HYDROCARBON DEGRADATION AND HEAVY METALS UPTAKE BY

SOIL POLLUTED WITH SPENT ENGINE

Ebere Omeje

UGWU, EMMANUEL CHIBUZO

REG. NO. PG/M.Sc./13/64875

HYDROCARBON DEGRADATION AND HEAVY METALS UPTAKE BY SENNA ALATA (L.)

SOIL POLLUTED WITH SPENT ENGINE

PLANT SCIENCE AND BIOTECHNOLOGY

FACULTY OF BIOLOGICAL SCIENCE

Ebere Omeje Digitally Signed by: Content

DN : CN = Webmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

UGWU, EMMANUEL CHIBUZO

REG. NO. PG/M.Sc./13/64875

HYDROCARBON DEGRADATION AND HEAVY (L.) ROXB. IN

SOIL POLLUTED WITH SPENT ENGINE OIL

PLANT SCIENCE AND BIOTECHNOLOGY

FACULTY OF BIOLOGICAL SCIENCE

: Content manager’s Name

Webmaster’s name

a, Nsukka

OU = Innovation Centre

HYDROCARBON DEGRADATION AND HEAVY METALS UPTAKE BY Senna alata (L.) Roxb. IN SOIL POLLUTED WITH SPENT

ENGINE OIL

BY

UGWU, EMMANUEL CHIBUZO

REG. NO. PG/M.Sc./13/64875

A PROJECT REPORT SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF SCIENCE [M.SC.] IN

ENVIRONMENTAL PLANT ECOLOGY

IN THE

DEPARTMENT OF PLANT SCIENCE AND BIOTECHNOLOGY, UNIVERSITY OF NIGERIA, NSUKKA

DATE: OCTOBER, 2015

CERTIFICATION

UGWU, EMMANUEL CHIBUZO, a postgraduate student in the Department of Plant Science and

Biotechnology with the registration number PG/M.Sc./13/64875 has satisfactorily completed the

requirement for the award of Master of Science Degree in Environmental Plant Ecology. The work

embodied in this project report is original and has not been submitted in part or full for any diploma or

degree of this or any other institution.

Approved By

------------------------------------ ---------------------------------------

Assoc. Prof. A. O Nwadinigwe Assoc. Prof. N. O. Nweze

Supervisor Head of Department, Plant Science and Biotechnology

------------------------------------------------------

External Examiner

DEDICATION

This write up is dedicated to God Almighty; my supervisor, Associate Professor A. O. Nwadinigwe;

my late mother, Mrs. Josephine L. Ugwu and the entire family.

ACKNOWLEDGEMENT

I am giving God all the glory for the successful completion of Masters Degree program in the

Department of Plant Science and Biotechnology, Faculty of Biological Sciences, University of Nigeria,

Nsukka. My heartfelt gratitude also goes to my loving and only living mummy, director and educator,

Associate Professor A. O. Nwadinigwe. Mummy, I do not know how to appreciate your kind gestures

in all my endeavors. May God keep blessing you for me in Jesus name, Amen. I also want to thank the

academic and non academic staff of the Department of Plant Science and Biotechnology as well as the

School of Postgraduate Studies, University of Nigeria, Nsukka for your immense support in the course

of the study. Also in my mind, is my late mother, Mrs. Josphine Ugwu who out of her passion and

desire to see the best in her children, encouraged me to take higher degree. Mummy, I do not have any

doubt about where you are now and I hope your prayer will lead us to the promise land. Also of great

importance to me are my family members headed by Mr. Wilfred Ugwu and my siblings – Fr. Ugwu,

Cpl. Ugwu, Dr. Ugwu, Mr. Emeka Ugwu and Mrs. Ogechi Mbah for their wonderful assistance in the

course of the study. Thank God for the gift of you people. My gratitude also goes to my lovely one,

Miss Ugwoke, Angela Chinenye and all M.Sc. classmates and friends who guided me in so many areas

of importance. I owe you all thanks. May God see us through in this program in Jesus name, Amen.

God bless you all.

ABBREVIATIONS

SEO --------------------------------------Spent engine oil

% -----------------------------------------Percentage

Wt --------------------------------------- Weight

Etc----------------------------------------Etcetera

TBN--------------------------------------Total base number

˚C-----------------------------------------Degree Celsius

W-----------------------------------------Weight

NPK--------------------------------------Nitrogen, Phosphorous, Potassium

g------------------------------------------Gram

l-------------------------------------------Liter

PCs--------------------------------------Phytochelatins

GSH--------------------------------------Glutathione

ml-----------------------------------------Millilit er

V/W--------------------------------------Volume per weight

GLC/MS---------------------------------Gas Liquid Chromatography/Mass Spectroscopy

FAAS-------------------------------------Flame Atomic Absorption Spectroscopy

mm--------------------------------------Millimeter

m-----------------------------------------Meter

μm---------------------------------------Micrometer

cm/min---------------------------------Centimeter per minute

μl-----------------------------------------Micro lit er

AAS--------------------------------------Atomic Absorption Spectrophotometer

Cm3--------------------------------------Centimeter cube

C14-C22-----------------------------------Fourteen carbon atoms to twenty-two carbon atoms

Mg/ml-----------------------------------Milligram per milliliter

THC--------------------------------------Total hydrocarbons

Ppm--------------------------------------Part per million

Cm2---------------------------------------Centimeter square

<------------------------------------------Less than

LIST OF TABLES

Table 1 - Studies on phytoremediation of PAH contaminants in soil----------------------------------------12

Table 2 - Composition and quantities of hydrocarbons in the unused spent engine oil, unvegetated and

vegetated soil samples of Senna alata polluted with spent engine oil----------------------------------------21

Table 3 - Composition and quantities of hydrocarbons in the unused spent engine oil and vegetative

plant parts of Senna alata polluted with spent engine oil------------------------------------------------------23

Table 4 - Percentage composition of total degraded hydrocarbons in the analyzed soil samples of Senna

alata polluted with spent engine oil ------------------------------------------------------------------------------25

Table 5 - Percentage accumulation of hydrocarbons in Senna alata polluted with spent engine oil ----27

Table 6 - Composition, quantity (ppm) and percentage of heavy metals in the vegetated and

unvegetated soils of Senna alata polluted with spent engine oil----------------------------------------------30

Table 7 - Composition, quantity (ppm) and percentage of heavy metals accumulated in the root, stem

and leaf samples of Senna alata polluted with spent engine oil-----------------------------------------------32

Table 8 - Vegetative parameters of Senna alata before and after pollution (57 and 163 days after

planting, respectively) ----------------------------------------------------------------------------------------------35

Table 9 - Root parameters of Senna alata 163 days after planting ------------------------------------------40

Table 10 - Reproductive parameters of Senna alata 294 days after planting ------------------------------50

LIST OF PLATES

Plate 1 - Senna alata seedlings, 57 days after planting (DAP) -----------------------------------------------16

Plate 2 - Senna alata plants 163 days after planting (106 days after pollution) ----------------------------38

Plate 3 - Control Senna alata showing no aerial roots produced (56 days after pollution) ---------------41

Plate 4 - Control Senna alata produced adventitious roots that entered the soil instead of aerial roots

(106 days after pollution) ------------------------------------------------------------------------------------------42

Plate 5 - 0.15% v/w (30 ml) treatment showing aerial roots produced by Senna alata (56 days after

pollution) -------------------------------------------------------------------------------------------------------------43

Plate 6 - 0.15% v/w (30 ml) treatment showing aerial roots produced by Senna alata (106 days after

pollution) -------------------------------------------------------------------------------------------------------------44

Plate 7 - 0.75% v/w (150 ml) treatment showing aerial roots produced by Senna alata (56 days after

pollution) -------------------------------------------------------------------------------------------------------------45

Plate 8 - 0.75% v/w (150 ml) treatment showing aerial roots produced by Senna alata (106 days after

pollution) -------------------------------------------------------------------------------------------------------------46

Plate 9 - 3.75% v/w (750 ml) treatment showing numerous aerial roots produced by Senna alata (56

days after pollution) ------------------------------------------------------------------------------------------------47

Plate 10 - 3.75% v/w (750 ml) showing numerous aerial roots produced by Senna alata (106 days after

pollution) -------------------------------------------------------------------------------------------------------------48

Plate 11 - Senna alata polluted with spent engine oil at flowering stage------------------------------------51

Plate 12 - Pods of Senna alata polluted with spent engine oil------------------------------------------------52

Plate 13 - Seeds of Senna alata polluted with spent engine oil-----------------------------------------------53

LIST OF FIGURES

Figure 1: Percentage degraded hydrocarbons by Senna alata polluted with spent engine oil------------26

Figure 2: Percentage accumulation of hydrocarbons in the root, stem and leaf samples of Senna alata

polluted with spent engine oil -------------------------------------------------------------------------------------28

Figure 3: Percentage quantities of heavy metals in the unvegetated soil and soil vegetated with Senna alata-------------------------------------------------------------------------------------------------------------------31

Figure 4: Percentage quantities of accumulated heavy metals in the root, stem and leaf samples of Senna alata polluted with different concentrations of spent engine oil--------------------------------------33

Figure 5: Vegetative parameters of Senna alata before pollution (57 DAP) -------------------------------36

Figure 6: Vegetative parameters of Senna alata after pollution (163 DAP) -------------------------------37

ABSTRACT

The aim of the study is to use Senna alata L. to remediate soil polluted by spent engine oil (SEO). One

hundred and twenty polythene bags filled with 20 kg of soil were separated into two groups A (60) and

B (60). Group A contained S. alata seedlings while group B had no plant. They were set up in

completely randomized design. Both parts were polluted with different concentrations (0.15% v/w,

0.75% v/w and 3.75% v/w) of SEO 57 days after planting (DAP). One hundred and six days after

pollution, the hydrocarbon and heavy metal contents of the vegetated and unvegetated soil, the unused

SEO, leaves, stems and roots of S. alata were analyzed. Also, vegetative and reproductive parameters

of S. alata were recorded and analyzed. Results showed that percentage of total hydrocarbons

degraded/removed from 0.15% v/w, 0.75% v/w and 3.75% v/w vegetated soils were 99.95%, 99.68%

and 99.28%, respectively. S. alata alone removed 0.06%, 0.18% and 8.05% hydrocarbons for the same

pollution concentrations, respectively. Polycyclic aromatic hydrocarbons accumulated in the leaves,

stems and roots of S. alata. Percentage of total hydrocarbons accumulated in the leaves, stems and roots

of S. alata in 3.75% v/w polluted vegetated soils were 112.47%, 1.49% and 1.35%, respectively. Heavy

metals such as Copper (Cu), Lead (Pb), Zinc (Zn), Iron (Fe) and Aluminium (Al) were detected in the

unused spent engine oil. There were higher concentrations of each of the heavy metals in the polluted

unvegetated soils than the vegetated soils. Heavy metals accumulated in various vegetative parts of S.

alata. Copper was found more in the stems than in the leaves and roots while Fe and Pb were found

more in the leaves than in the stems and roots. Zinc and Al were found more in the roots than in the

leaves and stems. Moreover, heavy metal concentrations (ppm) were more in the vegetative parts of S.

alata than in the polluted soil. Also, plant height, number of leaves, number of pinnules per leaf, leaf

area, stem circumference and number of roots increased significantly (P ≤ 0.05) after pollution. Root

circumference decreased significantly (P ≤ 0.05), with increase in the concentrations of SEO applied

but root length did not vary among the treatments and control. Number of inflorescences and dry

weight of seeds decreased significantly (P ≤ 0.05) but number of flowers, pods and seeds did not vary

among the treatments and control. Hence, S. alata is an ideal plant for the removal (phytoremediation)

of hydrocarbons and heavy metals in SEO contaminated soil. The plant can be regarded as a hyper

accumulator for some polycyclic aromatic hydrocarbons and heavy metals.

