Environment, Vol. 3, Issue 2, Sep  2019, Pages 1-10; DOI: 10.31058/j.envi.2019.32001 10.31058/j.envi.2019.32001

Germination, Phytochemical Composition and Oxidation Products of Abelmoscheus Esculentus (Okra) Plant Cultivated on Spent Engine Oil Polluted Soil

, Vol. 3, Issue 2, Sep  2019, Pages 1-10.

DOI: 10.31058/j.envi.2019.32001

Onyegeme Okerenta Blessing Minaopunye 1* , Alozie Sunday Chukwuemaka 1

1 Department of Biochemistry, Faculty of Science, University of Port Harcourt, Port Harcourt

Received: 30 July 2019; Accepted: 20 August 2019; Published: 31 August 2019

Abstract

The indiscriminate disposal of spent engine oil (SEO) drained from engines after maintenance has been found to affect plant growth. This study evaluated the effect of SEO on growth, phytochemical composition, and oxidation products of Abelmoscheus esculentus (Okra) plant. A potted experiment simulated environment was developed at the University of Port Harcourt, Choba, Rivers State, to investigate the minimum concentration of SEO that could be inhibitory to the growth of A. esculentus (Okra) seed. The plastic containers filled with 4900g of humus soil were contaminated with various volumes Of SEO (0, 49, 147 and 294ml/4900g of humus soil) to give a percentage concentration of 0%, 1%, 3%, and 6% respectively. Each treatment had three replicates arranged in a complete randomized block design. Germination studies carried out showed the number of seeds of okra that grew into seedling decreased with the increasing level of the contaminant. Percentage germination was found to decrease from 100% in 0ml to 66% in 49ml, 33% in 147ml and no growth in 294ml. Phytochemical composition of the leaves after 3 months showed a significant decrease (p<0.05) in vitamin C, saponin and alkaloid and a significant increase (p<0.05) in flavonoid, cyanogenic glycoside and oxalate concentrations with a respective increase in SEO pollution. The result obtained in this study shows the phytotoxic effect of SEO and suggests that SEO at a concentration of 49ml (1%) /4900g of humus soil could be inhibitory to growth and seedling of Okra.

Keywords

Abelmoschus Esculentus, Spent Engine Oil, Oxidation, Phytochemical, Polluted Soil

1. Introduction

Oil spills and indiscriminate disposal of spent engine oil into gutters, sewers, drainage ditches and open vacant plots by automobile mechanics that change the oil from motor vehicles and generators are hazardous to both plants and animals in Niger-delta region of Nigeria. It has been shown that the existing model of indiscriminate disposal of spent engine oil (SEO) does not only increase pollution incidents in the environment but it is equally more prevalent than crude oil pollution [1]. Reasons that could be adduced for this scenario are derived from an upsurge of vehicle owners and epileptic power failure that necessitated the use of generators in most homes, shop owners and industries that make use of this lubricant. The environmental effect of oil on the growth and performance of plants has been reported by many researchers. Germination of A. esculentus seeds was significantly affected in SEO polluted soil. [2,3,4] respectively demonstrated that crude oil application to soil significantly reduced crop growth and yield in okra, five cultivars of soya bean and six cultivars of cowpea. [5] reported a significantly higher mean plant height, leaf area and dry weight of Commelina bengalensis (dry flower) at 0 mg g-1 oil pollution than at 50 mg oil g-1 pollution level. [6] reported that vegetative cutting of Paspalum conjugation (Sourgrass) grew well in the absence of oil and salinity and that 75% of the test plant survived in low oiling but heavy oiling resulted in mortality. Similarly, [7] in their paper titled “effects of spent engine oil on germination and seedling growth of groundnut (Arachis hypogaea L.)” reported that spent engine oil as low as 25 mL is capable of becoming deleterious to groundnut growth.

