International Journal of Scientific Research in Environmental Sciences (IJSRES), 1(9), pp. 250-262, 2013 Available online at http://www.ijsrpub.

com/ijsres ISSN: 2322-4983; 2013 IJSRPUB http://dx.doi.org/10.12983/ijsres-2013-p250-262

Full Length Research Paper Phytoassessment of an Enhanced Naturally Attenuated Oil-Polluted Soil after Exposure to Various Concentrations of Sodium Bicarbonate Solution
Beckley Ikhajiagbe1*, Abibat S. Unuagbokhe2
1 2

Dept. of Plant Biol. and Biotechnology, University of Benin, Benin City, Nigeria Dept. Science Laboratory Technology, University of Benin, Benin City, Nigeria *Corresponding Author: ikhaj@yahoo.com
Received 24 July 2013; Accepted 25 August 2013

Abstract. The present study investigated the effects of sodium bicarbonate-induced soft water on the intrinsic qualities of a bioremediated petroleum hydrocarbon-polluted soil. this was also subjected to phytoassessment using African yam bean (Sphenostylis stenocarpa). Into already perforated buckets were measured 5kg sun-dried top soil (0-10cm). Waste engine oil (WEO) was added to soil and mixed thoroughly to obtain similar concentrations of 5 % w/w oil in soil. The entire setup was divided into 5 treatment sets, depending on the concentration of NaHCO3 solution used (0, 25, 50, 100 and 200mg/l). Each set was wetted daily with 200ml of different solutions for 3 months. Results showed significant reductions in heavy metal concentrations particularly in the 200mg/l of NaHCO3-wetted soils. Copper reduced to a range of 4.84 - 6.08mg/kg in the NaHCO3-wetted soil, compared to 9.32mg/kg in the control. However, total polyaromatic hydrocarbon (PAH) content of soil was lowest when the soil was wetted with 25mg/l NaHCO3 (96.43mg/kg), indicating a 90.51% efficiency, compared to the control with an efficiency of 79.15%. There were significant reductions in pollution indices (contamination factor, hazard quotient, toxicity equivalency factors, and probable effect concentration) used in the study; an indication that water softness positively impacted on the intrinsic remediation of the waste engine oil-polluted soil. Results of phytoassessment showed the improved plant growth and yield response in the oil-polluted soils upon addition of lower concentrations of NaHCO3 in soil. Grain yield per plant in the oil-polluted soil was 8.33g, compared to 29.29g in the control and 25.87g when oil-polluted soil was wetted with 25mg/l solution. Key words: Bioremediation, heavy metals, intrinsic qualities, petroleum hydrocarbon, polyaromatic hydrocarbons, sodium bicarbonate, Sphenostylis stenocarpa, water hardiness, water softness

1. INTRODUCTION The increase and development of various industries in time past has greatly increased the amount and concentration of toxic waste, which has resulted in the need for bioremediation using plants and microbes. The indiscriminate disposal of petroleum hydrocarbon wastes into the ecosystem occurs mostly as a result of human activity, and this has led to severe environmental contamination (Ikhajiagbe, 2010). For example, spillage of oil into ocean retards the amount of oxygen released from the water and the quantity of sunlight that penetrates the water, hence affecting the rate of photosynthesis and inhibiting growth and development of phytoplankton in the ocean leading to death of these plants and malnourishment to the zooplankton that depends on these plants for feeding. Similarly, the presence of little amount of oil can cause physical damage to animals such as skin irritation, altering of the immune system, reproductive or developmental damage, and liver disease. When large amount of oil gain access into water body chronic effects like cancer and death of wildlife are likely to occur.

Oil in soil creates conditions that seriously hamper plant growth and development. Oil-polluted soil causes nutrient deficiency in plants, soil appears waxy, there is little or no water penetration, soil forms a crust. All these inhibit plant growth due to lack of water and nutrients, which cannot penetrate the soil (Udo and Fayemi, 1975). Whisman et al. (1974) stated that there is rise in toxicity of certain heavy metals such as Manganese, Nickel, lead and Vanadium in used oil product. This rise in toxicity has made oil pollution a major problem and has motivated the deriving of various methods of remediation. Spills from waste engine oil is a paramount problem that requires immediate attention because this waste is not attributed only to service stations, oil drained from automobile but also from generator engines which results in great amounts of WEO dumped into the ecosystem (Anoliefo and Vwioko, 2001). The demoralizing nature of public power supply in many developing countries like Nigeria has greatly prompted the use of private generators, which has increased the need for engine lubricants. Nigeria accounts for more than 87 million litres of WEO annually (Anonymous, 1985). All these

