CHAPTER ONE

1.0 INTRODUCTION
Africa as a continent is faced with environmental degradation, un-controlled population growth
and low productivity in agriculture. The degradation of the environment is mostly caused by
spills from oil and this in turn affects agricultural productivity. Nigeria is a food deficit country
just like other countries in the continent and most families in rural and even urban communities
are not able to provide in their diet, adequate nutrient and thousands of children suffer retarded
physical growth and development. The deficiencies in nutrient, affects the health and economic
productivity of the adult population directly or indirectly (Ikhajiagbe, 2003). Pollution caused by
oil spill is a predominant feature in Nigeria because it is a major oil-producing country. The
spills could either directly pollute the environment i.e. from crude oil or indirectly pollute the
environment, i.e. from waste engine oil (Ikhajiagbe et al., 2013).
Over 734 cases of oil spills were reported between 1978 and 1980 in Nigeria (Awobayo, 1981).
The spills from oil are destructive both to vegetation and animal population found in the soil due
to their contact toxicity and hydrocarbon, which reduces the level of oxygen and causes
anaerobic conditions to set in. This causes harm to the roots of plants (Bossert and Bartha, 1984).
Generally, oil spillage on soil causes retarded growth in plants (Gill and Sandota, 1976, Glouse
et al., 1980, Atuanya, 1980, Ekpo and Nwankpa, 2005). In 1980, DeJong reported that oil spill
causes unsatisfactory growth in plants. Anoliefo and Edegbai (2000) reported that oil pollutedsoil affects root elongation, plant height and emergence. Atlas and Bartha in 1973 reported that
crude oil is a complex mixture of hydrocarbon, classified into aromatic, aliphatic and alicyclic
compound. The constituents of hydrocarbon are known to adversely affect the different biomass

(Walker and Colwell, 1976), and thus attracting much attention to the subject of oil spill and
pollution on agricultural lands. An essential product of petroleum which helps to reduce
frictional forces and contacts between metal surfaces of engine is lubricating oil, produced by the
vacuum distillation of crude oil (Kalichevsky and Peter, 1960). Lubricating oil is used by motor
mechanics and artisans in vehicles, heavy duty machines and generating sets. WEO is obtained
after servicing engines, and subsequently extracted (Anoliefo and Vwioko, 2001; Ogbo et al.,
2006). The improper disposal of waste engine oil (WEO) in drainages, sewages, plots and lands
by service stations results in soil pollution (Odjegba and Sadiqi, 2002). While new lubricating oil
contains only very low concentrations of polyaromatic hydrocarbon (PAH) (Wang et al., 2000,
Huang et al., 2004), waste engine oil may contain foreign substances such as polychlorinated
biphenyls.
Engine oil collected from automobile which has covered up to 3,000km contains higher
concentrations of polyaromatic hydrocarbon (PAH). Polyaromatic hydrocarbons have very low
water solubility and often tightly bound to soil particles. Heavy metals such as Al, V, Pb, Ni and
Fe which were undetected in unused engine oil, gave a high milligram per liter values in used or
waste engine oil (Whisman et al., 1974). Soil pollution as a result of WEO, an indirect source of
pollution, is more devastating and widespread than that caused by crude oil, a direct source of
pollution, as a result of explorative activity (Atuanya, 1980). A study carried out showed that
crude oil pollutant caused a shortage of air in the soil due to the displacement of oxygen from the
spaces between particles of soil. It also caused retard growth, results in dehydration of plants and
chlorosis of leaves (Rowell, 1977). Some plants have however shown resistance to Waste Engine
Oil (Anoliefo and Vwioko, 2001) and Waste Engine Oil at minimal concentration in soil,
stimulate growth (Anoliefo and Edegbai, 2000). When microorganisms in the soil come in
2

contact with oil, the initial reaction is a reduction of the activities as a result of reduced air
availability. This results from the selective destruction of aerobic fungi and bacteria, allowing
only the resistant and adaptive strains to thrive (Odu, 1981). Studies by Roscoe et al., (1989);
showed that there was an increase in anaerobic microorganisms in crude oil polluted soil.
In 1970, Baker reported that when oil penetrates and accumulates in plants, it causes damage to
cell membrane and leakage of cell contents. Erhenhi and Ikhajiagbe (2012) reported that
morphological observations of plants in oil polluted soil showed leaf chlorosis and necrosis.
Fertile plots for home gardens are fast disappearing and peasant gardeners have resorted to
roadside cultivation despite its consequent erosion risks. Large amounts of inorganic fertilizers
are needed to sustain reasonable growth for subsequent yield. WEO polluted soil is of great
concern not only because it makes agricultural land unsuitable but because it also contaminate
sources of drinking water (Schwab et al., 1999). The spillage of oils causes the release of heavy
metals and hydrocarbons which eventually get absorbed into plants tissue (Zaman and AlSidrawi, 1993) and at certain levels of intake, constitute serious health hazards to humans
(Martin and Griswold, 2009). Oil spill causes the top soil to appear laminated or layered and
prevent water from penetrating the soil from above but could enter easily from the sides (Adams
and Jackson., 1996). In 1994, Ross reported that soil polluted with Waste Engine Oil becomes
water logged; inducing several stresses on the plant; ranging from changes in structure and
configuration of enzymes, leading to changes in tissue contents of proline and ABA and induces
the closure of stomata along with its effects. Polluted soil could also become unsuitable due to
increase in the toxic levels of elements (Udo and Fayemi, 1975). Despite the numerous
challenges confronting the exploration of oil in Nigeria, the petroleum industry remains one of
the biggest income earners for the country. It is therefore necessary to explore plant resources to
3

