Evaluating the phytoremediation potential of ... - Springer Link
Environ Sci Pollut Res (2014) 21:1304–1313 DOI 10.1007/s11356-013-1997-y
RESEARCH ARTICLE
Evaluating the phytoremediation potential of Phragmites australis grown in pentachlorophenol and cadmium co-contaminated soils Nejla Hechmi & Nadhira Ben Aissa & Hassen Abdenaceur & Naceur Jedidi
Received: 4 February 2013 / Accepted: 10 July 2013 / Published online: 31 July 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Pot-culture experiments were conducted to evaluate the phytoremediation potential of a wetland plant species, Phragmites australis in cadmium (Cd) and pentachlorophenol (PCP) co-contaminated soil under glasshouse conditions for 70 days. The treatments included Cd (0, 5 and 50 mg kg−1) without or with PCP (50 and 250 mg kg−1). The results showed that growth of P. australis was significantly influenced by interaction of Cd and PCP, decreasing with either Cd or PCP additions. Plant biomass was inhibited and reduced by the rate of 89 and 92 % in the low and high Cd treatments and by 20 and 40 % in the low and high PCP treatments compared to the control. The mixture of low Cd and low PCP lessened Cd toxicity to plants, resulting in improved plant growth (by 144 %). Under the joint stress of the two contaminants, the ability of Cd uptake and translocation by P. australis was weak, and the BF and TF values were inferior to 1.0. A low proportion of the metal is found aboveground in comparison to roots, indicating a restriction on transport upwards and an excluding effect on Cd uptake. Thus, P. australis cannot be useful for phytoextraction. The removal rate of PCP increased significantly (70 %) in planted
Responsible editor: Philippe Garrigues N. Hechmi : H. Abdenaceur : N. Jedidi Laboratory of Wastewater Treatment, Water Research and Technologies Centre (CERTE), Technopole of Borj Cedria, BP 273, Soliman 8020, Tunisia N. Hechmi (*) : N. B. Aissa National Agronomic Institute of Tunisia, 43 Avenue Charles Nicolle, Mahrajene 1082, Tunisia e-mail: [email protected]
soil. Significant positive correlations were found between the DHA and the removal of PCP in planted soils which implied that plant root exudates promote the rhizosphere microorganisms and enzyme activity, thereby improving biodegradation of PCP. Based on results, P. australis cannot be effective for phytoremediation of soil co-contaminated with Cd and PCP. Further, high levels of pollutant hamper and eventually inhibit plant growth. Therefore, developing supplementary methods (e.g. exploring the partnership of plant–microbe) for either enhancing (phytoextraction) or reducing the bioavailability of contaminants in the rhizosphere (phytostabilization) as well as plant growth promoting could significantly improve the process of phytoremediation in co-contaminated soil. Keywords Phytoremediation . Wetland plant . Phragmites australis . Co-contamination . Pentachlorophenol . Cadmium
Introduction Soils have been submitted to several contaminants that vary in concentration and composition. Among all, organic chemicals such as persistent organic pollutants and heavy metals are recognized as two major chemical families that cause water and soil pollution (Sun et al. 2011). In many parts of the world, agricultural soil is slightly or moderately contaminated by cadmium (Cd) (Li et al. 2010). Cd is a heavy metal of great concern in agricultural ecosystems because of its high toxicity to animals and human health
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(Huang et al. 2011). Pentachlorophenol (PCP) has been released into the environment as a wood preservative, pesticide, herbicide and antiseptic. This compound is a major soil pollutant due to its toxicity and recalcitrance (Miller and Dyer 2002), and it is regulated as one of the priority pollutants by the U.S. Environmental Protection Agency. Cocontamination of PCP and Cd often occurs in vegetable fields due to the conventional use of pesticides (Lin et al. 2007) as well as increased application of sewage sludge and wastewater irrigation (Li et al. 2010). Hence, it is critical to develop efficient and cost-effective approaches to simultaneously remove multiple contaminants from co-contaminated soils. Phytoremediation is the use of plants to remediate polluted soils, an eco-friendly and cost-effective technology that is currently receiving considerable global attention (Glick 2010). A large number of plant species are capable of hyperaccumulating heavy metals and organics in their tissues; however, phytoremediation in practice has several constraints at the level of sites as these are with a variety of different contaminants (Wu et al. 2006). Further, the success of phytoremediation depends upon a plant's capacity to tolerate and to accumulate high concentrations of the pollutant, while yielding a large plant biomass (Grčman et al. 2001). The mechanisms of phytoremediation systems comprise at least four pathways to reduce soil contaminants, such as abiotic losses (leachate, volatilization, photodegradation, irreversible sorption, chemical degradation and so on), indigenous