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The Environmentalist ISSN 0251-1088Volume 31Number 3 Environmentalist (2011) 31:288-298DOI 10.1007/s10669-011-9333-x

Morphological and anatomical effects ofcrude oil on Pistia stratiotes

Akintunde Abdul-Rasaq Akapo,S. O. Omidiji & A. A. Otitoloju

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Morphological and anatomical effects of crude oil on Pistiastratiotes

Akintunde Abdul-Rasaq Akapo • S. O. Omidiji •

A. A. Otitoloju

Published online: 15 June 2011

� Springer Science+Business Media, LLC 2011

Abstract Fresh whole plants of Pistia stratiotes were

exposed to varying doses of crude oil (0–100 ppm) for

28 days at normal temperature of 30 ± 2�C. Samples were

taken weekly during this period for determination of

changes in leaf area, root length, number of leaves, and

number of sprouts. The cross-section of one terminal end of

the major roots and cellular distribution of the meristematic

region were also examined. The results show that crude oil

was toxic to the plant at all concentrations in all investi-

gated parameters for as low as 10 ppm. Association was

also observed between crude oil toxicity and certain metals

inherent in the crude oil such as manganese and lead. Cell

shape disruptions, changes in mitotic indices, and the dis-

tortion of cellular anatomy and structure at the apical

region also characterized the presence of crude oil in the

environment of P. stratiotes. P. stratiotes may not be a

good bio-accumulator of crude oil but may be used for the

detection of pollution.

Keywords Crude oil pollution � Pistia stratiotes �Cytotoxicity � Morphological aberrations in Pistia

stratiotes � Anatomical aberrations � Crude oil pollution

in Nigeria

1 Introduction

Almost all activities involved in the exploration and

exploitation of crude oil result in the discharge of crude oil

into the environment. Recently, the volume of crude oil

being spilled into the environment has increased signifi-

cantly, especially now that oil has taken the center stage as

the major source of energy to mankind. Crude oil-induced

pollution is dependent on the nature and type of crude oil,

the level of oil contamination, type of environment, and

selective degree of sensitivity of the individual organisms

(Garrity and Levings 1990). Oil interferes with the func-

tioning of the various organs and systems of plants and

animals and also creates environmental conditions unfa-

vorable for life. Various scientists have reported the adverse

effect of crude oil pollution on plant growth (De Jong 1980;

Murphy and Riley 1929; Stafford 1973; Udo and Fayemi

1975). The effect is proportional to the level, as well as the

concentration of crude oil in the environment (Ogboghodo

et al. 2004). Anoliefo (1991) reported morphological aber-

rations to include cell disruption in roots and other organs

and the presence of oil films in the epidermal and cortical

regions of the root, stem, and leaves. Amakiri and Onafeg-

hara (1983) also demonstrated that oil deposited on leaves of

plants penetrated the leaves and reduced transpiration rate

and photosynthesis. Growth of plants in oil-polluted soils

has also been reported to be generally retarded, and chlorosis

of leaves resulted coupled with dehydration of the plant,

indicating water deficiency (Udo and Fayemi 1975). The

performance of maize plant and other plants has also been

found to be seriously affected by oil pollution, that is, growth

was generally poor as pollution level increases (Toogood

and Rowell 1977; Odu 1981). The retardation in plant

growth is said to be due to a hindrance of transpiration and

photosynthesis (Amakiri and Onafeghara 1983; Odu 1978).

A. A.-R. Akapo (&) � S. O. Omidiji

Department of Cell Biology and Genetics, Faculty of Science,

University of Lagos, Akoka, Yaba, Lagos, Nigeria

e-mail: akapsy@yahoo.com

S. O. Omidiji

e-mail: kunjo2002@yahoo.co.uk

A. A. Otitoloju

Department of Zoology, Ecotoxicology Laboratory, Faculty

of Science, University of Lagos, Akoka, Yaba, Lagos, Nigeria

e-mail: bayotitoloju@yahoo.com

123

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DOI 10.1007/s10669-011-9333-x

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The impact of oil on organisms depends on the char-

acteristics of the oil spill such as its viscosity and toxicity,

the amount of oil and the time for which it was in contact

with the organism. Spilled oil on the surface of water

bodies’ limits gaseous exchange, entangles and kills sur-

face organisms, and coats the gills of fish (Wells et al.