meet the growing human needs and our focus should be to identify plants that can survive in an
oil-polluted soil. One of such plant is fluted pumpkin (Erhenhi and Ikhajiagbe, 2012).
Pumpkin (Telfairia occidentalis, Hook.) is a vegetable crop belonging to the family
Cucurbitaceae. The crop is grown in South Eastern and some parts of South- South Nigeria. T.
occidentalis has only two species; T. pedata hooker of East Africa and T. occidentalis hooker of
West Africa. It is a perennial woody plant, grown for its seeds and leaves which are very
nutritious (Odoemena, 1991). They have stems that are puberulous and cylindrical at maturity
with branched and spirally coiled tendrils. It is propagated by seed and common names include
Fluted gourd, Fluted pumpkin and Ugu. It does not occur in the wild but when encountered,
could be as a result of an escape from cultivation (Irvine, 1969) the female plants have longer
vegetative growth and development and the flower is solitary while the male inflorescence is a
raceme. The female bears the pod that carries the highly nutritious seeds. The fruit of the plant
can weigh up to 13kg. It is a creeping plant and grows very well when stacked to bamboo sticks.
Tindall in (1983) reported that pumpkin leaf contains 6% nitrogen, 0.6% phosphorus, 0.4%
potassium, 37% protein and 180ppm manganese while the seed contains 30% protein, 10g
carbonhydrate, 50g fat, 2g fibre, 10mg iron, 0.1mg vitamin A, 0.2mg thiamine, 40mg calcium
and 2mg nicotinamide. The lack of alternative lands and inorganic fertilizer are some of the
constrain facing the cultivation of pumpkin in Nigeria (Odiaka et al., 2008).
There is an alteration of both agricultural activities and ecosystem, when the soil is polluted and
the processes that could be used to remove these hydrocarbon and heavy metals are the physical,
chemical and biological processes respectively (Okoh, 2006). The most widely used procedures
are the chemical and physical methods. These methods are however not favorable as they
introduce harmful materials into the environment (Davis and Wilson, 2005). The most suitable
4

technology for cleaning spills is the bioremediation method, which must be specific for a
particular site; haven met some conditions like the type, quantity and toxicity of contaminant
chemicals present and the indigenous microbial population (Ikhajiagbe and Anoliefo, 2011).
Other remediation technologies include the addition of nutrient to stimulate the activities of host
microbial community. In the presence of favorable environmental condition, there is an increase
in the growth of microbial population which results in faster degradation of poisonous materials.
Some other technologies (phytoremediation and fungal remediation) have been used to clean up
polluted soils and underground water (Ikhajiagbe and Anoliefo, 2011). Apart from remediation,
it is even more important to understand whether local plants, particularly crops, are tolerant to
the inherent environmental problem.
Telfairia occidentalis, Hook. a very commonly cultivated crop by resource poor persons, who
cultivate them on plots that may have been contaminated with waste engine oil (WEO), has been
observed (undocumented) by the researcher that in certain areas like Ogida and Uselu, may use
organic manure (poultry droppings) to improve their yields. In the final analysis, this study is
aimed at highlighting the effect of Waste Engine Oil (WEO), on the physico-chemical
parametres of the soil and on Telfairia occidentalis, Hook. The improved growth and survival of
plants in soil media depends greatly on its inherent genetic capabilities. Some of these genetic
attribute have been reported (Mensah, 2012) to be enhanced by the use of mutagenic agents like
hydroxylamine hydrochloride.
Mutation is a tool used to study the function of genes as the building block of plant
development and growth to produce raw materials for the improvement of the genetic make-up
of economic crops, (Adamu and Aliyu, 2007). Favorable mutations at high frequencies (ionizing
radiation and chemical mutagens) are induced using various mutagenic agents. (Ahloowalia and
5

Maluszynski, 2001) Chemical mutagens have both positive and negative effects and are the one
cause of mutations in living organisms. Many of these chemicals have clastogenic (chromosome
damaging) effects on plants through reactive oxygen-derived radicals (Yuan and Zhang, 1993).
These effects can occur both artificially and spontaneously. Chemical mutagens produce changes
in the function of protein but do not abolish their functions as deletions mutations do (Van der
Veen, 1966). Coe and Neuffer, 1977; Mashenkov, 1986; Ricardo and Ando 1998, reported that
many workers have confirmed the role of chemical mutagens in enhancing the genetic variability
of higher plants which is fundamental to successful breeding programs in sexually and
vegetatively propagated plants. The mutagens produce resistance in crop that is susceptible,
improving their yield and quality against pathogens (Van et al., 1990 and Bertagne-Sagnard et
al., 1996). (Ikhajiagbe et al., 2006) reported that crop improvement with mutagens helps
researchers to understand the mechanism of mutation induction, quantify the frequency as well
as the pattern of changes in different selected plants. Mutagenic agents have been used to
improve major crops such as wheat, barley, peanut, cowpea and cotton, which are seed
propagated. Studies by Ikhajiagbe et al, 2012, shows that sodium azide at low concentrations of
0.016 % and 0.031 % gave significant changes in vegetative and yield parameters of cowpea but
at high concentrations of 0.125 % and 0.25 %, were deleterious. Rao and Siddiq (1977), Mensah
and Akomeah (1992, 1997), Srivastava and Singh (1996) also reported that mutagenesis is a
potential tool for the improvement of Oryza sativa, Vigna unguiculata, and Cajanus cajan
respectively. A proven way of creating variation within crop variety and introducing desired
attributes that were either lost to evolution or cannot be found in nature is induced mutation
(Ikhajiagbe et al., 2012).

