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Jun 5, 2010 - metals and by up to 80% in the heavy metal-treated soil. The control experiments showed a reduction of up to 47% in crude oil concentration, ...
Water Air Soil Pollut (2011) 215:261–271 DOI 10.1007/s11270-010-0476-z

Bioremediation of Co-contamination of Crude Oil and Heavy Metals in Soil by Phytoremediation Using Chromolaena odorata (L) King & H.E. Robinson Harrison Ifeanyichukwu Atagana

Received: 29 November 2009 / Accepted: 12 May 2010 / Published online: 5 June 2010 # Springer Science+Business Media B.V. 2010

Abstract The capability of Chromolaena odorata (L) to grow in the presence of different concentrations of three heavy metals in crude oil-contaminated soil and its capability to remediate the contaminated soil was investigated using pot experiments. C. odorata plants were transplanted into contaminated soil containing 50,000 mg kg−1 crude oil and between 100 and 2,000 mg kg−1 of cadmium, nickel, and zinc and watered weekly with water containing 5% NPK fertilizer for 180 days. C. odorata did not show any growth inhibition in 50,000 mg kg−1 crude oil. Plants in experiments containing 2,000 mg kg−1 Cd showed little adverse effect compared to those in Zn-treated soil. Plants in 1,000 and 2,000 mg kg−1 Ni experiments showed more adverse effects. After 180 days, reduction in heavy metals were: 100 mg kg−1 experiments, Zn (35%), Cd (33%), and Ni (23%); 500 mg kg−1, Zn (37%), Cd (41%), and Ni (25%); 1,000 mg kg−1, Zn (65%), Cd (55%), and Ni (44%); and 2,000 mg kg−1, Zn (63%), Cd (62%), and Ni (47%). The results showed that the plants accumulated more of the Zn than Cd and Ni. Accumulation of Zn and Cd was highest in the 2,000 mg kg−1 experiments and Ni in the 500 mg kg−1 experiments. Crude oil was reduced by 82% in the experiments that did not contain heavy

H. I. Atagana (*) Institute for Science and Technology Education, University of South Africa, P.O. Box 392, Pretoria, South Africa e-mail: [email protected]

metals and by up to 80% in the heavy metal-treated soil. The control experiments showed a reduction of up to 47% in crude oil concentration, which was attributed to microbial action and natural attenuation. These results show that C. odorata (L) has the capability of thriving and phytoaccumulating heavy metals in contaminated soils while facilitating the removal of the contaminant crude oil. It also shows that the plant’s capability to mediate the removal of crude oil in contaminated soil is not significantly affected by the concentrations of metals in the soil. Keywords Crude oil-contaminated soil . Heavy metals . Phytoremediation . Chromolaena odorata

1 Introduction Phytoremediation of soil containing various environmental contaminants have been carried out by many researchers and commercial operators around the world with diverse results (Clombi et al. 2000; Cong et al. 2002; Lasat 2002; Dominguez-Rosado and Pichtel 2004; Tanhan et al. 2007; Muratova et al. 2008). Crude oil contamination has been a major environmental concern for more than two decades with little solution to the problem due to increased dependence on petroleum products around the world. Although, biological processes for remediating crude oil-contaminated soil are slow they have continued to

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be attractive because of low cost and the lack of toxic by-products, which are commonly associated with other treatment types. Co-contamination of crude oil and metals is a common feature in nature. Studies have shown that high levels of metals in oilcontaminated environment impede the rate of oil degradation in the environment (Mattina et al. 2003; AL-Saleh and Obuekwe 2005). Studies of plants capable of remediating soil contaminated with crude oil and metals are not well documented in the literature due to the limited number of studies conducted in this field of research. Chromolaena odorata (L) King & Robinson, an asteraceae (compositae) is an invasive weed of wasteland that has demonstrated capabilities to grow in harsh environments. Earlier studies have shown that C. odorata has the capability to grow in soil contaminated with oil (Anoliefo et al. 2003). It has also been reported to accumulate metals from the soil (Tanhan et al. 2007). The aim of this study is to investigate the capability of C. odorata to phytoremediate soil contaminated with crude oil in the presence of metals. It is also aimed at investigating the ability of the plant to take up the metals used in the experiments.