TABLE OF CONTENT

Title page---------------------------------------------------------------------------------------------------------------i

Certification page-----------------------------------------------------------------------------------------------------ii

Dedication-------------------------------------------------------------------------------------------------------------iii

Acknowledgement---------------------------------------------------------------------------------------------------iv

Abbreviations----------------------------------------------------------------------------------------------------------v

List of tables----------------------------------------------------------------------------------------------------------vi

List of plates----------------------------------------------------------------------------------------------------------vii

List of figures-------------------------------------------------------------------------------------------------------viii

Abstract----------------------------------------------------------------------------------------------------------------ix

Table of content-------------------------------------------------------------------------------------------------------x

CHAPTER ONE: INTRODUCTION

1.0 Background information-----------------------------------------------------------------------------------------1

1.1 Spent engine oil---------------------------------------------------------------------------------------------------2

1.2 Senna---------------------------------------------------------------------------------------------------------------3

1.3 Objectives of the study-------------------------------------------------------------------------------------------3

CHAPTER TWO: LITERATURE REVIEW

2.0 Engine oil----------------------------------------------------------------------------------------------------------4

2.1 Engine oil additives ----------------------------------------------------------------------------------------------4

2.2 Properties of engine oil------------------------------------------------------------------------------------------6

2.3 Regeneration of used engine oil--------------------------------------------------------------------------------7

2.4 Effects of spent engine oil on the ecosystem------------------------------------------------------------------8

2.5 Nutrient requirements of Senna plant--------------------------------------------------------------------------8

2.6 Medicinal uses of Senna-----------------------------------------------------------------------------------------9

2.7 Phytoremediation-------------------------------------------------------------------------------------------------9

2.8 Phytoremediation of hydrocarbons---------------------------------------------------------------------------10

2.9 Phytoremediation of heavy metals----------------------------------------------------------------------------13

2.10 Phytoremediability of Senna---------------------------------------------------------------------------------14

CHAPTER THREE: MATERIALS AND METHODS

3.0 Planting and pollution of Senna plant------------------------------------------------------------------------15

3.1 Total hydrocarbon analysis------------------------------------------------------------------------------------17

3.2 Heavy metals analysis------------------------------------------------------------------------------------------18

3.3 Determination of vegetative parameters of Senna alata---------------------------------------------------18

3.4 Determination of reproductive parameters of Senna alata------------------------------------------------19

3.5 Data analysis-----------------------------------------------------------------------------------------------------19

CHAPTER FOUR: RESULTS

4.0 Result of the total hydrocarbon analysis---------------------------------------------------------------------20

4.1 Percentage compositions of total hydrocarbons in the samples-------------------------------------------24

4.2 Result of the heavy metal analysis----------------------------------------------------------------------------29

4.3 Result of the vegetative parameters of Senna alata --------------------------------------------------------34

4.4 Result of the reproductive parameters of Senna alata -----------------------------------------------------49

CHAPTER FIVE: DISCUSSION AND CONCLUSION

5.0 Discussion--------------------------------------------------------------------------------------------------------54

5.1 Conclusion-------------------------------------------------------------------------------------------------------59

REFERENCES

APPENDIX

CHAPTER ONE

INTRODUCTION

1.0 Background information

The disposal of spent engine oil (SEO) into gutters, water drains, open plots and farms is a common

practice in Nigeria especially by motor mechanics. These oils, also called spent lubricating or waste

engine oil, is usually obtained after servicing and subsequently drained from automobile and

generator engines (Anoliefo and Vwioko, 2001) and much of this oil is poured into the soil. This

indiscriminate disposal of spent engine oil adversely affect plants, microbes and aquatic lives (Nwoko

et al., 2007; Adenipekun et al., 2008) because of the large amount of hydrocarbons and highly toxic

polycyclic aromatic hydrocarbons contained in the oil (Wang et al., 2000; Vwioko and Fashemi, 2005).

Heavy metals such as vanadium, lead, aluminium, nickel and iron which are found in large quantities

in used engine oil may be retained in soil, in form of oxides, hydroxides, carbonates, exchangeable

cation and/or bound to organic matters in the soil (Ying et al., 2007). These heavy metals may lead to

build up of essential organic (carbon, phosphorous, calcium, magnesium) and non-essential

(magnesium, lead, zinc, iron, cobalt, copper) elements in soil which are eventually translocated into

plant tissues (Vwioko et al., 2006). Although heavy metals in low concentration are essential

micronutrients for plants, but at high concentrations, they may cause metabolic disorder and growth

inhibition for most of the plant species (Yadav, 2010). According to Nwadinigwe and Onwumere

(2003), contamination of soil arising from oil spills affect the growth of plants and causes great

negative impacts on food productivity (Onwurah et al., 2007). Therefore, these indiscriminate

disposals of spent engine oil on the environment and the adverse effects on living organisms were the

main reason for this research and so, there is a dire need to adopt a control measure that employs

environmentally friendly methods. One of these methods is the use of plants to extract or degrade the

pollutants into harmless chemicals. The use of plants to reclaim a damaged environment is called

phytoremediation. In this work, attempt was made to use Senna alata L. to phytoremediate

hydrocarbons and heavy metals present in SEO-polluted soil.

1.1 Spent engine oil

Spent engine oil contains complex mixtures of paraffinic, naphthalenic and aromatic petroleum

hydrocarbons and various contaminants that may contain one or more of the following: carbon

deposits, sludge, wear metals and metallic salt, aromatic and non aromatic solvents, water (as water-

in-oil emulsion), glycols, silicon based antifoaming compounds, fuel, polycyclic aromatic hydrocarbons

[PAHs] and miscellaneous lubricating oil additive materials (Ayoola and Akaeze, 2012). Engine oil

becomes contaminated as a result of physical and chemical reactions. Metals from engine from time

to time erode into the engine oil forming impurities. Oxidation of hydrocarbon chains bond together

to form sludge due to high temperature. Incombustible gasoline up to about 5% wt often leak from

fuel injector line, contaminating the oil (Fedak, 2001). Some additives such as multiple sulfur-based

detergents which keep materials from depositing on the engine piston often begin to break down as

sludge and accumulate in motor oil (Fedak, 2001). Used motor oils are also characterized by high

concentrations of PAHs. Dominguez-Rosado and Pichtel (2003) found that the PAHs content of used

motor oil was often between 34 and 90 times higher than new oil. PAHs belong to a group of over 100

hazardous substances of organic pollutants consisting of two or more fused-benzene aromatic rings

(Obini et al., 2013). In nature, PAHs may be formed by high temperature pyrolysis of organic materials

or low to moderate temperature diagenesis of sedimentary organic materials to form fossil fuel or

direct biosynthesis by microbes and plants (GFEA, 2012 and USGS, 2014). Sources of PAHs can be both

natural and anthropogenic. Natural sources include forest and grass fire, oil seeps, volcanoes,

chlorophyllous plants, fungi and bacteria. Anthropogenic sources include petroleum, power

generation, refuse incineration, home heating, internal combustion engine etc. (GFEA, 2012 and

USGS, 2014). PAHs have low solubility in water and are highly lipophilic. In water or when adsorbed on

particulate matter, PAHs can undergo photodecomposition in the presence of ultraviolet light from

solar radiation (Obini et al., 2013). Heavy PAHs (C16-C50) are more stable and toxic than the light PAHs

(C6-C16) (ATSDR, 1995). According to Comprehensive Environmental Response, Compensation and

Liability Act (CERCLA) list of hazardous substances, PAHs ranked 7th in 2005 in the biennial ranking of

chemicals deemed to pose the greatest possible risk to human health (Christopher, 2008). Some PAHs

have been demonstrated to be mutagenic and carcinogenic in humans and those that have not been

found to be carcinogenic may, however, synergistically increase the carcinogenicity of other PAHs

(Obini et al., 2013).

1.2 Senna

Senna alata (L.) Roxb. (syn. Cassia alata L.) (Aigbokhan, 2014) commonly known as candle stick

senna, wild senna, ringworm cassia and king of the forest, is a medium-sized flowering shrub

belonging to the Family Fabaceae (Mansuang et al., 2010). It is widespread in warm areas of the

world. Senna is native to Amazon rain forest but spread widely in the tropical and subtropical regions.

It starts its life mainly through seeds, though an in vitro propagation which induces maximum number

of shoots and beneficial shoot length by nodal and hypocotyl explants was proposed by Thirupathi

and Jaganmohan (2014). The leaves which often fold at night are large, bilaterally symmetrical and

even-pinnate. Leaflets are 4-26 (two to thirteen pairs) with lanceolate shape and smooth margin. It

reaches a height of about 2.5 meters and produces yellow flowers in the leaf axils. The inflorescence is

an erect waxy yellow spike that resembles fat candle before the individual blossom opens. The flower

is covered with orange bracts which fall off when the flower opens. The flower buds are rounded with

five overlapping sepals and five free but less equal petals narrowed at the base. The flower is bisexual

and zygomorphic. The ovary is superior with marginal placentation. The fruit is a winged black pod

and seeds are small, square and rattle in the pod when dry. The pericarp is dry when mature and

dehisces along the suture. Due to the beauty of the plant, it has been cultivated around the world as

an ornamental plant. The leaves of Senna plant are often attacked by foliage eating caterpillars while

the seeds are attacked by weevil in storage. No disease is of major concern, though some species are

attacked by virus (Wikipedia, 2015).

1.3 Objectives of the study

The objectives of this work are:

1. To determine the quantity of hydrocarbons degraded by Senna alata.

2. To ascertain the changes in hydrocarbon contents of soil unvegetated and vegetated with S.

alata and polluted with spent engine oil.

3. To ascertain the type and quantity of heavy metals that can be removed or accumulated by S.

alata in soil contaminated with spent engine oil.

4. To determine the vegetative and reproductive parameters of S. alata growing on different

concentrations of spent engine oil.

CHAPTER TWO

LITERATURE REVIEW

2.0 Engine oil

Engine oil contains two major components, which include base stock and additive packages

(Udonne, 2011). The base fluid, usually make up the bulk of the oil (70-95%) while the additive

chemicals are added to enhance the positive qualities of the base stock (Ogbeide, 2011). Engine oil

base stocks are made from petroleum or produced synthetically to desired quality. Petroleum base

stocks are purified from crude oil while the synthetic base stocks, on the other hand, are chemically

engineered from pure compounds (Ogbeide, 2011). Engine oil is made of branched alkanes,

cycloalkanes, polyaromatic hydrocarbons (PAHS), linear alkanes, zinc, phosphorus, calcium, sulfur and

additives (Ayoola and Akaeze, 2012). Generally, lubricating oil helps to protect rubbing surfaces,

reduce friction between moving and connected parts, eliminate build up of temperature on the

moving surfaces and keep engine clean (Udonne, 2011).

2.1 Engine oil additives

Engine oil additives are chemical compounds added to lubricating oils to impart specific properties

to the finished oils (Leslie, 2003; Rizvi, 2009). Some additives impart new and useful properties to the

lubricant; some enhance properties already present, while others act to reduce the rate at which

undesirable changes take place in the product during its service life. Moreover, engine oil became

specialized so that requirements for diesel engine oils began to diverge from requirements for

gasoline engines, since enhanced dispersive capability is needed to keep soot from clumping in the oil

of diesel engines. Some additives are multifunctional, as in the case of zinc dialkyl dithiophosphates

which function as antiwear, oxidation and corrosion inhibitors. The additive blended with these base

stocks according to Olufemi and Oladeji (2008) include:

� Friction modifiers additives: These are additives that usually reduce friction. The mechanism

of their performance is similar to that of the rust and corrosion inhibitors in that they form

durable low resistant lubricant films through adsorption on surfaces or association with the

oil. Common materials that are used for this purpose include long-chain fatty acids, their

derivatives, and molybdenum compounds (Masabumi et al., 2008).