Specific objectives of the present study include the following:

1. To investigate the mutagenic effect of hydroxylamine hydrochloride on some growth
and yield parameters of T. occidentalis, Hook. under environmental stress occasioned
by oil pollution.
2. To ascertain whether the pretreatment of T. occidentalis, Hook. seeds with
hydroxylamine hydrochloride before sowing, can enhance its survival on oil polluted
soil.

CHAPTER TWO
2.0 MATERIALS AND METHODS
2.1 Study Area
The study was carried out on the field behind the Animal House in the Department of Animal
and Environmental Biology, University of Benin, Benin City.

2.2 Seed Collection

The seeds of Telfaria occidentalis, Hook. were purchased from Oba market in Benin City, Edo
state. After purchase, they were stored on open tray and left to the open air.

2.3 Soil Collection and Preparation

Soil used was collected from an area measuring 50 m x 50 m marked on a farmland on the main
campus of the University of Benin, Benin City. Top soil of about 0-10 cm was collected for the
present study (Ikhajiagbe and Anoliefo, 2012) behind the Animal House, Department of Animal
and Environmental Biology, University of Benin, Ugbowo campus, Benin City. The plot
consisted mainly of Panicum maximum, Sida acuta prior to the cultivation. The soil
physiochemical parameters were determined to ascertain the physiochemical nature of the soil.
Seven (7) kg of the soil was weighed into 21 plastic buckets. Uniform perforations were made at
the bottom of each bucket. This was to allow excess water to drain out of the soil.

2.4 Amendment of soil with waste engine oil

Soils in the buckets were divided into two sets. To the first set was applied waste engine oil
(WEO) in one concentration on the weight basis of 2.5w/w oil in soil. This was done by mixing
0.175g WEO in 7kg soil. The second set (3 buckets) was left un-polluted to serve as control.
There were 3 replicates for each exposure time. These buckets were left exposed to prevailing
weather condition for 2 months before sowing. This was to allow for a natural recovery process
of the soil to take place. The field was constantly cleared to remove un-wanted weeds during the
period of fallow until the buckets were ready for use.

2.5 Preparation of Mutagenic Agent

Hydroxylamine hydrochloride solution was used for the present study as a mutagenic agent. The
solution was prepared on weight basis by dissolving 0.312g of hydroxylamine hydrochloride in
1000ml of water.

2.6 Pre-treatment of Seeds with Hydroxylamine Hydrochloride

Seeds of pumpkin were submerged in hydroxylamine hydrochloride for 1 hour, 3 hours, 6 hours,
12 hours, 18 hours and 24 hours respectively. The treated seeds were washed in running water to
remove the excess chemicals and were taken to the field immediately for sowing. They were
sown directly into the 2-month old waste engine oil polluted soil. Un-treated seeds were sown
into the 3 un-polluted buckets which served as control. Planting was done in the evening, just
beyond sunset, Klu et al, (2000).
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2.7 Experimental Design

The experimental design chosen was the complete randomized design (CRD) following the
assumption of homogeneity of the experimental plot in use. Treatment was randomized over the
whole plot. Each treatment consisted of 3 replicates. The buckets containing the polluted soil
were properly labeled with the hours of exposure of the seeds to the mutagenic agent, to avoid
misidentification.
Exposure of seeds to mutagenic agent for 1 hour .. x 3 reps
Exposure of seeds to mutagenic agent for 3 hours.. x 3 reps
Exposure of seeds to mutagenic agent for 6 hours ...x 3 reps
Exposure of seeds to mutagenic agent for 12 hours.. x 3 reps
Exposure of seeds to mutagenic agent for 18 hours.. x 3 reps
Exposure of seeds to mutagenic agent for 24 hours.. x 3 reps

2.8 Parameters Considered

2.9 Plant height
A seed was sown per bucket and the height of the plant (length of the main axil of the plant from
the soli level to the tip of the plant) was taken with the aid of a transparent calibrated ruler,
(Erhenhi and Ikhajiagbe, 2012). This was done periodically.

10

2.10 Number of leaves

Telfaria occidentalis, Hook. is a trifoliate plant and only trifoliate leaves were considered.
(Erhenhi and Ikhajiagbe, 2012). This was done periodically by counting.

2.11 Number of leaf branches

The number of branches emerging from the main stem was recorded periodically.

2.12 Total leaf area

The total leaf area (surface area of leaflet) was calculated as
Leaf area= Maximum length x width x 0.65
The maximum length and width of the leaflet was determined by aid of a transparent calibrated
ruler.

2.13 Soil Physiochemical Analyses

Soils were dried at ambient temperature (22-25oC), crushed in a porcelain mortar and sieved
through a 2-mm (10 meshes) stainless sieve. Air-dried <2mm samples were stored in polythene
bags for subsequent analysis. The <2mm fraction was used for the determination of selected soil
physiochemical properties and the heavy metal fractions as well as PAH.