Water Air Soil Pollut (2011) 215:261–271 Table 1 Characteristics of the soil used for the experiments Sand [% wt]

63.5

Silt [% wt]

18.5

Clay [% wt]

8

Texture

Loamy sand

pH (H2O)

5.9

Total organic carbon

9

Total N [% wt]

0.04

Total P [mg kg−1]

4.5

K [mg kg−1]

21

Ca [mg kg−1]

81

Mg [mg kg−1] Al [meq 100 g

17 −1

soil]

0.1

Total exchangeable cations

4.55 cmol L−1

Exchange acidity

0.20 cmol L−1

stem cutting in a greenhouse. The plants were allowed to grow for 6 weeks before being used in the experiments. The stem cuttings were watered twice a week without the addition of fertilizers. The temperature of the greenhouse was maintained at about 25°C by thermostat controlled fans.

2 Materials and Methods

2.3 Experimental Design

2.1 Soil

Portions of crude oil-contaminated soil (1 kg) amended with different concentrations of metal were then put in PVC pots and one 6-week-old plant of C. odorata was transplanted into each of the soil pots. The experiments were set up in triplicates. The pots were kept in a greenhouse with automatic temperature control set at 25°C. The plants were watered thrice a week with water containing 5% NPK fertilizer. The experiments were monitored for 180 days. Soil samples (100 g) were collected at 30 days intervals for analysis of total petroleum hydrocarbons (TPH) and concentrations of metals. Two set of control experiments were set up in triplicates. One set was without metal contamination but planted with C. odorata and was used to compare the effects of the presence of metals on the removal of crude oil in the experiments containing metals and a second set with metal contamination but not planted with C. odorata and was designed to compare the effects of the plants on the removal of crude oil and metals in the experiments that were planted with the level of removal by other unmeasured sources in the unplanted soil.

Fifty kilograms of soil, of which the characteristics are shown in Table 1 was obtained from a vegetable garden and homogenized with hand and air dried for 24 h before being artificially contaminated with crude oil to give a final concentration of 50,000 mg kg−1 oil obtained from a petroleum refinery in South Africa. The crude oil-contaminated soil was allowed to stand for 6 weeks to allow for partial aging of the contamination and to allow for the adsorption of the oil to the soil particles, as is found in matured contamination before amendment of portions of it with zinc sulfate, cadmium nitrate, and nickel nitrate to give final concentrations of 100, 500, 1,000, and 2,000 mg kg−1 of the respective metals. 2.2 Plants C. odorata plants were obtained from the botanic garden of Department of Botany, University of KwaZulu-Natal, Pietermaritzburg and propagated by