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find their way into the soil, particularly wastelands and even nearby farms, which receive these wastes or water-soluble fractions of the wastes. Apart from control measures involving enlighten campaigns to put in place preventive measures, it is also important that measures are put in place to remediate already polluted soils. Apart from the physical method of remediation, chemical measures are also used, but these methods have been reported in literature as not ecofriendly (Ikhajiagbe, 2010). Bioremediation therefore becomes imperative. Bioremediation is powered by the soils microbial diversity, richness and activity. Moisture in soil greatly influences this microbial activity. Payne and Wiebe (1978) opined that changes in soil water content could lead to changes in microbial growth efficiency. A decrease in water availability may cause a change in the physiology of microbes as they adjust to more desiccating conditions. What would be the effects be on the intrinsic qualities of the bioremediated soil, when instead of ordinary water (Ikhajiagbe et al., 2013), soft water was used to moisten the soil? Hard water contains high concentrations of magnesium and calcium. It can be a nuisance to homeowners because of mineral build-up on water fixtures and poor soap and detergent performance. They also affect soil fertility particularly with increase in soil alkalinity. Water softeners are used to continuously remove calcium and magnesium from the water source via an exchange process, particularly by introducing sodium bicarbonate (NaHCO3). The introduction of sodium and bicarbonate ions in soil moisture greatly influences changes in soil pH, thereby affecting soil microbial activity and bioremediation efficiency (Vidali, 2000; Ikhajiagbe, 2010; Ikhajiagbe et al., 2013). The present study therefore investigates the effects of sodium bicarbonate-induced water softness on the natural attenuation of a waste engine oil-polluted soil. 2. MATERIALS AND METHODS 2.1. Soil pollution using varying levels of WEO in each bowl Top soil (0-10cm), of known physicochemical properties (Table 1), was collected randomly from a fallow land situated near the Department of Plant Biology and Biotechnology Botanic Garden, University of Benin, Benin City, Nigeria, and pooled together. Thereafter, 10kg sun-dried soil was each placed into large perforated bowls with 5 random perforations made with 2 mm diameter nails at the bottom of each bowl. Waste engine oil (WEO) was

added to soil in the bowls and mixed thoroughly to obtain similar concentrations of 5% w/w oil-in-soil. 2.2. Preparation of sodium bicarbonate (NaHCO3) solution Four different concentrations of solutions of NaHCO3, which included 25mg/l, 50mg/l, 100mg/l and 200mg/l, were obtained by dissolving the salt in distilled water. The control was distilled water. 2.3. Application of various Treatments The polluted soils in the bowls were subjected to wetting with the various concentrations of NaHCO3 solutions on a daily basis. Having previously determined the soils water holding capacity to be 220 ml/kg soil, each bowl carefully received 2000ml of the solution on the very first day the soils were mixed with WEO. This was to ensure that the soils were primarily moistened to near saturation by soft water only. Subsequently each bucket received 2000 ml of solution in week (Ikhajiagbe et al., 2013). This was equally divided into 285ml of solution daily. Care was taken to ensure that the soils in the bowls were properly and equally moistened, while ensuring that the solutions as well as water soluble fractions of oil in soil did not drain out from the perforations at any time throughout the experimental period. The soils in the bucket were turned and properly mixed once every week to enhance relatively equal surface area of exposure of both pollutant and NaHCO3 solutions in the soil. The control soil was wetted with distilled water. The experiment was carried out in a well ventilated screen house with inherent room temperature. After three months of soil exposure to treatments, soils were taken to the laboratory for analysis. 2.4. Extraction of Micronutrients in Soils by Hydrochloric Acid Method Ten (10) g of soil was weighed into a 250 ml plastic bottle. 100 ml of 0.1 m HCl was added, stoppered, and then shaken for 30 minutes. The mixture was filtered through Whitman filter paper No.42 and then Fe, Cu, Mn, Zn, Cd, Cr, Pb, Ni, and V were determined in the filtrate by atomic absorption spectrometry. 2.5. Determination of Polyaromatic Hydrocarbon Contents of Polluted Soil by Gas Chromatography (GC) A 10 g sample was extracted with methylene chloride (DCM). The extract was filtered through anhydrous sodium sulphate to remove any trapped water

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molecule. This was followed by a clean- up/ fractionation of the sample extract into aliphatic and aromatic (PAH) components. Finally, the components were concentrated using a rotary evaporator for GC analysis, using FID as detector. Model of GC used

was AGILENT 6890. The GC analysis began by first injecting 1 L of the sample extract into the GC, and the results calculated as follows:

Where, Rf = Response factor = Total Area / Total Concentration, obtained from instrument calibration with standards; Area is obtained from the chromatogram output; F.vol is the final volume of the concentrated extract (in ml); Wt is the initial weight of the homogenized sample (in grams) 2.6. Identification of Soil Microorganisms The soil samples were air-dried and sieved through a 2 mm mesh to remove undesirable material. The dilution series for the soil sample was done by transferring 1 gram of the soil to nine (9 ml) millimetres of sterile distilled water in sterile glass containers as blank. The glass containers were shaken for 5 minutes and was taken as l0-1 dilution factor, 10 ml were then transferred from the 10-1 dilution into another 9 ml blank to obtain a 10-2 dilution and same process of transfer was repeated twice to obtain a dilution factor of 10-4. The spread plate method was employed in taking the heterotrophic bacteria counts. One (1) ml of the serially diluted portion of 10-4 of each soil sample was inoculated onto nutrient agar plates for bacteria and Potato dextrose agar plates for fungal counts. The plates were inoculated at room temperature for 24 hours and 72 hours respectively, for bacteria and fungi growth. After incubation colonies were then counted and the colony forming unit (cfu/g) of the soil samples determined.