microbial degradation, root tissues-enhanced dissipation and rootexudates-enhanced biodegradation (Sun et al. 2010). Up to now, there are numerous promising results indicating that the technique might become viable alternative to mechanical and chemical approaches in decontamination of metal polluted sites or a final polishing solution for organic contamination. However, little information was still available regarding the effectiveness and processes of phytoremediation of sites cocontaminated with organic and metal pollutants which have become more prevalent (Chigbo et al. 2013). The efficiency of phytoremediation of organic pollutants co-existing with heavy metals is complex and quite different from that in the singlepollutant system because different compounds may interact among themselves and/or with plants and their rhizosphere biota (Almeida et al. 2008). For instance, interaction of metals and organics with respect to degradation of organics and/or metal uptake can be negative or positive depending on type and concentration of both metals and organic pollutant (Zhang et al. 2012b). In a study of Cd and polycyclic aromatic hydrocarbons (PAHs) (phenanthrene (PHE) or pyrene (PYR)) remediation in co-contaminated soil planted with hyperaccumulator plant Sedum alfredii, removal rate of PYR decreased at the elevated Cd level (6.38 mg kg−1) in the soil. S. alfredii could effectively extract Cd from Cd-contaminated soils in the presence of PHE or PYR (Wang et al. 2012). Furthermore, planting of S. alfredii is an effective technique
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for phytoextraction of Cd and dichlorodiphenyltrichloroethane (DDT) in Cd–DDT co-contaminated soil with low Cd (0.895 mg kg−1) and high Cd ( 3.225 mg kg−1) concentrations, with 0.715 mg kg−1 DDT (Zhu et al. 2012). This uptake capability equaled that of the pot-grown crop Ricinus communis planted in spiked soils with DDT and Cd at the rate of 1.7 and 2.8 mg kg−1 soil. The total uptake of DDT and Cd varied from 83.1 to 267.8 and 66.0 to 155.1 μg pot−1, which indicates that R. communis (castor) has great potential for removing DDT and Cd from co-contaminated soils attributed to its strong absorption and accumulation for both DDT and Cd (Huang et al. 2011). The uptake and translocation of metals in various wetland plants have been studied (Liu et al. 2010; Zhang et al. 2010a), but few studies have focused on the uptake, translocation and/or metabolism of organic pollutants (e.g. PCP) in wetland species (Roy and Hänninen 1994) especially under co-contaminated conditions. However, the possible stimulatory, antagonistic (less than additive toxicity) and competitive uptake and metabolism of the combined PCP and Cd received little exploration (Hechmi et al. 2013). The growth and pollutant removal by emergent wetland plants may be influenced by Cd and PCP interaction. The knowledge about the pressure of these interactions between co-contaminants on the phytoremediation potential of wetland plants is relatively poor. Emergent aquatic macrophytes represent a diverse group of plants with an immense potential for removal/degradation of variety of contaminants (Dhir et al. 2009). Besides, macrophytes are more suitable for wastewater treatment than terrestrial plants due to their faster growth, production of more biomass and relative higher ability of pollutant uptake (Ali et al. 2013). Wetland plants can enhance metal removal and/or stabilization (Marchand et al. 2010) and may also relieve organic pollutant biodegradation (a) directly in the rhizosphere by the release of root exudates (Toyama et al. 2011) and (b) indirectly by improving soil biology via build up of organic carbon (Pilson-Smits 2005). The emergent wetland species such as Phragmites australis (Poaceae family) had numerous characteristics (e.g. cosmopolitan distribution, easy growth, inexpensive…) that make it a favoured test species. Previous studies reported that P. australis is one of the best plant organisms for detecting heavy metals and harmful compounds such as herbicides (Bonanno 2011). Furthermore, plant screening for phytoremediation of uranium, thorium, barium, nickel, strontium and lead contaminated soils showed that P. australis had the greatest removal capabilities for uranium (820 μg), thorium (103 μg) and lead (1,870 μg) (Li et al. (2011). Hence, effectiveness of P. australis in heavy metal filtration has prompted us to undertake this study. The main aims for the current investigation were (1) to examine the growing response of P. australis to single contamination of PCP and co-contamination of Cd–PCP, (2) to evaluate the plant uptake, accumulation, translocation and
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Material and methods
into shoots, roots and rhizomes. After drying with filter paper, the subsamples were freeze-dried and weighed to determine the biomass. The soil samples from the pots were separated into the rhizosphere and non-rhizosphere soils. Both types of soil samples were air-dried at room temperature (approximately 20–23 °C) and ground sufficiently to pass through a 100 mesh sieve.