11

2.14 Total organic carbon (TOC) and total organic matter (TOM) contents
Half a gram (0.5 g) of each air-dried soil sample was put in a conical flask and 2.5ml of 1N
potassium dichromate solution K2Cr2O7 was added and swirled gently to disperse the sample in
the solution. Five milliliters (5ml) of concentrated tetraoxosulphate (VI) acid was added rapidly,
into the flask and swirled gently until sample and reagents were mixed and finally swirled
vigorously for about a minute. The flask was allowed to stand in a fume cupboard for 30
minutes. Five to ten (5- 10) drops of indicator were added and the solution titrated with 0.5N
FeSO4 to maroon colour. A blank determination was carried out to standardize the dichromate
(Nelson and Sommers, 1982). TOC and TOM contents were calculated as follows (Osuji and
Nwoye, 2007):
TOC (%) = (meq K2Cr2O7 meq FeSO4) x 0.003 x 100 x 1.3
Weight of sample (g)
Where:

meq K2Cr2O7 = 1N x 2.5 ml

meq FeSO4 = 0.5N x Volume of titrant in ml
0.03 = Milliequivalent weight of carbon
1.30 = Correction factor

TOM (%) = TOC (%) x 1.724

Where: 1.724 = Conversion Factor, i.e. % TOM = % TOC x 100 /58
Since TOC is 58 % of TOM

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2.15 Soil PH
Twenty grams (20 g) of air- dried soil were sieved and 20 ml of distilled water was added to it
and allowed to stand for 30 minutes. The mixture was stirred occasionally with a glass rod. The
PH was determined by inserting the pH meter into the suspension.

2.16 Mechanical Analysis (Particle Size Distribution)

One hundred grams (100 g) of the soil was weighed out. If it was very clayey, 50 g was used
instead. This was placed in the 1 litre- shaking bottle, and then 50 ml Calgon solution, 3 ml of
sodium hydroxide and 200 ml of water were added. The mixture was then properly shaken on the
machine for 3 hours. It was then removed and transferred quantitatively to the mechanical
analyses cylinder. The volume was made up to the first (1130 ml) mark with water. The cylinder
was shaken by inverting it a few times and later placed on the bench and the time read.
After 41/2 minutes the hydrometer was inserted, and at 5 minutes the scale was read. Whenever
there was more froth on the surface of the liquid, one or two drops of amyl alcohol were added
before inserting the hydrometer. The hydrometer was then withdrawn and the process repeated 5
hours later. With 100 g soil sample being used for the determination, the result gave directly the
percentage silt and clay (1st reading) and clay (2nd reading).

2.17 Determination of Soli Nitrogen

Digestion of Soil Nitrogen by Micro- Kjeldahl Digestion
Nitrogen in the soil was determined by kjeldahl digestion, and the resulting ammonium ion
measured colorimetrically. Elements such as iron and manganese, which may interfere in the

13

alkaline medium during colorimetric determination, were first complexed with sodium potassium
tartrate. The ammonium was determined colorimetrically as the indophenols blue complex by
reaction with alkaline sodium phenate and sodium hypochlorite.
Some 0.2 g of finery ground soil was weighed into 30 ml kjeldahl digestion flask; and one tablet
of catalyst and 4.0 ml of conc. H2SO4 were added to it. This was properly shaken to ensure
complete mixing of the soil and catalyst mixture. The flask was placed on the heater and digested
for 45 minutes. At the completion of the digestion, the mixture was clear. The flask was then
removed from the heater. It was cooled until just warm to touch, and then 10 ml of the distilled
water was added. It was important that the mixture in the kjeldahl flask did not solidify before
the addition of water, as it was time consuming re-dissolving the solids. Solution was decanted
through a Whatman filter paper No. 42 into 100 ml volumetric flasks. The kjeldahl flask was
washed with 3 small aliquots of distilled water, adding all the washings into the volumetric flask
via the filter paper and made up to volume. Nitrogen was determined in the filtrate.

2.18 Colorimetric Determination of soil Nitrogen

Forty five (45) g of Potassium Sodium Tartrate and 15 g of Sodium Citrate were dissolved in 200
ml of water. 10 g of NaOH was added, and left to dissolve, and then made up to 300 ml. This
was filtered when cloudy. The reagent was prepared fresh each time before use.

14

2.19 Standard Nitrogen Stock Solution

Ammonium Sulphate was dried for 2 hours in an electric oven at 900 10 oC. Some 4.7 g of
dried salt was accurately weighed and dissolved in 1 litre volumetric flask with water and made
up to volume, and properly shaken. The solution contained 1000 mg/l nitrogen. A 100 mg/l
working stock solution was prepared from this.

2.20 Nitrogen working standard

From the 100 mg/l Nitrogen stock solution, standards containing 5, 10, 15, 20 and 25 mg/l
Nitrogen were prepared. To each standard, before making to mark (100 ml volumetric flask), 4
ml conc. H2SO4 and 0.95 g of anhydrous Sodium Sulphate were added. A solution containing no
Nitrogen (blank) was also prepared, but having the same quantity of acid and anhydrous Sodium
Sulphate.
Procedure
Five (5) ml of the filtrate was pipette from the digest into a 25 ml flask. Some 2.5 ml of the
alkaline phenol was added, and shaken well. 1 ml Sodium Potassium Tartrate was also added
afterwards, and also shaken well. Later 2.5 ml of Sodium Hypochlorite (bleach) was added. This
mixture was also properly shaken and the colour was left to develop, and then made to mark.
This was read colorimetrically at 630 nm. The N standards were treated as in the steps followed
above.
Calculations:
Instr. Reading x Slop Recip. x Colour Vol. x Digest. Vol. x 10-6 x 100 x Cf 1 %N
Weight of Sample x Aliquot Taken

15

Where:
Instr.
Recip
Vol.
Wt.
Cf

= Instrument
= Reciprocal of slope
= volume
= weight
= Correction factor

2.21 Exchange Acidity

Five (5) g of air dried soil was weighed into a 150 ml plastic bottle and then 50 ml of the M KCL
was added and shaken mechanically for 1 hour. This was filtered using Whatman filter paper
no.1 into a 250 ml conical flask. 3 drops of the indicator were then added, and titrated against the
0.05 M NaOH until the colourless solution turned to pink. The pink colour was neutralized with
0.05 M HCL. Then 10 ml of 1 m NaF was added to restore the pink colour. The set up was
titrated against 0.05 m HCL until colourless.
Calculations:
Exchange Acidity = 0.05 m x Titre x 20 meq/100 g soil
Al =
0.05 m x 20 x 26.98 meq/100 g soil
8.99