In Nigeria, the existing model of indiscriminate disposal of SEO increases pollution incidents in the environment and it has been shown that this is more widespread than crude oil pollution [8]. [9] reported the deleterious effects of Spent Lubricating Oil pollution on crop productivity which include growth and germination inhibition and reduction in other growth parameters and that SLO pollution also interferes with the availability of micro and macronutrients and increases the heavy metal content of soils, thereby posing a grievous public health challenge. Similarly, [10] reported that spent auto engine oil above 1 % v/w soil contamination significantly deteriorated water movement into and within the experimental soil and consequently the growth and yield of soybean was adversely affected; however, the effect naturally weaned beyond a year depending on the quantity spilled. Spills arising from improper disposal of SEO generated by service stations and other users now pollutes the land which ultimately affects the quality of crop production. The problem arises from the fact that service stations in many parts of the country have not devised a suitable and environmentally friendly means of disposing of SEO. As a result, considerable volumes of SEO are dumped in open plots of land, into sewages, and drainage ditches. Continuous degradation of the environment by indiscriminate dumping of SEO can lead to the pollution of streams and rivers as well as underground water. In this study, Germination, phytochemical composition and oxidation products of Abelmoscheus esculentus (Okra) plant cultivated on spent engine oil polluted soil were evaluated.

2. Materials and Methods

2.1. Soil Preparation

Top garden soil was collected from Omuihuechi, Aluu, in Rivers State. Using an industrial scale, 4900g of the soil was weighed and added into four containers labelled A, B, C and D. Forty-nine milliliters, 147mls and 294mls of SEO was added and stirred thoroughly into A, B and C to represent 1%, 3%, and 6% polluted soil samples respectively and poured into nursery containers. D had no SEO and served as the control for the experiment. The SEO was obtained from a roadside mechanic workshop where electric generating sets are repaired or serviced. The entire set up was kept for 3 days and adequately mixed and watered to allow the SEO to distribute evenly in the various polluted soil samples.

2.2. Planting of Seed

Six viable seeds were planted in each of the labeled nursery bags containing the polluted soil samples and observed for germination. After germination and seedling, the plants were allowed to stand for three months. The leaves were thereafter harvested using a clean knife and taken to the laboratory for analyses.

2.3. Preparation of Extract

The leaves of A. esculentus were thoroughly washed with distilled water and then homogenized separately for their percentage of SEO pollution. Extraction was done as described by [11] with a prepared sodium-phosphate buffer at the pH of 7.4 using Kenwood homogenizer. The homogenates were stored at 4 °C.

2.4. Phytochemical Analysis

Phytochemical constituents of the leaves of A. esculentus (Okra) plant such as Flavonoids, Saponins, Alkaloids, Oxalates, and Cyanogenic Glycosides were determined according to the methods of [12,13].

2.5. Ascorbic acid (Vitamin C) Concentration

The leaves were separately homogenized in 4 parts of the homogenizing buffer for one hour. One ml of 2, 4- dinitrophenyl hydrazine and 20μl of thiourea were added to the test tube containing 4ml of the supernatant, this served as the test solution. The blank was prepared by adding 1ml of 2, 4 dinitrophenyl hydrazine reagents to 4ml of 6% trichloroacetic acid (TCA) and 10μl of 10% thiourea. The preparation of the standard solution involved the addition of 1ml 2, 4 dinitrophenyl hydrazine (2%) into a test tube containing 4ml ascorbate and mixed properly. These tubes containing the test, blank and standard solutions were kept in boiling water for 15 minutes, cooled in a water bath and 5ml of 85% H2SO4 layered along the side of the tubes. The absorbance of the test and standard solutions was read against the blank at 540nm after 30 minutes [14].

2.6. Glutathione (GSH) Concentration

This method is based on the formation of a relatively stable yellow color when Ellman' s reagent is added to a sulfhydryl compound. The colored chromophoric product, 2-nitro-5-thiobenzoic acid resulting from the above reaction of Ellman' s reagent with GSH is measured at 412nm. The absorbance is proportional to GSH concentration [15].

2.7. Protein Thiol (PSH) Concentration

Protein carbonyl groups react with 2,4-dinitrophenylhydrazine (DNPH) to generate chromorphic dinitrophenyl hydrazones that absorb light maximally at 360nm. The method of the reaction of carbonyls with 2,4-dinitrophenylhydrazine (DNPH) was used to determine the amount of protein thiol, as described by [16].