Soil characterization
Extraction and analysis of PCP in soils and plants
The tested soil without detectable PCP and Cd was collected from the top layer (0–20 cm) of an agricultural field in NE of Tunisia. The soil was sandy loam containing 60 % sand, 30 % silt and 10 % clay. Selected soil properties were as follows: pH 6.4(1:2 soil/water), EC 0.012 dSm−1, total organic carbon 3.02 g kg−1, total nitrogen 0.22 g kg−1 and total phosphorus 0.12 g kg−1 . The soil was air-dried and sieved through a 2-mm sieve prior to spike with PCP and Cd.
PCP is extracted according to a slightly modified method previously described by Sharma et al. (2009). Soil (2 g) was ultrasonically extracted (45 kHz, 300 W) in 5 ml of methanol for 30 min followed by 4,000 rpm centrifugation for 10 min. As for plant tissues analysis, about 0.5 g of dried plant fragment was weighed into a 25-ml centrifuge tube and extracted by ultrasonication for 30 min with a 10-ml solution of methanol for three successive extractions. The extraction was centrifuged at 4,000 rpm for 5 min to separate the supernatant from the soil or plant. The solvent was removed gently by blowing under a stream of N2. The residue was derivatized in 400 μl of ethyl acetate and analyzed immediately on a gas chromatography–mass spectrometry (GC– MS). The GC–MS analyses were performed in electron ionization (EI) mode (70 eV) with an Agilent 6,890 N gas chromatograph, equipped with 5973 MSD (Agilent Technologies, Palo Alto, CA, USA). An HP-5MS (Agilent, USA) column was used at a temperature programme of 45 °C for 1.5 min, increased to 100 °C at 10 °C/min, increased to 180 °C at 4 °C/min and finally increased to 300 °C at 40 °C/min and held at 290 °C for 5 min. Helium was used as the carrier gas at a constant flow of 1.2 ml/min. The samples were analyzed in split mode (1:10) at an injection temperature of 250 °C, an EI source temperature of 230 °C and a quadrupole analyzer temperature of 150 °C, unit mass resolution, scan range m/z 35–500, with a scan cycle of three scans. The injected volume was 1 μl. The recovery rate of PCP was 89 %.
dissipation behaviours of Cd and PCP and (3) the interactive effects of Cd, PCP and plant on soil dehydrogenase activity (DHA) which is good indicators of soil microbial activity.
Experimental procedure Experiments were set up by artificially co-contaminating the soil sample with PCP and Cd. PCP>97 % purity (Sigma– Aldrich) is dissolved in acetone and added to 25 % by weight of the required amount of soil at concentrations of 0, 50 or 250 mg kg−1 (P0, P1 and P2) PCP. Cd (as CdCl2 ×21/2H2O, analytical grade, AJAX Chemicals) was dissolved in Milli-Q water and added to the PCP spiked soils at concentrations of 0, 5 or 50 mg kg−1 (C0, C1 and C2), resulting in a total of nine treatments including C0P0 (T1), C0P1(T2), C0P2(T3), C1P0 (T4), C1P1(T5), C1P2(T6), C2P0 (T7), C2P1(T8) and C2P2(T9). A modified Hoagland nutrient solution contained (mg kg−1 soil) N 30, P 20, K 89, S 30, Ca 40, Cl 73, Mg 4, Mn 3.26, Zn 2, Cu 0.50, B 0.11, Co 0.10 and Mo 0.08 was added to all treatments at the same rates and was mixed uniformly to the soils. The modified Hoagland nutrient solution was prepared as described by Dordio et al. (2009). The spiked soils were then sieved again through a 2-mm sieve to ensure homogeneity and stored for analysis. A cylindrical water-permeable polyethylene rhizobags (7.5 cm in diameter and 13 cm in length) were used to separate rhizosphere from non-rhizosphere soil. Six hundred fifty grams of soil was added to each rhizobag, and the space outside the bag was filled with 350 g of the same soil. In this system, the surfaces of the soil inside and outside the rhizobag were in the same level. The seedlings of P.australis (Cav.) were collected from an uncontaminated field site in NE of Tunisia and transplanted (with initial plant fresh weight 5.0±0.5 g per pot) into each rhizobag. All treatments were irrigated with the running water every day, and the depth of water was kept at 2– 3 cm high at the surface of the soil. The exposure experiment was carried out in a glasshouse with controlled temperature of 30–35/20–25 °C (day/night) under natural light conditions from early July to mid-September 2010). The plants were harvested after 70 days and washed with deionized water and separated
Cd content determination The soil samples and plant were digested with a solution of 3:1 HNO3:HClO4 (v/v). The concentrations of Cd were determined using the atomic absorption spectrophotometry. DHA Soil DHA was measured by the reduction of 2,3,5-triphenyl tetrazolium chloride (TTC) to 1,3,5-triphenyl formazan (TPF) using a modification of the methods of (Mills et al. 2006). Five gram soil samples, collected from each pot, were individually placed in 30 ml plastic vials with 0.1 g of CaCO3 and 3 ml of TTC solution (5 g L−1 in 0.2 M Tris–HCl buffer, pH 7.4) and were incubated for 24 h at 37 °C. After incubation, 20 ml of
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methanol was added, the sample shaken and then filtered through Whatman 41 filter paper to extract the TPF. The absorbance of the filtered solutions was measured at 485 nm using a spectrophotometer (Beckman DU 640).
