6pH9-90(-)).
The process of hydration involved rotation at a low speed at 43ºC in the rotary evaporator (with no
vacuum) for 30 minutes followed by hand shaking for 15 minutes at 43ºC in a thermostatically controlled
water bath. The resulting liposomal dispersion was left to mature over night at 4ºC. This method was
reported by Bangham, Stansfield and Walkins (30).
Separation of free dibucaine

This was achieved by centrifugation of the prepared dispersions at 16500 rpm (27800 Xg) for 90 minutes at
-5ºC (Beckman model J2-21 centrifuge). The resulting liposomal concentrate was washed twice each with
5 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. Dibucaine
concentration was determined spectrophotometrically at 241 nm (31) using the lysis mixture as blank as
described by Mezei et al.(12).
Dibucaine liposomal dispersions were examined by transmission electron microscopy using 2% ammonium
molybedate aqueous solution as a negative stain. Liposome vesicle size was determined using the laser
diffraction particle size analyzer.
Preparation of dibucaine liposomal gel

The calculated amount of hydroxypropyl methylcellulose powder was dusted slowly over phosphate buffer
solution of pH 5.6 under continuous agitation in an ice bath, and then the mixture was strongly agitated for
45 minutes. The prepared dispersion was then left in the refrigerator (4-5 0C) overnight. A clear solution
was obtained which gelled at room temperature (32, 33). Gel was degassed by centrifugation if necessary (34).
Dibucaine base liposomal concentrate (D6pH9-90(-)) was added to the prepared gel by trituration so as to
yield 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 dibucaine
liposomal 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 room
temperature for 12 hours to remove the preservative followed by rinsing thoroughly in distilled water. In
31
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 dialysis
bags 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 mm
diameter dialysis bag. The bag was closed at both ends with cotton thread and tested for leakage. The final
length of the bag after tying was 8±0.2 cm. The dialysis bag was attached horizontally fully stretched to the
paddle which was then immersed in the tablet dissolution tester beaker containing 250 ml of the release
medium (phosphate buffer pH 5.6 containing 7% propylene glycol and 25 % methanol). The bag was fully
immersed under the surface. The temperature was set at 32±0.2 0C and the speed of rotation of the paddles
at 100 rpm.
Control bags were prepared and tested along with the liposomal dispersions. The control bags each
contained 2 mg Dibucaine base dissolved in one ml of the release medium. A representative liposomal
dispersion 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. A
known weight (equivalent to 10 mg dibucaine base) of each gel formulation shown in Table 1 was filled in
stainless steel cups (Radius 1.5 cm, height 0.5 cm). The surface of the gel was made flat; the cups were
fitted with the dialysis sheet by a rubber band and immersed in the bottom of the beakers of the tablet
dissolution 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 dialysis
medium. The temperature was set to 32 ± 0.2 0C and the speed of rotation of the paddles of the dissolution
tester was set to 100 r.p.m. Aliquots of the release medium were periodically withdrawn for analysis and
replaced with equal volume of fresh release medium (adjusted at 32 ± 0.2 0C). Each release experiment was
continued for 12 hours.
Aliquots of the release medium were withdrawn for analysis at different time intervals and replaced with
fresh medium. Each release run was continued for 12 hours. The absorbance of the collected samples, after
dilution if necessary with release medium, was measured at λmax 244 nm. The results recorded were the
mean value of three runs carried out for each liposome concentrate.
Release stability of dibucaine liposomal dispersions and gels