� Anti-wear and extreme-pressure additives: Anti-wear agents have a lower activation

temperature than the extreme-pressure agents (Leslie, 2003; Masabumi et al., 2008; Rizvi,

2009). The latter are also referred to as anti-seize and anti-scuffing additives. Organosulfur

and organo-phosphorus compounds such as organic polysulfides, phosphates,

dithiophosphates, and dithiocarbamates are the most commonly used anti-wear and extreme

pressure agents (Leslie, 2003; Rizvi, 2009). Extreme pressure additives form extremely durable

protective films by thermo-chemically reacting with the metal surfaces.

� Anti-oxidant additives: One of the most important aspects of lubricating oils is the maximizing

of the oxidation stability. Exposure of hydrocarbons to oxygen and heat will accelerate the

oxidation process forming fatty acids, fatty alcohols, fatty aldehydes and ketones, fatty esters

and fatty peroxides. All these compounds form the solid asphaltic materials including resins

and lacquers (Masabumi et al., 2008). The main classes of oil-soluble organic and organo-

metallic antioxidants are aromatic amine, phenolic compounds, organo-zinc, organo-copper

and organo-molybdenum compounds (Leslie, 2003; Rizvi, 2009).

� Anti-foam agents: The foaming of lubricant is a very undesirable effect that can cause

enhanced oxidation by the intensive mixture with air, cavitations damage as well as

insufficient oil transport in circulation systems that can even lead to lack of lubrication (Leslie,

2003; Rizvi, 2009). Beside negative mechanical influences, the foaming tendency depends very

much on the lubricant itself and is influenced by the surface tension of the base oil and

especially by the presence of surface-active substances such as detergent-corrosion inhibitors

and other ionic compounds.

� Rust and corrosion inhibitors: Rust inhibitors are usually compounds with high polar

attraction toward metal surfaces (Leslie, 2003; Rizvi, 2009). By physical or chemical interaction

at the metal surface, they form a tenacious, continuous film that prevents water from

reaching the metal surface. Typical materials used for this purpose are amine succinates and

alkaline earth sulfonates.

� Detergent and dispersant additives: These additives are designed to control deposit

formation, either by inhibiting the oxidative breakdown of the lubricant or by suspending the

harmful products already formed in the bulk lubricant (Leslie, 2003; Rizvi, 2009; Ming et al.,

2009 and Alun et al., 2010). Oxidation inhibitors intercept the oxidation mechanism while

dispersants and detergents perform the suspending part (Kyunghyun, 2010). Sulfonate,

phenate, and carboxylate are the common polar groups present in detergent molecules.

� Viscosity index improvers: Probably the most important single property of lubricating oil is its

viscosity. Viscosity affects heat generation in bearings, cylinders, and gears; it governs the

sealing effects of the oil and the rate of consumption or loss and it determines the ease with

which machines may be started under cold conditions (Leslie, 2003; Rizvi, 2009; Margareth et

al., 2010). Oil viscosity must be high enough to provide proper lubricating films but not so high

that friction losses in the oil will be excessive. Viscosity improvers are long chain, high

molecular weight polymers that function by causing the relative viscosity of an oil to increase

more at high temperatures than at low temperatures. Among the principal viscosity improvers

are methacrylate polymers/copolymers, acrylate polymers, olefin polymers/copolymers and

styrene butadiene copolymers.

� Pour point depressant: The lowest temperature at which engine oil will pour or flow when it

is chilled without disturbance under prescribed conditions is known as its pour point (Leslie,

2003; Rizvi, 2009). Two general types of pour point depressant are alkylaromatic and

polymethacrylate.

2.2 General properties of engine oil

The general properties of engine oil according to Wikipedia (2015) include the following:

� Viscosity index: Oils behave differently at different temperatures. As temperatures drop, the

hydrocarbon molecules in mineral oils start to line up and stick together. This causes the

viscosity of the oil to increase, which makes it harder for it to lubricate an engine. At high

temperatures, the opposite happens and the oil's viscosity decreases, making it less effective

at protecting moving parts.

� TBN (total base number): TBN is a measure of the oil's alkalinity. Alkalinity in oil is important

because the combustion process produces acids which can attack metals and other materials

in an engine, increasing wear. When oil is new, the TBN is highest. Over time, TBN decreases

until finally the oil reaches a point where it cannot absorb any more acids and the acidity of

the oil in the engine will start to rise. Most often, it is this depletion of TBN which signals that

oil is 'worn out' and due to be changed.

� Pour point: Pour Point is the lowest temperature at which the oil can still be poured out of a

container.

� Flash point: Flash point is the temperature at which the vapor of the oil will start to combust,

but not continue to burn when mixed with air.

� Noack volatility: Noack volatility is an oil property testing in which oil is heated to a

temperature of 250˚C for one hour, after which the percentage of weight lost by the oil is

measured. This indicates the extent to which the lighter-weight fractions of the oil are

volatilized and lost to the atmosphere. Any oil that volatilizes easily performs poorly because it

quickly becomes thick.

� Shear stability: Shear stability is an expression of how well the oil stands up to mechanical

shear loads. In an internal combustion engine, oil is subjected to extreme shear loads as parts

slide past each other. Oils with poor shear stability will 'shear out' and lose viscosity.

� Oil weight: Most oils used in automotive and truck applications are multi-grade oils. This is

indicated by the familiar nomenclature like 10W-30 or 10W-40. The first number is the winter

weight of the oil. It indicates how the oil behaves when cold. The second number (30, 40, etc)

is the nominal viscosity of the oil at 100˚C. Thus, a '10W' indicates how the oil behaves as

“straight” 10 weight oil when cold. A 10W-30 behaves the same as “straight” 30 weight oil

when it is hot.

2.3 Regeneration of used engine oil

Most countries are paying serious attention to the menace of environmental degradation caused

by the disposal of wastes including used lubricating oil. Hence the need to proffer ways of reducing

the effects of used oil. Regeneration is one way of doing this. This is done by using the standard

method of testing and characterization of hydrocarbons as recommended by American Society for

Testing and Materials (ASTM) (Isah et al., 2013). The raw materials used in this include sulfuric acid,

fresh engine oil (SAE 40), waste engine oil, activated carbon (wood charcoal) and phenolphthalein.

Apart from regeneration of oil from spent engine oil, diesel fuel can also be produced from spent

engine oil (Beg et al., 2010). This can be carried out by pretreatment of used engine oil, blending and

filtration. The whole process use the following materials – used engine oil, fresh diesel, concentrated

sulfuric acid (98% H2SO4), caustic soda and activated clay (Beg et al., 2010).

2.4 Effects of spent engine oil on the ecosystem

Agbogidi (2010) reported that a marked change in properties occurs in soil polluted with

petroleum hydrocarbons, affecting the physical, chemical and microbiological properties of soil.

Changes of soil properties due to contamination of petroleum derived substances can lead to water

and oxygen deficit as well as shortage of available nitrogen and phosphorous (Wyszokowska and

Kucharski, 2000). Abdulhadi and Kawo (2006) also reported a significant decrease in germination,

plant height, leaf area index and yield of groundnut and maize (Arachis hypogaea L. and Zea mays L.)

in soil polluted with used engine oil. Okonokhua et al. (2007) also reported that plant height, root

number and root length of maize grown in spent engine oil-contaminated soil were adversely

affected. According to Adenipekun et al. (2008), engine oil affects the moisture content in Corchorus

olitorius Linn. Ogbuehi et al. (2011) reported a significant decrease in biochemical parameters

including fiber and carbohydrate content in cowpea (Vigna unguiculata) growing in soil contaminated

with spent engine oil. Agbogidi and Ilondu (2013) stated that soil contaminated with spent engine oil

has significant effect on reducing the germination response and subsequent performances including

the biomass production of Moringa oleifera seedlings. Moreover, Nwadinigwe and Oyiga (2009)

reported a significant decrease in height, number of leaves, leaf area, number of flowers, fruits and

dry weight of Solanum gilo with increase in foliar spray of petroleum hydrocarbon. Nwadinigwe and

Olawole (2010) also stated that crude oil pollution reduced the dry weight and number of seeds of

Sorghum vulgare. Nwadinigwe and Onwumere (2003) reported that pod production in Glycine max

was inhibited by petroleum hydrocarbons.

2.5 Nutrient requirements of Senna plant

Senna grows in wide variety of soil that is slightly alkaline and well drained. Chemical fertilization

improves the growth of Senna (Shaimaa et al., 2012). The nitrogenous nutrition is necessary for the

various biochemical processes that occur within the plant and these are essential for the plant growth

and development (Taiz and Zeiger, 2002). Pratibha et al. (2010) pointed out that nitrogen application

improves the yield of Cassia angustifolia. Kamel and Sakr (2009) showed that plant height, stem

diameter, number of branches per plant and dry weight of shoots, total chlorophyll in leaves, total

carbohydrate, nitrogen, phosphorous, potassium, copper and lead were favorably affected by NPK

fertilization on S. occidentalis. Al-Menaic et al. (2012) revealed that NPK at 1 g/L gave the highest

height of Cassia nodosa and Cassia fistula.

2.6 Medicinal uses of Senna

Many species of Senna have been known to perform notable functions in the ecosystem. The

plant contains chemicals such as anthraquinone which is well known for its laxative effect and

treatment of various skin diseases including Pityriasis versicolor (Idu et al., 2007). Decoction of the

flower is used as an expectorant in bronchitis and dysphoria, as an astringent and also mouth wash in

stomatitis (Idu et al., 2007). Villasenor et al. (2002) and Lewis and Levy (2011) also reported that

hexane extract of Cassia alata leaves exhibit anti-inflammatory activities in rat.

2.7 Phytoremediation

Phytoremediation basically refers to the use of plants and associated soil microbes to reduce the

concentrations or toxic effects of contaminants in the environment. Phytoremediation is widely

accepted as a cost-effective environmental restoration technology and an alternative to engineering

procedures that are more destructive to the soil (Greipsson, 2011). Phytoremediation may be in the

form of the following:

� Phytosequestration also called phytostabilization involves absorption by the root, adsorption

to the surface of root or the production of biochemical compounds by the plant which are

released into the soil or ground water in the immediate vicinity of the roots. These biochemical

compounds can sequester, precipitate, or otherwise immobilize nearby contaminants

(Greipsson, 2011).

� Rhizodegradation- This takes place in the soil or ground water immediately surrounding the

plant root. Exudates from plants stimulate rhizosphere microorganisms that enhance

biodegradation of soil contaminants (Vidali, 2001).

� Phytohydraulics- This involves the use of deep-rooted plants (usually trees) to contain,

sequester or degrade ground water contaminants that come into contact with their roots

(Greipsson, 2011).

� Phytoextraction- This is also called phytoaccumulation and involves plants taking up or hyper

accumulating contaminants through their roots and storing them in the tissues of the stems or

leaves. This is particularly useful for removing metals from the soil and in some cases, the

metals can be recovered for reuse by incinerating the plant in a process called phytomining

(Vidali, 2001).

� Phytovolatilization- This is the process in which a plant takes up volatile compounds through

their roots and transpire the same compounds or their metabolites through the leaves,

thereby releasing them into the atmosphere (Vidali, 2001).