2.7. Isolation of Degraders

Bacterial

and Fungal Oil

Bushnell- Haas (BH) medium (MgSO4, 0.20 g/I; CaCl2, 0.02 g/l; K2H,P04, 1 g/l; NH4NO3, 1 g/l; FeCl3, 0.05 g/l; KH2PO4, 1 g/l; pH 7.0, was used as the enrichment medium with 8 % (v/v) filter sterilized oil as the sole carbon source. The medium was dispensed into in 100 ml Erlenmeyer flasks and autoc1aved at 121 0C for 15 minutes. Thereafter, 5 g of each soil sample was inoculated into each flask of the medium and incubated at 130 rpm at room temperature in a HY-4 multifunctional shaker (B. Bran Scientific and Instrument Company, England). After 10 days, 1 ml of enriched media was transferred into freshly prepared enrichment media and incubated under the same conditions as described above. Serial dilutions from the third enrichment process were inoculated onto nutrient agar plates and potato dextrose agar plates for oil-degrading bacterial and funga1 counts respectively using the methods described by Cowan and Steel (1974) and Cheesebrough (1998). 2.8. Computation of Contamination Factor (CF) CF expresses the ratio between the eventual concentrations of pollutant against its precontamination reference.

Pre-contamination concentration is the concentration of each element or PAH components of the soil just prior to exogenous application of the contaminant (WEO) (Ikhajiagbe, 2010). When CF > 1, inherent concentration element in soil was due to exogenous application of contaminant (i.e. WEO), in which case the metal concentration was higher than values obtained in the original unpolluted soil.

2.9. Computation of Hazard Quotient (HQ) HQ expresses the possibility of the contaminant being an ecological risk or a contaminant of potential ecological concern. The hazards Quotient is expressed by the following equation:

HQ=

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Ikhajiagbe and Unuagbokhe Phytoassessment of an Enhanced Naturally Attenuated Oil-Polluted Soil after Exposure to Various Concentrations of Sodium Bicarbonate Solution

When HQ > 1: Harmful effects are likely due to contaminant in question; When HQ = 1: Contaminant alone is not likely to cause ecological risk; When HQ < 1: Harmful effects are not likely; Screening benchmarks were available at Efroymson et al. (1997).

2.10. Computation of Probable Effect Concentration Quotient (PECQ) of PAH Compounds PECQ predicts the presence or absence of toxicity. PEC value for PAH components were obtained from Honeywell (2011). Where mean PECQ > PEC, toxicity is indicated.

2.11. Computation of Concentration of Toxic Equivalency (TEQ) for Polycyclic Aromatic Hydrocarbons (PAH) Toxic Equivalency factors (TEF) are toxicity potency factors used as a consistent method to evaluate the toxicities of variable mixtures of organic compounds. TEQ = Ti x PEF Where, TEQ = Toxic equivalency; Ti = PAH concentration in soil; PEF= Potency equivalency factor (Cal-EPA, 2005). 2.12. Some Growth and Yield determined in African Yam Bean 2.12.1. Germination Experiments Percentage emergence was calculated as the percentage of seeds that sprouted above soil level of Analysis of variance in completely randomized design was done using the SPSS-15 statistical software, and means were separated by using the Least Significant Difference. 3. RESULTS AND DISCUSSIONS The physiochemical properties of soil and waste engine oil used for the study have been presented on Table 1. Immediately after soil was contaminated with WEO, heavy metal concentrations of the soil were determined and Cu in soil was 9.32mg/kg, but decreased to 7.35 mg/kg in the control. Cu in the 200mg/l NaHCO3-treated soil was 4.85 mg/kg. Generally, results showed a gradual decrease in heavy metal contents of soil with increase in bicarbonate concentrations. Efficiency of heavy metal remediation was highest in the 200mg/l NaHCO3 treated soil (74.95%), compared to 30.01% in the control. a near possible explanation may be simple displacement reactions. Metals are remediated from soil by a number of mechanisms including immobilization, leaching as Parameters

the 5 seeds originally sown per bucket. Heights were also taken by aid of a transparent calibrated ruler at 9 days after sowing (DAS). Dry weights of seedlings were taken at 9 DAS after drying seedlings in an oven at 30oC for 3 days. Percentage survival was also calculated as the percentage of successfully emerged seedlings compared to total number of seeds sown per bowl. 2.12.2. Other Measurable Growth and Yield Parameters Other measurable growth parameters included shoot height, no. of primary branches, stem width, leaflet area, root length, no. of root nodules/plant (> 3mm in diameters), no. of flowers/plant, no. of pods/plant, no.of seeds/pods, 100 seed wt (g), and grain yield (g/plant) calculated thus,

well as by microbial action. The role microorganisms play in the fate of toxic metals and metalloids in the environment cannot be overemphasized. These roles range from physiochemical to biological mechanisms that affect the transformations between soluble and the insoluble phases of these inorganic contaminants. Even though metals are readily leached in the soil particularly due to their solubility in the soil moisture, the role of microorganisms in bioleaching has been reported (Bosecker, 1997). Metals can be mobilized by microorganisms through autotrophic and heterotrophic leaching, methylation as well as chelation by microbial metabolites and siderophores. Volatilization which is also a mechanism of microbial-assisted metal remediation usually results from methylation. However, Gharieb et al. (1998) reported that immobilization can result from sorption to cell components or exopolymers, transport into cells and intracellular sequestration or precipitation as insoluble organic and inorganic compounds, e.g. oxalates, sulphides or phosphates.