Possible leakage of dibucaine base from multilamellar vesicles liposomal dispersions (D6pH5-90(-) and
(D6pH9-90(-)) or liposomal gels (DLG) was monitored during storage of two of the liposomal concentrates.
Changes in release profile of dibucaine were taken as indicator of instability. Release profile was
determined as previously detailed directly after preparation of the liposomal concentrate or gel and after
one, two and four weeks storage in well closed tubes at 4 0C.
In vivo evaluation local anesthetic effect of different dibucaine gel formulations applying "Pin Prick Test"
on guinea pigs
A weighed quantity of the prepared gel formulations (0.8 gm containing 8 mg of dibucaine) was applied on
a circular area of 12.57 cm2 (radius of 2 cm) located on the shaved back of a guinea pig(35). The tested area
was then covered with parafilm® to provide occlusion for 30 minutes. The pin prick test was then used to
assess the local anesthetic effect. The device for the pin prick test consisted of a pin pushed through a
rubber stopper obtained from a vacutainer, which prevents the pin from penetrating too deep into the skin.
A length of pin 0.4 mm protruding out of the rubber stopper was adequate to produce a sensation of pain
without producing injury. The pin was held perpendicular to the skin, and the rubber stopper was pushed
flush to the skin. This device has been used by anesthetists for testing sensory neural blockade during
regional anesthesia (28). The advantage of the device over an unrestricted pin is more uniform stimulus
intensity and prevention of skin injury. Testing was done immediately after removal of the occluding tape
and at 15, 30, 45, 60, 120, 180, 240, 300 and 360 minutes. The same guinea pig was used for each of the
selected formulations and for drug-free control gel. The animal was allowed to recover after each
32
(2, 4, 12, 26, 28, 36)
experiment for at least 24 hours . Hydroxypropyl methylcellulose gel containing no drug
served as a control.
Each formulation was tested on five male guinea pigs applying randomized crossover design. Ten pricks
covering the whole test area were applied at each time period, the number of painless scores out of ten
observed by the absence of skin shivering was used as a measure of anesthesia. The nonparametric data
obtained were transformed by square rooting to normally distributed data, on which ANOVA and paired t-
test could be applied (37, 38). Data were analyzed statistically to test significance by one way analysis of
variance followed by Tukey-Kramer multiple test.
Results and discussion
In this study, we have investigated only negatively charged liposomes as they showed high entrapment
efficiency with good visual stability after 12 months. The trends observed in these results were lower
entrapment efficiency for the phosphate buffer pH 5.6 (as the hydration medium) compared to the
bicarbonate pH 9.0 medium, the entrapment efficiency of D6pH5-90(-) was 91.76 ± 1.82 % while the
entrapment efficiency of D6pH9-90(-) was 40.45 ± 0.80%
Resulting electron microscope photos obtained for dibucaine liposomal dispersion D6pH9-90(-) at
magnification power 75,000 X using ammonium molybedate as negative stain is shown in Figure 1. The
multilamellar nature of the prepared liposomes is evident.
The computed median vesicle sizes for D6pH5-90(-) and D6pH9-90(-) (1.2 and 1.7 µm respectively) are
within the reported vesicle size for multilamellar liposomes (39, 40).
Regarding the in vitro drug release of dibucaine liposomes, compared to the control data, dibucaine release
from liposomes is sustained and prolonged. The release profile of dibucaine base from the liposomal
dispersions D6pH5-90(-) and D6pH9-90(-) showed that 44.20 % and 42.37% of the drug was released in 12
hours respectively. Low release rates exhibited by negatively charged liposomes, and this could be
contributed to the negatively charge imparting agent being conjugated with the phospholipids bilayer
structure undergoing an electrostatic attraction forces with the ionized part of the drug (41-44). One of the
liposomal concentrates included in the release study was examined using the electron microscope before
and after the release run to detect effects, if any, of release study conditions on the integrity of the
liposomes. No evidence of large scale lysis or coalescence of the vesicles could b found. The retention of
sealed vesicular structures after the release run is evident.
Looking into the kinetics of dibucaine release from the liposomes, the release data from time zero to 12
hours appear to best fit the Higuschi diffusion model based on the magnitude of correlation coefficient