2.22 Determination of Available Phosphorus

Extraction of Available Phosphorus:
An extracting solution (0.03 M NH4 Fin 0.025 M HCL) was first prepared by dissolving 1.1 g of
NH4F in water and adding 4.16 ml of 6 M HCL and then made up to 1 liter. 5 g of the soil was
weighed into the plastic bottle. 40 ml of the extracting solution (0.03 M NH4 Fin 0.025 M HCL)
was added, and was stopped. This was shaken manually for 1 minute and then filtered with
Whatman filter paper No. 42. The filtrate was reserved for P determination.

16

2.23 Determination of Phosphorus

Twelve (12) g of Ammonium Molybdate was dissolved in 250 ml of water. Some 0.2908 g
Antimony potassium tartrate was also dissolved in 100 ml of water. Some 2.5 M H2SO4 was
prepared by making 136 ml of conc. H2SO4 to 1 liter. The ammonium molybdate and antimony
potassium tartrate were then added to 1000 ml of 2.5 M H2SO4; mixed thoroughly, made 2000ml
and stored in a plastic container in a cool dark compartment.
A small amount (0.53) g of Ascorbic Acid was dissolved in 200 ml of reagent as prepared above,
and then the mixture was prepared as required since it does not keep for more than 24 hours.
Other reagent required were 0.25 % p-Nitrophenol, 2 M HCL, 2 M NH4OH and a P-Standard
Stock (100 mg/l) that was prepared by dissolving 0.4394 g of KH2PO4 in water and made to
1liter. Pipetting 0, 1, 2, 3, 4 and 5 ml from the 100 ml stock solution, intermediate standards of 0,
2, 4, 6, 8 and 10 mg/l were then prepared each in 50 ml flask.
Five (5) ml of the filtrate or supernatant was pipette into a 50 ml flask, while the pH of the
solution was adjusted to 5 by adding 3 drops of the p-nitrophenol, and when a yellow colour was
not obtained, some drops of 2 M NH4OH were added until yellow. Then 2 M HCL was added
drop-wise until colourless (the pH was now between 3 and 5).
Water was added to 30 ml, and then 10 ml of the Ascorbic Acid reagent was added. This was
made to volume and read spectrophotometrically at 660 nm.
Calculation:
P (mg/l) =

Inserting x Slope recip. x Colour Vol. x Extract Vol.

Weight of x Sample x Aliquot Taken

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2.24 Determination of Exchangeable Bases

Extraction of Exchangeable Bases:
Five (5) g air-dried soil was weighed into a 5 g plastic bottle. 100 ml of neutral 1 M ammonium
acetate was added, and the mixture was shaken mechanically for 30 minutes and filtered
thereafter, using a No. 42 Whatman filter paper, into a 100 ml volumetric flask. This was made
up with the accurate to the mark. Na (589-nm wavelength) and K (766.5nm wavelength) were
determined with a Flame Photometer, and then Ca and Mg by Atomic Absorption
Spectrophotometer.

Calcium:
Ten to twenty (10- 20) ml soil saturation extract was pipette, having not more than 1.0 meq Ca,
into a 250- ml Erlenmeyer flask. This was diluted to 20 30 ml with distilled water; and 2- 3 ml
2 N NaOH solution was added; and about 50 mg ammonium purpurate indicator. This was
titrated with 0.01 N EDTA. The color change was from red to purple. Near the end point, EDTA
was added, one drop every 10 seconds, since the color change was not instantaneous.

2.25 Calcium plus Magnesium

Ten to twenty (10 20) ml soil saturated extract was pipette into a 250 ml flask, and diluted to
20 30 ml with distilled water. Then 5 ml buffer solution was added, and a few drops of
Eriochrome Black Indicator. This was titrated with 0.01 NEDTA until the colour changed from
red to blue.
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Calculations:
Ca or Mg (meq/L) = (V B) x N x R x 100)
Wt
Mg (meq/L) =
Ca + Mg (meq/L) Ca (meq/L)
Where:
V = Volume of EDTA titrated for the sample (mL)
B = Blank titration volume (mL)
R = Ratio between total volume of the extract and extract volume used for titration.
N= Normality of EDTA solution
Wt = Weight of air- dry soil (g)

2.26 Determination of Total Hydrocarbons

Five (5) g soil was weighed into a 100 ml plastic bottle. 25 ml of n- hexane was added, and
mechanically shaken for 10 minutes and let stand covered. It was then filtered and the filtrate
was read at 460 nm.

Preparation of THC Standard Stock, 1000 mg/l:

Some 1.18 ml of Forcados Blend Crude Oil was pipette and made to 1 litre with n- Hexane.
From this, 0, 10, 20, 40, 60, 80 and 100 mg/l working standards were prepared.
Calculations:
THC (mg/l) = Instr. Reading x Slope Reciprocal x 25
5g
This method of soil THC determination was also applicable for determining THC of plant
materials (dried).

