Attenuation of Petroleum Hydrocarbons by Weathering: A Case Study

Abstract

Possible alterations in the distribution and composition of total petroleum hydrocarbon (TPH), polycyclic aromatic hydrocarbons (PAHs), and benzene, toluene, ethyl benzene, and xylene isomers (BTEX) in the released oil at Idu-Ekpeye in Niger Delta (Nigeria) were studied within two seasonal variations of two months and six months, with a view to assessing the level of attenuation of these hydrocarbons in impacted soils. Although there were significant contaminations in the kerosene range (n-C10-n-C14) two months after, especially of the n-C12 and n-C13 fractions, the complete disappearance of the n-C8 to n-C23 hydrocarbons, including the acyclic isoprenoids (pristane and phytane), and the reduced amounts of PAHs, and BTEX, six months after, provided substantial evidence of attenuation as indicated in the reduction in total hydrocarbon content (THC) from 61.17 to 42.86%. Soil physicochemical properties such as pH, moisture content, heavy metal, TOC, and TOM, all provided corroborative evidence of hydrocarbon attenuation. The pristane/phytane ratio of the spill samples suggests that the spilled oil was genetically oxic.

Full text

Attenuation of Petroleum Hydrocarbons by Weathering: A Case Study
by Leo C. Osuji*,Inimfon A. Udoetok, and Regina E. Ogali
Department of Industrial and Pure Chemistry, University of Port Harcourt, PMB 5323, Choba,
Port Harcourt, Nigeria (e-mail: osujileo@yahoo.com)
Possible alterations in the distribution and composition of total petroleum hydrocarbon (TPH) ,
polycyclic aromatic hydrocarbons (PAHs), and benzene, toluene, ethyl benzene, and xylene isomers
(BTEX) in the released oil at Idu-Ekpeye in Niger Delta (Nigeria) were studied within two seasonal
variations of two months and six months, with a view to assessing the level of attenuation of these
hydrocarbons in impacted soils. Although there were significant contaminations in the kerosene range
(n-C10 –n-C14) two months after, especially of the n-C12 and n-C13 fractions, the complete disappearance
of the n-C8to n-C23 hydrocarbons, including the acyclic isoprenoids (pristane and phytane) , and the
reduced amounts of PAHs, and BTEX, six months after, provided substantial evidence of attenuation as
indicated in the reduction in total hydrocarbon content (THC) from 61.17 to 42.86%. Soil
physicochemical properties such as pH, moisture content, heavy metal, TOC, and TOM, all provided
corroborative evidence of hydrocarbon attenuation. The pristane/phytane ratio of the spill samples
suggests that the spilled oil was genetically oxic.
Introduction. – Petroleum, in common parlance, refers to the three major forms of
hydrocarbons, namely crude oil, natural gas, and condensates. Crude oil, the liquid form
of petroleum, is a complex mixture of hydrocarbons or substituted hydrocarbons in
which thousands of compounds of the two major elements, carbon and hydrogen,
combine with those of three minor elements, nitrogen, sulfur, and oxygen (NSO
compounds) , as well as trace metals like nickel, vanadium, and cadmium. Structurally,
the predominant constituents of crude oils include its total petroleum hydrocarbon
(TPH ) content, the polycyclic aromatic hydrocarbons (PAH), and the volatile
aromatics, i.e., benzene, toluene, ethylbenzene, xylene (BTEX). Thus, a molecule of
crude oil contains the paraffins, naphthenes, aromatics, and asphaltics. The composi-
tional variation in crude oils comes from the geohistory of the particular crude oil
source and confers on it marked peculiarity in chemical and physical characteristics [1].
When crude oil spills, it releases hydrocarbons onto the environment, and one of the
problems usually associated with profiling such hydrocarbons at contaminated sites is
the phenomenon known as weathering. Weathering is the change in composition of
hydrocarbons with time, through the action of volatilization, leaching, chemical
reaction, and biotransformation [2] ; such alterations might attenuate the hydrocarbon
content of the affected site. Thus, substantial evidence of hydrocarbon attenuation can
be obtained by the direct monitoring of the disappearance of hydrocarbon fractions
from the affected area. Such monitoring programmes usually involve repetitive
observations, measurements, and evaluation of site and hydrocarbon content according
to pre-arranged schedules.
CHEMISTRY & BIODIVERSITY – Vol. 3 (2006)422
 2006 Verlag Helvetica Chimica Acta AG, Zrich