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Total pollutant accumulation ¼ ðpollutant concentration in shoots shoot DWÞ þ ðpollutant concentration in roots root DWÞ
ð1Þ
Data analysis Total accumulation of Cd or PCP in plants, expressed as microgram pot−1 and was calculated as:
The percentage removal of Cd or PCP by plants was calculated as:
Removal of Cd or PCP by plantsð%Þ . ¼ total Cd or PCP accumulation in plants total added Cd or total measured PCP in soil 100
ð2Þ
Statistical analyses were carried out using SPSS13.0. A one-way analysis of variance followed by a Tukey's multiple comparison test (at p ≤0.05) was employed.
high PCP treatments compared to the control. Compared to the low Cd treatment alone, the total plant biomass was significantly (p ≤0.05) increased (by 144 %) in the mixture of low Cd and low PCP.
Results
Cd uptake in P. australis under joint stress of Cd and PCP
Plant growth
The amount and distribution of Cd absorption in the tissues of P. australis are shown in Table 1. The concentrations of Cd increased with Cd additions and decreased with PCP additions. The total Cd accumulation in plant tissues increased by 165 % in the mixture of low Cd and low PCP compared to the low Cd treatment alone. Although the accumulation of Cd decreased by 22 and 14 %, respectively, no significant difference was observed in the mixtures of either low or high Cd or high PCP
Figure 1 shows the dry matter yield of P. australis under different soil PCP and Cd rates. The growth of P. australis was significantly (p ≤0.05) influenced by Cd, PCP and their interaction. The influence of Cd on biomass was much stronger compared to PCP revealed that the occurrence of Cd had prohibitive effects on the growth of P. australis. Plant biomass was inhibited by the rate of 89 and 92 % in the low and high Cd treatments and by 20 and 40 % in the low and
Table 1 The concentrations of Cd in plant tissues, BF and TFs under combined contamination of Cd and PCP after 70 days of plant growth Treatment
T4 T5 T6 T7 T8 T9
Fig. 1 Plant biomass influenced by Cd and PCP treatments after 70 days of growth. Bars (means ± SE, n =3). Treatment: C0P0 (T1), C0 P1(T2), C0P2(T3), C1P0 (T4), C1P1(T5), C1P2(T6), C2 P0 (T7), C2P1(T8) and C2P2(T9). C0, C1 and C2 represent 0, 5 and 50 mg Cd kg−1; P0, P1 and P2 represent 0, 50 and 250 mg PCP kg−1
Concentration of Cd (mg kg−1) Root
Rhizomes
Shoot
456±61d 436±105d 135±54e 1,278±18a 1,099±81b 824±25c
287±14e 386±39e 231±0.04b 1,109±154a 857±152e 611±88e
114±21c 89±12c 70±2c 545±137a 363±25b 280±41b
BF
TF
0. 43 0.77 0.63 0.56 0.43 0.13
0.31 0.20 0.52 0.42 0.34 0.33
Treatment: C1P0 (T4), C1P1(T5), C1P2(T6), C2 P0 (T7), C2P1(T8) and C2P2(T9). C0 and C2 represent 0, 5 and 50 mg Cd kg−1 ; P1, P1 and P2 represent 0, 50 and 250 mg PCP kg−1 . Means (±SE, n =3) followed by the same lowercase letter within columns are not significantly different according to LSD (p ≤0.05). Bioaccumulation factor (BF) is defined as the ratio of the metal concentration in shoots to that in the soil, and transfer factor (TF) is defined as the ratio of the metal concentration in shoots to that in roots
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in plants was significantly (p ≤0.05) influenced by Cd additions (Table 2).