1995; Spies et al. 1996). It also depresses phytoplankton

photosynthesis, respiration, and growth; kills or causes

developmental abnormalities in zooplankton and the young

stages of many aquatic organisms; and causes tainting of

fish, shellfish, etc. (Afolabi et al. 1985; National Research

Council (NRC) 1985; Otitoloju and Adeoye 2003; Powell

et al. 1985). The speed and extent of recovery of plants

from crude oil pollution appears to be species specific and

may also depend on some factors, which include the type

of oil, the mode of delivery, the timing of oiling, and the

amount of oil (Pezeshki et al. 2000).

Oil pollution of soil leads to the build up of essential

(organic carbon, phosphorus, calcium, magnesium) and

non-essential (manganese, lead, zinc, iron, cobalt, copper)

elements in soil and the eventual translocation in plant tis-

sues (Vwioko et al. 2006). The pollution of soil by spent

lubricating oil has been reported to cause growth retardation

in plants (Anoliefo and Vwioko 1995; Odjeigba and Sadiq

2002). This reduction in plant growth has been attributed to

the presence of heavy metals at toxic concentrations in soil

(Anoliefo and Vwioko 1995;). Whismann et al. (1974)

observed that most heavy metals such as vanadium, lead,

aluminum, nickel, and iron, which were usually below

detection in unused lubricating oil, gave high ppm values in

used oil. Elements such as copper, molybdenum, nickel,

manganese, chlorine, and zinc are essential for plant growth

in low concentrations (Reeves and Baker 2000). Never-

theless, beyond certain concentrations, these same elements

become toxic for most plant species (Monni et al. 2000).

In this study, Pistia stratiotes L., a perennial free-

floating invasive weed, was investigated because it has

been widely reported as a good bio-indicator of pollution

and a potential candidate plant for bioremediation purpose.

For example, Ghavzan et al. (2006) demonstrated that

occurrence of Pistia stratiotes L. was associated with high

pollution rates in water bodies, while Niaz and Rasul

(1998) showed that Pistia stratiotes L. and Eichhornia

crassipes could be used as biological indicators in aquatic

habitats if the nature of the polluting salt was known. When

exposed to salt shocks, Pistia stratiotes L. showed distur-

bances in cell size and number in the meristematic region,

which resulted in a suppressed growth of the plant (Hanif

and Daves 1998). It has been shown to present differential

accumulation and tolerance levels for different metals at

similar treatment condition coupled with trace element

accumulation in tissues, and the bio-concentration factors

were proportional to the initial concentration of individual

metals in the growth medium and duration of exposure

(Odjeigba and Fasidi 2004). It has also been indicated as a

good accumulator of zinc, chromium, copper, cadmium,

lead, silver, and mercury, and it meets with the phyto-

remediation perspective as a good metal accumulator

(Odjeigba and Fasidi 2004).

On the basis of the above, the objective of this study is

to assess the possibility of using P. stratiotes as a marker of

oil and heavy metal pollution in freshwater ecosystems by

evaluating the toxic effect of crude oil and its metal content

on the morphological, anatomical, and physiological

changes on the plant.

2 Materials and methods

2.1 Collection of samples

Whole plants of Pistia stratiotes were collected from the

Ogbe stream located beside the Distance Learning Institute

(DLI) road, University of Lagos, Akoka, Lagos. The water

used to cultivate the plants was obtained from a well at

Community Road, Akoka, Lagos State.