As levels of attenuation are somewhat synonymous with the degree of alteration(s)
exhibited by the petroleum hydrocarbons, the severity of the processes should depend
on factors such as the physico-chemical properties of the substrate. Oxygen, for
instance, is an obligate requirement for the biotransformation of hydrocarbons via a
general mechanism that involves the oxidation of hydrocarbons to alcohols, ketones,
and acids. Again, photo-oxidation may increase the solubility of oils due to the
formation of polar compounds such as carboxylic acids [3] [4]. Therefore, the fates that
befall the hydrocarbon molecule in soil modify the observed composition of the
hydrocarbon.
The present study takes a look at the preferential consumption of these hydro-
carbons vis-a
`-vis some bulk indices of attenuation within two seasonal variations (two
months and six months) of recorded incidence of oil spillage. The study site was
selected from the petroliferous Niger Delta of southern Nigeria, following reconnais-
sance surveys of oil impacted areas in the region. Our interest was focused on the
various inputs to the overall hydrocarbon contamination situation at the site (within the
said seasonal fluctuations) which should be taken into account in the site-recovery
management scheme.
Results and Discussion. –TPH, PAH,and BTEX. Results of the total petroleum
hydrocarbon (TPH ) content of the oil-impacted soils are presented in Tables 1 –3.The
chromatograms (cf.Fig. 1) are representations of the adsorptions and desorptions of
the hydrocarbons and their resultant separations by molecular sizes. The gas-
chromatographic (GC) analyses conducted on the first set of samples collected two
months after the incidence of oil spillage showed fuller chromatograms of n-C8–n-C23
as depicted in Fig. 1; it also showed a substantial concentration of polyaromatic
hydrocarbons (PAHs; Table 2). The benzene, toluene, ethylbenzene, and xylene
(BTEX ) contents (Table 3) of the first samples (collected two months after oil spill)
were also substantial, though volatile and easily degraded under aerobic conditions. In
compromise, the results show that the spilled oil was still fresh on site, by the time of the
first sampling (two months after) with significant hydrocarbon contamination in the the
kerosene range (n-C10 –n-C14 ), especially of the n-C12 and n-C13 fractions. However, six
months later, the results showed complete disappearance of the n-C8–n-C23 hydro-
carbon fractions including the isoprenoids (pristane and phytane), leaving the n-C13,n-
C14 ,n-C19 , and n-C30 hydrocarbons (Fig. 1) . The results obtained for the polyaromatic
hydrocarbons showed that naphthalene, acenaphthalene, benz[a]anthracene, benzo[k]-
fluoranthrene, and indeno[1,2,3-cd]pyrene were likely most prone PAHs to biode-
gradation and the other forms of attenuation as observed in the marked reduction in
the concentration of the fractions (Table 2). Similarly, a mass reduction of the BTEX
fractions was observed in each of the spill samples analyzed, which corroborates the
significant alteration adduced from the other parameters. Thus, the hydrocarbon
distributions have provided evidence of attenuation.
Complete disappearance of the acyclic isoprenoids (pristane and phytane) provides
similar evidence of hydrocarbon attenuation. Prince et al. [5] reported that pristane and
phytane, which were originally thought to be resistant to biodegradation, can only be
used to monitor the earliest stages of biodegradation. Also the amounts of hydrocarbon
fractions expressed as ratios such as the pristane/phytane, n-C17/pristane and n-C18/
CHEMISTRY & BIODIVERSITY – Vol. 3 (2006) 423