PCP removal from soil
Fig. 2 Cd accumulation in plant tissues and percentage of Cd removal from soils under stress of Cd and PCP after 70 days of growth. Bars, means ± SE, n =3 with different letters are significantly different based on LSD (p ≤0.05). Treatment: C0P0 (T1), C0P1(T2), C0P2(T3), C1P0 (T4), C1P1(T5), C1P2(T6), C2 P0 (T7), C2P1(T8) and C2P2(T9). C0, C1 and C2 represent 0, 5 and 50 mg Cd kg−1; P0 P1 and P2 represent 0, 50 and 250 mg PCP kg−1
compared to either low or high Cd treatment alone (Fig. 2). Cd accumulation in different plant tissues was in the sequence root > rhizome > shoot among the treatments with added Cd. The highest percent Cd removal from soils by plants was in the mixture of low Cd and low PCP, which increased significantly (p ≤0.05) (2.2-fold) compared to the low Cd treatment alone. PCP concentration and partitioning in P. australis The concentration of PCP in the tested plants could strikingly increase with an increase in the content of PCP in soil. The highest amount of PCP concentrated in roots was 3.10–7.6 times higher than those in shoots. The concentration of PCP
The percentage of PCP removal from soils was significantly influenced by the interaction of Cd, PCP and planting/nonplanting treatments (Fig. 3). The percentage of PCP removal was significantly higher (p ≤0.05) in the low than high PCP treatments. The removal of PCP decreased with Cd additions, which was more obvious in the non-planted treatments. Compared to the non-planted treatments, PCP removal significantly increased (p ≤0.05) by 16, 17 and 19 %, respectively, in the planted mixtures of low Cd and either low or high PCP and mixture of high Cd and low PCP.
Desydrogenase activity in soil The DHA in unspiked, and unplanted soil was taken as the control value of 100 %. As shown in Fig. 4a, DHA in unplanted soils co-contaminated by Cd and PCP was significantly lower (p ≤0.05) than the control value, decreasing to an average of 64 % of the control value. It tended to decline along with increase in soil PCP and Cd concentration in soil. It was observed that a low concentration of PCP alone contamination can increase DHA slightly. However, with an increase in the Cd level, DHA decreased to 64 % of the control value in 5 mg kg−1 Cd treatment and 55 % of the control value in 50 mg kg−1 Cd treatment, respectively. DHA in planted soils was increased significantly (p ≤0.05) by 11 % than in unplanted soil.
Table 2 Concentrations of PCP in plant tissues, BF and the root-toshoot TFs influenced by Cd treatments after 70 days of plant growth Treatment
T2 T3 T5 T6 T8 T9
Concentration of PCP (mg kg−1) Root
Rhizomes
Shoot
40±2.61d* 45±1.75d 47±0.54e 48±1.28a 39±0.61b 37±2.05b
2.6±14a 4.06±39e 3.11±0.11d 1.2±0.15a 6.5±0.2b 6.11±0.3d
7.2±0.11a 11±0.11c 10±0.2c 21±0.13b 6.9±0.15b 6±0.31b
BF
TF
0.36 0.44 0.56 0.63 0.76 0.72
0.18 0.24 0.22 0.43 0.18 0.16
Treatment: C0 P1(T2), C0P2(T3), C1P1(T5), C1P2(T6), C2P1(T8) and C2P2(T9). C0 and C2 represent 0, 5 and 50 mg Cd kg−1 ; P1, P1 and P2 represent 0, 50 and 250 mg PCP kg−1 . Bioaccumulation factor (BF) is defined as the ratio of the metal concentration in shoots to that in the soil, and transfer factor (TF) is defined as the ratio of the metal concentration in shoots to that in roots *p ≤0.05 (means (±SE, n =3) followed by the same lowercase letter within columns are not significantly different according to LSD)
Fig. 3 Percentage of PCP removal from soils. Bars, means ± SE, n =3. Treatment: C0P0 (T1), C0P1(T2), C0P2(T3), C1P0 (T4), C1P1(T5), C1P2(T6), C2 P0 (T7), C2P1(T8) and C2P2(T9). C0, C1 and C2 represent 0, 5 and 50 mg Cd kg−1; P0, P1 and P2 represent 0, 50 and 250 mg PCP kg−1
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Fig. 4 Changes in dehydrogenase activity in unplanted and planted soils with different treatments (a). Correlation between the dehydrogenase activity and removal ratio of pentachlorophenol in unplanted and planted soils (b). Bars, means ± SE, n =3 (p ≤0.05). Treatments: C0P0
(T1), C0P1(T2), C0P2(T3), C1P0 (T4), C1P1(T5), C1P2(T6), C2P0 (T7), C2P1(T8) and C2P2(T9). C0, C1 and C2 represent 0, 5 and 50 mg Cd kg−1; P0, P1 and P2 represent 0, 50 and 250 mg PCP kg−1
Discussion
