EFFECT replicated five times. Groups 1 to 5





oil was discovered in the southern part of Nigeria in the 1950s.Till date,
activities involve in its production continue to liberate hydrocarbons into the
environment, including farmlands. The aim of this
study was to determine the
effect of petroleum products contaminated soil at different concentrations on
the biomass and levels of protein in the leaves of both cowpea and maize
seedlings. Improved
varieties of maize (Zea mays) and Vigna unguiculata (L)
Walp were mobilized from approved sources and planted in soil contaminated at
different concentrations six groups replicated five times. Groups 1 to 5
contained 0.1%, 0.25%, 0.5%, 1.0% and 2.0% (v/w) respectively of each of the
petroleum products while group six served as control (0.0%). Three seeds were
planted in each bag and watered daily. The biomass of the whole seedling and
levels of protein were determined four days, eight days and twelve days
after germination. The
results indicated that the petroleum products caused significant (P<0.01) decrease in the biomass as well as the level of protein in both cowpea and maize seedlings. The petroleum products exhibited differential effects on the biomass and the levels of protein in the two seedlings .In addition, cowpea seedlings were affected more than the maize seedlings by the petroleum products. It is obvious that petroleum products toxicity is dependent on the type of product as well as being specie dependent         Key words:  Biomass, Cowpea, Maize, Protein, Petroleum product     INTRODUCTION In recent times the environmental impact crude oil or its refined products have been a popular area of interest in experimental research (Nwaogu and Onyeze, 2010, 2014; Achuba and Ogwumu, 2014; Achuba and Nwokogba, 2015) Petroleum toxicity in plants is observed at multiple levels, from reduced yield, through effects on leaf and root growth (Peretiemo-Clarke and Achuba, 2007). Plant growth and development depend on resources present in soil and air environment, which consists of growth factors (Shanker et al., 2005).The presence of petroleum in the external environment leads to changes in the growth and metabolic pattern of plant (Peretiemo-Clarke and Achuba, 2007; Achuba and Okoh, 2015; Achuba and Asagba, 2015). Petroleum compounds are highly toxic to plants and are detrimental to their growth and development (Amadi et al., 1996; Achuba, 2006; Peretiemo-Clarke and Achuba., 2007).  Petroleum in soil depresses seed germination and there are reports that crude oil, water soluble fraction of crude oil and spent engine oil inhibited seed germination (Eriyamremu, 1999; Akaninwor et al., 2007; Anoliefo and Edegbai, 2000; Njoku, 2008).  Petroleum products have been shown to induce metabolic modifications in plants (Achuba, 2006; Peretiemo-Clarke and Achuba, 2007; Achuba and Okoh, 2015), increased production of metabolites such as glucose, total carbohydrate as well as proteins and amino acids in plants (Achuba, 2006). Achuba, (2010) and Nwaogu and Onyeze (2010) observed an increase in lipid peroxidation product in plant exposed to petroleum. Various authors reported that exposure of plant to petroleum products in soil and other forms of pollution inhibited starch metabolism as well as peroxidase activity in plant species (Eriyamremu et al 1999; Achuba 2006; Aki, 2009; Achuba and Okoh 2015). One of the macromolecules that perform crucial role in every living organism is protein. This is because it constitutes most of the enzymes that catalyze biochemical reactions (Nelson and Cox, 2005).The aim of the present investigation was to determine the effect of petroleum products on the level of protein and biomass of cowpea and maize seedlings. MATERIALS AND METHODS Materials used Petroleum products (specific gravities: kerosene = 0.81; diesel = 0.85; engine oil=0.87; petrol = 0.75) were obtained from Warri Refining and Petrochemical Company, Warri, Nigeria. Maize (Zea mays) seeds were obtained from Delta Agricultural Development Project (DTADP) Ibusa Delta State, Nigeria while cowpea seeds (Vigna unguiculata L Walp ) were obtained from International Institute of Tropical Agriculture IITA, Ibadan, Nigeria. The soil sample used for the experiment (sand 84%, silt 5.0%, clay 0.4% and organic matter 0.6%, pH 6.1) was obtained from an uncultivated land in the premises of Delta State University, Abraka, Nigeria. Reagents used were of analytical grade.  