obtained for zero order, first order and Higuschi's model (45).
The release profile of the free unentrapped dibucaine base from its gel (DG) showed that 95% of the drug
was released within four hours. In comparison, the release profile of dibucaine base from the liposomal gel
(DLG) showed that 27.8% of the drug was released in 12 hours reflecting the sustained release of dibucaine
from the liposomal gel. The release profiles of the combination gel showed that 56.04% were released in 12
hours from the combination gels containing 87.5% respectively of the drug content entrapped in the
liposomes indicating the effect on release of changes in proportion of free to liposome-entrapped drug in
the prepared gels (Figure 6). The release profiles and the slopes of the Higuschi release plots obtained for
the various freshly prepared gels (Figures 6 & 7) indicated the potential to tailor dibucaine release from the
gels by varying the proportion of free and liposome-entrapped drug in the gels. The Higuschi release plots
intercept values (Figure 7) were a direct function of the gel composition again reflecting the potential to
tailor initial release (burst effect) through varying free drug content in the gels.
Intercept and slope values were calculated and plotted against the proportion of free to liposome-entrapped
dibucaine in the gel (Figure 7). Lowest slope and intercept values were shown by gels containing mainly
33
liposome-entrapped drug among the gels prepared. The correlation coefficient was calculated for each of
the two sets of data.
Possible drug leakage out of liposomes during storage of liposome concentrate was monitored by recording
changes in release rates of negatively charged drug-loaded liposomal dispersions or gels as a function of
storage time. Results of monitoring changes in release rates of these liposomal dispersions and gels as a
measure of instability are given in Figures 2, 3 & 8. The results indicate progressive increase in release rate
particularly evident by the fourth week of storage.
Comparing release profiles before and during storage indicated a rise in the percent drug released during
the initial faster phase of release (zero-2 hours) evidenced by the gradual increase in Higuschi plot intercept
as a function of storage time (Figure 4 & 5).
Higuschi plot intercept, considered in the present part as a measure of unentrapped drug associated with the
liposomes was found to increase as a function of storage time (Figures 4 & 5) suggesting progressive drug
leakage out of liposomes stored at 4 °C in the form of aqueous liposome dispersions.
The results of monitoring release at 32 0C of one of the prepared gels (DLG) as a function of storage time at
4 0C for 4 weeks indicated good stability as evidenced by the lack of change in release profile of the stored
gel (Figure 8). This in contrast to the relative instability of the same liposomes stored as an aqueous
concentrate. Figure 9 compares the percent drug retained in the liposomes after one hour release as a
function of storage time for both the liposomal dispersion and the liposomal gel. The gel as a structured
vehicle apparently enhances liposome stability and delays or prevents drug leakage from the liposomes.
In an attempt to identify the rate limiting step in the release process of the drug from the liposomal gel, the
release results for DLG were compared with the release profile generated in chapter one of this part for a
similar liposomal system (D6pH9-90(-)) but as an aqueous concentrate not a gel (Figure 10 & 11). The
similarity between the two liposome profiles suggests that the gelling agent in the concentration used had
little effect on the release and that the rate limiting step was the diffusion of the drug out of the liposomes.
A similar observation was reported in a study of lidocaine HCl release from topical liposomal formulation
(46)
.
Regarding the in vivo evaluation of the local anaesthetic effect of the different dibucaine base gels
prepared, the pin prick test performed in guinea pigs over a period of 6 hours proved capable of
distinguishing between the anaesthetic effect of the prepared dibucaine gels particularly in terms of
duration of effect. Results of applying the pin prick test to guinea pigs 30 minutes after application of
different dibucaine topical formulations are shown in Table 2 and Figure 12. Judging by the results in Table
2, dibucaine combined liposomal gel (DCG) appeared to afford highest anaesthetic effect over 6 hours in
each of the five animals while the anaesthetic effect of liposome-free gel (DG) was apparent only in the
first hour.