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2.4 Measurement of Total Petroleum Hydrocarbons in Soil Analysis of total petroleum hydrocarbons in the crude oil-contaminated soil used was done using the USEPA 4181.1 (1982) method. Triplicate crude oil-contaminated soil samples (2 g) and 2 g anhydrous Na2SO4 were placed in 30 ml amber glass vial. Carbon tetrachloride (10 ml) was added and the vial was sealed with a teflon-lined screw cap. The sealed vial was vortexed for 15 s and then placed in a sonicating bath (Whaledent Biosonic) for 15 min before remixing on the vortex mixer for about 15 s. It was then placed in the sonicating bath for another 15 min. The solvent was transferred to a clean, dry vial containing 1 g activated FlorosilTM (Sigma) and 0.6 ml water [i.e., 6% water (w/w)]. The sealed vial was shaken for 1 min and allowed to stand overnight at ambient temperature. This silica “clean-up” procedure was used to remove interfering humic materials (EPA 1985). The extract was finally filtered through a Whatman GF/C glass fiber filter. The filtrate was made up to 10 ml in a volumetric flask and the absorbance determined with a Nicolet Avater 320 Infra-red Spectrophotometer at wave numbers between 2,760 and 3,070 cm−1 and an integration value for the absorbance peak area was automatically generated. 2.5 Measurement of TPH in Plant Tissues Whole plants were harvested from the experimental pots after 180 days of treatment and washed with distilled water to rid them of soil materials. The plants were separated into shoots and roots and chopped before homogenizing with a blender. The plants shoots and roots from the different treatments were then separately extracted in carbon tetrachloride. The extracts were then analyzed using IR spectroscopy for TPH. 2.6 Measurement of Concentrations of Metals in Contaminated Soil and Plant Tissues Samples of contaminated soil (10 g each) in triplicate were homogenized by grinding in a mortar and digested in nitric acid (69% HNO3) before analyzing for metals with atomic absorption spectrophotometer. Portions of air-dried samples (10 g) of plants harvested from the experiments were homogenized and digested in nitric acid (69% HNO3) before analyzing for metals with atomic absorption spectrophotometer.

2.7 Chemicals All chemicals and reagents used in the experiments were of analytical grade. 2.8 Statistical Analysis Statistical analysis was done by analysis of variance using three replicates and the level of significant difference was at p=0.05.

3 Results 3.1 Total Petroleum Hydrocarbons in Soil The contaminated soil material contained 50,000 mg kg−1 crude oil at the start of the experiments. Results of the analysis of the soil for total petroleum hydrocarbon showed that the effects of the metals on the reduction in TPH levels in the experiments amended with different concentrations of zinc (Zn), cadmium (Cd), and nickel (Ni) varied with the metals and the concentrations applied. Reduction in TPH in the Znamended experiments was rapid within the first 30 days ( 10 0 m g kg − 1 = 4 6 % , 5 0 0 m g k g − 1 = 40 % , 1,000 mg kg−1 =16%, and 2,000 mg kg−1 =15.3%; Fig. 1a). There was no significant difference in TPH reduction between treatments containing 100 and 500 mg kg−1 Zn. There was also no significant difference in TPH reduction in treatments containing 1,000 and 2,000 mg kg−1 Zn. There were, however, significant differences at p=0.05 between the experiments containing 100 and 500 mg kg−1, and those containing 1,000 and 2,000 mg kg−1. TPH reduction became relatively slower and stable in all treatments until day90 before increasing rapidly in the 500 and 1,000 mg kg−1 treatments on day120 and in all the treatments until the end of the experimentation. The reduction continued to be slow and stable to the end of the experimentation in the control experiment. TPH reduction in the Cd- and Ni-amended soil are shown in Fig. 1b and c. Reduction in both treatments was much slower compared to the Zn-amended soil. TPH reduction in the 100 and 500 mg kg−1 Cd-amended soil are not significantly different from the reduction in similar treatments in the Zn-amended soil. TPH reduction in the Ni-amended treatments was lower in all treatments compared to the Cd and Zn treatments.

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Fig. 1 Changes in TPH levels in metal-amended soil with Chromolaena odorata plants. Values are means of three ±SE

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Fig. 2 Residual TPH after 180 days in unplanted soil containing different concentrations of Zn, Cd, and Ni. Values are means of three ±SE

A comparative analysis of the residual levels of TPH in soil amended with metals but not planted with C. odorata after 180 days of treatment showed that the treatments amended with Ni contained higher amounts of TPH than those amended with Cd and Zn, and those amended with Cd consistently had higher residual TPH than the Zn-amended soil (Fig. 2). The amount of recoverable TPH in the control was significantly lower at p=0.05 compared to all the metal-treated soil. 3.2 Total Petroleum Hydrocarbons in Plant Tissues Measurements of TPH in the plant tissues showed that there was reasonable amounts of the hydrocarbons present in the plant tissues at the end of the experimental period. There was more accumulation of the hydrocarbons in the tissues of the plants grown in soil containing lower concentrations of metals compared to those containing higher concentrations of metals (Fig. 3). The amounts of hydrocarbons accumulated in the plants grown in the experiments containing the three different metals were similar. However, plants grown in the 1,000 and 2,000 mg kg−1 Zn-amended soil showed slightly lower amounts of hydrocarbons in the shoots when compared to those grown in Cd- and Ni-amended soil. There was no significant difference at p=0.05 in the level of metal accumulation in the shoots when compared to the roots.