� Phytodegradation- This is the process in which contaminants are taken up into the plant

tissues where they are metabolized or bio-transformed. Where the transformation takes place

depends on the type of plant and it can occur in the root, stem or leaves (Greipsson, 2011).

2.8 Phytoremediation of hydrocarbons

Plants can enhance bioremediation processes by absorbing, translocating or sequestering the

organic contaminants and removing them from the soil compartment. These hydrocarbons can be

degraded by various processes, including photooxidation, microbial action and natural rhizosphere

action (Greenberg et al., 2007). Plant enzymes aid in the degradation of contaminants during

phytoremediation, but during natural attenuation or bioaugmentation, indigenous microbial

population performs the degradation. However, the hydrocarbon assemblage is resistant to soil

micro-organisms under normal conditions and persist in the subsurface for decades (Srujana and

Anisa, 2011). Since plant roots can supply readily available carbon sources for microorganisms, they

influence the soil microbial community by increasing microbial numbers (Nwadinigwe and Obi-Amadi,

2014), humification and adsorption of pollutants onto the rhizosphere and also improve physical and

chemical conditions of soil (Srujana and Anisa, 2011). Plant endophyte partnerships are beneficial to

improve phytoremediation of a mixture of contaminants (Weyens et al., 2009). Wolf et al. (2007)

attribute rhizosphere effect to the very characteristics of the roots. However when trees establish

rhizosphere colonization, the hydrocarbons become amenable to biodegradation (Phillips, 2004).

Dominguez-Rosado and Pichtel (2004) reported that Clover (Trifolium) plant sown in the soil treated

with used motor oil in green house, removed all the oil after 150 days. Nwadinigwe and Obi-Amadi

(2014) also observed that Pennisetum glaucum significantly reduced the percentage of hydrocarbons

in crude oil polluted soil. Meudec et al. (2006) reported that PAHs distributions in plant tissues were

predominantly low to medium weight hydrocarbons that are volatile with higher molecular weights at

the upper parts of the plants. Joelle et al. (2002) reported that root uptake was the main pathway for

high molecular weight PAHs which are non volatile. Some plants which are used in the

phytoremediation of soils contaminated with polycyclic aromatic hydrocarbons [PAHs] are in Table 1.

Table 1: Studies on phytoremediation of PAH contaminants in soil

Common name of

plant

Scientific name of plant

Research findings References

Cocks foot

Tall fescue

Red fescue

Rye grass

Birdsfoot-trefoil

Red clover

White clover

Dactylis glomerata

Festuca arundinacea

Festuca rubra

Lolium perenne

Lotus carniculatus

Trifolium pretense

Trifolium repens

Naphthalene decreased to about 20% and other

PAHs decreased with an increase in molecular

weight, except with pyrene, the only PAH which

did not show any significant decrease.

Smith et al. (2006)

Cat claw Mimosa monancistra

Dissipation of benzo[a]pyrene significantly faster

in vegetated soil. Bernal et al. (2007)

Baby bear Cucurbita pepo

As soil moisture increase, plant density increased

rates of contaminant accumulation in their roots.

Concentrations of the compound in plant roots

were inversely related to plant age whereas no

change in the bioavailability of the compound was

observed in successive generations of plants

grown in the same contaminated soil.

Kelsey et al. (2006)

Annual ryegrass

Tall Fescue

Yellow Sweet

Clover

Lolium multiflorum

Festuca arundinacea

Schreb.

Melilotus officinalis Lam

Maturity of plant root contributes to reduction in

the bioavailability of target PAHs. Parrish et al.

(2005)

Sunflower Helianthus annuus L. Vegetation increases total numbers of beneficial Olson and Fletcher

Bermuda grass

Southern

crabgrass

Cynodon dactylon L.

Digitaria ciliaris (Retz.)

Koeler

fungi and bacteria in contaminated soil. (2000)

Maize Zea mays L.

Increase in hydrocarbon bioavailability, stimulates

bacterial population

Radwan et al.

(1995)

Chaineau et

al.(2000)

Switch grass

Little bluestem

grass

Alfalfa

Panicum virgatum

Schizachyrium scoparium

Medicago sativa L.

Reduction in total PAH concentration after six

months of treatment

Pradhan et al.

(1998)

Wiltse et al. (1998)

Rice

Naked Spinach

Devil's beggartick

Oryza sativa L.

Spinacia oleracea L.

Bidens frondosa L.

Significant decrease in TPH concentration under

vegetated conditions Kaimi et al. (2007)

Slender oat Avena barbata

A large phenanthrene degrader population in

rhizosphere is related to root debris and soil

exudates

Miya and

Firestone (2001)

Alfalfa

Alpine blue grass

Medicago sativa L.

Poa alpine L.

Stimulation of bioremediation around plant roots

due to higher number of organic chemical

degraders indicates potential

Nichols et al.

(1997)

Tall fescue Festuca arundinacea

Greater total bacterial numbers and PAH-

degrading bacteria in rhizosphere soil. Ho and Banks

(2006)

Alfalfa

Reed

Medicago sativa L.

Phragmitis australis

Rhizosphere microflora of alfalfa was less

inhibited by hydrocarbon contamination with

higher degradative potential compared to reed.

Muratova et al.

(2003)

(Source: Srujana and Anisa, 2011)

2.9 Phytoremediation of heavy metals

Unlike organic wastes, heavy metals present in SEO are non-biodegradable and needed to be

removed from the environment (Alluri et al., 2007). Heavy metals may be retained in the polluted soil

from season to season but at higher concentrations in the dry seasons than in the wet seasons

(Nwadinigwe et al., 2014ab

). Plants and the associated microbes have been found to be effective in

remediation of heavy metal polluted site (Ghose and Singh, 2005). However, the ability to accumulate

heavy metals varies significantly between species and among cultivars within species, as different

mechanisms of ion uptake are operative in each species, based on their genetic, morphological,

physiological and anatomical characteristics (Mohammad et al., 2008). One of the most deleterious

effects induced by heavy metals exposure in plants is lipid peroxidation, which can directly cause

biomembrane deterioration (Yadav, 2010). Malondialdehyde (MDA), one of the decomposition

products of polyunsaturated fatty acids of membrane is regarded as a reliable indicator of oxidative

stress (Demiral and Turkan, 2005). However, plants have developed a very potential mechanism to

combat such adverse environmental heavy metal toxicity problems. Plants produce low molecular

weight thiols that show high affinity for toxic metals (Bricker et al., 2001). The most important/critical

low molecular weight biological thiols are glutathione (GSH) and cysteine. GSH is a substrate for

phytochelatins (PCs) synthesis and crucial for detoxification of heavy metals such as cadmium and

nickel (Freeman et al., 2004). PCs form complexes with toxic metal ions in the cytosol and

subsequently transport them into the vacuole (Yadav, 2010) hence, protect plants from the

deleterious effect of heavy metals. Moreover, hyperaccumulation of metals in various plant species

has been extensively investigated and has become clear that different mechanisms of metal

accumulation, exclusion and compartmentalization exist in various plant species (Mohammad et al.,

2008). In Thlaspi caerulescens, zinc (Zn) is sequestered preferentially in vacuoles of epidermal cells in a

soluble form (Frey et al., 2000) while in Arabidopsis halleri, Zn was found to be accumulated in the

mesophyll cells of the leaves (Sarret et al., 2002). Cosio et al. (2004) reported the existence of

regulation mechanism on the plasma membrane of T. caerulescens that resulted in the storing of

heavy metals in the root vacuoles and thus became unavailable for loading into the xylem and

subsequent translocation to shoots.

2.10 Phytoremediability of Senna

Some investigations have been carried out on the phytoremediability of Senna species on soil

pollutants. Ghose and Singh (2005) and Gupta and Sinha (2008) reported that Cassia tora

accumulated high concentrations of lead, copper, nickel, aluminium, zinc, cadmium, iron, manganese

and chromium in their leaves and root, hence, reducing their negative effect on the ecosystem.

Siringoringo (2000) also reported that Cassia multijuga was capable of absorbing and accumulating

lead. Kumar et al. (2002) and Raju et al. (2008) reported that Cassia siamea accumulated high

concentrations of nickel, manganese, chromium, lead, copper, iron and zinc in their leaves and shoots.

Also, Hanif et al. (2007) stated that Cassia fistula played an important role in phytoremediating soil

contaminated with potassium, iron, nickel, zinc, manganese, chromium, lead, cobalt, copper and zinc.

Cassia siamea was found to accumulate iron, manganese, zinc, copper, nickel, chromium, lead and

cadmium at high concentrations and it could be used as hyper accumulator plant for bioremediation

(Jambhulkar and Juwarkar, 2009). Al-Qahtani (2012) indicated that Cassia italica is an accumulator of

iron, zinc, chromium, copper, lead, nickel, cobalt and cadmium. However, little work has been carried

on the phytoremediation of organic compounds especially SEO using Senna species and this work was

done to bridge this gap.

CHAPTER THREE

MATERIALS AND METHODS

3.0 Planting and pollution of Senna plant

Ten centimeter top soil from Botanic Garden, Department of Plant Science and Biotechnology,

University of Nigeria, Nsukka was mixed with poultry manure at a ratio of 6:2 and watered for two

weeks. Poultry manure was collected from a poultry farm in University of Nigeria, Nsukka. One

hundred and twenty polythene bags filled with 20 kg of the mixture of soil and poultry manure were

separated into parts A (60 bags) and B (60 bags). Seeds of Senna alata were collected from Botanic

Garden, Department of Plant Science and Biotechnology, University of Nigeria, Nsukka. Part A had one

seed of S. alata sown in each bag while part B had no seed. The experiment was completely

randomized and carried out in 3 replicates. The bags were kept under the sun and rain fed since the

experiment was carried out during the rainy season. To simulate spillage, each one of 15 soil bags was

polluted with 0.15% v/w (30 ml) of spent engine oil, 57 days after planting (DAP) (Plate 1). Pollution

was repeated with 0.75% v/w (150 ml) and 3.75% v/w (750 ml) of spent engine oil, separately on

other soil bags, instead of 0.15% v/w. Both parts A and B were polluted in the same manner. The

control had no spent engine oil.

Plate 1: Senna alata seedlings, 57 days after planting (DAP)

3.1 Total hydrocarbon analysis

The unused spent engine oil, vegetated and unvegetated soils, leaf, stem and root samples of

the polluted and unpolluted Senna alata were analyzed for total hydrocarbons using gas liquid

chromatography/mass spectroscopy (GLC/MS) 106 days after pollution. Soils and vegetative samples

were oven dried at 45oC in Memmert 854 Schwabach Oven. Dried plant organs were crushed into fine

powder using mortar and pestle. Twenty grams of each homogenized sample was mixed with 60 g of

anhydrous sodium sulphate (Na2SO4) in agate mortar to absorb moisture. The homogenate was

placed into extraction cellulose thimble (3394 mm), covered with a Whatman filter paper and placed

in a Soxhlet Extraction chamber of the Soxhlet unit. Extraction was carried out with 200 ml of n-

hexane at 340˚C for eight hours (US EPA, 1996). The crude extract obtained was evaporated to

dryness using a Ribby RE 200B rotary vacuum evaporator at 40˚C. One gram of activated florisil (60-

100 mesh) was added to an 8 ml florisil column plugged with glass wool followed by 0.5 g of

anhydrous Na2SO4. Five ml of n-hexane was added to the packed column for conditioning. The

stopcock was opened to allow n-hexane to run out into a receiving vessel until it just reaches the top

of the Na2SO4 while gently tapping the top of the column till the florisil settled well in the column. The

residue or spent engine oil was then transferred to the florisil column with disposable Pasteur pipette

from an evaporating flask to clean it up. The evaporating flask was rinsed twice with 1 ml of n-hexane

and added into the column. Eluate was collected in an evaporating flask and rotary evaporated to

dryness (US EPA, 1996).