high treatment). In the same way, Zhang et al. (2012a) revealed that the growth of Scirpus triqueter tends to decrease (including height, diameter, shoot number and biomass) in the relatively high concentration of 80 and 160 mg kg−1 PYR. These results were different to those of Hechmi et al. (2013), who reported that with the initial concentration of 50 mg kg−1 PCP, no outward sign of phytotoxicity or even better growth for the crop Z. mays was shown with the increment of soil PCP level. An interactive effect of Cd and PCP on plant growth was observed in the present study. In the mixture of low Cd and low PCP, plants grew better than in the low Cd treatment alone (Fig. 1), implying that PCP could at least partly alleviate Cd toxicity effects. The available study investigating the combined toxicity of metals and organics have indicated that toxicity may be independent, additive, synergistic (greater than additive toxicity), or antagonistic (less than additive toxicity) (Zhang et al. 2012b). For instance, PCP present in a 50 mg kg−1 alleviated also Cu toxicity to ryegrass (Lolium perenne) and radish (Raphanus sativus), while high concentrations intensify that toxicity (Lin et al. 2006). Similarly, Hechmi et al. (2013) reported that in Cd co-contaminated soil with the initial PCP concentration of 50 and 100 mg kg−1, Z. mays grew better with the increment of soil Cd level (0, 2 and 6 mg kg−1), implying that combinations of Cd and PCP exerted antagonistic toxic effects on plant growth. Besides, Lin et al. (2008) observed an increase in shoot yield of Z. mays with copper and PYR co-contamination, whereas our results showed that the yield of both root and shoot of P. australis decreased significantly (p ≤0.05) with the increase of soil PCP and Cd concentrations. This suggests a synergistic effect of metals and organics in co-contaminated soil and is supported by Zhang et al. (2009) which showed that PYR did not alleviate the toxicity Cd to Z. mays. These diverse results suggest that
Interactive effects of Cd and PCP on plant growth Several studies have illustrated symptoms and responses of wetland plants upon toxic metal exposure (Bonanno and Giudice 2010). The inhibiting effects of Cd on biomass accumulation, height, root length and other biometric parameters of plants were reported in most investigations. For instance, Zhang et al. (2010a) observed an adverse effect of Cd on the relative growth rates of four wetland species. The relative growth rates of the three species (Baumea articulata, Schoenoplectus validus and Juncus subsecundus) changed little in various Cd treatments, but was severely inhibited for Baumea juncea at 20 mg l−1of Cd, whereas an increase of growth for S. validus was observed at 5 mg l−1. In the present study, dry weight of P. australis was significantly reduced by Cd additions after 70 days of growth in soils (Fig. 1). The effects of Cd toxicity have been studied for other plants. Hechmi et al. (2013) reported nonsignificant inhibitory effects of Cd (up to 6.00 mg kg−1) on shoot growth of Zea mays. However, significant growth enhancement of S. alfredii was observed at level 6 mg kg−1 of Cd (Wang et al. 2012). This different response to Cd in the degree of expressed phytotoxicity could be due to various Cd levels as well as to different plant species and its tolerance. The effect of organic pollutants on wetland plant growth could be species specific regardless of pollutant types and media (Zhang et al. 2010b). For instance, exposure to low doses of PCP can stimulate growth of Phragmites communis Trin, Typha orientalis Pres and Scirpus validus Vahl, but high doses of PCP (>2 μg kg−1 dry weight) hamper and eventually inhibit plant growth (Zhao et al. 2011). In the present study, biomass of P. australis was significantly reduced (p ≤0.05) by PCP additions (40 % in PCP
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growth response of plants to joint toxicity of metal and organic pollutants are dependent on certain factors including plant species, age, type and concentration of contaminants (Zhang et al. 2012b; Chigbo et al. 2013). Cd/ PCP uptake and partitioning in P. australis Our results showed that whether soil Cd was present in single or mixed contamination, higher Cd concentrations were always observed in the root. The Cd removal efficiency by P. australis was both relative to biomass production and tissue concentration. The observed increase in total Cd accumulation with increasing soil Cd concentration (Table. 