effect of exposure time to a mutagenic agent on the growth and performance of pumpkin (telfairia...

8/10/2019 EFFECT OF EXPOSURE TIME TO A MUTAGENIC AGENT ON THE GROWTH AND PERFORMANCE OF PUMPKIN (TELFAIRIA OCCIDENTALIS) IN AN OIL-P

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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, DeJongreported that oil spill

causes unsatisfactory growth in plants. Anoliefo and Edegbai (2000) reported that oil polluted-

soil affects root elongation, plant height and emergence. Atlas and Bartha in 1973reported 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


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(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 etal., 2000,

Huang etal., 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 etal., 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


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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 etal., 1999).The spillage of oils causes the release of heavy

metals and hydrocarbons which eventually get absorbed into plants tissue (Zaman and Al-

Sidrawi, 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, Rossreported 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


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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.

occidentalishas only two species; T.pedata hooker of East Africa and T. occidentalishooker 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 etal., 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


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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


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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 etal., 2012).


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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 Telfariaoccidentalis, 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.


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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




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.


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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


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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 K2Cr2O7was 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 K2Cr2O7meq 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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Where:

Instr. = Instrument

Recip = Reciprocal of slope

Vol. = volume

Wt. = weightCf = 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 soil8.99

2.22 Determination of Available Phosphorus

Extraction of Available Phosphorus:

An extracting solution (0.03 M NH4Fin 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 NH 4Fin 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.


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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 H 2SO4was

prepared by making 136 ml of conc. H2SO4to 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 NH 4OH 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 (1020) 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) = (VB) 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 solutionWt = 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

5 g

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.


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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, NO2and 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


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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.


NO2(mg/l as mg/l for soil) = IR x SR x Colour Vol.

Aliquot taken


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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 GelatineBaCl2reagent 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.


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


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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 riskWhen 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 accumulantConcentration 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.


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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

K 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, ECelectric conductivity,

TNtotal nitrogen


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Table 2 shows the heavy metal composition and total hydrocarbon contents of the soil before

Telfariaoccidentalis, 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 V THC

mg/kg

337.9 12.2 27.3 15.9 9.49 8.60 10.1 7.09 5.91 943.21


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

Telfariaoccidentalis, Hook. in an oil polluted soilare 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 6.50cm2for 1 hour exposure time to NH2OH HCL

to 12.56cm2for 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.


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Fig 1: Impact of NH2OH HCL on the rate of development in plant height (cm) of Telfairia

occidentalisin an oil polluted soil

Fig 2: Impact of NH2OH HCL on the rate of development in number of leaf branches of Telfairia

occidentalisin an oil polluted soil.

0

10

20

30

40

50

60

12 16 22 29

Plantheight(c

m)

Days after sowing

1hr

3hr

6hr

12hr

18hr

24hr

control

0

2

4

6

8

10

12

14

16

12 16 22 29

Noofleafbranches

Days after sowing

1hr

3hr

6hr

12hr

18hr

24hr

control


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28

Fig 3: Impact of NH2OH HCL on the rate of development in number of leaves of Telfairia


Fig 4: Impact of NH2OH HCL on the rate of development in leaf area (cm2) of Telfairia


0

5

10

15

20

25

30

35

12 16 22 29

Noofleaves

Days after sowing

1hr

3hr

6hr

12hr

18hr

24hr

control

0

2

4

6

8

10

12

14

16

18

20

12 16 22 29

Leafareacm

2

Days after sowing

1hr

3hr

6hr

12hr

18hr

24hr

control


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29

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

A= 1 hour treatment

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

B= 2 hours treatment

A

B


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30

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

C= 6 hours treatment

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

F= Control

C

F

35.50cm


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31

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

D= 12 hours treatment

50.40cm

D


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32

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) andLens 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,


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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


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34

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 vulgarisL. in an oil-polluted soil showed that P. vulgarisexhibited

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).


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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.


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APPENDICES

APPENDICE 1

Table 3: Mean for height of sprouted Telfairia occidentalis,Hook.

Table 4: Mean for number of leaf branches of sprouted Telfairia occidentalis, Hook.

Time of exposure

to treatment

12 16

Days after sowing

22 29

1hour 1.33 2.00 3.00 9.33

3hours 2.00 3.67 5.33 8.33

6hours 2.00 3.00 4.67 7.00

12hours 4.33 6.67 9.00 14.6718hours 4.00 5.33 5.33 8.67

24hours 4.00 4.67 5.67 8.67

Control 4.67 5.33 6.33 6.67

Time of exposure

to treatment

12 16

Days after sowing

22 29

1hour 5.43 9.17 16.37 29.17

3hours 9.33 20.13 28.70 38.07

6hours 8.47 15.07 17.70 24.6712hours 18.00 28.90 38.63 50.40

18hours 12.10 15.63 14.73 27.00

24hours 12.23 16.43 22.33 35.50

Control 17.20 22.90 28.40 35.67


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APPENDICE 2

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

Time of exposureTo treatment

12 16Days after sowing

22 29

1hour 4.33 6.00 6.00 8.33

3hours 6.33 10.67 10.67 15.33

6hours 7.00 8.00 8.00 14.3312hours 11.33 19.33 19.33 31.00

18hours 10.33 12.67 12.67 17.00

24hours 11.00 13.00 13.00 15.00

Control 14.67 16.67 16.67 20.33

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

Time of exposureto treatment

12 16Days after sowing

22 29

1hour 1.13 1.99 6.50 6.643hours 2.29 5.01 12.56 11.41

6hours 2.93 5.33 10.75 10.63

12hours 5.31 10.58 17.28 17.96

18hours 4.92 8.36 11.15 14.8424hours 2.82 3.79 5.74 13.79

Control 5.05 7.31 12.92 16.01

effect of exposure time to a mutagenic agent on the growth and performance of pumpkin (telfairia...

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