2.2 Physicochemical assessments of plants samples

2.2.1 Determination of the total hydrocarbon content

(THC) in test samples

The THC of the plant samples and water sample used during

the experiment were determined by gravimetric analysis.

About 90 g of each sample were refluxed in redistilled

methanol (100 ml) containing potassium hydroxide (3.0 g)

for about 90 min. The suspension was filtered while the

methanol extract was cooled and extracted twice with

n-hexane (2.5 ml). The n-hexane extracted was reduced to

about 0.5 ml by distillation and chromatographed on a

column of silica gel using n-hexane (12 ml) and dichloro-

methane (15 ml) to elute the aliphatic and aromatic frac-

tions, respectively. The two eluates were evaporated, and

the residues were weighed to obtain the gravimetric con-

centrations of the aliphatic and aromatic fractions. The sum

of weights of the two fractions was taken as the total

hydrocarbon concentration of each sample.

2.2.2 Determination of the metal ion content in Pistia

stratiotes

Plant samples were collected from the various culturing

bowl and taken to the laboratory. At day seven (week 1),

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the plants in the various water media were harvested and

taken to the laboratory for physicochemical assays (to

detect selected metal ion concentration) using the AAS.

The leaves and root/stem were assayed. The heavy metals

of interest are manganese, cadmium, and lead.

After collection, the leaves and the root samples were

detached and dried in the oven at 105�C for 1 h and ashed.

They were then weighed after drying before transferring to

the furnace (550�C). They were allowed to cool and

weighed. Nitric acid (HNO3) was added to dissolve the

particles after which it was warmed slightly on the water

bathe to allow for further dissolving. Distilled water

(50 ml) was added, and it was then allowed to cool. After

cooling, it was filtered (to remove particles) into a 100-ml

standard flask and this was used for metal ion content

determination. From the readings (of the AAS), the metal

ion content was determined by using the formula below:

AAS

10� 1000

Sample Weight

2.3 Growth monitoring experiment

The culturing of Pistia stratiotes was carried out using

crude oil as the pollutant. The crude oil was obtained from

Port Harcourt and was identified as Forcados Mix by Shell

Petroleum Nigeria Limited. Five concentrations of crude

oil were used (0, 10, 20, 50, and 100 ppm). For the various

concentrations, 4.5 l of water was measured into each bowl

and the crude oil concentrations were adjusted to correlate

with the volume of water as follows:

100 ppm = 100 mg/l = 0.1 g/l 9 4.5 = 0.45 g

50 ppm = 50 mg/l = 0.05 g/l 9 4.5 = 0.225 g

20 ppm = 20 mg/l = 0.02 g/l 9 4.5 = 0.09 g

10 ppm = 10 mg/l = 0.01 g/l 9 4.5 = 0.045 g

The crude oil was weighed using an analytical weighing

balance and mixed with the water in the various culture

media as described above.

Ten viable young rosettes of Pistia stratiotes were

obtained from an initial bowl (where the plants had been

grown earlier to study their growth rate and other param-

eters) and transferred to bowls containing the water sup-

plemented with crude oil. The physically observed

parameters, which included leaf length, leaf breath, root

length, number of leaves, and number of sprouts, were

observed and recorded every week for 4-week duration.

The control experiment consists of a bowl with water

containing no crude oil. The water obtained from a well

was assayed for the total hydrocarbon content (THC)

content and metal ion concentration using the Atomic

Absorption Spectrophotometer (AAS). Ten plants were

placed in each bowl, and parameters mentioned above were

observed and values recorded.

2.4 Anatomical changes assessment

2.4.1 Anatomical assessments of the root cross-sections

Newly growing root sample was carefully detached from

one of the plants in the various concentrations and placed

in Petri dishes filled with water. Each root was taken one

after the other, transversely cut into the possible thinnest

sections, and the pieces were put in water in another Petri

dish. The cross-sections were carefully picked using a pair

of forceps or a dropping pipette to suck it up and were

placed on a clean-labeled slide. A drop of safranine stain

was put on it for 30 s, and the excess stain was removed by

adding 2–3 drops of 50% alcohol. This was dried off using

a filter paper, and 2 drops of 50% glycerine were put on it

to prevent drying of the tissue. It also served as the

mountant for the specimen and the slides were then

observed under the light microscope using 940 and 9100

magnification objective lens.

