EFFECT times the environmental impact crude oil
EFFECT OF HYDROCARBON POLLUTED SOIL ON THE BIOMASS ANDLEVELS OF PROTEIN IN COWPEA AND MAIZE SEEDLINGS Achuba ,F.
I . Department ofBiochemistry, Delta State University, PMB 1, Abraka Nigeria. [email protected] ABSTRACTCrudeoil was discovered in the southern part of Nigeria in the 1950s.Till date,activities involve in its production continue to liberate hydrocarbons into theenvironment, including farmlands. The aim of thisstudy was to determine theeffect of petroleum products contaminated soil at different concentrations onthe biomass and levels of protein in the leaves of both cowpea and maizeseedlings.
Improvedvarieties of maize (Zea mays) and Vigna unguiculata (L)Walp were mobilized from approved sources and planted in soil contaminated atdifferent concentrations six groups replicated five times. Groups 1 to 5contained 0.1%, 0.25%, 0.5%, 1.0% and 2.0% (v/w) respectively of each of thepetroleum products while group six served as control (0.
0%). Three seeds wereplanted in each bag and watered daily. The biomass of the whole seedling andlevels of protein were determined four days, eight days and twelve daysafter germination. Theresults indicated that the petroleum products caused significant (P<0.01)decrease in the biomass as well as the level of protein in both cowpea andmaize seedlings. The petroleum products exhibited differential effects on thebiomass and the levels of protein in the two seedlings .
In addition, cowpeaseedlings were affected more than the maize seedlings by the petroleum products.Itis obvious that petroleum products toxicity is dependent on the type of productas well as being specie dependent Key words: Biomass,Cowpea, Maize, Protein, Petroleum product INTRODUCTIONIn recent times the environmental impact crude oilor its refined products have been a popular area of interest in experimentalresearch (Nwaogu and Onyeze, 2010, 2014; Achuba and Ogwumu, 2014; Achuba andNwokogba, 2015) Petroleum toxicity in plants is observed at multiple levels,from reduced yield, through effects on leaf and root growth (Peretiemo-Clarkeand Achuba, 2007). Plant growthand development depend on resources present in soil and air environment, whichconsists of growth factors (Shanker et al., 2005).The presence of petroleum inthe external environment leads to changes in the growth and metabolic patternof plant (Peretiemo-Clarke and Achuba, 2007; Achuba and Okoh, 2015; Achuba andAsagba, 2015). Petroleum compounds are highly toxic to plants and aredetrimental 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 thatcrude oil, water soluble fraction of crude oil and spent engine oil inhibitedseed germination (Eriyamremu, 1999; Akaninwor et al., 2007; Anoliefo andEdegbai, 2000; Njoku, 2008). Petroleum products have been shown to inducemetabolic modifications in plants (Achuba, 2006; Peretiemo-Clarke and Achuba,2007; Achuba and Okoh, 2015), increased production of metabolites such asglucose, total carbohydrate as well as proteins and amino acids in plants (Achuba,2006). Achuba, (2010) and Nwaogu and Onyeze (2010) observed an increase inlipid peroxidation product in plant exposed to petroleum.
Various authorsreported that exposure of plant to petroleum products in soil and other formsof pollution inhibited starch metabolism as well as peroxidase activity inplant species (Eriyamremu et al 1999; Achuba 2006; Aki, 2009; Achuba and Okoh2015).One of themacromolecules that perform crucial role in every living organism is protein.This is because it constitutes most of the enzymes that catalyze biochemicalreactions (Nelson and Cox, 2005).The aim of the present investigation was todetermine the effect of petroleum products on the level of protein and biomassof cowpea and maize seedlings. MATERIALSAND METHODSMaterialsusedPetroleumproducts (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 DeltaAgricultural Development Project (DTADP) Ibusa Delta State, Nigeria whilecowpea seeds (Vigna unguiculata L Walp ) were obtained from InternationalInstitute of Tropical Agriculture IITA, Ibadan, Nigeria.
The soil sample usedfor the experiment (sand 84%, silt 5.0%, clay 0.4% and organic matter 0.6%, pH6.1) was obtained from an uncultivated land in the premises of Delta StateUniversity, Abraka, Nigeria. Reagents used wereof analytical grade. Planting of seedsThesoil (1.60 kg) was added to each planting polybags and divided into six groupsof five replicates.
Groups 1 to 6 contained 0.1%, 0.25%, 0.5%, 1.0%, 2.
0% and0.0 % (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 producttreated soil sample was mixed vigorously with hand to obtain homogeneity of themixture. 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 weredetermined by pouring sizable quantity into bowl containing water. Seeds thatdid not float on water were taken to be damaged seeds were discardedothers used for planting.Three seeds were planted in each polybag to a depth of 2cm immediately aftertreatment of soil with the respective petroleum products and kept under shade.
Water was added daily (80 cm3) to keep the soil moist for twelvedays Seeds which failed to germinate after 12 days were regarded as not germinable.The experiment was carried out under laboratory conditions of temperature 28oCand 12hr day/ night regime.Preparation of homogenate and assay for proteinThe leafhomogenate was prepared by collecting leaves (1.0 g) from each treatment. Theleaves were placed in a mortar containing 0.5 g acid washed sand. This wasfollowed by grinding with a pestle.