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Table 1: Physical and chemical properties of soil and waste engine oil used for the study
Parameters pH Electrical Conductivity (s/cm) Total Org. Matter (%) Total Nitrogen (%) Exchangeable Acidity (meq/100 g soil) K (meq/100 g soil) Ca (meq/100 g soil) Mg (meq/100 g soil) P (mg/kg) Clay (%) Silt (%) Sand (%) Copper, Cu (mg/kg) Manganese, Mn (mg/kg) Nickel, Ni (mg/kg) Vanadium, V (mg/kg) Chromium, Cr (mg/kg) Lead, Pb (mg/kg) Naphthalene (mg/kg) Acenaphthylene (mg/kg) 2-bromonaphthalene (mg/kg) Acenaphthene (mg/kg) Fluorene (mg/kg) Phenanthrene (mg/kg) Anthracene (mg/kg) Fluoranthene (mg/kg) Pyrene (mg/kg) Benzo(a)anthracene (mg/kg) Chrysene (mg/kg) Benzo(b,j,k)fluoranthene (mg/kg) Benzo(a)pyrene (mg/kg) Indeno(1,2,3-cd)pyrene (mg/kg) Dibenzo(a,h)anthracene (mg/kg) Benzo(g,h,i)perylene (mg/kg)
BDL beyond detectable limit (<0.0001 mg/kg), NA not available

Soil 6.19 312 0.59 0.14 0.26 1.21 12.07 11.24 128.11 7.90 13.90 78.20 BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL 0.85 BDL BDL BDL BDL BDL BDL 40.28 5.24 12.25 19.24

WEO 5.56 NA NA NA NA NA NA NA NA NA NA NA 7.92 BDL BDL BDL 16.85 9.96 26.58 7.98 29.54 26.32 42.04 3.68 21.57 32.68 23.98 42.05 106.54 41.68 129.87 129.54 34.68 63.25

Chemolithotrophic, acidophilic bacteria have been known to carry out most autotrophic leaching. These bacteria can fix carbon dioxide and obtain energy from the oxidation of ferrous iron or reduced sulphur compounds (Bosecker, 1997). As a result of sulphur and iron-oxidation by these bacteria, metal sulphides are solubilized and the pH of their immediate environment is decreased, which enhances the solubilization of other metal compounds. Heterotrophic metabolism can also lead to leaching as a result of the efflux of protons, organic acids and siderophores (Burgstaller and Schinner, 1993; Gadd, 1999). Organic acids provide both protons and a metal-chelating anion to complex the metal cation. Citrate and oxalate anions can form stable complexes with a large number of metals. Microbial methylation of metalloids to yield volatile derivatives, e.g. dimethylselenide or trimethylarsine, can be effected by a variety of bacteria, algae and fungi (Gadd, 1993). Microbial reduction of heavy metals by biomethylation is made possible by a number of different bacterial species including Pseudomonas sp., Bacillus sp., and Clostridium sp. (Ikhajiagbe et al.,

2013), and these organisms may have been involved in the methylation of Fe, Cr and Mn (Tinoko et al., 1985). Contamination factor (CF) for heavy metals at 3 months after pollution have been presented on Table 3. Contamination factor provides that the inherent concentration of the elements were not necessarily due to exogenous application of the contaminant. When CF < 1, it implied that the concentration of heavy metal was not due to exogenous concentration of waste engine oil in the soil. CF values for Cu, Cr and Pb were all greater than unity. Hazard Quotient (HQ) expresses the tendency of the contamination posing an ecological risk or contamination of economic concern (Table 4). HQ was greater than unity for Cu and Cr; an indication that these metals still could pose an ecological risk. Comparatively however, there was decrease in HQ values with increased concentration of bicarbonate applied to soil. this meant that the increased incidence of water hardiness decreased the risk posed by the heavy metals both to the environment (Table 4) and to microbial processes (Table 5).