2.4.2 Cytological assessments of the root tips

Newly growing roots were cut and placed in fixative in a

Petri dish that had been carefully labeled and were taken to

the laboratory. HCL (1 N) was added to a Petri dish and the

root tip immersed in it for a minute. The root was then

taken and further trimmed to have only the tip left. It was

then gently placed on a tile and macerated and left for

about 15–20 min. The slide was then covered with a cover

slip and left for about 3 min. The slide covered with a

cover slip with the mount on it was then wrapped with a

filter paper (to remove excess staining from the mount).

Tissue paper was further used to mop up the stains. Cortex

was then added to seal the cover slip with the slide. It was

then mounted on the microscope for examination (to see

the chromosomes).

3 Results

3.1 Physicochemical assessments of plants samples

3.1.1 Determination of the total hydrocarbon content

(THC) in test samples

Table 1 is the result of analysis of metal ion content and

total hydrocarbon content (THC) of the plants after the

experiments had been conducted for 28 days.

The total hydrocarbon content (THC) was low in the

control and high at 10 ppm as compared to the remaining

concentrations at the end of the experiment with 5.5 and

49.62 mg/kg in the leaves, respectively, and 0.0 and

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117.96 mg/kg in the roots, respectively. The THC values

were seen to be higher in the roots than what obtains in

the leaves and it has a correlation with increasing

concentration.

3.1.2 Determination of the metal ion content in Pistia

stratiotes

Table 1 shows the heavy metal ion (manganese, cadmium

and lead) content and total hydrocarbon content of the

water from the site of collection of experimental plants,

crude oil sample, water used for the experiment, and

analysis of the plant sample before experiment.

For the heavy metals analyzed, manganese accumulation

was relatively higher in both leaves and roots at the other

concentrations as compared with the control that had 101.79

and 491.12 mg/kg in the leaves and roots, respectively.

Lead accumulation was seen to be uniform in the entire

growth medium regardless of the concentration of the test

pollutant. However, the rate of accumulation in the

roots was higher than in the leaves for the entire growth

medium.

Cadmium, initially present (Table 1), was observed to

have completely disappeared from the entire experimental

set up except for the little dosage (1.04 mg/kg) present in

the leaves of the 100-ppm crude oil-supplemented growth

media. Except for the leaves of plants in the 100-ppm

growth media, Cadmium was not detected in all the other

concentration (Table 2).

3.2 Growth monitoring experiment

Figures 1, 2, 3, and 4 show the effect of the different

crude oil concentrations on the growing plant rosettes of

P. stratiotes. Growth is generally expressed in terms of

changes in leaf area, number of leaves, root length, and

number of sprouts. The weekly increase in the leaf area at

different concentrations of crude oil is shown in Fig. 1. For

the control, the leaf area had increased by 204% over starting

value by the end of the fourth week. This increase was still

linear up to 28 days. Other concentrations (10, 20, 50, and

100 ppm) showed increase in leaf area up to day 14 before

declining up to the 28th day. The reduced increase in leaf

area in treated plants was dose dependent up to 50 ppm. The

change in root length of the control and experimental plants

is shown in Fig. 2. The number of leaves in the control plants

increased by 95.8% by the end of the fourth week as com-

pared to the treated plants (25.5% in 10 ppm and 16% in

100 ppm). The phytotoxic effect was manifested in both the

20 and 50 ppm as the values had fallen below zero.