2.27 Extraction of Ammonium Nitrate Nitrite Nitrogen and Sulphate in Soils

The extracting solution was first prepared by dissolving 100 g of the sodium acetate in 500 ml of
water, and 30 ml of acetic acid was added, and made to 1 liter with water.
19

Ten (10) g of soil sample was weighed into a 150 ml plastic bottle. 40 ml of the extracting
solution was added and shaken for 30 minutes. This was then filtered, and the filtrate reserved
for the determination of NH4-N, NO3, NO2 and SO4-2, Cl

2.28 Determination of Ammonium Nitrogen

Five (5) ml of the filtrate from the sodium acetate extract was pipette. 2.5 ml alkaline phenol,
1ml sodium potassium tartrate and 2.5 ml of sodium hypochlorite (bleach) were added; and
shaken well in between each addition. Standard was treated similarly. This was finally read
colorimetrically at 636 nm against the mg/l as blank.
NH4-N (mg/l as /g for soil) = IR x SR x Colour Vol. x Extra. Vol.
Wt. Of sample x Aliquot taken

NH4-N (mg/l as mg/l for soil) = IR x SR x Colour Vol.

Aliquot taken

2.29 Determination of Nitrate

Ten (10) ml of the filtrate was pipette into a 50 ml flask, and 2 ml of Brucine was added and
then, rapidly 10 ml of conc. H2S04. This was mixed well and let to stand for 10 minutes. The
standards were treated similarly and thereafter the samples and standards were made to mark.
This was read spectrophotometrically at 470 nm.
Calculations:
NO3 (mg/l as /g for soil) = IR x SR x Colour Vol. x Extra. Vol.
Wt. of sample x Aliquot taken
NO3 (mg/l as mg/l for soil) = IR x SR x Colour Vol.
Aliquot taken

20

2.30 Determination of Nitrite

Ten (10) ml of the filtrate was pipette into a 50 ml flask and 2 ml of 2 m HCL was added. This
was diluted to about 30 ml with water. Two (2) ml of sulphailic acid was added and stirred, and
then allowed to stand for 5 minutes. The standards were treated in a similar manner as the
samples. 10 ml of alpha-naphthylamine was added, stirred, and made to volume. Colour
development occurred at a few minutes later. The absorbance was read at 520 nm after 20
minutes.

2.31 Standard NO2 Solution (100 mg/l)

Some 0.15 g of the naphthylamine was dissolved in 100 ml of 30 % acetic acid. This was gently
heated to aid the dissolution.

Working NO2 Solution (10 mg/l):

Ten (10) ml of the stock was pipette into 100 ml flask and made to mark. From this solution, 0,
1, 2, 3, 4 and 5 ml were taken each into 5 ml flask to have 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/l when
made to mark after adding the colour-developing reagents, Note: the 0 was the extractant for
NO2
Calculations:
NO2 (Mg/l as /g for soil) = IR x SR x Colour Vol. x Extra. Vol.
Wt. of sample x Aliquot taken
NO2 (mg/l as mg/l for soil) = IR x SR x Colour Vol.
Aliquot taken

21

2.32 Determination of Sulphate

Ten (10) ml of the filtrate was pipette into a 50 ml flask. Water was added to bring the volume to
about 20 ml. 1 ml of the Gelatine BaCl2 reagent was also added to the solution. This was let to
stand for 30 minutes and then made to mark with water. The solution was properly mixed. The
standard was treated similarly. Turbidity was read at 420 nm in a spectrophotometer.
Calculation:
SO4-2 (mg/l as /g for soil) = IR x SR x Colour Vol. x Extra. Vol.
Wt. of sample x Aliquot taken
SO4-2 (mg/l as mg/l for soil) = IR x SR x Colour Vol.
Aliquot taken

2.33 Extraction of Chloride

One gram (1 g) of sample (previously dried for 1 hour at 180oC) was weighed and then mixed
with 0.2 g of calcium oxide. The mixture was then made wet with water to form a thick paste.
This was later ashed in a silica dish at 500Oc for 2 hours until all the organic matter had become
complete charred. The residue was extracted with successive 30 ml portion of hot water, filtering
each portion into a 100 ml flask. This was then diluted to the 100 ml mark. The filtrate was
reserved for chloride determination.

2.34 Computation of Contamination Factor (CF)

CF expresses the ratio between the eventual concentration of pollutant against its pre-industrial
concentration (Ikhajiagbe, 2010; Ikhajiagbe and Anoliefo, 2012b).
CF =

Concentration of pollutant
Pre- contamination reference

22

Pre-contamination reference refers to the initial concentration of a particular contaminant

(mg/kg) in the soil prior to exogenous application of source of contaminant (Ikhajiagbe, 2012)

2.35 Computation of Hazard Quotient (HQ)

HQ expresses the possibility of the contaminant being an ecological risk or a contaminant of potential
ecological concern. The hazards Quotient is expressed by the following equation:
HQ =

Measured concentration
Toxicity reference value or selected screening benchmark
When HQ > 1: Harmful effects are likely due to contaminant in question
When HQ = 1: Contaminant alone is not likely to cause ecological risk
When HQ < 1: Harmful effects are not likely

2.36 Computation of Bioaccumulation Quotient (BQ)

BQ expresses the possibility of the contaminant being significantly accumulated in plant parts, thereby
posing health threats.
The Bioaccumulation Quotient is expressed
BQ=

Concentration of accumulated pollutant in the accumulant

Concentration of accumulated pollutant in Soil (Source)

When BQ > 1=

Significant accumulation in of the pollutant is implied.

When BQ < 1=

Bioaccumulation is not of significant effect.

23

CHAPTER THREE
3.0 RESULTS
Physiochemical properties of the soil used in the present study are presented on Table1. Soil used
in the study had a pH of 5.47. Total nitrogen was 0.02%, whereas exchangeable acidity was
0.7mg/100g. Heavy metal contents of soil include Fe (337.9mg/kg), Mn (12.2mg/kg), Zn
(27.3mg/kg), Cr (9.49mg/kg), Pb (10.1mg/kg), Ni (7.09mg/kg) and V (5.91mg/kg).

