Effect of detergent and sawdust addition on hydrocarbon reduction and growth of Abelmoschus esculentus L ( Okra ) in a petroleum-contaminated soil

A study to investigate the effect of sawdust and detergent on hydrocarbon reduction and the growth of okra (Abelmoschus esculentus L.) in petroleum polluted soil was carried out. The study was conducted at the Centre for Ecological Studies, University of Port Harcourt. Bonny light crude (300 ml) was used to pollute 10 kg soil. Detergent (20 g), sawdust (200 g) and sawdust: detergent (200 g: 20 g) were used as the amendment materials alongside a control (pollution but un-remediated) arranged in a completely randomized design of 12 replications. After 30 days post-amendment, 3 seeds of okra (var: V35-45 days) were grown in each treatment pot for 6 weeks. Soil Total Hydrocarbon Content (THC) and okra growth performance (shoot length, leaf number, root length, total fresh weight, dry weight, leaf area and leaf area ratio) were analyzed. Results showed significant (p=0.05) reductions in THC of 44.23%, 26.5%, 70.80% and 10.79%, in detergent (20 g), sawdust (200 g), sawdust (200 g) + detergent (20 g) and control, respectively. Improved growth performances of okra were observed in the amended soil as compared to the control. Okra growth performance was in the order of: 200 g sawdust + 20 g detergent combination > 200 g sawdust > 20 g detergent. This implies that these two materials are good bio-stimulation materials especially when used in combined form for THC reduction and