For gas liquid chromatography/mass spectroscopy (GLC/MS) analysis, 1 ml of dried eluate was

diluted with 1 ml of hexane. The gas flow columns (the inlet, the detectors and the split ratio) of a

Buck 530 (910 Model, USA) Gas Chromatograph equipped with an on-column, automatic injector,

flame ionization detector, HP 88 capillary column (100 m x 0.25μm film thickness) were adjusted. The

injector and detector temperatures (detector A: 250˚C, injector: 22˚C, integrator chart speed: 2

cm/min, oven: 180˚C) were also adjusted and allowed to warm up (initial temperatures 70˚C-220˚C

and final temperatures 220˚C-280˚C). The detectors were generally held at the high end of the oven

temperature range to minimize the risk of analyte precipitation. When the “NOT READY” light was off,

1 microliter (μl) of diluted dry eluate was injected onto column A of GLC/MS using proper injection

technique and allowed to run for 45 minutes. The type and quantities of hydrocarbons present

together with the resulting gas chromatograph were collected from Peak426-32 bit (PC/Window 7)

software (US EPA, 1996).

3.2 Heavy metals analysis

Samples were analyzed for the accumulation of heavy metals using flame atomic absorption

spectroscopy (FAAS), 106 days after pollution. Samples were dried at 45oC using Memmert 854

Schwabach Oven. After drying, individual sample was crushed into fine powder. One gram of fine

powdered sample was heated for 8 hours in a furnace and latter cooled in desiccators. Five ml of

trioxonitrate (v) acid (HNO3) solution was added to the left-over ash and evaporated to dryness on a

hot plate and returned to the furnace for heating at 400˚C for 15-20 minutes until perfect grayish-

white ash was obtained and then allowed to cool in desiccators. Fifteen ml of hydrochloric acid (Hcl)

was added to the ash to dissolve it. The solution was filtered into 100 cm3 volumetric flask and made

up to 100 cm3 with distilled water. Unused SEO sample was also prepared by digestion method

(Adrian, 1973). This was done by putting 2 g of SEO into a digestion flask. This was followed by

addition of 20 ml of acid mixtures (650 ml conc. HNO3, 80 ml perchloric acid and 20 ml sulfuric acid)

into the flask and heated till a clear solution was obtained. Hexane was then added to the flask to the

mark of 25 ml for dilution (Adrian, 1973).

Flame atomic absorption spectroscopic (FAAS) analysis was carried out according to the

method adopted by American Public Health Association (1995). A series of standard metal solutions in

the optimum concentration range was prepared by diluting the single stock element solutions with

water containing 1.5 ml concentrated nitric acid/liter. A calibration blank was also prepared using all

the reagents except for the metal stock solution. The sample was aspirated using Varian AA240

Atomic Absorption Spectrophotometer (AAS) into the flame and atomized when the AAS’s light beam

is directed through the flame into the monochromator. The atomized sample was directed onto a

detector that measured the amount of light absorbed by the atomized element in the flame.

Calibration curve for each metal was prepared by plotting the absorbance of standard versus their

concentration using Spectra AA scan (PC/Window 7) software.

3.3 Determination of vegetative parameters of Senna

The vegetative parameters of the plant such as plant height, number of leaves per plant,

number of pinnules per leaf, leaf length, leaf width, area of the leaves and stem circumference were

determined before pollution, that is, 57 days after planting (DAP) (Plate 1). After pollution, the same

vegetative parameters mentioned above, in addition to the number of roots, root length, root

circumference and aerial roots [formed 56 days (initial number) and 106 days (final number)] were

also measured at 163 DAP. Plant height, root length, leaf length and width were measured with meter

rule. Plant height was obtained by measuring the plant from soil level to the terminal bud. Length of

each leaf was measured from the base of the petiole to the end of the rachis while the width of each

leaf was obtained by taking measurement of two opposite pinnules of the leaf at the widest point.

Gaps between the pinnules were regarded as non-existent because of overlapping of some pinnules.

Stem and root circumferences were measured using thread and meter rule. Stem circumference was

measured at 6 cm from soil level. Number of roots, leaves, pinnules per leaf and initial and final

number of aerial roots were determined by counting. Leaf area was calculated from the product of

leaf length and width multiplied by a correction factor (0.75) following the procedure of Francis et al.

(1969).

3.4 Determination of reproductive parameters of Senna

Reproductive parameters: number of inflorescences per plant, flowers, pods, seeds and dry

weight of seeds were measured at maturity (294 DAP). Number of inflorescences, flowers, pods and

seeds were determined by counting while dry weight of seeds was measured with digital balance,

after drying.

3.5 Data analysis

Data from gas liquid chromatography/mass spectroscopy (GLC/MS) and flame atomic

absorption spectroscopy (FAAS) were collected from Peak426-32 bit (PC/Window 7) and Spectra AA

scan (PC/Window 7) software respectively. Data from vegetative and reproductive parameters were

analyzed using GenStat Release 10.3DE (PC/Window 7), copyright 2011, VSN international Ltd.

(Rothamsted Experimental Station) to generate the means and variance at p ≤ 0.05. Means of each

parameter were compared using Fisher’s Least Significance Difference and T-test.

CHAPTER FOUR

RESULTS

4.0 Result of the total hydrocarbon analysis

Polycyclic aromatic hydrocarbons (PAHs) of various concentrations were detected in unused

SEO and range from C14-C22 (Table 2). These hydrocarbons include Phenanthrene (C14H10),

Flouranthene (C16H10), 1,2-Benzanthracene (C18H12), Benzo(a)pyrene (C20H12) and Indeno(1,2,3-

cd)pyrene (C22H12). The concentrations of total hydrocarbons in the unvegetated and vegetated soils

increased with increase in the concentrations of SEO applied among the treatments. Unvegetated

soils had more concentrations of total hydrocarbons than those of the vegetated soils (Table 2).

Hence, the concentration of total hydrocarbons in 0.15% v/w unvegetated soils was 4.15 mg/ml, while

that of 0.15% v/w vegetated soils was 1.81 mg/ml. All other soil treatments followed the same trend:

0.75% v/w unvegetated soils (18.52 mg/ml); 0.75% v/w vegetated soils (12.66 mg/ml); 3.75% v/w

unvegetated soils (345.78 mg/ml) and 3.75% v/w vegetated soils (28.24 mg/ml). However, different

PAHs such as Acenaphthylene (C12H8), Flourene (C13H10), Benzo (k) flouranthene (C20H12) and Benzo(g-

h-i) perylene (C22H12) which were not detected in unused SEO were detected in 0.75% v/w and 3.75%

v/w polluted unvegetated soils. Some PAHs found in unused SEO were retained in some polluted

vegetated and unvegetated soils in comparatively smaller quantities.

Table 2: Composition and quantities of hydrocarbons in the unused spent engine oil, unvegetated

and vegetated soil samples of Senna alata polluted with spent engine oil.

HYDROCARBON

COMPOSITIONS

CONCENTRATIONS (mg/ml)

Unused

SEO

Polluted unvegetated soil samples

Polluted vegetated soil samples

0%

v/w

0.15%

v/w

0.75%

v/w

3.75% v/w 0%

v/w

0.15%

v/w

0.75%

v/w

3.75%

v/w

Acenaphthylene

(C12H8)

- - - - 335.84 - - - -

Flourene

(C13H10)

- - - - 0.02 - - - -

Phenanthrene

(C14H12)

1205.35 - - - - - - 0.30 -

Flouranthene

(C16H10)

460.01 - - - - - - - 27.42

1,2-

Benzanthracene

(C18H12)

1063.30 - - - - - - - -

Benzo(a)pyrene

(C20H12)

1207.11 - 4.15 - 7.41 - 1.56 - 0.82

Benzo(k)flouran

thene (C20H12)

- - - 18.52 - - - - -

Benzo(b)flouran

thene (C20H12)

- - - - - - 0.24 - -

Indeno(1,2,3-

cd)pyrene

(C22H12)

5.23 - - - 2.31 - - 12.36 -

Benzo(g-h-

i)perylene

(C22H12)

- - - - 0.21 - - - -

THC 3941.00 - 4.15 18.52 345.78 - 1.81 12.66 28.24

Legend: THC = Total hydrocarbon (in each sample)

- = Absence

Polycyclic aromatic hydrocarbons found in polluted unvegetated soils were also detected at

higher concentration in vegetative parts of S. alata (Table 3). For example, Acenaphthylene and

Benzo(g-h-i)perylene which were neither detected in unused SEO nor in any of the soil samples except

in 3.75% v/w polluted unvegetated soil (335.84 mg/ml and 0.21 mg/ml respectively) were increased

to 781.42 mg/ml and 642.45 mg/ml respectively in the leaves of 3.75% v/w treatments (Table 2 and

3). Moreover, many hydrocarbons that were not detected in the unused SEO and polluted

unvegetated soils were detected in the polluted vegetated soils and vegetative parts of the plant.

These hydrocarbons include Anthracene (C14H10) which was found in 0.15% v/w stem samples (0.03

mg/ml) and 3.75% v/w leaf samples (12.11 mg/ml); Benzo (b) flouranthene (C20H12), found in 0.15%

v/w vegetated soil samples (0.24 mg/ml) and 3.75% v/w leaf samples (1200.95 mg/ml); Pyrene

(C16H10), found in 0.15% v/w leaf samples (7.63 mg/ml), 0.75% v/w stem samples (11.35 mg/ml) and

3.75% v/w leaf samples (417.69 mg/ml); Naphthalene (C10H8), found only in 3.75% v/w root samples

(19.36 mg/ml) and Chrysene (C18H12), found only in 3.75% v/w stem samples (15.62 mg/ml) (Table 2

and 3). Other plant samples showed comparatively lower quantities of accumulated hydrocarbons

when compared with the hydrocarbons in the unused SEO (Table 3). Anthracene, Flouranthene, Benzo

(b) flouranthene and Benzo (a) pyrene were detected in the control (0% v/w) but their concentrations

were small compared to other treatments. As in all the treatments, hydrocarbon accumulation in the

controls were more on the stems and leaves while the concentrations in soils and roots (0% v/w) were

negligible (Table 3).