2.8. Total Thiol (TSH) Concentration

This was estimated by the algebraic summation of the protein thiol (PSH) concentration and the non-protein thiol (GSH) concentration. The sum of the two gives the total thiol concentration of the leave of A. esculentus. Protein thiol (PSH) + Glutathione (GSH) = Total thiol (TSH).

2.9. Malondialdehyde (MDA) Concentration

Malondialdehyde (MDA) is an end product of lipid peroxidation, which reacts with thiobarbituric acid to form pink chromogenthiobarbituric acid reactive substance which absorbs light maximally at 532nm. The determination of MDA was carried out according to the method of [17].

2.10. Statistical Analysis

Data generated from the above tests were analyzed using one-way student’s T-Test while results were expressed as mean ± SEM and the probability tested at 95% level of significance (p< 0.05).

3. Results and Discussions

3.1. Effect of SEO on Growth and Seedling of A. esculentus Plant

Growth studies carried out showed the number of seeds of okra that grew into seedling decreased with the increasing level of the contaminant ( Table 1 ). Percentage growth was found to decrease from 100% in 0ml to 66% in 49ml, 33% in 147ml and there was no growth in 294ml or 6% polluted soil sample.

Table 1. Effect of SEO on growth and seedling of A. esculentus plant.

Treatment (ml/4900gm)

% of Pollution

No of viable seed sown

No of seeds that sprouted at the end of 11 days

Plants with leaves present after 3 months

% Growth and seedling after 3 months

0

0

6

6

6

100

47

1

6

6

4

66

147

3

6

5

1

33

294

6

6

4

0

0

3.2. Effect of SEO on the Phytochemical Composition of the Leaf Extract of A. esculentus

The percentage phytochemical composition of the leaf extract of A. esculentus grown on SEO after 3 months were compared to the unpolluted control soil sample (Figure 1). The result obtained showed that there was a significant decrease (p< 0.05) in saponin and alkaloid and a significant increase (p< 0.05) in flavonoid, cyanogenic glycoside and oxalate concentrations with a respective increase in SEO pollution.

Saponins are important therapeutically plant chemicals necessary for the activity of cardiac glycosides. The impact of the pollution manifested as a marked reduction in saponin concentration in A. esculentus as the level of pollution increased respectively. The responsive reduction in the saponin content could be attributed to the high level of phytotoxic damage that might have been caused by SEO pollution on the plant.

Flavonoids are an important group of polyphenols widely distributed among the plant flora and numerous reports support their use as antioxidants or free radical scavengers [18]. As antioxidants, they scavenge the damage caused by free radicals in the plant cells. There was a corresponding increase in the percentage of flavonoid concentrations of A. esculentus leaves for increasing SEO pollution. Similarly, Cyanogenic glycoside concentration was responsive to the degree of SEO pollution in A. esculentus leaves as there was a progressive rise in its concentration with increased SEO pollutants (p> 0.05). These observations are in harmony with the report of [19] who noted that oil in soil has deleterious effects on the biological, chemical and physical properties of the soil depending on the dose, type of the oil and other factors. However, results obtained for alkaloid concentration in this study showed no significant difference (p> 0.05) in all levels of pollution. Alkaloids exist in large proportions in the seeds and roots of plants and often in combination with vegetable acids and have pharmacological applications as anesthetics and central nervous system stimulants [20].

On the other hand, the result obtained for the percentage oxalate concentration of respective SEO polluted soils showed that there was no significant difference (p> 0.05) in the experimental groups. Oxalates are naturally-occurring substances found in plants, animals, and humans. It could thus be inferred that oxalate is an integral content of A. esculentus hence the observed trend irrespective of the SEO pollution level.

Figure 1. Phytochemical concentration of A. esculentus leaves harvested from SEO polluted soil samples.

3.3. Effect of SEO on the Oxidation Products of the Leaf Extract of A. esculentus

The results obtained for oxidation products - Malondialdehyde (MDA), Protein thiol (PSH), Glutathione (GSH), Total thiol (TSH) and Vitamin C concentration of A. esculentus leaves in various percentages SEO polluted soil samples are shown in Figure 2- Figure 6. The results showed that there was a significant difference (p> 0.05) between all the levels of the percentage of SEO pollution. The concentration of MDA in the leaves increased progressively with increasing SEO pollution while PSH, GSH, TSH, and Vitamin C concentration in the leaves decreased progressively with increasing SEO pollution.