These results showed that the onset and duration of action of local anaesthetic effect could be controlled
through the proper choice of percentage of free to liposome-entrapped local anaesthetic loaded in the
prepared hydroxypropyl methylcellulose gel.
Also, no obvious in-vivo in-vitro correlation was observed. In this respect, it has been suggested in the
literature that in vitro release studies in liposome research are not meant to mimic in vivo conditions but
may be considered a measure of instability (47).
CONCLUSIONS
Negatively charged dibucaine base liposomes showed high entrapment efficiency and sufficient visual and
physical stability. Incorporation of these liposomes in hydroxypropyl methylcellulose gel highly improved
their physical stability. On consideration of the in-vivo performance of the liposomal gel, the tailor
dibucaine base gels formulation through varying the proportion of free and liposome-entrapped drug in the
34
gels was a key step in obtaining a fast onset and a sustained duration of action of local anaesthetic effect of
dibucaine base.
REFERENCES
1). Kundu, S. and Achar, S., Principles of office anesthesia: part II. Topical anesthesia. Am Fam
Physician, 2002. 66(1): p. 99-102.
2). Foldvari, M., In vitro cutaneous and percutaneous delivery and in vivo efficacy of tetracaine from
liposomal and conventional vehicles. Pharm Res, 1994. 11(Nov): p. 1593-1598.
3). Hallen, B., Calsson, P., and Uppfeldt, A., Clinical study of lignocaine-prilocaine cream to relieve
pain of venepuncture. Br J Anaesth, 1985. 57: p. 326-328.
4). Nagarsenker, M.S. and Joshi, A.A., Preparation, characterization, and evaluation of liposomal
dispersions of lidocaine. Drug Dev Ind Pharm, 1997. 23(12): p. 1159-1165.
5). sweetman, S. Martindale, The complete drug reference. [CD] 2003 [cited 2003.
6). Lai, P.M. and Roberts, M.S., Epidermal iontophoresis. Part 2. Application of the ionic mobility
pore model to the transport of local anesthetics. Pharm Res, 1998. 15(Oct): p. 1579-1588.
7). Masters, D.B., Berde, C.B., Dutta, S., Turek, T., and Langer, R., Sustained local anesthetic release
from bioerodible polymer matrices: potential method for prolonged regional anesthesia. Pharm
Res, 1993. 10(Oct): p. 1527-1532.
8). Thoma, K. and Schlutermann, B., Relationships between manufacturing parameters and
pharmaceutical-technological requirements on biodegradable microparticles. Part 6. In vitro
release characteristics of cinchocaine and bupivacaine from biodegradable microparticles.
Pharmazie, 1992. 47(Jun): p. 436-439.
pharmaceutical-technological requirements on biodegradable microparticles. Part 4. Interactions
between drug loading, matrix properties and manufacturing parameters. Pharmazie, 1992.
47(Apr): p. 269-272.
pharmaceutical-technological requirements on biodegradable microparticles. Part 2. Preparation of
injectable microparticles of biodegradable polyesters. Pharmazie, 1992. 47(Feb): p. 115-119.
pharmaceutical-technological requirements on biodegradable microparticles. Part 3.
Pharmaceutical and technological investigations on the particle size distribution of local
anesthetic-containing biodegradable microspheres. Pharmazie, 1992. 47(Mar): p. 198-202.
12). Mezei, M., Foldvari, M., Gesztes, A., Cardinal, L., Behl, M., and Kowalczyk, I., Topical
liposomal local anesthetics: design, optimization and evaluation of formulations. Drug Dev Ind
Pharm, 1993. 19(19): p. 2499-2517.
13). Schafer-Korting, M., Korting, H.C., and Braun-Falco, O., Liposome preparations: a step forward
in topical drug therapy for skin disease? A review. J Am Acad Dermatol, 1989. 21(6): p. 1271-5.
14). wholrab, W., Lasch, J., Taube, K.M., and Wozniak, K.D., Hautpermeation von liposomal
incorporirtem hydrocortison. Pharmazie, 1989. 44: p. 333-335.
15). Gesztes, A. and Mezei, M., Topical anesthesia of the skin by liposome encapsulated tetracaine.
Anesth Analg, 1988. 67(11): p. 1079-81.
16). Foldvari, M., Gesztes, A., and Mezei, M., Dermal drug delivery by liposome encapsulation:
clinical and electron microscopic studies. J Microencapsul, 1990. 7(4): p. 479-89.
17). Gabrijelcic, V. and Sentjurc, M., Influence of hydrogels on liposome stability and on the transport
of liposome entrapped substances into the skin. Int J Pharm, 1995. 118(May 16): p. 207-212.
18). El-Ridy, M.S. and Khalil, R.M., Free versus liposome-encapsulated lignocaine hydrochloride
topical applications. Pharmazie, 1999. 54(9): p. 682-684.
19). Glavas-Dodov, M., Fredro-Kumbaradzi, E., Goracinova, K., Calis, S., Simonoska, M., and
A.Hincal, A., 5-Fluorouracil in topical liposome gels for anticancer treatment-Formulation and