CHEMISTRY & BIODIVERSITY – Vol. 3 (2006)424
Fig. 1. A chromatogram of one of the spill samples from Idu-Ekpeye oil-polluted site in Niger Delta,
Nigeria, showing n-C8to n-C23 hydrocarbon fractions of the released oil
Table 1. Seasonal Variation of Total Petroleum Hydrocarbon (TPH ) Content of Soils Two Months and
Six Months after Oil Spill
Total Petroleum Hydrocarbon ( TPH [mg/kg] ) S.E.a)
Hydrocarbon Fraction 1st Sampling (two months after) 2nd Sampling (six months after)
Amount [mg/kg] 103Amount [mg/kg]103
C81.100.07 –
C90.430.02 –
C10 0.220.02 –
C11 0.040.00 –
C12 1.070.04 –
C13 5.340.01 0.130.01
C14 0.480.00 0.030.01
C15 0.840.01 –
C16 0.140.00 –
C17 1.650.00 –
Pristaneb) 0.48 0.05 –
C18 0.270.00 –
Phytanec) 0.02 0.01 –
C19 0.020.01 0.270.00
C20 0.070.01 –
C21 0.030.01 –
C22 0.090.01 –
C23 0.030.00 –
a) S.E. is standard error at 95% confidence limit. b) 2,6,10,14-Tetramethylpentadecane. c) 2,6,10,14-
Tetramethylhexadecane.

phytane suggests increasing attenuation of hydrocarbons as observed from the
decreasing ratios. The pristane/phytane ratio decreased from 21.41 to almost zero,
the n-C17/pristane and n-C18/phytane ratio also decreased from 3.45 and 12.14,
respectively, to almost zero. These ratios may also serve useful purposes in locating the
source of the contaminant. The fingerprints, n-alkanes, and the acyclic isoprenoids
(pristane and phytane) are diagnostic and have far reaching implications on correlation
studies [6] . The quantitative expression of the fingerprints shows that pristane was
much more abundant than phytane. This, therefore, implies a very high pristane/
CHEMISTRY & BIODIVERSITY – Vol. 3 (2006) 425
Table 2. Seasonal Variation in Polycyclic Aromatic Hydrocarbon (PAH) Content of Soil Samples
2 Months and 6 Months after Oil Spill
Hydrocarbon Fraction 1st Sampling (two months after) 2nd Sampling (six months after)
Amount [mg/kg] Amount [mg/kg]
Naphthalene 7.7 1.2 BDLa)
Acenaphthylene 70.95.7 BDL
Acenaphthene 98.310.0 0.30.1
Fluorene 13516.8 2.91.4
Phenanthrene 1378.0 BDL
Anthracene 13424.5 39.10.5
Fluoranthene 977.8 18.66.2
Pyrene 1358.0 21.812.4
Benz[a]anthracene 22126.4 BDL
Chrysene 1382.4 27.48.0
Benzo[b]fluoranthene 231 10.2 22.42.1
Benzo[k]fluoranthene 79.6 9.6 BDL
Benzo[a]pyrene 31813 80.80.8
Indeno[1,2,3-cd]pyrene 24648 BDL
Dibenz[a,b]anthracene 13112 16.14.4
Benzo[ghi]perylene 103 6.4 69.1 7.0
a) BDL¼Below Detectable Limit (where detectable limit is 1 106mg/kg)
Table 3. Seasonal Variation of Benzene, Toluene, Ethylbenzene, and Xylene (BTEX ) Contents of Soils
2 Months and 6 Months after Oil Spill
Hydrocarbon Fraction 1st Sampling (two months after) 2nd Sampling (six months after)
Amount [mg/kg] Amount [mg/kg] a)
Benzene 48.212.5 BDL
Chlorobenzene 32.34.6 BDL
1,2-Dichlorobenzene 20.64.8 BDL
1,3-Dichlorobenzene 412.866.5 BDL
1,4-Dichlorobenzene 4.52.1 BDL
Ethylbenzene 11.71.9 BDL
Toluene 18.16.6 BDL
m,p-Xylene 22.112.2 BDL
o-Xylene 3.81.6 BDL
a) BDL¼Below Detectable Limit (where detectable limit is 1 106mg/kg).