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Ikhajiagbe and Unuagbokhe Phytoassessment of an Enhanced Naturally Attenuated Oil-Polluted Soil after Exposure to Various Concentrations of Sodium Bicarbonate Solution Table 2: Heavy metals in oil-polluted soil after three months of treatment application
Oil-polluted soil at day 1 Heavy metal Cu (mg/kg) Mn (mg/kg) Ni (mg/kg) V (mg/kg) Cr (mg/kg) Pb (mg/kg) Total (mg/kg) Efficiency (%)
Detectable limit > 0.0001 mg/kg

3 Months after pollution/treatment application Control 7.35 BDL BDL BDL 12.11 7.95 25mg/l NaHCO3 6.98 BDL BDL BDL 10.68 4.65 50mg/l NaHCO3 6.08 BDL BDL BDL 8.13 2.11 100mg/l NaHCO3 5.64 BDL BDL BDL 2.81 2.46 200mg/l NaHCO3 4.85 BDL BDL BDL 2.68 2.28

9.32 BDL BDL BDL 18.52 11.32

39.16 -

27.41 30.01

22.31 43.03

16.32 58.32

10.91 72.14

9.81 74.95

Table 3: Contamination factor for heavy metal contents of oil-polluted soil after three months of treatment application
Heavy metal (mg/kg) Oil-polluted soil at start Day 1 >104 0 0 0 231.5 >104 3 months after pollution Control >104 0 0 0 151.38 >104 25mg/l NaHCO3 >104 0 0 0 133.50 >104 50mg/l NaHCO3 >104 0 0 0 101.63 >104 100mg/l NaHCO3 >104 0 0 0 35.13 >104 200mg/l NaHCO3 >104 0 0 0 33.5 >104

Cu Mn Ni V Cr Pb

Table 4: Hazard quotient to determine ecological toxicity of heavy metal components of waste engine oil-polluted soil at 3 months after pollution
Day 1 Unpolluted soil Heavy metal Cu [40.00] Mn [100.00] Ni [30.00] V [2.00] Cr [1.00] Pb [50.00] 0 0 0 0 0.08 0 Oilpolluted soil 0.23 0 0 0 18.52 0.23 Control 3 months after pollution 25mg/l NaHCO3 0.17 0 0 0 10.68 0.09 50mg/l NaHCO3 0.15 0 0 0 8.13 0.04 100mg/l NaHCO3 0.14 0 0 0 2.81 0.05 200mg/l NaHCO3 0.12 0 0 0 2.68 0.05

0.18 0 0 0 12.11 0.16

Values presented in bracket are ecotoxicological benchmark values (Efroymson et al., 1997)

Polyaromatic hydrocarbon contents of the oilpolluted soil after treatment application are presented on Table 6. Results show that total PAH content of hard water-wetted soil was lowest in the 25mg/l NaHCO3 treated soil, indicating a 90.51% remediation efficiency, compared to 59.93% efficiency recorded in the 200mg/l NaHCO3 treated soil. This means that increasing the concentration of bicarbonate in the hardwater decreased the efficiency of PAH remediation in the oil-polluted soil. Total PAH in the control was 211.73mg/kg (i.e. 79.15% efficiency), compared to values of 268.68 mg/kg and

406.96mg/kg for both 100mg/l and 200mg/l salttreated soils respectively. Remediation of most polyaromatic hydrocarbon compounds begins first with volatilization for most low molecular weight PAHs. However, as the molecular sizes increase and when exposure to soil particles is prolonged, bioavailability is reduced greatly and, biodegradation rates become slower. Naphthalene, acenaphthylene and fluorine with lower molecular weight (two to four ringed compounds) are degraded easily. This probably explains their lower concentrations in the study. In general, the molecular

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size and topology of compounds determine the fates of PAHs in the soil (Kanaly and Harayama, 2000). This perhaps accounts for their rapid degradability compared to other PAH components. It is essential that the bioavailability of PAHs in soil be increased in order to increase the biodegradation processes and making it economically realistic and speedy (Piskonen

and Itavaara, 2004). Moisture level greatly determines the rates at which PAHs are degraded because water is needed for microbial growth and enzymatic/biochemical activities (Leahy and Colwell, 1990). In the present study the buckets received adequate amount of moisture.

Table 5: Hazard quotient to determine toxicity heavy metal components of waste engine oil-polluted soil against soil microbes and microbial processes at 3 months after pollution
Day 1 Unpolluted soil Heavy metal Cu [10.00] Mn [100.00] Ni [90.00] V [20.00] Cr [10.00] Pb [900.00] 0 0 0 0 0.01 0 Oilpolluted soil 0.09 0 0 0 1.85 0.01 Control 3 months after pollution 25mg/l NaHCO3 0.07 0 0 0 1.07 0.01 50mg/l NaHCO3 0.06 0 0 0 0.81 0 100mg/l NaHCO3 0.06 0 0 0 0.28 0 200mg/l NaHCO3 0.05 0 0 0 0.27 0

0.07 0 0 0 1.21 0.01

Values presented in brackets are benchmarks for toxicity of heavy metal components of waste engine oil-polluted soil against soil microbes and microbial processes (Efroymson et al., 1997). HQ > 1 indicates toxicity.