1) could be as a result of increased plant biomass. The ability of plants to tolerate and accumulate heavy metals is useful for phytoextraction and phytostabilization purpose measured using translocation factor (TF) and bioconcentration factor (BF). BF and TF are usually >1 (or≫1) in hyperaccumulators and leaf > stem > root and all the parameters (BF and TF reached 3.28 and 1.51, respectively) were very low (Table 1). Hence, P. australis was inappropriate for Cd phytoextraction. An added experiments carried out by Zhang et al. (2012b) showed an order of Cd accumulation shoot > root > rhizome among the treatments with added Cd (5, 10 and 20 mg kg−1) and TF between 144 and 307 %, suggesting the phytoextraction potential of wetland plant J. subsecundus. This inconsistency may be due to differences in the levels, durations of exposure to Cd and the different growth media as well as plant species which can differ in rates of metal uptake, allocation and excretion in metal dynamics. Plant uptake of hydrophobic organics, such as PCP, could be described as partitioning between the soil aqueous solution and plant roots, with translocation from root to shoot being highly restricted (Gao et al. 2011). Obviously, PCP uptake is not a significant pathway of dissipation for PCP in this experiment. PCP was undetectable in the aboveground part of the P. australis and BF and TF values were 0.36–0.76 and 0.16–0.34, respectively (Table 2). Therefore, the mechanisms of dissipation evaluated were biodegradation and incorporation into soil. In the present investigation, P. australis plants were able to accumulate xenobiotics such as PCP only in their root which corroborate with the previous research on aquatic macrophyte and rice demonstrating that plant could remediate the PCP-contaminated soil by uptake PCP into root tissue. The maximum BF of PCP in the roots of
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the macrophyte P. communis Trin was 0.71 at sediment PCP concentration of 2 μg mg−1 (Zhao et al. 2011). The root fraction of the rice treatment had the highest PCP concentration, but accounted for only 0.015 μg of the added PCP of 75 μg (Terry and Elisa 2005). The main reason of this substantiation might be the physicochemical characteristics (the octanol–water partition coefficient K ow) of PCP. Since the log Kow of PCP was 5.01, which might make it difficult to transport from root tissue to upper part tissue (Schröder et al. 2008). Since uptake of PCP in shoots was negligible, PCP may be catabolized by plant enzymes either mineralized completely to inorganic compounds (e.g. CO2, H2O) or degraded partially to a stable intermediate that is stored in the plants (Pilson-Smits 2005). There is a trend that antagonistic and synergistic responses of the plant reflected bioaccumulation patterns in the cocontamination soil. For instance, Sun et al. (2011) reported that under the co-contamination of heavy metals and PAH, the accumulation of Cd in shoots of T. patula was favourably influenced by the presence of PAH in soil. Nevertheless, adding PAH with two levels (25 and 150 mg kg−1) of PHE or PYR could restrain Cd translocation in S. alfredii (Wang et al. 2012). On the other hand, in the present investigation, whether soil Cd was present in single or mixed contamination, higher Cd concentrations were always observed in the root and PCP addition increased significantly (p ≤0.05) the uptake of Cd by the roots of P. australis. This could be as a result of the influence on plant growth as observed in the present study where co-contamination reduced shoot dry weight significantly (p ≤0.05). Chigbo et al. (2013) observed that PYR significantly affected Cu uptake as a result of reduction in growth of B. juncea. As a potential remediation plant, the basic characteristic is high tolerant capability and exhibits a variety of responses to mechanical stresses that enable them to tolerate and evolve resistance to adverse conditions that are toxic to most other plants (Sun et al. 2008). Based on the results in this investigation and in the previous one, we can point out that under the coexistence of Cd and PCP in contaminated soil, the interaction between metal and organic pollutant could influence plant growth, metal uptake and accumulation by plants. The phytoextraction of Cd by P. australis is very restricted, and it could be that PCP altered the way P. australis influenced Cd sorption and solubility. Z. mays unlikely has higher capability to phytoextract Cd and could be useful for remediation of Cd even at co-contamination of PCP–Cd, but the ability of Cd phytoextraction would be inhibited under co-contamination of high level of PCP (PCP>100 mg kg−1 DW) in highly Cd polluted soil (Cd>6 mg kg−1 DW) (Hechmi et al. 2013). Z. mays as well can normally grow in the co-contaminated soil with high level Cd and PCP and can effectively remedy sites co-contaminated with these two types of pollutant.