Introduction
such as land, water and air.The continual changes occurring in the Oil pollution is a serious multi-dimensional natural environment could be man-made or natural environmental problem in Nigeria as its impact on the (Dacko and Dacko, 2009;Anyanwu et al. 2014).
entire ecosystem cannot be over-emphasized (Tanee Man exploits the resources of the environment for and Albert, 2015).Various activities of the oil industries his personal benefits and as a result causes (crude oil exploration, refining, transporting and alterations in the natural balance of the ecosystem marketing) in addition to artisanal refining and oil theft (Goodwin et al., 2008).These changes or have resulted in many oil spills and other oil discharges alterations caused by the interactions between man into the environment.The effects of these activities and his immediate environment might introduce have been reported to be disastrous to the ecosystems substances into the environment which in most and thus become an issue to the living organisms of cases may be undesirable to man and other living contaminated areas, firms or organizations organisms (Evelyn and Tyav, 2013).Pollution participating in oil prospects and exploration.appears to be one of the environmental problems (Ambituuni et al., 2014).Wilcox, (2013) plaguing human existence on earth.Pollution reported that crude oil and its by-products are the most occurs and affects every aspect of the environment important organic pollutants of land and water in Nigeria.Crude oil or petroleum hydrocarbon hydrocarbon hence increase the rate of remediation contamination affects the environment in many process (Millioli et al., 2009;Couto et al., 2010).ways.Such include: disruptions of natural This study is carried out to compare the effect equilibrium between the living species and their of sawdust and detergent in the biostimulation of natural environment (Das and Chandran, 2010); petroleum contaminated soil for growth of okra.It is contamination of surface and groundwaters expected that results obtained from this study will give (Balasubramaniam et al., 2007); stunted growth us insight on other suitable methods for ameliorating and death of plants (Anyanwu and Tanee, 2008); crude oil contaminated soil for enhanced growth of reduction in species diversity (Tanee and Albert, plants. 2015); alteration of physical and chemical status of soil with subsequent abandonment of such land Materials and methods (Kibblewhite et al., 2008;Gighi et al., 2012) and; Experimental site negative effect on animals and human health The experiment was conducted at University of (Abowei, 1996;Chekroun et al., 2014 Soil was collected from a demonstration farm biological (bioremediation) methods (Okoh, 2006).
within the university environment for the experiment.The method to be adopted for the remediation of a Loamy soil was collected at the surface with a spade.polluted site depends on the circumstances and Plant particles were removed and the soils thoroughly how suitable or efficient the method is, in each mix to homogeneity.The method of Tanee and Jude case.However, most of these methods have some (2017) was used for the pollution application in which drawbacks in completely remediating hydrocarbon 10 kg of soil was weighed into each perforated contaminated soil.Among the techniques used for experimental bag.Each bag filled with soil was ameliorating or remediating crude oil polluted soils, artificially polluted with 300 ml of bonny light crude oil bioremediation (e.g biostimulation) and chemical obtained from Port Harcourt Refinery Company, Eleme.remediation (e.g.use of detergent) are effective in A total of 48 bags filled with experimental soil were used reducing the impact of crude oil on contaminated for the experiment.The crude oil was thoroughly mixed sites, although they have their limitations (Kumar with the soil.The set up was allowed for 2 weeks for full et al., 2011).
acclimatization of the oil with the soil under natural Biostimulation is a bioremediation strategy environmental (climatic) conditions of sunshine, that has to do with the addition of materials to rainfall, relative humidity and temperature.stimulate the growth of indigenous microorganisms (Kumar and Gopal, 2015).Input of large Addition of Amendments quantities of carbon sources such as crude oil, used Detergent (brand name: Zip) bought from lubricating oil, and diesel oil tends to result in a Eleme market and sawdust obtained from timber mill rapid depletion of the available pools of major Rumuosi were applied as the amendment materials inorganic nutrients such as N and P. Hence, the according to the method of Jude and Tanee (2016b).addition of nutrients, either organic or inorganic, The chemical ingredients of the detergent used are will replenish the soil with these nutrients and surfactant, enzymes, photo bleach, optical brightening enhances the activities of indigenous microbes.
agent, builder, perfume and anti-redeposition agent.The concept of biostimulation is that, by adding The concentration levels (amounts) of sawdust and more nutrients, microorganisms replicate, increase detergent used as amendment were as follows: in number and grow rapidly and thus increase the T1: 10 kg soil + 300 ml crude + 20 g detergent), rate of biodegradation (Thieman and Palladino T2: 10 kg soil + 300 ml crude + 200 g sawdust, 2009).Addition of detergent to the oil T3: 10 kg soil + 300 ml crude + 200 g sawdust + 20 g contaminated soil aids to demulsify the detergent), T4: 10 kg soil + 300ml crude oil (Control).
( 4 b ags planted with okra of each treatment) were The amendments were thoroughly mixed to obtain harvested.The poly bags were shaken in order for the a uniform mixture.The amendment set-up was plants to lose grip, and to make the removal of the allowed for 30 days before planting of the okra.
plants easier.The plants were harvested by destroying the bags, scattering the soil and the plants in the poly bags were gently and carefully removed to ensure they Planting of Okra were intact without destroying the roots.Using an Okra (Abelmoschus esculentus L) (var: V35electronic weighing balance Model EK5350, the total 45 days) obtained from the Centre for Ecological fresh weights of the plants (per pot) were determined Studies of the University of Port Harcourt, Rivers by weighing the whole plants in each treatment pots State, Nigeria were planted after 30 days of immediately after harvest and the values obtained were amendment addition.The okra seeds were planted recorded.in the experimental bags at the rate of five seeds The root length was determined by using metre per bag at the depth of 4 cm.After two weeks of rule calibrated in centimetre (cm).Lengths of three planting, the seedlings were thinned to three (3) longest roots on each plant sampled/bag were per bag after germination.The growth performance measured (cm) and average values calculated were of the test plant (okra) was monitored for 6 weeks recorded.using some selected growth parameters.
Total dry weight was determined after oveno drying for a period of 48 hrs at 75 C.The dried sampled Determination of Total Hydrocarbon content and plants were then weighed on an electronic weighing okra growth parameters balance Model HX-Z232 to obtain the weights.The Soxhlet Extraction Method (APHA, The formula: Leaf area (LA) = 0.89 × Length of the leaf 1995) was used for the determination of Total × width of the leaf (broadest part) and 0.89 as a Hydrocarbon Content of the treatment soils.The constant (Asoegwu, 1988) was used for the calculation percentage reduction in THC was calculated using of leaf area and the total leaf area was calculated.the formula: The leaf area ratio was calculated using the % THC reduction = formula Leaf Area Ratio (LAR) = Selected plant growth parameters were investigated.These include germination Statistical evaluation percentage, shoot length (plant height), number of From the data generated, means and standard leaf, leaf area, root length, total fresh weight, total mean error (SEM) were calculated.The data were dry weight and leaf area ratio (LAR).These further analyzed using Analysis of variance (ANOVA) parameters were determined at 2 week interval for and Least Significant Difference (LSD) tools according 6 weeks with the exception of germination to SPSS data analysis package (2014 version).The percentage which was determined by counting the results were presented in graphs with difference number of seedlings that germinated within 5 days superscripts indicating significant difference @ P = after planting.0.05.Germination percentage was calculated as: % Germination =