phytane ratio, which is far greater than unity (>1) (pristane/phytane 21.41), and thus
gives a clue on the spilled-oils depositional environment. Pristane represents a product
of decarboxylation, therefore, the pristane/phytane ratio tends to be high in more
oxidizing environments such as peat swamps and low in strongly reducing environ-
ments [7][8] . Hence, the pristane/phytane ratio of 21.41 for the spilled oil might well
suggest an oxic depositional environment and a phytoplankton input.
Total Extractable Hydrocarbon Content. The total hydrocarbon content (THC ) of
soils is frequently used to assess and ascertain the extent of hydrocarbon contamination
at an oil-spill-polluted site. Results obtained show that the samples collected two
months after the oil spill had higher hydrocarbon content than the control. Although
the concentration of hydrocarbons at the time of spillage, barely two months prior to
sampling, was not known, the paucity of the data obtained for the THCs at surface and
subsurface depths (Fig. 2) presuppose that there was a high level of hydrocarbon
contamination on site, at least an amount far exceeding the compliamce limit of 50
parts per million set for petroleum industries in Nigeria [9].
The decrease in THC of 61.17 and 42.86% (Fig. 2) obtained between first and
second sample collection may be attributable to the increase in the number of
hydrocarbon degraders in the soil, and their adaptation to hydrocarbons. The severity
of hydrocarbon depletion is observed in the disappearance of the isoprenoids (pristane
and phytane) , which were previously thought to be resistant to degradation. The
decrease in the hydrocarbon concentration at the site might also be attributed to the
Fig. 2. Seasonal variation in the total hydrocarbon content of soils two months and six months after the oil
spill at Idu-Ekpeye in Niger Delta, Nigeria
CHEMISTRY & BIODIVERSITY – Vol. 3 (2006)426

tilling of the soil and the construction of secondary windows, which might have
enhanced microbial activities on the petroleum hydrocarbons due to aeration of the soil
and physical abrasion of the spilled oil. Aeration enhances the oxidation of the
substrate by oxygenases, which might have led to the formation of alcohols, ketones,
and acids [10] . This was probably why soil-pH decreased on second sampling as
observed from the physico-chemical collected six months after the spillage (Fig. 3).
Though there is evidence of extensive petroleum hydrocarbon attenuation at the
site, the level of hydrocarbon contamination of the soil as indicated in this study is still
significantly high compared to the control site. Usually, the presence of high
concentration of petroleum hydrocarbons in soils is detrimental to the growth and
productivity of plants and animals. This calls for more stringent depollution measures
towards facilitating the removal of these hydrocarbons from the affected site.
Soil Physicochemical Properties. The moisture content of soils from the oil-polluted
and control areas are represented by Fig. 4. The result shows higher moisture in the oil-
impacted samples (on first and second sampling, resp.) . This may be attributed to
insufficient soil aeration due to the displacement of air arising from water logging and
reduced rate of evaporation. The higher moisture content may also be as a result of the
hydrophobic nature of the soil. The higher moisture content at subsurface depth can be
explained by the evaporation that took place at the surface depth due to its exposure to
sunlight. The results from the control site (Fig. 4) corroborate this as the subsurface
control soils had higher moisture content. Moisture content of soils in % six months
after the oil spill indicated an increase of 11.8 and 25.0% for surface and subsurface
Fig. 3. Seasonal variation in soil-pH two months and six months after the oil spill at Idu-Ekpeye in Niger
Delta, Nigeria
CHEMISTRY & BIODIVERSITY – Vol. 3 (2006) 427