Table 3: Composition and quantities of hydrocarbons in the unused spent engine oil and vegetative

plant parts of Senna alata polluted with spent engine oil

HYDROCARBON

COMPOSITIONS

CONCENTRATIONS (mg/ml)

UNUSED

SEO

POLLUTED VEGETATED PLANT PARTS

LEAF STEM ROOT

0%

v/w

0.15

%

v/w

0.75%

v/w

3.75% v/w 0%

v/w

0.15

%

v/w

0.75%

v/w

3.75%

v/w

0%

v/w

0.15

%

v/w

0.75%

v/w

3.75%

v/w

Naphthalene

(C10H8)

- - - - - - - - - - - - 19.36

Acenaphthylene

(C12H8)

- - - - 781.42 - 2.16 - - - - - -

Flourene

(C13H10)

- - - - 0.25 - - - - - - - -

Anthracene

(C14H10)

- - - - 12.11 0.03 0.03 - - - - - -

Phenanthrene

(C14H12)

1205.35 - - - - - - - - - - - -

Flouranthene

(C16H10)

460.01 - - - 252.57 0.13 0.91 0.01 - - 2.36 0.18 -

Pyrene (C16H10) - - 7.63 - 417.69 - - 11.35 - - - - -

1,2-

Benzanthracene

(C18H12)

1063.30 - - 32.14 - - - - - - - - 29.36

Chrysene

(C18H12)

- - - - - - - - 15.62 - - - -

Benzo(a)pyrene

(C20H12)

1207.11 0.92 2.61 1.91 1134.89 - - 5.26 - - - 0.57 1.68

Benzo(k)flouran

thene (C20H12)

- - - - - - - - - - - - 2.67

Benzo(b)flouran

thene (C20H12)

- 0.19 - - 1200.95 - - - - - - - -

Indeno(1,2,3-

cd)pyrene

(C22H12)

5.23 - - - - - - - 42.97 - - 10.12 -

Benzo(g-h-

i)perylene

(C22H12)

- - - - 642.45 - - 4.53 - - - - -

THC 3941.00 1.11 10.24 34.04 4432.34 0.16 3.10 21.15 58.59 - 2.36 10.87 53.08

Legend: THC = Total hydrocarbon (in each sample)

- = Absence

4.1 Percentage composition of total hydrocarbons in the samples

Percentage composition of the total hydrocarbons in each sample was calculated by dividing the

total concentration of hydrocarbons in a sample by the total hydrocarbons in the unused SEO and

multiplied by 100 (Total hydrocarbons in sample/ 3941.00 (Total hydrocarbons in unused SEO) x 100).

This allowed for easy comparison of the quantities of total hydrocarbons that were undegraded in the

soil as well as those degraded and accumulated in the plant parts. Percentage compositions of total

hydrocarbons degraded in the polluted vegetated soil were higher than those of the polluted

unvegetated soil. Percentage of total degraded hydrocarbons in the polluted vegetated and

unvegetated soils were determined by subtracting the total hydrocarbons in the each soil sample

from the total hydrocarbons in unused SEO. The result was divided by the total hydrocarbons in the

unused SEO and multiplied by 100. The percentage of total hydrocarbons degraded by the plant alone

was obtained by subtracting the percentage of total hydrocarbons degraded in polluted unvegetated

soils from the percentage of total hydrocarbons degraded in the polluted vegetated soils. Hence, it

was 0.06%, 0.18% and 8.05% for 0.15% v/w, 0.75% v/w and 3.75% v/w concentrations, respectively

(Table 4). Figure 1 below compared the percentage of total hydrocarbons degraded by plant alone.

Also, percentage total accumulated hydrocarbons in the vegetative parts of S. alata were most in the

leaves when compared to the stems and roots (Table 5). Figure 2 compared the total accumulated

hydrocarbons in the vegetative parts of S. alata.

Table 4: Percentage composition of total degraded hydrocarbons in the analyzed soil samples of

Senna alata polluted with spent engine oil

Treatment Total

hydrocarbons in

unused spent

engine oil (%)

Total degraded

hydrocarbons in

the unvegetated

soil (%)

Total degraded

hydrocarbons in

the vegetated soil

(%)

Total degraded

hydrocarbons by

the plant alone

(%)

Spent engine oil 100.00 - - -

0% v/w - - - -

0.15% v/w - 99.89 99.95 0.06

0.75% v/w - 99.50 99.68 0.18

3.75% v/w - 91.23 99.28 8.05

Legend: – = Absence

Figure 1: Percentage degraded hydrocarbons by Senna alata polluted with spent engine oil.

0

1

2

3

4

5

6

7

8

9

0.15% v/w 0.75% v/w 3.75% v/w

To

tal

de

gra

de

d h

yd

roca

rbo

ns

(%)

Spent engine oil concentrations

Total degraded hydrocarbons by the

plant alone

Table 5: Percentage accumulation of hydrocarbons in Senna alata polluted with spent engine oil

Treatment Total

hydrocarbons in

unused spent

engine oil (%)

Total accumulated

hydrocarbons in

the root (%)

Total accumulated

hydrocarbons in

the stem (%)

Total accumulated

hydrocarbons in

the leaf (%)

Spent

engine oil

100.00 - - -

0% v/w - - 0.004 0.03

0.15% v/w - 0.06 0.09 0.26

0.75% v/w - 0.28 0.54 0.86

3.75% v/w - 1.35 1.49 112.47

Legend: - = Absence

Figure 2: Percentage accumulation of hydrocarbons in the root, stem and leaf samples of Senna

alata polluted with spent engine oil

4.2 Result of the heavy metal analysis

0

20

40

60

80

100

120

0% v/w 0.15% v/w 0.75% v/w 3.75% v/w

To

tal

acc

um

ula

ted

hy

dro

carb

on

s (%

)

Spent engine oil concentrations

Total accumulated hydrocarbons in

the root

Total accumulated hydrocarbons in

the stem

Total accumulated hydrocarbons in

the leaf

Heavy metals such as copper (Cu), lead (Pb), iron (Fe), zinc (Zn) and aluminium (Al) were

detected in unused SEO and in all the polluted vegetated and unvegetated soil samples (Table 6).

Percentage quantity of the heavy metals in each sample was determined for the clarity of the results.

These were obtained by dividing the concentration of heavy metal in a sample by the concentration of

heavy metal present in unused spent engine oil and multiplied by hundred (Concentration of heavy

metal in a sample/ Heavy metal in SEO x 100). Percentage concentration of each of the heavy metals

in unused SEO was higher than those of polluted vegetated and unvegetated soils. The quantities of

heavy metals that remained in the soil increased with increase in the concentrations of spent engine

oil (SEO) applied. Also, heavy metal concentrations in polluted unvegetated soils were higher in all the

treatments than those of the polluted vegetated soils. However, appreciable quantities of heavy

metals were detected in the vegetative parts of Senna alata. Copper was found to be stored most in

the stem of the plant; Pb and Fe in the leaves; Zn and Al in the roots (Table 7). Copper concentrations

were higher in the stems than in the roots and leaves. Lead concentrations were higher in the leaves

than in the roots and stems while zinc accumulated more in the roots followed by the leaves and

stems. Iron also showed high accumulation in the leaves, followed by the stems and roots while

aluminium showed highest accumulation in the roots, followed by the stems and the least were the

leaves. Like hydrocarbons present in the SEO, very small quantities of heavy metals were also

detected in the vegetative parts of unpolluted vegetated soils. The concentrations of each of the

heavy metals in the plant were within the tolerable/allowable limits in medicinal plants as stipulated

in WHO (1998) reports. The standard maximum allowable limits of these heavy metals include: Cu –

10ppm, Zn – 50ppm, Pb – 10ppm, Fe – 20ppm and Al – 5ppm (WHO, 1998). Hence, these allowable

limits were higher in comparison to the values of the analyzed heavy metals absorbed by the plant in

each treatment. The standard solutions of each of the heavy metals which was used for calibration

were included in Tables 6 and 7.

Table 6: Composition, quantity (ppm) and percentage of heavy metals in the vegetated and

unvegetated soils of Senna alata polluted with spent engine oil

Treatment Sample

type

Cu

(ppm) %

Pb

(ppm) %

Zn

(ppm) %

Fe

(ppm) %

Al

(ppm) %

SEO SEO 1.63 100.0 3.50 100.0 10.12 100.0 18.80 100.0 12.19 100.0

0% v/w

Vegetated

soil

0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0

Unvegetat

ed soil

0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0

0.15% v/w

Vegetated

soil

0.09 5.3 0.01 0.4 0.17 1.6 0.95 5.0 0.16 1.3

Unvegetat

ed soil

0.95 58.3 1.90 54.3 3.50 34.6 6.00 31.9 3.71 30.4

0.75% v/w

Vegetated

soil

0.10 6.1 0.74 21.3 1.26 12.5 2.34 12.4 1.50 12.3

Unvegetat

ed soil

1.00 61.3 2.01 57.4 5.47 54.1 9.97 53.0 6.49 53.3

3.75% v/w

Vegetated

soil

0.56 34.4 0.55 15.7 1.81 17.9 2.58 13.7 1.56 12.8

Unvegetat

ed soil

1.20 73.8 2.15 61.5 6.47 63.9 10.46 55.6 7.09 58.1

Standard 10.00 40.00 10.00 60.00 200.00

Legend: SEO = Unused spent engine oil.

% = Percentage

ppm = Part per million

Figure 3: Percentage quantities of heavy metals in the unvegetated soil and soil vegetated with

Senna alata

0

10

20

30

40

50

60

70

80

Ve

ge

tate

d s

oil

Un

veg

eta

ted

so

il

Ve

ge

tate

d s

oil

Un

veg

eta

ted

so

il

Ve

ge

tate

d s

oil

Un

veg

eta

ted

so

il

Ve

ge

tate

d s

oil

Un

veg

eta

ted

so

il

0 0.15 0.75 3.75

He

av

y m

eta

l a

ccu

mu

lati

on

(%

)

Spent engine oil concentrations (% v/w)

Cu

Pb

Zn

Fe

Al

Table 7: Composition, quantity (ppm) and percentage of heavy metals accumulated in the root,

stem and leaf samples of Senna alata polluted with spent engine oil

Treatment Sample

Type

Cu

(ppm) %

Pb

(ppm) %

Zn

(ppm) %

Fe

(ppm) %

Al

(ppm) %

SEO SEO 1.63 100.0 3.50 100.0 10.12 100.0 18.80 100.0 12.19 100.0

0% v/w

Root 0.00 0.0 0.00 0.0 0.005 0.04 0.00 0.0 0.001 0.008

Stem 0.001 0.06 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0

Leaf 0.00 0.0 0.001 0.02 0.00 0.0 0.001 0.005 0.00 0.0

0.15% v/w

Root 0.01 0.6 0.10 2.8 1.00 9.9 0.60 3.2 1.32 10.8

Stem 0.18 10.9 0.01 0.3 0.02 0.2 0.64 3.4 0.20 1.6

Leaf 0.003 0.2 0.15 4.3 0.05 0.5 3.67 19.5 0.19 1.6

0.75% v/w

Root 0.29 17.8 0.48 13.7 2.62 25.9 0.60 3.2 2.02 16.6

Stem 0.56 34.6 0.11 3.1 0.15 1.5 1.70 9.0 0.19 1.5

Leaf 0.03 1.6 1.06 30.4 0.43 4.3 4.96 26.4 0.14 1.2

3.75% v/w

Root 0.37 22.7 0.41 11.7 3.41 33.7 0.78 4.1 3.55 29.1

Stem 0.68 41.6 0.24 6.9 0.28 2.8 1.80 9.6 0.25 2.0

Leaf 0.08 4.7 1.66 47.5 0.58 5.7 6.74 35.8 0.20 1.7

Standard 10.00 40.00 10.00 60.00 200.00

Legend: SEO = Unused spent engine oil.

% = Percentage

ppm = Part per million

0

5

10

15

20

25

30

35

40

45

50

Root Stem Leaf Root Stem Leaf Root Stem Leaf Root Stem Leaf

0 0.15 0.75 3.75

He

av

y m

eta

ls a

ccu

mu

lati

on

(%

)

Spent engine oil concentrations (% v/w)

Cu

Pb

Zn

Fe

Al

Figure 4: Percentage quantities of accumulated heavy metals in the root, stem and leaf samples of

Senna alata polluted with different concentrations of spent engine oil

4.3 Result of the vegetative parameters of Senna alata

The vegetative parameters (plant height, number of leaves, number of pinnules per plant, area of

leaves and stem circumference) were not statistically different from each other before pollution

(Table 8, Fig. 5). After pollution, the mean height of plants, mean number of pinnules, average leaf

area and stem circumference decreased slightly with increase in the concentration of SEO applied but

these variations were not significant (Fig. 6). However, the mean number of leaves after pollution

decreased significantly (P ≤ 0.05) with increase in concentration of SEO applied especially for 3.75%

v/w treatment (Table 8). Multiple comparison of the mean number of leaves after pollution showed

that 3.75% v/w produced fewer number of leaves when compared to control and 0.15% v/w but not

with 0.75% v/w. Moreover, vegetative parameters determined before pollution were significantly (P ≤

0.05) lower than the vegetative parameters measured after pollution within the treatments. The only

exception was the number of leaves produced in 3.75% v/w treated plants whose value did not differ

before and after pollution (Table 8). Moreover, Senna alata plants were almost of the same height at

the end of the work (Plate 2) though, one plant from 3.75% v/w treatments died 84 days after

pollution.

