The effect of crude oil contaminated soil on physical and biochemical properties

of beans (phaseolus vulgaris) was investigated. Fifteen polythene pots with drainage holes at the bottom, each containing 10 kg of surface soil, were randomly placed on a table in the screenhouse in a factorial combination of five treatment levels (0.4%, 0.3%, 0.2%, 0.1% and 0% w/w) of crude oil and were designated P4, P3, P2, P1 and P0

respectively. Three seeds of beans per pot were planted. Growth parameters (plant height, stem girth, relative water content (RWC), and soluble protein content (SPC)) and antioxidant indices were determined in the beans over a period of nine weeks after planting (WAP). Results showed that growth of beans planted in contaminated soil was significantly lower (p<0.05) than that of control. Beans planted in the contaminated soil also showed a significant increase (p<0.05) in superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) and glutathione reductase (GR) activities in leaves when compared with control plants. The results suggest that crude oil contaminated soil hindered availability of water, air and

nutrients to beans roots, creating a drought condition which could induce oxidative stress in the plant and consequently retarding growth and yield of beans plant.


Title page        i

Certification        ii

Approval page        iii

Dedication        iv

Acknowledgement        v

Table of content        vi

List of figures                    xi

List of table        xii

Abstract       xiii


1.0 Introduction         1

1.1 Statement of study problem               3

1.2 Relevance of study         3

1.3 Aim & Objective of study         3


2.1 Soil contamination by crude oil         5

2.2 The effect of total petroleum hydrocarbon (TPH) on soil        7

2.3 Impact of crude oil spillage pollution on agricultural soil properties and crop growth        9

2.4 Effect of different crude oil fractions on growth and oxidative stress parameters of plant    12  

2.5 Effect of light crude oil contamination on the germination shoot growth of plant grown      14                   in sandy loam soil

2.6.0 Common beans (phaseolus vulgaris)        16

2.6.1 Bioactive components in common beans (phaseolus vulgaris)        19 Polyphenols        20 Lectins        22 Trypsin inhibitors        28

2.7.0 Magnesium deficiency and high light intensity enhance activities of       30

Superoxide dismutase, ascorbate peroxidase and glutathione reductase in beans leaves       

2.7.1 Superoxide dismutase       31

2.7.2 Catalase       33

2.7.3 Lipid peroxidase       36

2.7.4 Ascorbate peroxidase       40 Characteristics of ascorbate peroxidase       42

2.7.5 Peroxidase       44

2.7.6 Glutathione Reductase       45


3.1 Reagents       47

3.2 Crude oil Collection       47

3.3 Experimental design and agronomic details       47

3.4 Relative water content (RWC) determination       48

3.5 Soil analysis       49

3.5.1 Wet oxidation method for soil digestion (heavy metals)       49

3.5.2 Exchange acidity in soil       49

3.5.3 Exchangeable cations in soil       50

3.5.4 Determination % organic carbon and organic matter in soil       51

(using walkley-black method)

3.5.5 Available Phosphorus in soil       52

3.5.6 Available Nitrate in soil       53

3.6 Preparation of Homogenate       54

3.7 Determination of Protein content       55

3.8 Determination of Malondialdehyde (MDA) concentration       56

3.9 Determination of Reduced Glutathione (GSH) concentration       57

3.10 Determination of Superoxide Dismutase (SOD) activity       58

3.11 Determination of Catalase activity       59

3.12 Determination of Ascorbate Peroxidase activity       60

3.13 Peroxidase       61



5.0 Discussion       70

5.1 Conclusion       72


APPENDIX I: Result of analysis carried out on experimental soil       95

APPENDIX II: Standard calibration curve for protein determination       96

APPENDIX III: Standard calibration curve for GSH concentration       97

APPENDIX IV: Standard calibration curve for the determination of catalase activity       98


Figure 2.1: Chemical structure of some polyphenolic compounds present in common beans      23

Figure 2.2: Initiation step of lipid peroxidation         38

Figure 2.3: Initial phase of propagation step of lipid peroxidation process indicating the         39Oxygen uptake

Figure 4.1: Effect of crude oil-impacted soil on height of beans over a period of nine         63(9) weeks 

Figure 4.2: Effect of crude oil-impacted soil on stem girth of beans over a period of nine         64 (9) weeks. 

Figure 4.3: Effect of crude oil-impacted soil on the relative water content (RWC) of planted     65 Beans over a period of nine (9) weeks


Table 2.1: Characteristic of plant ascorbate peroxidase        42

Table 4.1: Effect of crude oil impacted soil on activities of superoxide dismutase (SOD),        66

Catalase (CAT), peroxidase (POD), ascorbate perioxidase (APX) and glutathione reductase

(GR) activities of beans leaves.                                     