In the present study, higher concentrations of sodium bicarbonate in the oil-polluted soil decreased the bioremediation efficiency of PAH in contrast to heavy metal remediation. No reason has been attributed to this trend. This perhaps leaves room for more research on the subject. One possible reason for decrease in efficiency of PAH bioremediation could be because of the antimicrobial activity of NaHCO3. Intrinsic bioremediation of oil-polluted soils have been found to be greatly influenced by its inherent rich microbial diversity and activity. Therefore, whatever hampers microbial activity reduces overall remediation efficiency. Newbrun et al. (1984) and Bell et al. (1997) reported bactericidal effects of NaHCO3 solutions. The higher the concentration of bicarbonate, the faster the lethality. Yao et al. (2004) investigated the possible influence of NaHCO3 on Candida albicans and reported antagonistic effects on the yeast cells. Another important effect of bicarbonate in the remediative process could be as a result of pH alteration. Soil pH is very important because most microbial species can survive only within certain pH range. NaHCO3 influences soil pH particularly by implication of its sodium and carbonate ions. Microbial activity can be prevented from occurring due to alteration in pH which can render essential microbe enzymes inactive and /or denature proteins within the cells. Changes in pH can also affect microbes in their access to metals and organics that react differently under varied pH regimes (Sylvia et al., 2005). Ikhajiagbe et al. (2012) reported enhanced PAH remediations at a pH of 5 and lower remediations at alkaline soil conditions.

Similarly, increase in pH causes deprotonation of metals ions binding site exposed to cellular surfaces (Chojnacka, 2010). While decrease in pH causes competition between protons and positively charged metal ions. This is however only for cations (Naja et al., 2010). Generally, an increase in soil pH tends to decrease the availability of calcium, magnesium, sodium, potassium, ammonia, nitrogen and phosphorus, whereas decrease in soil pH results in decreasing availability of nitrate and chloride (Selatnia et al., 2004). NaHCO3 is an alkali and as such has the capability for raising soil pH. Table 7 presents the hazard quotient to determine ecological toxicity of PAH components of waste engine oil-polluted soil at 3 months after pollution. HQ values for acenaphthene and fluorene were less than unity in the hard water-wetted soils (Table 7). Comparatively, the higher the concentration bicarbonate in the hardwater, the higher the HQ value, and hence the posibilioty for the PAH component to pose ecological threat. In Table 8 is the concentration of toxic equivalent (TEQ) of PAH components of waste engine oil-polluted soil at 3 months after polluted. Total TEC increased with increasing concentration of biocarbonate used for wetting the oilpolluted soil, from 26.41 mg/kg in 25mg/l NaHCO3treated soil to 96.83 mg/kg in 200% NaHCO3-treated soil respectively. The Total TEC for the c-PAH mixtures of the soil sample exceeded the Method B cleanup level for benzo[a]pyrene (0.137 mg/kg), and as such the cleanup level for benzo[a]pyrene was not met for this particular soil sample (Cal-EPA, 2005).

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Ikhajiagbe and Unuagbokhe Phytoassessment of an Enhanced Naturally Attenuated Oil-Polluted Soil after Exposure to Various Concentrations of Sodium Bicarbonate Solution Table 6: Polycyclic aromatic hydrocarbon contents of oil-polluted soil after three months of treatment application
Oilpolluted soil at start Day 1 29.25 10.54 35.21 35.46 45.22 5.62 29.24 42.52 36.20 53.87 129.54 59.48 209.16 169.54 52.20 72.65 1015.70 3 months after pollution and treatment application Control 25mg/l NaHCO3 BDL BDL 14.71 BDL BDL 0.33 15.26 BDL BDL BDL 0.94 0.24 26.02 3.75 12.50 22.68 96.43 90.51 50mg/l NaHCO3 15.63 BDL 15.66 BDL BDL 0.05 16.34 BDL BDL 20.30 0.51 10.47 37.01 8.06 3.94 22.05 150.02 85.23 100mg/l NaHCO3 13.50 8.34 14.86 BDL 0.96 0.42 15.34 11.81 1.65 21.54 32.84 19.54 56.85 15.08 26.48 29.47 268.68 73.55 200mg/l NaHCO3 12.62 12.41 12.90 13.04 12.85 1.61 13.57 13.26 13.90 26.68 42.53 19.52 90.95 27.76 55.90 37.46 406.96 59.93

PAH (mg/kg) Naphthalene Acenaphthylene 2-bromonaphthalene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(a)anthracene Chrysene Benzo(b,j,k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd)pyrene Dibenzo(a,h)anthracene Benzo(g,h,i)perylene Total PAH (mg/kg) Efficiency (%)

15.32 BDL BDL BDL 15.68 BDL 16.41 15.79 16.73 31.13 2.37 6.98 63.62 5.38 3.86 18.46 211.73 79.15

BDL beyond detectable limit(<0.0001 mg/kg)

Table 7: Hazard quotient to determine ecological toxicity of selected PAH components of waste engine oil-polluted soil at 3 months after pollution
PAH Oil-polluted soil at start Day 1 Control Naphthalene [0.10] Acenaphthene [20.00] Fluorene [30.00] Phenanthrene [0.10] Anthracene [0.10] Fluoranthene [0.10] Pyrene [0.10] benzo(a)pyrene [0.10] 0 0 0 8.5 0 0 0 402.8 292.5 1.77 1.51 56.2 292.4 425.2 362.0 2091.6 153.2 0 0.52 0 164.1 157.9 167.3 636.2 3 months after pollution 25mg/l NaHCO3 0 0 0 3.3 152.6 0 0 260.2 50mg/l NaHCO3 156.3 0 0 0.5 163.4 0 0 370.1 100mg/l NaHCO3 135.0 0 0.03 4.2 153.4 118.1 16.5 568.5 200mg/l NaHCO3 126.2 0.65 0.42 16.1 135.7 132.6 139.0 909.5

Values presented in brackets are ecotoxicological benchmark values (Efroymson et al., 1997). HQ > 1 indicates toxicity.