At the end of each homogenization 5.0 ml ofdistilled water was added and stirred with a glass rod. The homogenate wasfiltered through cheese cloth and the filtrate centrifuged at 1000 rpm for 30minutes.
The supernatant obtained was used for the determination of proteinaccording to Lowry’smethod (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 allowedto stand for 10 minutes. Then 0.5 ml of Folins-Ciocalteau reagent was thenadded to each tube, mixed well and allowed to stand for 30 minutes.
Absorbancewas then read at 660 nm using reagent blankDetermination of dry weight andplant parts relationships The dry weight of the seedlings was determinedby drying whole plants of each treatment to constant weight in an electric ovenat 105°C for 24hour. Relationship between the various parts of the seedlings wereanalyzed through the use of regression analysis (SPSS, version 20) RESULTS AND DISCUSSION Thelevels of protein in the leaves of cowpea and maize seedlings were affected bythe four petroleum products after four days, eight days and twelve days ofgermination (Figure 1).There was a petroleum products mediated increase inprotein in the leaves of cowpea and maize seedlings at lower levels of soilcontamination.
Petroleum products contaminated soil altered the levels ofprotein in the leaves of exposed cowpea and maize seedlings. This observationis in agreement with the increase in the level of protein in seedlings exposedto crude oil (Malallah, 1996; Achuba, 2006; Peretioemo – Clarke and Achuba,2007). The increase in the levels of protein in the leaves of seedlings exposedto crude oil has seen attributed to the presence of sulphur in whole crude oilas well as growth stimulating chemicals present in crude oil. However, therewas a petroleum products mediated decrease in the levels of protein in theleaves of exposed plants relative to the respective controls at higher levelsof soil contamination. Anoliefo and Edegbai (2000) reported that at low levelsof oil pollution that hydrocarbons could be easily degraded by naturalrehabilitation in soil, thereby increase organic matter in soil as well asimprove soil fertility, physical and chemical properties of the soil. This mayin part, explain why there was increase in protein at low levels of soilcontamination as against the reduction at high levels of petroleum products insoil.
Therewere decreases in the levels of protein in the stem after four days, eight daysand twelve days of germination as well as in the root after four days, eightdays and twelve days of germination in cowpea and maize seedlings ( Figs 2 and3). The petroleum products affected the levels of protein in the tissues ofexposed seedlings differently. Thisexplains why an inverse relationship exists between the level of protein in theleaves, stem and root of cowpea and maize seedlings after four and eight daysof growth in petroleum products treated soil (Table 1). Thismay be due to hydrocarbon induced differences in the metabolic states ofleaves, stem and root of a plant (Sadunishvili et al., 2009). In addition, itis a common feature of both dicot and monocot that an increasing proportion ofshoot carbon and nitrogen is allocated to non-photosynthetic tissues during thegrowth of the crop (Gastal and Lemaire, 2002).
It is documented that leaf andstem ratio decrease as crop biomass increases (Lemaire and Chartier, 1992; Belanger and McQueen,1999; Belanger and Richards, 2000). Therefore, a progressive great proportionof 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 ofprotein 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 thatin the stem. This may be attributed to the photosynthetic activity of the shootof cowpea seedlings in early stage of development. On the whole, the keroseneexhibited more toxic effect compared to the other petroleum products studied.However, the manner of toxicity in cowpea and maize seedlings were speciespecific Previousstudies have reported petroleum products mediated reduction in dry weight ofexposed plants (Omosun et al., 2008; Njoku et al 2009). This is consistent withthe present investigation in which increase in concentration of petroleumproducts in soil caused successive decrease in dry weight of cowpea and maizeseedlings ( Fig. 4.
0). According to Wyszkowski and Zoikowska (2008) growth ofplant is dependent on the content of soil nutrient. Therefore, the reduction indry matter in both cowpea and maize seedlings may be predicated on the effectof petroleum products on soil. The adverse effect could be due to thedisruption of the absorption and uptake of nutrients by petroleum products(Njoku, 2008).
Dimitrow and Markow (2000) showed that exposure hydrocarbondecrease the availability of phosphorous and potassium to plant. Thesenutrients are essential to plant growth and development; hence reduction in thebioavailability will lead to reduced plant growth (Njoku, 2009). This mayexplain why the petroleum products caused a reduction in the dry weight ofcowpea and maize seedlings.
Like the effect of these petroleum products on thelevel of protein, the biomass was lower in kerosene exposed seedlings than theseedlings exposed to the other petroleum products.Itis pertinent to conclude that different refined petroleum products havedifferent toxic effects on cowpea and maize seedlings. This is indicated by thedifferential responses of levels of protein and plant biomass to the respectivepetroleum products. REFERENCESAchuba F. I. and Asagba.
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Wyszkowski, M. and Ziolkowska, A. (2008). Effect ofpetrol 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 proteinin the leaves, stem and root of cowpea and maize seedlings after four days ofgermination 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