Table 4.2: Effect of crude oil impacted soil on activities of superoxide dismutase (SOD),         67

Catalase (CAT), peroxidase (POD), ascorbate perioxidase (APX) and glutathione reductase

(GR) activities of beans stems.

Table 4.3: Effect of crude oil impacted soil on soluble protein content (SPC)         68 and malondialdehyde (MDA) of beans leaves.

Table 4.4: Effect of crude oil impacted soil on soluble protein content (SPC)         69   and malondialdehyde (MDA) of beans stems.



Crude oil is a complex mixture of thousands of hydrocarbons and non-hydrocarbon compounds, including heavy metals. Nigeria is an established crude oil exporting nation producing medium and light crude oil, such as bonny light (Amund et al., 1993). Crude oil exportation is the main stay of Nigeria’s economy. Crude oil is a colloidal mixture of different hydrocarbons (90%) and non-hydrocarbon (10%) components (Cadwellaer et al., 1993). Various activities in crude oil exploration, exploitation, storage and transportation lead to spillage of oil to the environment (Agbogidi et al., 2007). Crude oil causes harmful effects on the environment, where it poses a serious threat to organisms and farmland that are linked in a complex food chain that includes humans (Lundstedt et al., 2003). The effects of crude oil on the growth and performance of plants have been reported in many studies (Njoku et al., 2008). Crude oil in soil makes the soil condition unsatisfactory for plant growth. It can reduce the level of available plant nutrient in contaminated soils (Jong et al., 1980) and can also raise the levels of certain elements such as iron and zinc to toxic amounts (Udo et al., 1975). Water and oil are usually considered to be immiscible. However, crude oil contains a very small soluble portion referred to as the water soluble fraction (WSF). The soluble constituents are dispersed particulate oil, dissolved hydrocarbons and soluble contaminants such as metallic ions. The components of crude oil that go into solution make up the WSF. The lower the molecular weight of the constituent hydrocarbon of crude oil, the higher is its concentration in the water-soluble fraction (Edema et al., 2012).

Exposure of plants to crude oil and heavy metal poisoning has been reported to produce reactive oxygen species (ROS) and other free radicals which induce oxidative stress and cause lipid peroxidation (Blokhina et al., 1999). Even at an early stage, it can cause a reduction in cell proliferation and growth. Various researchers have reported activation of lipid peroxidation in plants exposed to different pollutants (Chirkova et al., 1998). Increase in superoxide dismutase (SOD) activity has also been reported in oat, wheat and Arabidopsis thaliana in soils contaminated with various pollutants (Alscher et al., 2002). Several plant species have also been shown to have elevated peroxidase activities in response to increased pollutant concentrations (Jouili et al., 2003). Reactive oxygen species is thought to increase cellular damage through the oxidation of several macromolecules such as lipids and proteins (Ortega et al., 2005).

The common dry beans or Phaseolus vulgaris L., is the most important food legume for direct consumption in the world. Among major food crop, it has one of the highest levels of variation in growth habit, seed characteristics (size, shape, colour), maturity, and adaptation. It also has a tremendous variability (> 40,000 varieties). In Africa, beans are grown mainly for subsistence, where the Great Lakes region has the highest per capita consumption in the world. Beans are a nearly “perfect” food. Nutritionally rich, they are also a good source of protein, folic acid, dietary fibre and complex carbohydrates. Beans are also one of the best non-meat sources of iron, providing 23-30% of daily recommended levels (Pachico et al., 1993) from a single serving. Consumption of beans is high mostly because they are a relatively inexpensive food. For the poor of the world, they are a means of keeping malnutrition at bay (WHO. 1992).

This research work is geared towards outlining the effect of soil contaminated with crude oil (at various levels) on the biochemical properties and growth rate of beans (phaseolus vulgaris). 

1.1 Statement of the problem

In Niger Delta, oil pollution arising from oil spillages and gas flaring regularly occurs. Therefore, the environment has been destroyed, while the rivers and farmland which the inhabitants rely on for their farming and fishing activities have been rendered unwholesome. This environmental destruction has increased the poverty level of the inhabitants. The prevention of crude oil contamination on soil cannot be overemphasized.  Consequently, the problem of this study is to determine the effect of crude oil pollution on beans germination and growth rate with special reference to the effect on the biochemical characteristics of beans.

1.2 Relevance of study

The proposal study is an attempt to determine the effect the crude oil contaminated soil will have on the growth, relative water content and oxidative status of cultivated beans.

1.3 Aim & Objective of the study


The aim of this study was to clarify whether crude oil contaminated soil will not hinder the growth of beans at physiological levels.


To investigate effect of crude oil contaminated soil on the growth of cultivated beans.

To investigate the effect of crude oil contaminated soil on the relative water content of the cultivated beans.

To investigate the oxidative status of beans planted in crude oil contaminated soil at the molecular level.



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