Planting of seeds The soil (1.60 kg) was added to each planting polybags and divided into six groups of five replicates. Groups 1 to 6 contained 0.1%, 0.25%, 0.5%, 1.0%, 2.0% and 0.0 % (v/w) respectively of each of the petroleum products Kerosene (1.6 ml) was added to the first bag, which corresponds to 0.1%. The petroleum product treated soil sample was mixed vigorously with hand to obtain homogeneity of the mixture. The same procedure was adopted for 0.25%, 0.5%, 1.0%, 1.5% and 2.0%. Similar procedure was adopted for diesel, engine oil and petrol.             Potentially good seeds were determined by pouring sizable quantity into bowl containing water. Seeds that did not float on water were taken to be damaged seeds were discarded others used for planting. Three seeds were planted in each polybag to a depth of 2cm immediately after treatment of soil with the respective petroleum products and kept under shade. Water was added daily (80 cm3) to keep the soil moist for twelve days Seeds which failed to germinate after 12 days were regarded as not germinable. The experiment was carried out under laboratory conditions of temperature 28oC and 12hr day/ night regime. Preparation of homogenate and assay for protein The leaf homogenate was prepared by collecting leaves (1.0 g) from each treatment. The leaves were placed in a mortar containing 0.5 g acid washed sand. This was followed by grinding with a pestle. At the end of each homogenization 5.0 ml of distilled water was added and stirred with a glass rod. The homogenate was filtered through cheese cloth and the filtrate centrifuged at 1000 rpm for 30 minutes. The supernatant obtained was used for the determination of protein according to Lowry's method (1951) using bovine serum albumin as standard. The reagent (2.5 ml) was added, followed by 1.0ml of the leave homogenate in a testtube, mixed well and allowed to stand for 10 minutes. Then 0.5 ml of Folins-Ciocalteau reagent was then added to each tube, mixed well and allowed to stand for 30 minutes. Absorbance was then read at 660 nm using reagent blank Determination of dry weight and plant parts relationships  The dry weight of the seedlings was determined by drying whole plants of each treatment to constant weight in an electric oven at 105°C for 24 hour. Relationship between the various parts of the seedlings were analyzed through the use of regression analysis (SPSS, version 20)         RESULTS AND DISCUSSION   The levels of protein in the leaves of cowpea and maize seedlings were affected by the four petroleum products after four days, eight days and twelve days of germination (Figure 1).There was a petroleum products mediated increase in protein in the leaves of cowpea and maize seedlings at lower levels of soil contamination. Petroleum products contaminated soil altered the levels of protein in the leaves of exposed cowpea and maize seedlings. This observation is in agreement with the increase in the level of protein in seedlings exposed to crude oil (Malallah, 1996; Achuba, 2006; Peretioemo – Clarke and Achuba, 2007). The increase in the levels of protein in the leaves of seedlings exposed to crude oil has seen attributed to the presence of sulphur in whole crude oil as well as growth stimulating chemicals present in crude oil. However, there was a petroleum products mediated decrease in the levels of protein in the leaves of exposed plants relative to the respective controls at higher levels of soil contamination. Anoliefo and Edegbai (2000) reported that at low levels of oil pollution that hydrocarbons could be easily degraded by natural rehabilitation in soil, thereby increase organic matter in soil as well as improve soil fertility, physical and chemical properties of the soil. This may in part, explain why there was increase in protein at low levels of soil contamination as against the reduction at high levels of petroleum products in soil. There were decreases in the levels of protein in the stem after four days, eight days and twelve days of germination as well as in the root after four days, eight days and twelve days of germination in cowpea and maize seedlings ( Figs 2 and 3). The petroleum products affected the levels of protein in the tissues of exposed seedlings differently. This explains why an inverse relationship exists between the level of protein in the leaves, stem and root of cowpea and maize seedlings after four and eight days of growth in petroleum products treated soil (Table 1).  