Table 8: Concentration of toxic equivalency (TEQ) of PAH components of waste engine oil-polluted soil at 3 months after pollution
PAH Oil-polluted soil at start Day 1 Unpolluted Oilsoil polluted soil 0 5.39 0 40.28 1.30 209.16 3 months after pollution Control 25mg/l NaHCO3 0 0.01 26.02 50mg/l NaHCO3 2.03 0.01 37.01 100mg/l NaHCO3 2.15 0.33 56.85 200mg/l NaHCO3 2.67 0.43 90.95

Benzo(a)anthracene [0.10] Chrysene [0.01] Benzo(a)pyrene [1.00]

3.11 0.02 63.62

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International Journal of Scientific Research in Environmental Sciences (IJSRES), 1(9), pp. 250-262, 2013

Indeno(1,2,3-cd)pyrene [0.10] Total TEC

0.52

16.95

0.54

0.38

0.81

1.51

2.78

40.80

232.8

67.29

26.41

39.86

60.84

96.83

Values presented in bracket are potency equivalency factors (Cal-EPA, 2005)

Petroleum hydrocarbon-contaminated soils have been reported to have abundant growth of many microorganisms (April et al., 2000; Ikhajiagbe and Anoliefo, 2011, 2012; Ikhajiagbe et al., 2013). Bacterial isolates in the polluted soil at day 1 were Achromobacter sp, Micrococcus luteus, M. roseus, Bacillus pumilis and B. subtilis (Table 9), making up a total heterotrophic bacterial count of 3.4 x 105 cfu/g. Total hydrocarbon degraders at day 1 were 2.0 x 105 cfu/g. Percentage hydrocarbon degrading bacteria in the 25mg/l NaHCO3- treated soils was highest
Microorganisms Oilpolluted soil at Day 1

(65.5%), including M. luteus, B pumilis and B.subtilis. There was reduction in total bacterial count with increasing bicarbonate concentration in soil. This was similar to total fungal count. Fungal isolates present were Aspergillus niger, A. Flavus, Penicillium sp, Fusarium solani, Rhiszopus stolonifer and Trichoderma sp. Ikhajiagbe and Anoliefo (2010) reported the predominance of the fungi Aspergillus niger, Penicillium sp, Geotrichum sp, and Trichoderma sp. as well as bacteria B. subtilis, and B. pumilis in waste engine oil-polluted soils.
3 months after pollution/treatment application

Table 9: Microbial composition of treated and control soils after waste engine oil pollution and application of treatments

Control

25mg/l NaHCO3 + + + + +

50mg/l NaHCO3

100mg/l NaHCO3 + + 3.8 1.7 44.7 + + 1.8 1.0 55.5

200mg/l NaHCO3 + + 3.7 1.2 32.4 + + 1.6 0.3 18.7

Achromobacter sp Clostridium sp *Micrococcus luteus M. roseus *Bacillus pumilis *B. subtilis Heterotrophic bacteria (x 105 cfu/g) Hyd. Deg. bacteria (x 105 cfu/g) % Hydrocarbon degraders *Aspergillus niger A. Flavus *Penicillium sp *Fusarium solani Rhiszopus stolonifer Trichoderma sp Heterotrophic Fungi (x 105 cfu/g) Hyd. deg. Fungi (x 105 cfu/g) % Hydrocarbon degraders

+ + + + + 3.4 2.0 58.8 + + + + + 2.7 1.6 59.2

+ + + + + 5.2 3.4 65.4 + + + + + 2.6 1.0 38.4

Bacterial isolates + + + +

5.8 4.6 3.8 2.1 65.5 45.6 Fungal isolates + + + + + + 2.9 1.2 41.3 2.2 0.9 40.9

+ Present, - absent, * hydrocarbon degrader

Some fungi species play very important role in PAH degradations. Aspergillus niger and A. fumigatus have been implicated in metabolizing terpenes and PAHs (Yamazaki et al., 1988). A. niger has also been reported to cleave the rings of naphthalene, anthracene and phenanthrene (Yogambal and Karegoudar, 1997). Walker et al. (1975) compared the abilities of bacteria and fungi to degrade hydrocarbons. In the present study there are more bacteria than fungi. This is corroborated by the findings of Ikhajiagbe (2010). The degradative ability of certain microorganisms to petroleum is governed by environmental conditions and adaptive process