soils, respectively (when compared with results obtained two months after), the
difference being statistically significant (P<0.05). This indicates that, six months after
the oil spill, the moisture content of the polluted soil increased. This could be ascribed
to water droplets which initially adhered to the oil-in-water emulsion being released to
the soil, resulting in the wetting of the inner parts of soil aggregates [11]. The higher
moisture content of the subsurface depth of the affected soil on second sampling
confirms this.
Increased moisture content of the polluted soil six months after the spill might also
be attributed to the rainfall that was predominant at the region prior to the second
sampling. Moisture content of soils usually affects soil formation, erosion, and
aggregate stability. It is essential for photosynthesis and nutrient mobility. High
moisture content, for instance, may result in the leaching of organic nutrients. This,
therefore, means that high moisture content may prevent the full use of all organic
nutrients by the organisms involved in organic matter decomposition and thus
hydrocarbon attenuation.
The pH of the affected soils (Fig. 3) generally fell within the acidic range of the pH
scale, implying that the soils are acidic; however, soil samples collected six months after
the spill were more acidic with % decrease in pH of 15.22 and 14.37 for surface and
subsurface soils, respectively. This higher pH of the first samples and a reduction in the
soil-pH (increased soil acidity) of the second samples may be ascribed to the
introduction of petroleum hydrocarbons to the study site as a result of the oil spillage,
and subsequent degradation which might have led to the removal of these hydro-
carbons as corroborated by qualitative and quantitative results of the GC analyses
earlier discussed. These attenuation processes result in increased activity of hydro-
carbon utilizers whose hydrocarbon-oxidizing potentials might have already been
CHEMISTRY & BIODIVERSITY – Vol. 3 (2006)428
Fig. 4. Moisture content, total organic carbon (TOC) and total organic matter (TOM) of soil samples
from Idu-Ekpeye oil polluted site in Niger Delta, Nigeria

increased due to prior exposure to hydrocarbons (as depicted by the total hydrocarbon
content (THC ), total organic carbon (TOC), and total organic matter ( TOM) of the
control site. This will have led to an enhanced production of organic acids, as these
organisms feed on and oxidize the spilled oil. Again, this explains the fall in soil-pH.
The fall in pH may also be as a result of the evolution of carbon(IV) oxide (CO2) from
the organic acids produced and the release of hydrogen ion (Hþ) to the soil [12][13] .
That the pH of the control site is lower might be due to a history of pollution of the site
as depicted by its THC result. pH is indeed a master variable that affects nearly all soil
properties; chemical, physical, and biological [14–16]. Therefore, such an increased
acidity at the study site may have far reaching implications on agricultural productivity,
since the amounts of acid or alkali present in soils determine the availability of many
nutrients and minerals, which influence the growth and maintenance of crops [17– 19].
The TOC content of polluted and control areas are presented in Fig. 4. On the first
and second instances of sampling (i.e., two and six months after the oil spill), results
showed that the subsurface depth of the polluted soil had the highest organic carbon
content. A comparative analysis of the results showed that the samples collected two
months after had higher organic carbon content. The introduction of petroleum
hydrocarbons (as additional source of carbon) to the soil by the oil spillage may account
for this. The marked reduction in the organic carbon content of the soil six months after
the spill may be due to the enhanced action of hydrocarbon degraders, which feed on
the hydrocarbons preferentially as their sole source of energy. The percentage decrease
in organic carbon content of the soil, 84.05 and 86.30 for surface and subsurface depths,
respectively, implies that there was severe hydrocarbon depletion at the site [14].
Soil organic matter is obtained in the soil as a result of the death, and subsequent
decay of plants and animals. It contributes to soil productivity in several ways, although
there is no direct qualitative relationship between soil productivity and soil organic
matter. The TOM results (Fig. 4) indicate that the subsurface depth of the oil-polluted
area had the highest organic matter content at the first and second sampling instances.
This might be as a result of the impact of the oil spillage on plants and animals. The
death and decay of these organisms over time might have resulted in increased organic
matter content of the oil-spillage area. However, the added carbonaceous material in
the form of the released hydrocarbon would have contributed, at least in part, to the
relatively higher amount measured from the affected plots. Comparatively, the TOM
showed a percentage difference of 81.39 and 82.28 (at surface and subsurface soil
depths, resp.) within sampling intervals of two and six months after spillage. Again, this
has provided corroborative evidence on degradation and hydrocarbon attenuation at
the affected site. The hydrocarbons will be associated with sorbed organic matter in the
soil, and hence the strength of the hydrocarbon sorption will vary according to the
nature of the hydrocarbon and organic matter content of the soil. However, those
hydrocarbons that most strongly sorbed onto the soil organic matter will be most
resistant to loss or alteration by weathering.
Heavy Metals. Heavy metals were detected in varying concentrations in the oil-
impacted area (Fig. 5). The results show a mean concentration ( S.E) of 1.04 0.04
for nickel, 0.03 0.01 for lead, 2.93 0.04 for chromium and less than 1 103mg/kg
(below detectable limit) for vanadium, arsenic, and cobalt at surface depth on first
sampling. All metals were below detectable limit at subsurface depth on first sampling.
CHEMISTRY & BIODIVERSITY – Vol. 3 (2006) 429