The phytotoxic effect of crude oil pollution on root

length was concentration dependent as shown in Fig. 3. A

605% increase was seen in the control as compared to the

initial value by the end of the 28th day. However, the

decline in the growth of the plants in the 10-ppm crude oil-

supplemented medium was observed after the 21st day. By

this period, the plants had 264.4% increase. Other concen-

trations show decline by the end of the second week and had

fallen to a negative value by the end of the 28th day as seen

with the 50-ppm crude oil-exposed plants. Figure 4 shows

Table 1 Metal ion and THC of water, crude oil, and P. stratiotes before experiment

Element Water from

sample site (ppm)

Water used for

experiment (ppm)

Crude oil sample

(Forcados Mix) (ppm)

Leaf

(mg/kg)

Root/stem

(mg/kg)

Lead (Pb) ND ND ND 7.45 3.59

Cadmium (Cd) 0.14 0.1 58.14 0.62 0.99

Manganese (Mn) 0.05 ND 0.63 104.78 74.43

THC 56 0 100% 5.5 0

ND Not detected

Table 2 Metal ion and THC content of P. Stratiotes after 28 days of experiment

Conc. 0 ppm 10 ppm 20 ppm 50 ppm 100 ppm

Leaf Root Leaf Root Leaf Root Leaf Root Leaf Root

THC 5.5 0 49.62 117.96 4.88 55.62 6.37 70.78 10.09 33.21

Mn 101.79 491.12 137.16 704.96 467.97 1,084.86 206.67 1,268.88 225.73 641.62

Pb 62.95 97.89 65.39 154.97 60.22 161.39 57.59 153.32 44.70 129.35

Cd ND ND ND ND ND ND ND ND 1.04 ND

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the average number of young sprouts in the various con-

centrations. Although the sprouts appearance was by the

second week, phytotoxicity set in by the 21st day as seen in

the 10, 20, and 50 ppm. Apart from the above observations,

chlorosis of leaves together with shrinkage of the roots was

observed in all the crude oil-supplemented growth samples.

3.3 Morphological and anatomical changes assessment

3.3.1 Anatomical assessments of the root cross-sections

Plate 1 shows plant sections that stained evenly and

appeared normal in terms of structure and arrangement.

They were used to analyze the effect of crude oil on the root

cross-sections of plants in the supplemented media. All the

plates were taken by the 7th day of the experiment. The

sections were obtained at 5 mm from the tip of the main root.

The cortex has three layers of cells followed by two-layered

radiating plate of cortical parenchyma that separate the

intercellular cavity. The pericycle has three layers of cells

and is bounded externally by the endoderm that surrounds

the stele of the root. The metaxylem, circular structures that

are arranged in rings within the stele, and some other

structures that make up the vascular bundles are scattered

within the stele and has an average number of thirteen. The

cortical parenchyma has six cells arranged in a row.

The cytological effects of crude oil pollution on the

structures of the root section are shown in Plates 2, 3, 4,

and 5. These plates show cross-sections of the root of

plants grown in the crude oil-supplemented medium.

Plate 2 show the root cross-section of the plant in

10-ppm crude oil-supplemented media. In the plate, the

cortical parenchyma was seen to be elongated more than

what was observed in the control plate; endodermal cells

were still intact, and the epidermis was still clearly visible.

However, the cortex is seen to be gradually rupturing, and

the schlerenchyma is not well defined. Rupturing of the

intercellular cavity was associated with black deposits on

the walls of the intact ones.

Plates 3 and 4 show the anatomical structures of roots of

plants grown in water containing 20 and 50 ppm of crude

oil concentrations, respectively. Although the cells stained

well, black deposits are seen all over the walls of their

-100

-50

0

50

100

150

200

250

0 7 14 21 28

Growth period (days)

Lea

f ar

ea (

% c

on

tro

l)

Fig. 1 Change in leaf area during growth of Pistia stratiotes for a

period of 28 days. Leaf area of control on Day 0 = 559 mm2. Blacksquare Control, black triangle 10 ppm, black circle 20 ppm, whitetriangle 50 ppm, white square 100 ppm

