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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
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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
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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.
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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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