The results for the second samples collected six months after the oil spill indicate a
mean concentration of 0.490.44 for nickel, 1.790.28 for chromium, and below
detectable limit ( <1103mg/kg) for vanadium, lead, arsenic, and cobalt at surface
depth. At subsurface depth, nickel had a concentration of 0.56 0.20, chromium had
1.700.55, and vanadium, lead, arsenic, and cobalt were still below detectable limit.
These results show significantly higher concentration of nickel and chromium at surface
depth for the first samples, thus implying a difference of 52.28% for nickel and 38.90%
for chromium at this depth between the first and second samples. The heavy-metal
concentration at subsurface depth was <110 3mg/kg for the first samples but was
above this for the second samples. This may be a result of the tilling of the soil and
construction of secondary windrows, which might have led to the mixing of the top and
subsoils, thereby enhancing the penetration of these heavy metals [15].
Conclusions. – The composition and distribution of petroleum hydrocarbons at the
Idu-Ekpeye oil-spillage site in Niger Delta, Nigeria, showed that the spilled oil was still
fresh on site, by the time of the first sampling (two months after) with significant
hydrocarbon contamination in the kerosene range (n-C10 –n-C14 ), especially of the n-
C12 and n-C13 fractions. However, six months later, the results showed complete
disappearance of the n-C8–n-C23 hydrocarbon fractions including the isoprenoids
(pristane and phytane), leaving the n-C13,n-C14 ,n-C19 , and n-C30 hydrocarbons. These
Fig. 5. Seasonal variation in the heavy-metal concentration of soils two months and six months after oil
spill at from Idu-Ekpeye in Niger Delta, Nigeria
CHEMISTRY & BIODIVERSITY – Vol. 3 (2006)430