-100

0

100

200

300

400

500

600

700

0 7 14 21 28

Growth period (days)

Ro

ot

len

gth

(%

co

ntr

ol)

Fig. 2 Change in root length during growth of Pistia stratiotes for a

period of 28 days. Root length of control on Day 0 = 16.8 mm. Blacksquare control, black triangle 10 ppm, black circle 20 ppm, whitetriangle 50 ppm, white square 100 ppm

-80

-60

-40

-20

0

20

40

60

80

100

120

0 7 14 21 28

Growth period (days)Nu

mb

er o

f le

aves

(%

co

ntr

ol)

Fig. 3 Change in number of leaves of Pistia stratiotes during growth

for a period of 28 days. Average number of leaves of control on Day

0 = 5.0 ± 1.0. Black square control, black triangle 10 ppm, blackcircle 20 ppm, white triangle 50 ppm, white square 100 ppm

0

0.5

1

1.5

2

2.5

3

3.5

4

0 7 14 21 28

Growth period (days)

Nu

mb

er o

f S

pro

uts

Fig. 4 Change in number of sprouts during growth for a period of

28 days. Black square control, black triangle 10 ppm, black circle20 ppm, white triangle 50 ppm, white square 100 ppm

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Plate 1 Anatomy of root

cross-section of Pistia stratiotes(water lettuce) in Control

medium (a 9100 and b 940

magnification). This diagram

shows the structures of the

cross-section of a well-stained

and healthy plant

Plate 2 Anatomy of root

cross-section of plants grown in

10-ppm crude oil-supplemented

medium (a 9100 and b 940

magnification)

Plate 3 Anatomy of root

cross-section of plants grown in

20-ppm crude oil-supplemented

medium (a 9100 and b 940

magnification)

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cortical parenchyma and intercellular cavity. Although the

endoderm was still present, metaxylems seem to have been

lost. The intercellular cavity, schlerenchyma, cortical

parenchyma appear to be gradually rupturing away, and the

phytotoxic effects seen were more intense with high crude

oil concentration.

In Plate 5 (plant in 100 ppm crude oil), the root cross-

section had lost almost all the essential structures and the

intercellular spaces were seen to be clumping together. In

all, the severity of the phytotoxic effect of crude was

dependent on the concentration, and this was observed to

be more pronounced with increased concentration.

3.3.2 Cytological assessments of the root tips

Plates 6, 7, 8, 9, and 10 show the micrograph obtained after

squashing of the root tip of water lettuce (P. stratiotes)

grown in various concentrations of crude oil. Plate 6 is the

control and serves as the standard to which Plates 7, 8, 9,

and 10 are being compared. In all (Plates 7, 8, 9, 10), there

is distortion of cell morphology, that is, arrangement of the

cells in the root has been disrupted. The disruption is seen

to be concentration dependent as it increases with

increasing concentration. The disruption is also associated

with decreasing number of cells and also seen to be con-

centration dependent, that is, the number of cells reduces as

the concentration increases.

Although there were observed distortion, the phases of

cell division (mitosis) were normal qualitatively. Mitosis

was going on qualitatively, but quantitatively, it has been

affected. The mitotic index, from Table 3, has been affected

with increasing concentration of the pollutant. The cells

appear to be arrested at the prophase stage of cell division.

The shapes of some cells were also seen to be affected.

Some black deposits observed within Plates 7, 8, 9, and 10

were not seen in Plate 6.