Free radicals can stimulate the free-radical chain reaction known as lipid peroxidation which may be reflected in the production of malondialdehyde (MDA) [21]. The concentrations of MDA obtained from this study showed a corresponding increase in the leaves of A. esculentus for increasing SEO pollution. The observed increase in MDA concentrations in A. esculentus leaves may be attributed to the high level of polycyclic aromatic hydrocarbons present in the SEO polluted soil samples.

Figure 2. Malondialdehyde concentration of A. esculentus leaves harvested from SEO polluted soil samples.

Figure 3. Protein Thiol concentration of A. esculentus leaves harvested from SEO polluted soil samples.

Figure 4. Total Thiol concentration of A. esculentus leaves grown on SEO polluted soil samples.

Protein-carbonyl oxidation (PCO) or Protein thiol (PSH) has been reported as one of the early markers of protein oxidation [22]. It plays an essential role in the pathogenesis of an important number of petroleum hydrocarbon-induced changes in tissues of living organisms (plants and animals). These markers have attracted the attention of various investigators [23]. The results obtained in this research illustrates that the PSH of A. esculentus leaves decreased as the percentage of SEO pollution increased. This could be attributed to the increased oxidative protein damage caused by the SEO pollution on A. esculentus leaves as the concentration increased respectively in the various polluted soil samples. SEO induced pollution in the soil samples significantly (p> 0.05), reduced the mean values of Protein thiol of the respective soil samples.

Figure 5. Glutathione concentration of A. esculentus leaves grown on SEO polluted soil samples.

Plants tend to accumulate excessive amounts of reactive oxygen species (ROS) when exposed to abiotic stresses, which in turn react with cellular lipids, proteins, and DNA. Therefore, decreasing ROS accumulation is indispensable to survive under stress, which is accomplished by inducing enzymatic and nonenzymatic antioxidant defense pathways. Glutathione, particularly reduced glutathione (GSH), represents a principal antioxidant that could decrease ROS through scavenging them directly or indirectly through ascorbate–glutathione cycle or GSH peroxidases [24]. In this study, glutathione of A. esculentus leaves from the respective SEO polluted soils, was responsive to the pollution level in the soils when compared to that from the control sample. This was shown by a marked reduction in GSH concentration from the respective SEO polluted soils.

Figure 6. The effect of SEO on Vitamin C concentration of A. esculentus leaves from polluted soil samples.

As a powerful cellular reductant, ascorbic acid readily donates electrons to reduce free radicals in the cellular compartment and is converted to dehydroascorbic acid (DHAA), its oxidized form. Petroleum hydrocarbon-induced pollution by SEO significantly reduced the mean concentration of ascorbic acid from 342mg/ml in the control, to 315mg/ml in 1%, and 270 in 3% samples. This agrees with the findings of [25], who reported a substantial reduction in liver ascorbic acid concentration in fowls from petroleum hydrocarbon polluted Warri area in contrast to that of fowls from un-polluted Abraka environment. This observed a marked reduction in ascorbic acid concentration may be attributed to the involvement of ascorbic acid in oxidation reactions.

4. Conclusions

The impact of SEO pollution due to increased activities in automobile workshops can affect the ecosystem surrounding that area and this has contributed immensely to the poor yield and performance of plants and edible vegetables in such regions. However, this study was targeted on the effect of SEO on the growth, phytochemical composition and oxidation products of A. esculentus plant. The result from the study indicated that the indiscriminate dumping of spent engine oil has significant effects on the growth, phytochemical composition and oxidation products of A. esculentus.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Acknowledgments

The authors would like to acknowledge the support of the Laboratory staff of the Department of Plant Science and Biotechnology and also that of the Department of Biochemistry, University of Port Harcourt, Rivers State, Nigeria.

Copyright

© 2017 by the authors. Licensee International Technology and Science Press Limited. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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