provide substantial evidence of attenuation of hydrocarbons at the affected site. The
increase in moisture content, decrease in THC, TOC, TOM, and the fall in the soil-pH
of the affected area six months later were also positive indicators to the on-going
attenuation at the site. The pristane/phytane ratio of the spill samples suggested an oxic
depositional environment of a probable non-waxy, marine-derived organic matter. That
pristane hydrocarbon was more highly abundant than phytane affirmed that the spilled
oil was genetically oxic.
The authors thank the management of Shell Petroleum Development Company of Nigeria (SPDC),
Port Harcourt, for allowing the use of their Preparatory Chemistry Laboratory and facilities for chemical
analysis.
Experimental Part
Site Description and Sample Collection. The study site is located in Idu-Ekpeye, Ahoda West Local
Government Area of Rivers State, Nigeria. Sampling was carried out within intervals of two months and
six months after spill; first sampling exercise was on April 04, 2004. Sample area and technique were
adopted after Osuji and Adesiyan [14] .
Oil Extraction and Chromatographic Analysis. Homogenized samples (5 g) were accurately weighed
into clean, dry beakers. The weighed samples were extracted with 10 ml of hexane, resp., and passed
through a filter paper. The extract (the hydrocarbon/hexane mix) , now ready for GC, was injected into a
Varian model 3400 gas chromatograph with the following operational conditions: flow rate (H2: 30 ml/
min, air : 300 ml/min and N2: 30 ml/min) ; injection temp.: 508, detector temp.: 3208; recorders voltage
(ImV); and chart speed : 1 cm/min. For interpretation of results, the GC recorder was interfaced to a
Hewlett Packard (hp) Computer (6207AA Software, Kayak XA PIT/350 W/48 megabytes CD-ROM).
The chromatograms were quantified with respect to the internal standards.
Estimation of Hydrocarbon Content, Moisture Content, pH, Electrical Conductivity, and Soil
Nutrients. Total Extractable Hydrocarbon Content. Total extractable hydrocarbon content (THC ) was
estimated by the method of Osuji and Adesiyan [14] . Each soil sample (5 g) was weighed out and
transferred into a 500-ml volumetric flask. Into this was added 50 ml of xylene. The xylene/soil mixture
was shaken vigorously for 5 min and filtered into 400-ml cylinder. The volumetric flask and solid
materials were rinsed properly with 500 ml of xylene and filtered again into the cylinder. The xylene/oil
extract was thereafter placed in cuvette wells, and its absorbance was determined with a Hack DR/2010
particle data logging spectophotometer. A calibration curve was obtained by measuring the absorbance
of dilute standard solns. of lease oil (Bonny Light/Bonny Medium crude oils), prepared by diluting 2.5,
5.0, 10.0, 20.0, 25.0, and 30.0 ml of the lease oil with 50 ml of xylene soln. THC was calculated after reading
the absorbance of the extract from the spectrophotometer, extrapolating from the calibration curve and
multiplying by an appropriate dilution.
Soil-pH and Electrical Conductivity. To 5.0 g of each soil sample (in a sample cell) was added 50 ml of
dist. H2O. The lump of the soil was stirred to form homogenous slurry, then the pH-meter probe (Jenway
3015 model) was immersed into the sample and allowed to stabilize at 258, and pH of sample was
recorded. Conductivity was measured with the conductivitymeter (Jenway 4010 model) as described by
Osuji and Onojake [15].
Determination of Moisture Content. A constant weight of watch glass was obtained, and, thereafter,
20 g of sample was weighed into the watch glass, and transferred into the oven for 1 h at 1108. The samples
were cooled inside a desiccator for 30 min before a constant weight of the sample and watch glass after
heating and cooling was recorded. Moisture content was estimated as:
CHEMISTRY & BIODIVERSITY – Vol. 3 (2006) 431

% Moisture Content ¼[W1(W3W2)]100 / W1
where
W1¼weight of sample,
W2¼constant weight of watch glass, and
W3¼weight of sampleþwatch glass after heating and cooling.
Total Organic Carbon (TOC) and Total Organic Matter (TOM) . TOC and TOM were measured by
the modified Walkey– Black wet oxidation method [20] as follows : 1 g of soil sample was weighed into a
500-ml flask, and 10 ml of K2Cr2O7and 20 ml of conc. H2SO4were added. To the mixture were further
added 200 ml of dist. H2O, 10 ml of H3PO4, and five drops of diphenylamine indicator before titrating
with 0.5n(NH4)2SO4Fe. A blank titration (without 1 g of soil) was thereafter carried out, and percent
TOC was calculated as:
%TOC¼Blank TiterSample Titer0.003100 / Sample Weight
%TOM¼TOC [%]1.724
where: 1.724 ¼conversion factor;
(i.e. %TOM¼%TOC 100 / 58 ; since TOC is 58% of TOM) .
Statistical Analysis. Standard Error (SE ) was given as: SE ¼SD / N1/2 where SD is standard
deviation and Nis the number of replicates. SE was estimated at 95% Confidence Limit (CL) by
multiplying by 1.96
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Received December 28, 2005
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