Table 8: Vegetative parameters of Senna alata before and after pollution (57 and 163 days after planting, respectively)

Treatm.

(% v/w) Plant height (cm) No. of leaves No. of pinnules Leaf area (cm2) stem circum. (cm)

Before

pollution

After

pollution

Before

pollution

After

pollution

Before

pollution

After

pollution

Before

pollution

After

pollution

Before

pollution

After

pollution

0 36.4±2.4a

225.0±8.3b

13.5±0.6c

27.3±2.5d

9.6±0.3e

18.3±0.3f

240.5±21.5g

1012.6±83.4h

3.8±0.1j

9.7±0.4k

0.15 38.4±1.6a

215.3±9.9b

13.6±0.7c

26.0±3.0d

9.6±0.4e

17.3±1.1f

251.6±23.3g

992.0±59.3h

3.9±0.08j

9.5±0.4k

0.75 36.2±2.7a

214.20±7.8b

13.5±0.6c

21.7±2.2d

9.3±0.4e

18.2±0.5f

249.5±25.7g

910.9±60.8h

3.9±0.1j

9.0±0.4k

3.75 37.8±2.7a

203.4±9.3b

14.5±0.7c

18.3±2.2d,c

9.5±1.2e

16.7±1.3f

240.6±27.2g

842.6±92.1h

3.9±0.1j

8.6±0.4k

Values represent mean ± standard error. Means followed by the same letters in the same column are not significantly different at P ≤ 0.05

while means followed by different letters in the same row for each vegetative parameter are significantly different at P ≤ 0.05.

Legend: Treatm. = Treatment; No. = Number; Circum. = Circumference

xxxvi

Figure 5: Vegetative parameters of Senna alata before pollution (57 DAP)

Keys: No. = Number; Circum. = Circumference

0

50

100

150

200

250

300

Plant height No. of leaves No. of pinnules Leaf area Stem circum.

Me

an

va

lue

of

ea

ch p

ara

me

ter

Vegetative parameters

0% v/w

0.15% v/w

0.75% v/w

3.75% v/w

xxxvii

Figure 6: Vegetative parameters of Senna alata after pollution (163 DAP)

Keys: No. = Number; Circum. = Circumference

0

200

400

600

800

1000

1200

Plant height No. of leaf No. of pinnules Leaf area Stem circum.

Me

an

va

lue

of

ea

ch p

ara

me

ter

Vegetative parameters

0% v/w

0.15% v/w

0.75% v/w

3.75% v/w

xxxviii

Plate 2: Senna alata plants 163 days after planting (106 days after pollution)

xxxix

Results showed that the highest concentration of SEO applied significantly (P ≤ 0.05) increased the

number of roots (Table 9). Three point seventy five percent v/w treatments had the highest mean

number of roots (77.5±3.227) while 0.75% v/w treatments had the least (60.5±4.406). Multiple

comparison showed that control, 0.15% v/w and 0.75% v/w treatments did not vary with each other

but varied significantly (P ≤ 0.05) with 3.75% v/w treatments. Mean root lengths did not vary among

the treatments. The mean root circumference decreased significantly (P ≤ 0.05) with increase in the

concentrations of SEO applied. Multiple comparison of the average root circumference showed that

control did not vary with 0.15% v/w and 0.75% v/w but decreased significantly (P ≤ 0.05) with 3.75%

v/w treatments. Senna alata started showing signs of the pollutant by the production of aerial roots,

about 56 days after pollution. The aerial roots produced in each treatment increased with an increase

in spent engine oil (SEO) concentrations applied. Initial (at 56 days after pollution) and final (106 days

after pollution) number of aerial roots increased significantly (P ≤ 0.05) with increase in the

concentrations of SEO applied (Table 9). Multiple comparisons of the initial number of aerial roots

showed that control did not vary with 0.15% v/w but was significantly (P ≤ 0.05) less than those of

0.75% v/w and 3.75% v/w treatments. Also, 0.15% v/w was significantly (P ≤ 0.05) less than the

number in 0.75% v/w and 3.75% v/w while 0.75% v/w and 3.75% v/w varied significantly (P ≤ 0.05)

with each other and with the rest of the treatments. Also final number of aerial roots increased

significantly (P ≤ 0.05) with increase in SEO applied. For each treatment, the final number of aerial

roots at 106 days after pollution increased when compared with the initial number at 56 days after

pollution (Plates 3, 4, 5, 6, 7, 8, 9 and 10).

xl

Table 9: Root parameters of Senna alata 163 days after planting

Treatment

(% v/w)

Root number Root length

(cm)

Root

circumference

(cm)

Initial no. of

aerial root (56

DAP)

Final no. of

aerial root

(106 DAP)

0

61.2±6.009a

56.52±2.107b

1.300±0.058c

0.67±0.159h

0.00±0.000a

0.15 65.2±2.056a

58.23±0.716b

1.150±0.087c

3.13±0.904h

6.40±1.027b

0.75 60.5±4.406a

55.92±2.673b

1.125±0.025c,d

7.53±0.867e

13.60±1.287c

3.75 77.5±3.227b

54.67±2.175b

1.050±0.029d,e

27.87±1.973f

55.07±2.306d

Values represent mean ± standard error. Means followed by the same letters in the same column

are not significantly different at P ≤ 0.05

Legend: No. = Number; DAP = Days after pollution

Plate 3: Control Senna alata showing no aerial roots produced (56 days after pollution)

Control plant

showing no aerial roots produced (56 days after pollution)

Control plant

xli

showing no aerial roots produced (56 days after pollution)

Adventitious roots

Plate 4: Control Senna alata

roots (106 days after pollution)

Control plant

Adventitious roots

produced adventitious roots that entered the soil instead of aerial

roots (106 days after pollution)

Control plant

xlii

produced adventitious roots that entered the soil instead of aerial

Plate 5: 0.15% v/w (30 ml) treatment showing aerial roots produced by

pollution)

0.15% v/w (30 ml)

polluted plant

Aerial roots

Plate 5: 0.15% v/w (30 ml) treatment showing aerial roots produced by Senna alata

0.15% v/w (30 ml)

polluted plant

xliii

Senna alata (56 days after

Plate 6: 0.15% v/w (30 ml) treatment showing aerial roots produced by

pollution)

Aerial roots

ml) treatment showing aerial roots produced by Senna alata

0.15% v/w

(30 ml)

treated

plant

xliv

Senna alata (106 days after

Plate 7: 0.75% v/w (150 ml) treatment showing aerial roots produced by

pollution)

0.75% v/w (150 ml)

treated plant

Aerial roots

ml) treatment showing aerial roots produced by Senna alata

0.75% v/w (150 ml)

treated plant

xlv

Senna alata (56 days after

Plate 8: 0.75% v/w (150 ml) treatment showing aerial roots produced by

pollution)

0.75% v/w

(150 ml)

treated plant

Aerial roots

ml) treatment showing aerial roots produced by Senna alata

0.75% v/w

(150 ml)

treated plant

xlvi

Senna alata (106 days after

Plate 9: 3.75% v/w (750 ml) treatment showing numerous aerial roots produced by

days after pollution)

3.75% v/w (750 ml)

treated plant

Aerial roots

ml) treatment showing numerous aerial roots produced by

3.75% v/w (750 ml)

xlvii

ml) treatment showing numerous aerial roots produced by Senna alata (56

Plate 10: 3.75% v/w (750 ml) showing numerous aerial roots produced by

after pollution)

Aerial roots

ml) showing numerous aerial roots produced by

3.75% v/w (750 ml) treated

plant

xlviii

ml) showing numerous aerial roots produced by Senna alata (106 days

3.75% v/w (750 ml) treated

xlix

4.4 Result of the reproductive parameters of Senna alata

Spent engine oil (SEO) decreased the number of flowers, pods (Plate 12) and seeds (Plate 13)

produced by S. alata with increase in concentrations but these decreases were not significant (Table

10). Number of inflorescences (Plate 11) and dry weight of seeds decreased significantly (P ≤ 0.05)

with increase in the concentration of SEO applied. Multiple comparison of the mean number of

inflorescences showed that control varied significantly (P ≤ 0.05) with 0.75% v/w but not with the rest

of the treatments. Also, 0.15% v/w treatments produced significantly (P ≤ 0.05) highest number of

inflorescences when compared with 0.75% v/w and 3.75% v/w treatments. On the other hand,

multiple comparison of the dry weight of seeds showed that 3.75% v/w treatment varied significantly

(P ≤ 0.05) with control but not with 0.15% v/w and 0.75% v/w treatments.

l

Table 10: Reproductive parameters of Senna alata 294 days after planting

Treatm.

(% v/w)

No. of inflors. No. of flowers No. of pods No. of seeds Dry wt. of seeds

(g)

0 7.8±0.917a

155.1±14.02b

51.4±4.672c

30.7±1.075d

49.0±4.392e

0.15 8.5±0.898a

144.3±14.20b

43.8±5.337c

24.7±1.350d

34.6±5.418e,f

0.75 5.1±0.900b,c

131.5±23.76b

39.7±9.175c

21.4±4.951d

37.5±10.47e,f

3.75 5.2±1.104a,c

97.4±17.06b

27.7±6.112c

24.0±4.266d

21.3±4.764f

Values represent mean ± standard error. Means followed by the same letters in the same column

are not significantly different at P ≤ 0.05

Legend: Treatm. = Treatment; Inflors. = Inflorescences; No. = Number; Wt. = weight

li

Flowers

Plate 11: Senna alata polluted with spent engine oil at flowering stage

Inflorescences

lii

Pods

Plate 12: Pods of Senna alata polluted with spent engine oil

liii

Plate 13: Seeds of Senna alata polluted with spent engine oil

liv

CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.0 Discussion

Analysis of the unused spent engine oil (SEO) showed that polycyclic aromatic hydrocarbons

(PAHs) were the only hydrocarbons present. This suggests that vehicles in Nigeria overuse their

crankcase oil between each change with the result that more toxic and lethal pollutants were often

produced. This agreed with the findings of Dominguez-Rosado and Pichtel (2003) who reported that

the PAHs content of used motor oil was often between 34 and 90 times higher than new oil.