Table 1: Physiochemical properties of soil used for the present study

Parameters
Units

Soil

pH

5.49

Electric conductivity

S/cm

1420

Total org. carbon

0.22

Total Nitrogen

0.02

Exchangeable acidity

meq/100g of soil

0.7

Na

meq/100g of soil

3.51

meq/100g of soil

0.39

Ca

meq/100g of soil

8.77

Mg

meq/100g of soil

6.58

Cl

mg/100g of soil

120.1

Av. P

mg/100g of soil

2.51

NH4N

mg/100g of soil

6.89

NO2

mg/100g of soil

4.13

NO3

mg/100g of soil

5.99

SO4

mg/100g of soil

75.1

EA - exchangeable acidity, Org. C- organic carbon, EC electric conductivity,

TN total nitrogen

24

Table 2 shows the heavy metal composition and total hydrocarbon contents of the soil before
Telfaria occidentalis, Hook. was sown. Fe content of the soil was 337.9 mg/kg, Mn 12.2mg/kg,
Cd 8.60 mg/kg, Lead 10.1mg/kg and Vanadium 5.91mg/kg. The total hydrocarbon content was

943.21mg/kg.
Fe

Mn

Zn

Cu

Cr

Cd

Pb

Ni

THC

mg/kg
337.9

12.2

27.3

15.9

9.49

8.60 10.1 7.09 5.91

943.21

25

The impact of hydroxylamine hydrochloride (NH2OH HCL) on some selected parameters of

Telfaria occidentalis, Hook. in an oil polluted soil are presented in fig 1 4, appendix 1 and 2.
Plant height showed progressive growth after 12 days of planting from 5.43 9.33cm to 29.17
38.07cm at 29 days after planting (DAP). The rate of development of leaf branches in plants
exposed to mutagenic agent for 3hours increased from 3.67 at 16DAP to 8.33 at 29DAP.
At 22 DAP, leaf area of pumpkin ranged from 6.50cm2 for 1 hour exposure time to NH2OH HCL
to 12.56cm2 for 3 hours exposure time. Overall, plants exposed to mutagenic agent for 12 hours,
showed better plant height, leaf area and number of branches when compared to plants exposed
to thesame mutagen for 1, 3, 6 and 18 hours, irrespective of the number of days after planting.

26

Plant height (cm)

60
50
1hr
3hr

40

6hr
30

12hr
18hr

20

24hr
control

10
0
12

16

22

29

Days after sowing

Fig 1: Impact of NH2OH HCL on the rate of development in plant height (cm) of Telfairia
occidentalis in an oil polluted soil

16
14

No of leaf branches

12
1hr

10

3hr
8

6hr

12hr

18hr

24hr
control

0
12

16

22

29

Days after sowing

Fig 2: Impact of NH2OH HCL on the rate of development in number of leaf branches of Telfairia
occidentalis in an oil polluted soil.
27

35
30

No of leaves

25

1hr
3hr

20

6hr
15

12hr
18hr

10

24hr

control

0
12

16

22

29

Days after sowing

Fig 3: Impact of NH2OH HCL on the rate of development in number of leaves of Telfairia
occidentalis in an oil polluted soil.

20
18

Leaf area cm2

16
14

1hr

12

3hr

10

6hr

12hr

18hr

24hr

control

0
12

16

22

29

Days after sowing

Fig 4: Impact of NH2OH HCL on the rate of development in leaf area (cm2) of Telfairia
occidentalis in an oil polluted soil.

28

A
Plate 1: Pumpkin exposed to 1hours treatment of NH2OH HCL 12 days after planting.
A= 1 hour treatment

B
Plate 2: Pumpkin exposed to 3hours treatment of NH2OH HCL 12 days after planting.
B= 2 hours treatment

29

C
Plate 3: Pumpkin exposed to 6 hours treatment of NH2OH HCL 12 days after planting.
C= 6 hours treatment

35.50cm

F
Plate 4: Pumpkin exposed to 24 hours treatment of NH2OH HCL 29 days after planting
F= Control
30

50.40cm

D
Plate 5: Pumpkin exposed to 12 hours treatment of NH2OH HCL 29 days after planting
D= 12 hours treatment

31

CHAPTER FOUR
DISCUSSION
Induced mutations using chemical agents, have been successfully utilized to improve yield and
yield components of various crops like Oryza sativa (Rao and Siddiq, 1977; Awan et al. 1980;
Singh et al. 1998), Hordeum vulgare (Gustafsson 1963; Ramesh et al., 2001), Triticum durum
(Sakin and Yildirim, 2004), Vicia faba (Ismail et al., 1977), Vigna unguiculata (Mensah and
Akomeah, 1992), Cajanus cajan (Srivastava and Singh, 1996), Vigna mungo (Kundu and Singh,
1981; Singh and Singh, 2001) and Lens culinaris (Kumar et al., 1995; Rajput et al., 2001; Khan
et al., 2006).
In the present study, pumpkin was treated with hydroxylamine hydrochloride for 1, 3, 6, 12, 18
and 24 hours respectively. The control was also treated with the mutagen for 24 hours but was
sown in an un-polluted soil. After polluting 7kg of the soil with 2.5w/w of WEO (0.75g), the
soil was analyzed to ascertain the values of the essential nutrients. The essential soil nutrient,
NPK had values of 0.02%, 2.51 mg/kg and 0.39 meq/100g of soil respectively. The pH was 5.49.
The study revealed that plants exposed to mutagenic treatment for 12 hours showed better and
improved yield in height, number of leaves per plant, leaf area and number of branches per plant,
compared to plants exposed to 1, 3, 6, 18 and 24 hours treatments respectively. Some of the
replicates did not emerge for plants exposed to 1, 3 and 6 hours treatment. This could probably
be as a result of the inability of the mutagen to penetrate the seed coats.
Small molecules like aromatics that can enter and get across cell membranes could cause
a reduction in membrane integrity and/or death of the cell. In spite of WEO-pollution effects,