Results of phytoassessment using African yam bean showed significant impact of oil-in-soil on the growth and yield response of the plant (Table 10). Further wetting the oil-polluted soil with NaHCO3 solution improved plant parameters in lower concentrations compared to higher ones. At 2 weeks after sowing, percentage survival of the test plant in the oil-polluted soil was 53.98% in the unwetted soil, compared to 86.32% in the 25mg/l NaHCO3-wetted soil and 67.95% in the 200mg/l NaHCO3-wetted soil respectively. Shoot height at 8 weeks after sowing was 122.52 cm in the unpolluted soil. in the oil-polluted soil,

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Ikhajiagbe and Unuagbokhe Phytoassessment of an Enhanced Naturally Attenuated Oil-Polluted Soil after Exposure to Various Concentrations of Sodium Bicarbonate Solution

however, shoot height decreased from 110.85 cm in 25 mg/l NaHCO3-wetted soil to 69.68 cm in the 200 mg/l NaHCO3-wetted soil, compared to 74.68 cm in the unwetted soil. Number and size of root nodule per plant were reduced in the oil-polluted soils compared to the control. There was an average of 23.08 nodules per plant in the control compared to 12.98 in the unwetted

oil-polluted soil. Wetting the soil with NaHCO3 enhanced nodule formation majorly in the lower concentrations. Grain yield, represented in the present study as dry weight of total harvested grain per plant, was 8.33g in unwetted oil-polluted soil, compared to 29.29g in the control. In the NaHCO3-wetted soil, however, grain yield decreased from 25.87g to 8.99g; the 25mg/l solution impacting the highest yield.
Treatments 50mg/l NaHCO3 4.62a 72.32c 14.32bc 0.32ab 73.65b 87.65b 10.65bc 7.32bc 49.62a 5.32ab 30.65c 20.35a 4.65bc 82.65ab 92.33a 89.65bc 16.52bc 12.52abc 7.69a 15.91b

Table 10: The effects of treatments on some growth parameters of Sphenostylis stenocarpa
Parameters Unpolluted soil 3.54b 92.32a 18.02a 0.45a 88.09a 122.52a 16.05a 9.02a 52.63a 7.02a 53.21a 23.08a 9.35a 78.95b 83.65b 106.52a 21.85a 14.65a 9.15a 29.29a 0 mg/l NaHCO3 5.21a 62.35d 12.01cd 0.24b 53.98c 74.68c 9.85c 6.06c 43.21b 4.98ab 46.85b 12.98c 3.02c 82.65ab 93.35a 82.35c 10.22d 11.20c 7.28a 8.33c 25mg/l NaHCO3 4.21ab 87.62b 16.95ab 0.40a 86.32a 110.85a 13.66ab 8.95ab 50.32a 6.21a 49.65ab 19.95ab 8.35ab 81.29ab 90.62a 92.65b 18.65ab 15.33ab 9.05a 25.87a 100mg/l NaHCO3 4.89a 70.22c 9.25d 0.31ab 68.65b 82.36bc 9.98c 6.51c 50.32a 4.98ab 29.65c 18.32b 3.25c 83.65a 93.32a 90.51bc 12.21cd 10.52c 7.09a 9.11c 200mg/l NaHCO3 5.06a 68.95c 9.65d 0.32ab 67.95b 69.68c 9.52c 5.85c 42.89b 4.02b 28.22c 16.22b 3.98c 84.58a 92.87a 86.54bc 12.65cd 9.87c 7.20a 8.99c

No. of days taken for emergence Percentage emergence (%) @ 1 WAS Height of emergent in 9DAS (cm) Dry wt. of emergents at 9DAS (g) Percentage survival of emergents at 2WAS Shoot height (cm) @ 8WAP No. of primary branches@8WAP Stem width (mm) @8WAP Leaflet area (cm2) @8WAP No. of primary root branches/plant@8WAP Root length (cm) @8WAP No. of root nodules/plant (> 3mm in diameters) Av. Nodule dry wt. (x10-2 g) Days to 50% flowering (DAP) Days to 50% maturity (DAP) No. of flowers/plant No. of pods/plant No.of seeds/pods 100 seed wt (g) Grain yield (g/plant)

Values are means of 5 determinations. Means on the same row with similar alphabets do not differ significantly (p>0.05) from each other. DAP days after planting; WAP - weeks after planting; DAS days after sowing.

4. CONCLUSION The presence of ions in irrigation waters have been found to greatly affect soil pH, which is an important factor in bioremediation. Soils are most likely to become more alkaline. Hard water is widely known to affect agricultural production, hence the need for softening of such hard water by the application of NaHCO3. The present study investigated the effects of NaHCO3 solution in the intrinsic qualities of a naturally attenuated oil-polluted soil and showed that increasing concentrations of bicarbonate solutions in soil decreased bioremediative capacity. However, lower concentrations of 25mg/l relatively enhanced remediation.

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Dr. B. Ikhajiagbe, Environment Mgt Consultant and Lecturer at the Dept of Plant Biology and Biotechnology, University of Benin, Benin City, Nigeria. His area of specialty is Ecophysiology and Bioremediation Technology. He is a member of the Nigeria Environmental Society.

Miss Abibat Unuagbokhe, She is an undergraduate student in research training at the Dept. of Science Laboratory Technology, University of Benin, Benin City.

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