Soil Total Hydrocarbon content (THC) Shoot length determination was done by
The result for the total hydrocarbon content measuring the shoot of the plants in the bags from (THC) of the soil is presented in Fig 1 .The result showed the soil level to the terminal bud using a meter rule.a significant (p=0.05)reduction in the THC content in all Average mean shoot length of the plant was the treatments with T3 (amended with a combination of calculated.The leaf number count was done by sawdust and detergent) having the highest percentage counting the leaves, taking into consideration all reduction.The least Total Hydrocarbon Content (THC) leaves for each plant in the experimental pot.
reduction was observed in control (unremediated soil).Using the destructive method 4 replicates

Shoot length and leaf number between
recorded lower values in the shoot length but there was treatments show significant increment (p= 0.05) in significant difference in the leaf number between the the combined treatment of sawdust and detergent treatments in the order of 20 g + 200 g sawdust > 200 g than the other treatment options.The un-amended sawdust > 20 g detergent > unremediated soil (Table soil (control) recorded the least.At 6 WAP, there 1).Results show significant reduction in the leaf area in all was no significant difference between 200 g the treatment options from the 4 WAP to the 6 WAP.sawdust single treatment and 200 g + 20 g Between treatments, the combined treatment of sawdust detergent combined treatment while 20 g and detergent recorded the highest (P= 0.05) leaf area detergent treatment and unremediated soil from 2 WAP to 6 WAP (Table 1).
Table 1: Shoot Length (cm), leaf number and leaf area of okra in the different treatments Different superscripts within column represent significant difference @(p=0.05) There was a progressive increase in the treatment option recorded the highest (P = 0.05) root root length of okra in all the treatment options from length, followed by the sawdust + detergent the 2 WAP to 6 WAP.At the termination of the (combined) and the unremediated (control) recording experiment (6 WAP), 200 g sawdust single the least (Table 2).
Table 2: Root Length (cm) of okra in the different treatments Different superscripts within column represent significant difference @(p=0.05) The results of the fresh weight and Total trend was observed in the leaf area ratio (LAR) in which dry weight showed that the amended soil showed there was a progressive decrease in LAR with time in all an improvement in the fresh weight and dry weight the treatment options.Significant difference (P = 0.05) of okra as compared with the control (un-amended) was observed at 2 WAP between the treatments; with soil.It was also observed that combining sawdust the control (unremediated) treatment taking the lead and detergent had a greater positive impact (P = and the 20 g detergent recording the least.0.05) than the other treatments and control.
At the termination of the experiment (6 WAP), there was Although, at 6 WAP, there was no significant a drop in the LAR in all the treatments with no significant difference in both total fresh and dry weights difference between them (Table 4).between 200 g sawdust treatment and 20 g detergent treatment (Table 3).A reversed result

Discussion
these factors might have contributed to the highest rate of hydrocarbon degradation in the combined treatment Different biostimulation methods have as compared to other treatments.been proposed by researchers for the remediation Results also show significance reductions of of petroleum contaminated sites.The suitability of THC in the other amendment options (200 g sawdust any method depends on a number of factors such and 20 g detergent single treatments) when compare as type of oil, season of spill, amount of spill, types with the control.This suggests the abilities of sawdust of ecosystem and the environmental factors and detergent to reduce hydrocarbon in petroleum prevailing in that ecosystem.The suitability of polluted soil.The use of sawdust in crude oil remediation has been reported by Akonye and sawdust and detergent in the remediation of Onwudiwe, (2004); Boodoosingh et al., (2007) and hydrocarbon contamination tropical soil of the Niger Tanee and Albert, (2011) while that of detergent has Delta was confirmed by the decrease in Total been reported by Millioli et al., (2009) and Jude and Hydrocarbon Content (THC) in all the treatments.