32

pretreatment with NH2OH HCL minimally improved seedling emergence and establishment in
the polluted soil, compared to un-polluted soil, Plates 4 and 5.
The reduced seed emergence even in mutagenic treatment for 24 hours had been
explained to be due to delayed or inhibition of physiological and biological processes necessary
for seed germination which include enzyme activity, inhibition of mitotic processes and
hormonal imbalance. There were significant increases in the vegetative parameters between the
seedlings in the WEO polluted soil and those of the non-polluted soil (control). Previous research
shows that oil in the soil inhibits plant growth (Kayode et al., 2009; Ikhajiagbe and Anoliefo,
2010, 2012) and leads to decrease in biomass productivity (Amakari and Onofeghara, 1983).
Plants on oil-polluted soil could become suffocated as a result of the exclusion of air by the oil
(Udo and Fayemi, 1999). A report by De Jong (1980), also supports the claim that oil in soil,
creates unsatisfactory conditions for plant growth due to insufficient air in the soil. This may be
caused by the displacement of air from pore spaces by oil, and an increase in the demand for
oxygen brought about by the activities of oil-decomposing microorganism (Gudin and Syratt,
1975), limiting the normal diffusion processes.
Studies by Bossert and Bartha (1984) showed that crude oil penetrate the pore spaces of
terrestrial vegetation and subsequently impedes the plants photosynthetic and physiological
processes. In 1970, Baker reported that oil penetrated and accumulated in plants causing
membrane damage and leakage of the contents of the cell. The symptoms showed by plants sown
in oil polluted soil were typical of extreme nutrient deficiency in plants. Deficiency of nutrient in
plant could be directly proportional to water uptake, and as such, damages to plants were
probably due to shift in plant-water relations of the root in the polluted soil. A reduction in the

33

level of available nutrient and/ or a rise in the toxic level of elements such as manganese could
make oil-polluted soil unsuitable for plant growth (Udo and Fayemi, 1975).
Overall, plant yield was poor. Experiment carried out by Nwoko et al, (2007) on the
performance of Phaseolus vulgaris L. in an oil-polluted soil showed that P. vulgaris exhibited
reduced plant growth and yield. Mechanism of toxicity of metals tends to be dependent on the
nature of the reactivity of the metal itself. Efroymson et al., (1997), reported that heavy metal in
soil, alter or inhibit enzyme activity, interfere with deoxyribonucleic acid (DNA) synthesis or
electron transport, or may block the uptake of essential elements. The variability shown by plants
in response to the level of toxic metal may be due to a number of defenses: exclusion from the
roots, translocation in nontoxic form and the isolation of the toxic in nontoxic forms in roots or
other plant parts (Peterson, 1983).

34

CONCLUSION
Oil pollution of soil negatively impacted on the plant growth and performance of Telfaria
occidentalis, Hook. Plant performance in oil polluted soil was enhanced by pretreatment of seed
with hydroxylamine hydrochloride. Results also show that pumpkin had potentials for heavy
metal remediation, which may have also been enhanced by the pretreatment.

35

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42

APPENDICES
APPENDICE 1
Table 3: Mean for height of sprouted Telfairia occidentalis, Hook.
Time of exposure
to treatment
1hour
3hours
6hours
12hours
18hours
24hours
Control

12
5.43
9.33
8.47
18.00
12.10
12.23
17.20

16
Days after sowing
9.17
20.13
15.07
28.90
15.63
16.43
22.90

22

29

16.37
28.70
17.70
38.63
14.73
22.33
28.40

29.17
38.07
24.67
50.40
27.00
35.50
35.67

Table 4: Mean for number of leaf branches of sprouted Telfairia occidentalis, Hook.
Time of exposure
to treatment
1hour
3hours
6hours
12hours
18hours
24hours
Control

12
1.33
2.00
2.00
4.33
4.00
4.00
4.67

16
Days after sowing
2.00
3.67
3.00
6.67
5.33
4.67
5.33

22

29

3.00
5.33
4.67
9.00
5.33
5.67
6.33

9.33
8.33
7.00
14.67
8.67
8.67
6.67

43

APPENDICE 2

Table 5: Mean for number of leaves of sprouted Telfairia occidentalis, Hook.

Time of exposure
To treatment
1hour
3hours
6hours
12hours
18hours
24hours
Control

12
4.33
6.33
7.00
11.33
10.33
11.00
14.67

16
Days after sowing
6.00
10.67
8.00
19.33
12.67
13.00
16.67

22

29

6.00
10.67
8.00
19.33
12.67
13.00
16.67

8.33
15.33
14.33
31.00
17.00
15.00
20.33

Table 6: Mean for leaf area of sprouted Telfairia occidentalis, Hook.

Time of exposure
to treatment
1hour
3hours
6hours
12hours
18hours
24hours
Control

12
1.13
2.29
2.93
5.31
4.92
2.82
5.05

16
Days after sowing
1.99
5.01
5.33
10.58
8.36
3.79
7.31

22

29

6.50
12.56
10.75
17.28
11.15
5.74
12.92

6.64
11.41
10.63
17.96
14.84
13.79
16.01

44