This may be due to hydrocarbon induced differences in the metabolic states of leaves, stem and root of a plant (Sadunishvili et al., 2009). In addition, it is a common feature of both dicot and monocot that an increasing proportion of shoot carbon and nitrogen is allocated to non-photosynthetic tissues during the growth of the crop (Gastal and Lemaire, 2002).It is documented that leaf and stem ratio decrease as crop biomass increases (Lemaire and Chartier, 1992; Belanger and McQueen, 1999; Belanger and Richards, 2000). Therefore, a progressive great proportion of carbons and nitrogen is allocated to the stem (Gastal and Lemaire, 2002). This is consistent with the result of the current study in which the level of protein in the stem of maize seedlings is greater than that in the leaves. However, in cowpea seedlings, the level of protein in the leaf is equal to that in the stem. This may be attributed to the photosynthetic activity of the shoot of cowpea seedlings in early stage of development. On the whole, the kerosene exhibited more toxic effect compared to the other petroleum products studied. However, the manner of toxicity in cowpea and maize seedlings were specie specific   Previous studies have reported petroleum products mediated reduction in dry weight of exposed plants (Omosun et al., 2008; Njoku et al 2009). This is consistent with the present investigation in which increase in concentration of petroleum products in soil caused successive decrease in dry weight of cowpea and maize seedlings ( Fig. 4.0). According to Wyszkowski and Zoikowska (2008) growth of plant is dependent on the content of soil nutrient. Therefore, the reduction in dry matter in both cowpea and maize seedlings may be predicated on the effect of petroleum products on soil. The adverse effect could be due to the disruption of the absorption and uptake of nutrients by petroleum products (Njoku, 2008). Dimitrow and Markow (2000) showed that exposure hydrocarbon decrease the availability of phosphorous and potassium to plant. These nutrients are essential to plant growth and development; hence reduction in the bioavailability will lead to reduced plant growth (Njoku, 2009). This may explain why the petroleum products caused a reduction in the dry weight of cowpea and maize seedlings. Like the effect of these petroleum products on the level of protein, the biomass was lower in kerosene exposed seedlings than the seedlings exposed to the other petroleum products. It is pertinent to conclude that different refined petroleum products have different toxic effects on cowpea and maize seedlings. This is indicated by the differential responses of levels of protein and plant biomass to the respective petroleum products.                                           REFERENCES Achuba F. I.  and Asagba. S. O.( 2015) Glutathione-S-transferase activity in Cowpea (Vigna unguiculata) and Maize (Zea mays) seedlings exposed to petroleum products in soil. Biokemistri 27 (2) 117–122  Achuba F. I. and Ogwumu M. D (2014). Possible protective role of palm oil and beef liver on the kidney and liver of wistar albino rats fed diesel-contaminated diet. Biokemistri 26 (4) 124–129. Achuba F. I.,  and Okoh,  P. N. (2015).Effects of Petroleum Products in Soil on ?-amylase, starch phosphorylase and peroxidase activities in cowpea and maize seedlings. American Journal of Experimental Agriculture, 6(2): 112-120 Achuba, F I and Nwokogba,. C.C. (2015) Effects of honey supplementation on hydrocarbon-induced kidney and liver damage in wistar albino rats. Biokemistri 27 (1) 50–55 Achuba, F. I. (2006). The effects of sublethal concentrations of crude oil on the growth and metabolism of cowpea (Vigna unguiculata) seedlings. The Environmentalist. 26:17-20. Achuba, F.I. (2010). Spent engine oil mediated oxidative stress in cowpea (Vigna unguiculata) seedlings. Eletronic Journal of Environment, Agriculture and Food Chemistry. 9(5): 910-917  Akaninwor, J.O., Ayeleso, A. O. and Monago, C. C. (2007). Effect of different concentrations of crude oil (Bonny light) on major food reserves in guinea corn during germination and growth. Scientific Research Essay 2(4) 127-131 Aki, C, Guneysu, E and Acar, O (2009). Effect of industrial wastewater on total protein and the peroxidase activity in plants. African Journal of Biotechnology 8 (20) 5445 -5448 Amadi, A., Abbey, S.D. and Nma, A. (1996). Chronic effect of oil spill on soil properties and michroflora of rainforest ecosystem in Nigeria. Water, Air and Soil Pollution. 