3.3 Changes in Concentrations of Metals in Soil Results of the analyses of metal concentrations in soil are shown in Fig. 4. At the 100 and 500 mg kg−1 concentrations, changes in metal concentrations were relatively small with Ni showing the least reduction in concentration (100 mg kg−1 =27% and 500 mg kg−1 = 25%) compared to Cd and Zn (33% and 41% and 35% and 37%), respectively, which were relatively similar in the rate of reduction (Fig. 4). Reduction in metal concentrations in the 1,000 and 2,000 mg kg−1 treatments was larger compared to the former treatments. The differences in amount of reduction in these treatments are more evident (e.g., 1,000 mg kg−1 Zn =65%; Cd=55% and Ni=44%) than in the former treatments. 3.4 Concentrations of Metals in Plant Tissues Metal accumulation in the plant tissues was highest in the 1,000 and 2,000 mg kg−1 treatments with the exception of accumulations in the root tissues of 500 mg kg−1 Ni treatment where accumulation was 57 mg kg−1 compared to 8, 25, and 38 mg kg−1 in the 100, 1,000, and 2,000 mg kg−1 treatments, respectively (Fig. 5). Metal accumulation was higher in the Zntreated experiments than in the Cd and Ni treatments generally. Accumulation in the Ni treatments was significantly smaller at p=0.05 compared to those in Cd- and Zn-treated experiments.

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Fig. 3 TPH in plant tissues after 180 days of growth in crude oil-contaminated soil amended with different concentrations of Zn, Cd, and Ni. Values are means of three ±SE

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Fig. 4 Changes in concentrations of metals in crude oil-contaminated soil. Values are means of three ±SE

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Fig. 5 Concentration of metals in plant tissues after 180 days of treatment. Values are means of three ±SE

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4 Discussion The phytoremediation of soil contaminated with 50,000 mg kg−1 crude oil in the presence of different concentrations of Zn, Cd, and Ni by C. odorata in this study is attributed to a number of factors. The rapid reduction in TPH within the first 30 days of experimentation can be attributed to a number of activities. Although not measured, volatilization of lighter fractions of crude oil have been reported to contribute to such phenomena. Microbial actions, which were also not measured, have been reported in previous studies to contribute to reduction in crude oil in soil. Both these action constitute part of the natural attenuation phenomenon (Margesin and Schinner 2001; Pichtel and Liskanen 2001; Bento et al. 2005; Chaineau et al. 2005; Sarkar et al. 2005; Scow and Hicks 2005). The variation in the level of removal of TPH in the different treatments is therefore believed to be associated with microbial actions in the soil, most probably actions of the rhizosphere organisms, which were impeded to different extents by the different metal concentrations applied to the soil. The removal of compounds below C14 by volatilization during remediation studies within 21 days have been reported in the literature (Pichtel and Liskanen 2001). The slow rate of removal after day 30 is an indication that the volatile components had been removed and that the remaining components of the crude oil required intervention to be removed from the soil. A comparison of the TPH removal (80%) in the metal-amended treatments planted with C. odorata and those without plants (47%) clearly showed that phytoremediation by the plants was responsible for the removal of more than 30% of the oil (Figs. 1 and 2). TPH removal in the planted experiments was significantly higher than those in the unplanted experiments. C. odorata is known to adapt to very harsh environments, including disturbed environments and those with low moisture and poor nutrients. The plant has been reported to grow in oil-contaminated soil (Anoliefo et al. 2003; Atagana 2008). In this study, C. odorata grew well in the contaminated soil and extracted components of crude oil from the soil. Phytoremediation of hydrocarbon-contaminated environment has been demonstrated by a number of researchers using different plant species (Hutchinson et al. 2001;