Evaluation. Acta Pharm, 2003. 53(4): p. 241-250.
20). Foldvari, M., Effect of vehicle on topical liposomal drug delivery: petrolatum bases. J
Microencapsul, 1996. 13(5): p. 589-600.
35
21). Gabrijelcic, V., Sentjurc, M., and Kristl, J., Evaluation of liposomes as drug carriers into the skin
by one-dimensional EPR imaging. Int J Pharm, 1990. 62(Jul 15): p. 75-79.
22). Adriani, J. and Zepernick, R., Clinical Effectiveness of Drugs Used for Topical Anesthesia.
JAMA, 1964. 188: p. 711-6.
23). Orkin, L.R., Clark, R.E., and Rovenstine, E.A., Objective method in of evaluating topical
anesthesia in man utilizing laryngeal reflex. Anesthesiology, 1954. 15: p. 161-173.
24). Stanton Hicks, M., Murphy, T.M., Bonica, J.J., Berges, P.U., Mather, L.E., and et al., Effects of
peridural block. 5. Properties, circulatory effects, and blood levels of etidocaine and lidocaine.
Anesthesiology, 1975. 42(Apr): p. 398-407.
25). Planas, M.E., Gonzalez, P., Rodriguez, L., Sanchez, S., and Cevc, G., Noninvasive percutaneous
induction of topical analgesia by a new type of drug carrier, and prolongation of local pain
insensitivity by anesthetic liposomes. Anesth Analg, 1992. 75(4): p. 615-21.
26). Sharma, B.B., Jain, S.K., and Vyas, S.P., Topical liposome system bearing local anaesthetic
lignocaine: preparation and evaluation. J Microencapsul, 1994. 11(3): p. 279-86.
27). Fisher, R., Hung, O., Mezei, M., and Stewart, R., Topical anaesthesia of intact skin: liposome-
encapsulated tetracaine vs EMLA. Br J Anaesth, 1998. 81(6): p. 972-3.
28). Santos, D., Juneja, M., and Bridenbaugh, P.O., A device for uniform testing of sensory neural
blockage during regional anesthesia. Anesth Analg, 1987. 66: p. 581-582.
29). Bromage, P.R., Spread of analgesic solutions in the epidural space and their mode of action: a
statistical study. Br J Anaesth, 1962. 34: p. 161-177.
30). Bangham, A.D., Stansfield, M.M., and Walkins, J.C., Diffusion of univalent ions across the
lamellar of swollen phospholipids. J Mol Biol, 1965. 13: p. 238-252.
31). Padmanabhan, G.R., Dibucaine and dibucaine hydrochloride, in Analytical profiles of drug
substances, K. Flory, Editor. 1989, Academic Press,Inc. p. 105-134.
32). Sokkar, H.b.m., A physicochemical stability study of some anti-inflammatory drugs in different
formulations, in Pharmaceutics Department. 1996, Alexandria university: Alexandria.
33). Harwood, R., Hydroxypropyl MethylCellulose, in Hand book of pharmaceutical excipients. 1995.
p. 252-255.
34). Barry, B.W. and Meyer, M.C., The reological properties of carbopol gels: 1.continous shear and
creep properties of carpobol gels". Int J Pharm, 1979. 2: p. 1-25.
35). Vogel, H.G. and Vogel, W.H. Drug Discovery and evaluation. 1997 [cited.
36). Satoskar, R.S. and Bhandarkar, S.D., Pharmacology and pharmacotherapeutics. 12th edition ed.
1983, Bombay, India: Popular Prakashan.
37). Bolton, S., Pharmaceutical statistics: Practical and clinical applications. Third edition ed. Drugs
and the pharmaceutical sciences, ed. J. Swarbrick. 1997, Newyork Basel HongKong: Marcel
Dekker, Inc.
38). Muller, J., Statistics for advanced level. 1996: Cambridge university press.
39). Weiner, N., Martin, F., and Riaz, M., Liposomes as a drug delivery system. Drug Dev Ind Pharm,
1989. 15(10): p. 1523-1554.
40). Crommelin, D.J., Zuidam, N.J., and Vrueh, R.D., Chapter 2: Characterization of liposomes, in
Liposomes : a practical approach. 2003, Oxford University Press: NewYork. p. 66-67.
41). Khairy, L., Investigations on microcapsules as drug delievery systems., in Pharmaceutics
Department. 1983, Cairo University: Cairo.
42). Holaiel, S.M., Pharmaceutical studies on liposomes in topical drug formulation, in Pharmaceutics
Department. 2001, Cairo Universty: Cairo.
43). Kulkarni, S.B., Betageri, G.V., and Singh, M., Factors affecting microencapsulation of drugs in
liposomes. J Microencapsul, 1995. 12: p. 229.
44). Vemuri, S. and Rhodes, C.T., Development and validation of a drug release rate method for a
water soluble drug in a liposome preparation. Drug Dev Ind Pharm, 1995. 70(2): p. 95-111.
45). Arica, B., Ozer, A.Y., Ercan, M.T., and Hincal, A.A., Characterization, in-vitro and in-vivo
studies on primaquine diphosphate liposomes. J Microencapsul, 1995. 12(5): p. 469-485.
46). Glavas-Dodov, M., Goracinova, K., Mladenovska, K., and Fredro-Kumbaradzi, E., Release profile
of lidocaine HCl from topical liposomal gel formulation. Int J Pharm, 2002. 242(1-2): p. 381-4.
36
47). Mezei, M., Liposomes in the topical applications of drugs: a review, in Liposomes as drug carriers
: recent trends and progress, G. Gregoriadis, Editor. 1988, Wiley: Chichester. p. 663-675.