Hydrocarbons such as Acenaphthylene, Benzo (k) flouranthene, Flourene, Benzo (b) flouranthene,

Naphthalene, Pyrene, Chrysene, Anthracene and Benzo(g-h-i) perylene were not detected in unused

SEO but were detected in polluted vegetated and unvegetated soils. Their formation might perhaps be

as a result of microbial and plant activities acting on the SEO. This agreed with the findings of GFEA

(2012) and USGS (2014) who reported that PAHs may be formed by microorganisms and plants

through biosynthesis. Hence, SEO pollution enhanced the production of more hydrocarbons by

combined activities of microbes and Senna alata. Though, biosynthesis of PAHs by microbes and

plants is still a controversial issue but this result has also pointed to the possibility of biosynthesis of

PAHs by these organisms. However plants enhance microbial synthesis and degradation of organic

pollutants. Nwadinigwe and Obi-Amadi (2014) reported that plant roots can supply readily available

carbon sources for microorganisms and so, influence the soil microbial community by increasing

microbial numbers in the rhizosphere. In the present work, percentage hydrocarbons left undegraded

in the vegetated soil were less than those of the unvegetated soil. This expressed the plant’s ability to

remediate SEO polluted soil. Hence, S. alata has the ability to remove hydrocarbons from the soil. This

agreed with the findings of Dominguez-Rosado and Pichtel (2004) who reported that Clover plant

(Trifolium) sown in the soil treated with used motor oil in green house, removed all the oil after 150

days. Nwadinigwe and Obi-Amadi (2014) also observed that Pennisetum glaucum significantly reduced

the percentage of hydrocarbons in crude oil polluted soil. All the vegetative parts (roots, stems and

leaves) of S. alata accumulated hydrocarbons in the present work. The leaves were the major organs

of accumulation of hydrocarbons and recorded highest accumulation in all the treatments even

beyond the concentrations in the unused SEO. In a similar way Meudec et al. (2006) reported that

higher molecular weights PAHs were found in large quantities at the upper parts of Salicornia fragilis.

lv

Very little quantities of hydrocarbons were also detected in the vegetative parts of the unpolluted

plants (control or 0% v/w) in the present work. This was perhaps as a result of the volatility of the

lower molecular weight PAHs as well as the contacts made among the roots of various treatments,

since some of the roots of the plant grew beyond the polythene bags used and this made it possible

for the control plants to absorb some hydrocarbons from the treated soils. The stems and leaves of

the 0% v/w treatments had comparatively small concentrations of hydrocarbon accumulation like

other treatments. This agreed with the findings of Meudec et al. (2006) who reported that PAHs

distributions in plant tissues were predominantly low to medium weight hydrocarbons that are

volatile with higher molecular weights at the upper parts of the plants. Joelle et al. (2002) also

reported that root uptake was the main pathway for high molecular weight PAHs which are non

volatile. Moreover, some PAHs such as Benzo(a)pyrene and Benzo(k)fluoranthene have been

demonstrated to be mutagenic and carcinogenic when exposed to humans. Obini et al. (2013)

observed that those PAHs that have not been found to be carcinogenic may, however, synergistically

increase the carcinogenicity of other PAHs. Hence, there is need to reduce the rate of SEO exposure

especially by Nigerian auto mechanics to avoid the risk of cancer and mutation. This can be done by

planting a lot of S. alata near auto mechanic workshops to reduce this risk so that the plant can

absorb and accumulate these toxic hydrocarbons.

In the present experiment, the quantities of heavy metals that remained in the soil in each

treatment increased with increase in the concentrations of SEO applied. This implies that removal

efficiency of heavy metals decreased with increase in SEO applied. This agreed with the findings of

Carvalho and Martin (2001) and Keith et al. (2006) who reported that the phytoremediation efficiency

of metals greatly depend on the concentrations of such metals in solutions. The higher the

concentration of the metal in the solution, the lower the removal efficiency. However, the

concentrations of heavy metals in some vegetative parts of S. alata were higher than the

concentrations in the vegetated soil. This means that the plant organs might have taken up and

accumulated these heavy metals. This disagreed with the findings of Agbogidi and Ohwo (2013) who

reported that the concentrations of heavy metals in contaminated environments were always higher

in the source than in the sink. Higher concentrations of these heavy metals in the vegetative parts of

S. alata showed that some plants can hyper accumulate these substances and consequently, affect

other organisms along the food chain through bioaccumulation. However, the quantities of each of

the heavy metals in S. alata in each treatment fall below the standard allowable limits for medicinal

lvi

plants (Cu – 10 ppm, Zn – 50 ppm, Pb – 10 ppm, Fe – 20 ppm and Al – 5 ppm) (WHO, 1998). Even

though, the concentrations of these heavy metals in some vegetative parts of S. alata were higher

than those in the polluted soil, yet the heavy metals did not have negative effects on the vegetative

growth of the plant. This in contrast to the findings of Nwachukwu et al. (2010) who reported that

heavy metals in plants lead to degeneration of main cell-organelles and even death of plants. Hence,

S. alata might have contained a cysteine rich polypeptide known as phytochelatin that form

complexes with toxic metal ions in the cytosol and subsequently transport them into the vacuole

(Yadav, 2010). This polypeptide might have protected the plant from the deleterious effect of heavy

metals. In the present work, S. alata accumulated Cu, Pb, Zn, Fe and Al in the leaves, stems and roots.

Therefore, the plant can be used as a hyper accumulator plant for bioremediation of some heavy

metals. In this way, these heavy metals are removed from the environment. In a similar way

Jambhulkar and Juwarkar (2009) reported that Cassia siamea accumulated iron, zinc, copper and lead

at high concentrations and it could be used as hyper accumulator plant for bioremediation. Moreover,

Cu was higher in the stems than in the other parts of the plant in the present work. Pb and Fe were

found in large quantities in the leaves while Zn and Al were found in large quantities in the roots.

These suggested that different mechanisms of metal accumulation, exclusion and

compartmentalization exist in various plant species (Mohammad et al., 2008). In the present work,

more Zn and Al were stored in the roots than in the leaves and stems. Cosio et al. (2004) found that

more Zn was stored in the root vacuoles of Thlaspi caerulescens and thus became unavailable for

loading into the xylem because of the existence of regulation mechanism on the plasma membrane.

In the present investigation, more Pb and Zn were stored in the leaves and roots than in the stems of

S. alata. This agreed with the findings of Ghose and Singh (2005) and Gupta and Sinha (2008) who

reported separately that Cassia tora accumulated high concentrations of Pb, Cu, Al and Zn, in their

leaves and roots.

The vegetative parameters of the S. alata before pollution did not vary among the treatments

and within two months, the plant had attained the mean height of 38.4 cm with average leaf area of

252 cm2

for 0.15% v/w treatment. This showed that the environment was favorable to the plant.

However, within two months after pollution with SEO, the plant started producing aerial roots which

increased with an increase in the concentrations of SEO applied. This showed that the pollutant might

have disrupted the normal uptake of water, oxygen and mineral salt, as well as inhibited the

production of new lateral roots below the soil. This agreed with findings of Wyszokowska and

lvii

Kucharski (2000) who reported that contamination of soil with petroleum derived substances can

lead to water and oxygen deficit as well as shortage of available nitrogen and phosphorous.

Therefore, the aerial roots produced might have helped the plant to obtain water and oxygen from

the surface of the soil to compensate for the shortage in the soil. This showed that the bioavailability

of water and oxygen drastically reduced in the soil and the plant combated this by the production of

aerial roots while using the underground roots for the absorption of nutrients. The mean heights of

the plant in 163 days after planting (106 days after pollution) were below 300 cm (225 cm for control

and 203.4 cm for 3.75% v/w treatment). This is in contrast to the findings of Otto et al. (2014) who

reported that S. alata attained heights of 3 to 4 m (300 - 400 cm). However, since the variations

among the treatments (when compared with the control) were not significant and the vegetative

parameters after pollution were significantly higher than those obtained before pollution, it means

that SEO pollution had no adverse effects on the growth of the plant. The plant seemed to have

utilized the degraded SEO to grow. Also, the productivity of a plant is often measured by the area of

leaves that can absorb sun’s energy during photosynthesis. Spent engine oil pollution significantly

enhanced the height, number of pinnules, stem circumference and leaf area of S. alata since these

parameters significantly increased after pollution. This is in contrast to the findings of Abdulhadi and

Kawo (2006) who reported a significant decrease in the plant height, leaf area index and yield of

groundnut and maize (Arachis hypogaea L. and Zea mays L.) in soil polluted with used engine oil.

However, the number of leaves of S. alata polluted with 3.75% v/w was the same before and after

pollution. This somhow agreed with the findings of Nwadinigwe and Oyiga (2009) who reported a

significant decrease in number of leaves of Solanum gilo with increase in foliar spray of petroleum

hydrocarbons. Strong shoot systems (stem base, stem branches and leaves) in plants, increase their

ability to maintain constant position against mechanical forces of the wind. A well established shoot

system shows that the environment is favorable to the plant. In this work, stem base of S. alata

significantly increased after pollution even with increase in concentrations of SEO applied. Therefore,

SEO pollution had no adverse effect on the development of shoot systems of S. alata. There were

differences in the root systems especially on the number of roots produced and root circumference.

Root lengths were not affected by SEO pollution as the variation among treatments was not

significant. The effects on the number of roots produced were positive since the highest

concentration of SEO pollution increased the number of roots per plant, instead of inhibiting root

production. This in contrast to the findings of Okonokhua et al. (2007) who reported that root number

lviii

of maize grown in spent engine oil contaminated soil were adversely affected. Spent engine oil

pollution slightly retarded the circumference of the roots produced since root circumferences

decreased with increase in concentrations of SEO applied. This agreed with the findings of Masakorala

et al. (2013) who reported a significant reduction in root cross-section of Vigna radiata in soil

contaminated with petroleum hydrocarbons. However, the decrease in root circumference was

compensated for by the increase in the number of roots. Though, few lateral roots were produced in

S. alata below the soil when the concentrations of SEO applied increased but these were

compensated for by the production of aerial roots above the soil. Moreover, one plant from 3.75%

v/w treatment died 84 days after pollution. This could be as a result of foliage eating caterpillars

which often ate up the apical bud of the plants, reducing growth rate even as the plant reacted to the

presence of the pollutant.

In the present study, analysis of the reproductive parameters of S. alata showed that SEO had

effect on the number of inflorescences and dry weight of seeds produced which decreased

significantly (P ≤ 0.05) with increase in the concentration of SEO applied. This agreed with the findings

of Nwadinigwe and Oyiga (2009) who reported a significant decrease in number of flowers and dry

weight of seeds of S. gilo with increase in foliar spray of petroleum hydrocarbons. However, the

number of flowers, pods and seeds of S. alata were the same with the control. This is in contrast to

the findings of Nwadinigwe and Oyiga (2009) who reported a significant decrease in number of

flowers of S. gilo with increase in foliar spray of petroleum hydrocarbon. Nwadinigwe and Olawole

(2010) also stated that crude oil pollution reduced the number of seeds of Sorghum vulgare.

Nwadinigwe and Onwumere (2003) also reported that pod production in Glycine max was inhibited by

petroleum hydrocarbons.

lix

5.01 Conclusion

This work has proved that Senna alata L. is an ideal plant for the phytoremediation of soil

polluted with spent engine oil. This was done by phytodegradation and phytoaccumulation. The

hydrocarbons and heavy metals from the SEO were accumulated in the leaves, stems and roots of the

plant. Spent engine oil did not have any adverse effect on the plant rather the plant degraded it and

utilized it for growth. In the present work, S. alata accumulated Cu, Pb, Zn, Fe and Al in the leaves,

stems and roots. Therefore, the plant can be used as a hyper accumulator plant for bioremediation. In

this way, these heavy metals are removed from the environment. With the spate of oil pollution going

on in many parts of Nigeria especially in oil producing states and mechanic workshops, planting of this

perennial shrub will not only decontaminate these pollutants but also aerate the environment during

photosynthesis. Vehicle owners in Nigeria should avoid over usage of crankcase oil between each

change to reduce the production of toxic PAHs and heavy metals with the devastating effect on living

organisms. Bioaccumulation of hydrocarbons and heavy metals along food chain should be curbed

through recycling of SEO rather than indiscriminate pouring into the environment.

lx

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