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Dominguez-Rosado and Pichtel 2004; DominguezRosado et al. 2004; Kaimi et al. 2006; White et al. 2006; Muratova, et al. 2008). C. odorata’s rapid growth and high transpiration rates are characteristics that enhance the translocation of materials in solutions in the environment. In this study, the C. odorata tolerated the presence of different concentrations of Zn, Cd, and Ni in the contaminated soil. The higher concentrations of the metals inhibited oil removal in the early stages of the experiments but the inhibitory effect started to wane with time and the plants continued to thrive until the end of the experimentation while oil removal improved in the last quarter of the experimentation. C. odorata had been reported to grow in soil contaminated with metals (Tanhan et al. 2007). It is believed that the slight inhibition in growth caused by Cd and Ni, which resulted in the slow removal of oil in the soil containing these metals accounts for the higher amounts of oil in the tissues of the plants grown in these experiments. It can be argued that the metal affected the physiological processes responsible for movement of solutions in the plants such as imbibitions and transpiration, resulting in slow movement of water and solutes in the plant. This in turn allowed for larger accumulations of oil in the plant tissues (Vassil et al. 1998; van der Vliet et al. 2007; Shi et al. 2008). The high rate of metal removal from the soil in the treatments with higher concentrations of metals can be attributed to high concentration of the metals in the soil. This high rate of removal of metal ions may have interfered with the removal of oil in these experiments, as it was noted earlier that soil with higher concentration metals showed a lower oil removal. The higher accumulation of metals in the tissues of the plant in the 1,000 and 2,000 mg kg−1 treatments, further explains the slow oil removal in these treatments. Different plant species, including C. odorata have been reported to grow in metal-contaminated soil and bioaccumulate metals from the soil (Robinson et al. 1997; Cong et al. 2002; Lasat 2002; Tanhan et al. 2007), many of such reports were at relatively low concentrations. Few studies, however, have been able to demonstrate the ability of plants to remove high concentrations of metals, such as applied in this study. There is also a paucity of information on the removal of oil contaminants from the soil by plants in the presence of high concen-

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trations of metals, such as applied in this study. Phytoremediation of co-contamination of metals and hydrocarbons was reported by Palmroth et al. (2006). In their study, the plants did not accumulate the metals but hydrocarbons were degraded in fertilized soil. In the present study, Chromolaena removed oil and metal simultaneously, however, the metal was removed at a much lower rate compared to the oil. Although these experiments were conducted under greenhouse conditions, the profuse growth of the plants and the potential to continue to grow in the presence of the contaminant oil and metal suggests that the plants would survive a pilot trial outside the greenhouse. Earlier studies using the same plant species showed substantial removal of used engine oil from contaminated soil without the application of fertilizers (Atagana 2008).

5 Conclusions C. odorata (L) King & Robinson was able to grow in soil contaminated with 50,000 mg kg−1 crude oil in the presence of between 100 and 2,000 mg kg−1 Zn, Cd, and Ni. The growth of the plant was sustained for 180 days during which period the plant was able to cause the removal of both the contaminant oil by 82% and the present heavy metals by up to 65%. The ability of the plant to survive such high concentrations of crude oil and metals is an indication that it is a possible candidate for phytoremediation of soil contaminated with either crude oil or metals, or a co-contamination of both pollutants. Acknowledgment I wish to acknowledge the South Africa National Research Foundation (NRF) for providing the funds for this research. Dr. Pat Caldwell and Dr. Kwesi Yobo of the Plant Pathology Discipline, University of KwaZulu-Natal, Pietermaritzburg, South Africa for allowing the use of their tunnels and for providing technical assistance.

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