Plate 4 Anatomy of root

cross-section of plants grown in

50-ppm crude oil-supplemented

medium (a 9100 and b 940

magnification)

Plate 5 Anatomy of root

cross-section of plants grown in

100-ppm crude oil-

supplemented medium (a 940

and b 9100 magnification)

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4 Discussion

In this study, the trend of accumulation of lead and man-

ganese and the total hydrocarbon content was seen to be

greater at low concentrations and the values decreased as

concentration increased. The source of the plant was

slightly polluted with hydrocarbon (HC). After 28-day

growth in HC-free medium, only 5.5 ppm of HC was

detected in the leaves and with no HC in the roots. It is

suggestive that HC translocation upwards is probably

irreversible accumulating in the leaves. After treatment

with crude oil, the accumulation of HC in the plants was

highest at the lowest concentration of 10 ppm and lowest at

higher doses of 100 ppm. However, the THC accumulated

in the roots was greater than in the leaves for all treatment.

Crude oil treatment led to the build up of manganese and

lead in all parts of treated plants. Such observation

including the eventual translocation in plant tissues has

been observed earlier by Vwioko et al. (2006). Considering

the trend of accumulation of lead and manganese, this

study agrees with the work of Beauford et al. (1977). They

had observed that heavy metals such as mercury are

accumulated more in the roots of plants, and they associ-

ated the observation to the fact that the roots are in direct

contact with the metals in the soil environment.

Lead accumulation in the plants part was also seen to be

dose dependent up to 20 ppm before decline. There was a

higher amount of lead in the roots than in the leaves. This

was despite the non-availability of lead in the water sam-

ples and crude oil sample used.

Plates 6 and 7 Plate 6

showing micrograph of root tip

of the control plants, while

Plate 7 is showing micrograph

of the root tips grown in 10-ppm

crude oil-supplemented

medium. Plate 6 is the control,

and it shows the normal

arrangements of cells at the root

tips of P. stratiotes. Distortion

of cellular morphology had set

in Plate 7. Arrows show mitotic

processes

Plates 8 and 9 Plate 8 shows

micrograph of root tip grown in

20-ppm crude oil-supplemented

medium, while Plate 9 shows

micrograph of root tip grown in

50-ppm crude oil-supplemented

medium. Both plates show

drastic reduction in the number

of cells as compared to the

control in addition to distorted

shapes. Although mitotic

process could be identified

qualitatively, it has been

affected quantitatively. Arrowsshow mitotic processes

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According to Odunlami (1998), these could be as a

result of introduction into the environment through many

anthropogenic activities; it has also been reported to be

transferred from the atmosphere to soil, water, and vege-

tation by dry and wet depositions and from the exhaust of

petrol-driven vehicle.

The plant, water lettuce (Pistia stratiotes), could be

suggested to be a good accumulator of lead, and this agrees

with the work of Salim et al. (1993) and Odjeigba and

Fasidi (2004).

The result also conforms to the work Odjeigba and Fasidi

(2004), when they studied the implications of accumulation

of trace elements by water lettuce (Pistia stratiotes) for

phytoremediation purposes. They had shown that Pistia

stratiotes moderately accumulated zinc, chromium, copper,

lead, silver, and cadmium to a high concentration.

From this work, cadmium accumulation was relatively

not observed except for the little dosage that was analyzed

in the leaves of the plant in the 100-ppm crude oil treat-

ment. This could be attributed to atmospheric deposition.

The growth of water lettuce plant (Pistia stratiotes) is

retrogressively affected by crude oil, and the effect is

dependent on the concentration of crude oil in environ-

ment. This is similar to the report of Ogboghodo et al.

(2004). It was also observed that the dose of crude oil from

10 ppm was inhibitory to the growth of Pistia stratiotes.

All physical growth parameters measured (such as number

of leaves, root length, leaf area, and number of sprouts)

declined during growth in the presence of crude oil. The

decline was shown to be concentration dependent. There

was no recovery of growth during the period in crude oil-

treated plants.

The roots of plants exposed to various concentration of

crude oil were observed to shrink and detach. Shrinking

was also concentration dependent. This observation agrees

with the work of Udo and Fayemi (1975) who showed that

plants growing in oil-polluted soils were generally retarded

and showed chlorosis of leaves. They attributed some of

the effects to dehydration and general water deficiency.