The highest percentage (%) reduction of
The low hydrocarbon degradation in the control THC in soil was observed in treatment amended soil (without amendment) confirms the toxicity of crude with 200 g sawdust and 20 g detergent (combined).
oil to soil microbial degradation (Ogbo, 2009).Crude oil This is understandable since sawdust is an blocked the air pores (Essien and John, 2010) and water absorbent ((Pala et al., 2005;Trejo-Hernandez, et pores of soil (hydrophobic).This reduces water holding al., 2006) as well as containing nutrients (high in capacity and aeration in the soil (Abosede, 2013) organic matter) needed for microbial growth depriving the soil of oxygen and water; thus affecting coupled with the demulsification of crude oil by the the soil-water and soil-air relations for efficient detergent (Couto et al., 2010).
hydrocarbon degradation.The sawdust supplied nutrients which are Results showed a significance increase in always limited in a crude oil polluted soil (Akujobi et germination rate in 200g sawdust + 20 g detergent al., 2011) to energize the hydrocarbon degrading treatment followed by 200 g sawdust treatment and microbes while the detergent desorbs the recording the least at 20 g detergent and control.The hydrocarbon and provides enough surface area for reduction in hydrocarbon toxicity and modification of the microbe to degrade.Hence the combination of Table 3: Total biomass (fresh weight and dry weight) (g) of okra in the different treatments Different superscripts within column represent significant difference @(p=0.05)(2007).used in single (separate) form.This reveals that Effects of time of application of crude oil to soil on the mixture of organic and chemical treatments is growth of maize (Zea mays L.).Res.J. Environ.Toxicol.favourable in shielding and reducing the adverse 1(3): 116-123.effect of oil pollution to plants.This assertion is justifiable since there was above 70% reduction of Ajolagba, G.A. and Peter, K.D. (2013).Effect of THC in the combined treatment option.Also the remediation on growth parameters, grains and dry enhancement of the soil chemical properties such matter yield of soybean (Glycine max) in crude oil as soil aeration, soil moisture content and pH in a polluted ultisols in Ogoni land, south-east, Nigeria.Afri.crude oil polluted amended soil (Ikhajiagbe and J. Environ.Sci.Technol., 7(2): 61-67.Anoliefo, 2010) could have also contribute to the improved vegetative parameters of the okra.It is Akonye, L.A. and Onwudiwe, I.O.( 2004).Potential for possible that in the combined treatment, the Sawdust and Chromolaena leaves as soil amendments sawdust as an organic material with high nutrient for plants growth in oil polluted soil.Niger Delta content may have cushion the adverse effect of the Biologia, 4:50-60.detergent such as high pH (Chan, 2015) leading to favourable soil conditions for plant growth.

Fig. 1 :
Fig.1: Percentage Reduction in THC in the different treatments

Table 4 :
Leaf Area Ratio (cm2/g) of okra in the different treatments Different superscripts within column represent significant difference @(p=0.05) the soil chemical properties (Oyem and Oyem, Conclusion 2013) couple with access to air and water by the This study reveals the effect of detergent and seeds might have accounted for the high sawdust (either single or combine applications) as germination rate in the combined (sawdust and biostimulating agents for the degradation of petroleum detergent) and sawdust treatments, respectively.hydrocarbons in soils as confirmed by the significant The low germination rates in the 20 g reductions in Total Hydrocarbon Content (THC) with detergent treatment and control suggest the improvement in vegetative growth of okra.Agbogidi et al. 2007; Tanee and Anyanwu, Abosede, E.E.(2013).Effect of crude oil pollution on some 2007; Kelechi et al., 2008; Eze et al., 2013).soil physical properties.J. Agric & Vet.Sc., 6(3): 14-17.Significant improvements in these parameters were recorded in the amended treatments than the Abowei, M.F.N. (1996).Prediction and consequences of control.This may be attributed to the reduction in petroleum spills into the Nigerian aquatic environment the THC content in addition to nutrient enrichment in the year 2000.Int J. Environ.Pollut.6: 306-316.(especially the sawdust) of the amended soil.Thus the toxicity of crude oil to plant growth was Akujobi, C.O., Onyeagba, R.A., Nwaugo, V.O. and Odu, drastically reduced in the amended soil.N.N.(2011).Effect of nutrient amendment of diesel oil It was vividly observed that the vegetative polluted soil on plant growth parameters.Cur.Res.J.