86:1-11. Anoliefo, G.O. and Edegbai, B.O., (2000). Effect of spent engine oil as an oil contaminant on the growth of two eggplant species; Solanum melongena L. and S. incanum. Journal of Agriculture. Forestry and Fisheries, 1: 21-25 Belanger, G. and Richards, J. E. (2000). Dynamics of biomass and N accumulation of alifafia under three N fertilization rates. Plant and soil 219: 177-185.   Belanger, G and McQueen, R.E. (1999). Leaf and stem nutritive value of timothy grown with varying N nutrition in spring and summer. Canadian Journal of Plant. Science. 79: 223-229 Dimitrow, D.N. and Markow, E. (2000). Behaviour of available forms of NPK in soil polluted by oil products. Poczwoznanie, Agrochimija I Ekologia. 35(3): 3-8. Eriyamremu, G.E., Iyasele, J.U., Osubor, C.C., Anoliefo, G.O, Osagie, V.E. and Osa, M.O (1999). Bonny light crude oil alters protease and respiratory activities of germinating beans (Vigna unguiculata) (L) Seeds. Journal of Science, Engineering and Technology, 6(1): 1589 – 1600. Gastal, F. and Lemair,e G. (2002). N uptake and distribution in crops: an agronomical and ecophysiological perspective. Journal of Experimental Botony 53(370): 789-799. Lemaire, G. and Chartier, M. (1992). Relationships between growth dynamics and nitrogen uptake for individual sorghum plants grown at different densities. Proceeding of the 2nd ESA Congress, UK 92: 98-99. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randal, R.J. (1951) Protein measurement with the folin phenol reagent. Biological Chemistry 193: 265-275. Malallah, G., A. Fzal., M. Gulsham, S., Abraham, D., Kunan, M. and Dhami, M.S.I. (1996). Vicia faba as a bio indicator of oil pollution. Environmental Pollution. 92(2): 213-217. Nelson, D.L. and Cox, M.M., (2000). Principles of Biochemistry. Macmillan Press, London, UK. Njoku, K.L., Akinola M.O. and Oboh B.O. (2008). Growth and performance of glycine max L. (Merill) in crude oil contaminated soil augmented with cow dung. Nature and Science, 6(1):48-58. Njoku, K.L., Akinola M.O. and Oboh, B.O. (2009). Phytoremediation of crude oil contaminated soil: the effect of growth of glycine max on the physico-chemitry and crude oil contents of soil. Nature and Science. 7(12): 22-30. Nwaogu, L.A and Onyeze, G.O. (2014). Effect of chronic exposure to petroleum hydrocarbon pollution on oxidative stress parameters and histology of liver tissues of native fowl (Gallus domestics). International Journal of Biochemistry Research and Review, 4(3)233-242  Nwaogu, L.A. and Onyeze, G.C.O. (2010). Effects of spent engine oil on oxidative stress parameters of Teferia occidentalis leaves. Nigerian Journal of Biochemistry and. Molecular Biology, 25(2) 98-104. Omosum, G., Markson, A. A. and Mbanasor, O. (2008). Growth and anatomy of amaranthus hybridus as affected by different crude oil concentrations. American- Eurasian Journal of Scientific Research 3(1): 70-74. Peretiemo-Clarke, B.O. and Achuba, F.I. (2007). Phytochemical effect of petroleum on         peanut (Arachis hypogea) seedlings. Plant Pathology Journal, 6:179-182. Sadunishvili, T. Kvesitadze, E., Betsiashvili, M., Kuprava, N., Zaalishvili G. and Kvesitadze, G. (2009). Influence of Hydrocarbons on plant cell ultrastructure and main metabolic enzymes. World Academy of Science, Engineering and. Technology. 57: 271-276. Shanker, A.K. Carlos Cervantes, T., Loza-Tavera, H, and Avudainayagam, S. (2005) Chromium toxicity in plants. Environment International. 31: 739-735. Wyszkowski, M. and Ziolkowska, A. (2008). Effect of petrol and diesel content of organic carbon and mineral components in soil. American- Eurasian Journal of Agriculture 2(1): 54-60.                                 Table1 Relationship between the levels of protein in the leaves, stem and root of cowpea and maize seedlings after four days of germination in petroleum products treated soil Physiological region of plant/ Days of germination      Kerosene Cowpea     Maize        Diesel Cowpea   Maize Engine oil Cowpea     Maize    Petrol Cowpea    Maize 4 days after germination         Leave/ Stem  -0.623 -0.385 -0.623 -0.286 -0.028 -0.265 0.155 0.218 Leave/ Root -0.207 -0.415 -0.459 -0.425 -0.301 -0.269 -0.162 -0.656 Stem/ Root -0.275 0.169 -0.038 0.317 -0.307 -0.90 -0.123 -0.713 8 days after germination                 Leave/ Stem -0.034 -0.252 -0.626 -0.027 -0.732 0.546 -0.034 -0.656 Leave/ Root 0.017 -0.735 0.011 -0.153 -0.410 -.0607 0.017 -0.851 Stem/ Root 0.712 0.735 0.615 0.700 0.466 -0.231 0.712 0.680 12 days after germination                 Leave /Stem 0.997 0.997 0.998 0.989 0.851 0.421 0.941 0.990 Leave/ Root 0.957 0.981 0.938 0.975 0.794 0.818 0.400 0.666 Stem/ Root 0.954 0.983 0.932 0.976 0.643 0.615 0.644 0.680