The decline in growth parameters that had been observed

earlier by Odjeigba and Fasidi agrees with the present

study. Retardation of growth at high levels of crude oil

treatment was observed by Toogood and Rowell (1977)

and Odu (1981) although using terrestrial plants. Accord-

ing to these workers, growth retardation may be due to

hindrance of transpiration rate and photosynthesis (Odu

1978). Similar observations were reported by Atuanya

(1987), Ghouse et al. (1980), and Gill and Sandota (1976);

all observed a positive relationship between the extent of

retardation in growth and concentration of crude oil in the

soil. According to McLaughlin and Norby (1991), phyto-

toxicity of a contaminant depends on the uptake potential,

biochemical reactivity, and exposure dose; a given expo-

sure dose may correspond to different degrees of ‘‘internal

dose’’ in different species or individual according to rates

of entry, distribution within the plants, environmental

conditions, and many other factors. Therefore, toxicity to

crude oil observed in this study is likely to be due to

Plate 10 Micrograph of root tip grown in 100-ppm crude oil-

supplemented medium. Cellular arrangements are no longer well

defined. Arrows show mitotic processes

Table 3 Rough estimate effect of various concentrations of crude oil on the mitotic index of the root tip of P. stratiotes (the cell counts were

made on the slide as opposed to direct microscopic view)

Crude oil

medium

Cells in

interphase

Cells in

prophase

Cells in

metaphase

Cells in

anaphase

Cells in

telophase

Total no. of

cells in mitosis

Total no.

of cells on slide

Mitotic

index %

Control 40 8 1 0 0 9 49 18.4

10 ppm 30 7 2 0 0 9 39 23.1

20 ppm 13 1 1 0 0 2 15 13.3

50 ppm 9 3 1 0 0 4 13 30.8

100 ppm 9 3 0 1 0 4 13 30.8

296 Environmentalist (2011) 31:288–298

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combination of effects that may be separated by further

studies.

From the cytology assessments, cell disruptions in the

roots were readily observed. Cell shape changed in treated

cells and the mitotic index was affected, suggesting that

more cells were in the mitotic phase in treated plants. The

mitotic indices increased with increasing concentration of

crude oil, suggesting enhanced entrance into mitosis and

thus cell division. This, however, did not reflect in the

growth of the roots of the plants treated with crude oil. On

the other hand, treatment with higher dose of crude oil

resulted in reduced root elongation. It is likely therefore

that mitotic index determination suffered from the small

number of cells counted on the microscope per view. It is

to be noted that the very small size of P. stratiotes root

cells necessitated analysis with 9100 objectives and this

probably contributed to the problem. It is ideal that many

more cells are grouped to evaluate mitotic index as was

done by Howell et al. (2007) in onion roots.

Most of the cells in healthy plants were in the prophase

and metaphase stages of cell division. This agrees with the

observation of Howell et al. (2007) in normal onions. They

had shown that most cells in the control onion roots are in

prophase and metaphase stages. However, there is the

observation of lower mitotic index after treatment with

20 ppm of crude oil while treatment with 10 ppm appeared

to be stimulatory. It is seen that the plant may be able to

tolerate crude oil treatment up to 10 ppm.

The treated plants show the presence of dark zones in

the cortical and epidermal regions of the root cross-sec-

tions. Similar observations have been reported by Anoliefo

(1991) to occur in the roots, stems, and leaves of plants

exposed to crude oil.

Cytotoxicity at 10 ppm was pronounced and suggested

that lower doses of less than 10 ppm might have shown

drastic effects too. A strong relationship between the degree

of disruption and concentration of crude oil was observed

and this agrees with the work of Ogboghodo et al. (2004).

Cytotoxicity was observed at all doses of hydrocarbons

and this increased with high crude oil treatment. The results

suggests Pistia stratiotes as a possible bio-indicator for the

detection of crude oil pollution in water bodies using the

growth and cell disruption at lower doses as markers.

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