Nounou, M.M.
Department of Pharmaceutics, Faculty of Pharmacy, University of Alexandria, Egypt
Nounou@uh.edu
37
38
39
40

Vol 2 - Continental J. Applied Sciences.

Hochgeladen von

Dokumentinformationen

Originaltitel

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

Vol 2 - Continental J. Applied Sciences.

Hochgeladen von

Copyright:

Verfügbare Formate

Continental J.

Applied Sciences 2:1 - 6, 2007

ASSESSMENT OF ORGANOCHLORINE AND POLYCHLORINATED PESTICIDES RESIDUES IN

P.A. Egwaikhidea, S.O. Okeniyib S.O. Ohikhenac and C.E. Gimbad

KEYWORDS: Contamination, pollution, organochlorine pesticides, polychlorinated

MATERIALS AND METHODS

Samples collection and Preservation

Extraction and clean – up procedures

Quantification of Pesticides Residue

RESULTS AND DISCUSSION

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

Received for Publication: 11/10/2007

KINETICS OF THE ADSORPTION OF MUCIN ONTO COMMERCIALLY PURE TITANIUM

OMONIYI K. ISRAEL1*. LORI A. JOSEPH2 AND OGBODOBRI O. JUDE2

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

MATERIALS AND METHODS

Protein Adsorption Study

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

100mgTi 7.81 x 10-2 7.25 x 10-2

100mgTi 7.25 x 10-2 7.07 x 10-2

RESULTS AND DISCUSSION

Kinetics of adsorption of mucin onto Ti

The two models are the Lagergren and BhattaCharya/Venkobacharya.

The Lagergren equation is given as Log (qe – qt) = Log qe – (Kad/2.303)t

Log [1 – (U)T] = (Kad/2.303)t

Where U (T) = Ci – Ct/Ci-Ce

Mode of Particle diffusion

0 Series1 0.2 Series1

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

y = -0.0346x + 0.1961 -0.2

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

Received for Publication: 07/09/2007

SPATIAL VARIATION IN HEAVY METAL CONCENTRATION IN SOIL PROFILES IN EMENE

KEYWORDS: Heavy metals, Spatial variation, Depth variation, Soil profiles,

MATERIALS AND METHODS

Location: Emene is in Enugu East. (Longitude 70

Soil Taxonomic classification: Typic Paleudult (FDALR, 1985).

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

Integral 572 7100/7150 Read out

Fig 1. Equipment Arrangement for the Experiment.

Soil pH Determination Studies.

RESULTS AND DISCUSSION.

PERCENTAGE INCREASE IN HEAVY METAL CONCENTRATION OVER CONTROL:

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

Mobility: The results showed the following sequence of mobility:

STATISTICAL ANALYSIS/ COMPARISON OF pH AT THE VARIOUS SPATIAL DISTANCES AND

Table 1: Table of unpaired t - test for soil pH

6m 7.0 0.342 5.820 <0.0001

3m 6.9 0.404 4.759 0.0001 Sig

12m 7.0 0.233 2.405 0.0230 Sig

12m 7.0 0.233 8.113 <0.0001 Sig

15m 6.8 0.348 3.674 0.0010 Sig

18m 6.8 0.331 3.8912 0.007 Sig

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

TEST OF HYPOTHESIS ON SOIL pH AND ARSENIC, CHROMIUM AND LEAD

CONCENTRATION OF ARSENIC, CHROMIUM AND LEAD AT THE STUDY LOCATION:

Heavy Permissible Permissible Factory Control

Arsenic 75 75 75.80 46.79

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

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

Received for Publication: 11/10/2007

INFLUENCE OF MATERNAL PARITY ON TRANSPLACENTAL TRANSMISSION OF MALARIA

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

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