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PCP removal PCP removal in planted soil was greater than in no planted soil except in co-contaminated soils which showed similar rates of removal (Fig. 3). The removal rate of PCP in soils excelled 70 % for all the planted soils after 70 days of remediation, which was far more than that in the control. Likewise, Zhao et al. (2011) reported that the removal rates of PCP in the sediments planted with three macrophytes P. communis Trin, T. orientalis Pres and S.validus Vahl were 90.35, 99.23 and 99.33 %, respectively, while the rate in the control sediment was only 29.87 %. The removal rates are also more than that in previous reports of PCP phytoremediation using terrestrial , such as Chinese chive (40 %, 28 days) and rice (63 %, 82 days) (Takashi et al. 2004; Terry and Elisa 2005). During phytoremediation, organic contaminants could be removed by plant uptake, plant accumulation and plant enhanced dissipation; however, plant enhanced dissipation was the main way (Cheema et al. 2010). In the current investigation, enhanced dissipation of PCP in planted versus unplanted soil would overwhelmingly derive from P. australis promoted biodegradation which is the dominant contribution in this present study. Similarly, Liste and Alexander (2000) investigated the capability of nine plant species to promote the degradation of PYR in soil and the results showed that more PYR was degraded in the presence of roots of all nine species than in unplanted soil. The enhanced dissipation of PCP in planted soil could be attributed to the increased microbial activity in the rhizosphere due to presence of plant root exudates leading to increased microbial transformation and/or mineralization of contaminants (Bolan et al. 2011). This explanation can be supported by the results of He et al. (2005) reported that the largest microbial biomass carbon was found at each PCP concentration treatment in the near-rhizosphere soils and the degradation gradient followed the order: near rhizosphere > root compartment > far-rhizosphere soil zones. Wide investigations have demonstrated the soil environment influenced by plant roots or rhizosphere represents an interesting potential to stimulate biodegradation of organic contaminants, including PYR (Xie et al. 2012). In the present investigation, the removal rate of PCP decreased significantly (p ≤0.05) with the addition of Cd. A negative effect of Cd on dissipation of PCP was observed mainly in higher Cd level (50 mg kg−1) suggesting the change of the microbial numbers and activity and/or the root physiology under Cd stress which was unfavourable to PCP dissipation. Hechmi et al. (2013) reported also that with the increment of Cd level, residual PCP in the planted soil with Z. mays tended to increase. The interactive effect on pollutant removal was also observed for other organic pollutants with metals. For instance, Wang et al. (2012) revealed that removal rate of PYR decreased at the elevated Cd level
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(6.38 mg kg−1) in the soil planted with hyperaccumulator plant S. alfredii. Furthermore, Olaniran et al. (2011) reported that heavy metals have different impacts on the degree of 1,2-dichloroethane degradation and suggested that the degree of inhibition is metal specific. Therefore, an interactive effect of heavy metals and organic pollutants on contaminants removal from co-contaminated soil can either cause a negative or positive effect depending on the type and concentration of both PAHs and metals (Zhang et al. 2012b). Microbial activity in rhizosphere of P. australis As a vital constituent part, soil enzymes are direct participants of biochemical reactions and the activities of which designate the degree of biochemical processes such as material transformation, energy metabolism and pollutant degradation (Xie et al. 2012). Organic pollutants can be catabolized by enzymes either mineralized completely to inorganic compounds (e.g. carbon dioxide and water) or degraded partially to a stable intermediate (Zhu and Zhang 2008 ). Among all enzymes, the dehydrogenase is an important oxidoreductase in soils, which is the catalyst for important metabolic processes, including the decomposition of organic inputs and the detoxification of xenobiotics (Margesin et al. 2000). In the study presented here, a stimulation of the metabolic soil processes (increase in DHA) was observed in planted soils (11 %) compared to unplanted soil (Fig. 4a). By comparison, it was found that DHA depressed by pollutant addition and increased (p ≤0.05) remarkably with the plantation of P. australis, which might be attributed to the increased microbial activity as a result of root exudation of plants (Cheema et al. 2009). The results suggested that this P. australis can effectively alleviate the inhibitory effect of pollutants on soil microbial activity, and that cultivation could improve the activities of some enzymes (DHA). Similar reports by Yang et al. (2011) during phytoremediation of butachlor contaminated soil by different wetland plants: P. australis, Zizania aquatica and Acorus calamus which indicated that throughout the incubation periods (35 days), the rhizosphere soil of A. calamus showed higher enzymatic activities followed by P. australis and Z. aquatica. Significant positive correlations were found between the DHA and the removal ratio of PCP in soils (Fig. 4b). R2 values for linear regressions were 0.8828 and 0.9658 for DHA in unplanted and planted soil, respectively. Such results indicated that the dehydrogenase was a dominating enzyme involved in PCP biochemical transformation. Similarly, Kaimi et al. (2006) reported that there was a significant correlation between the dissipation rate of diesel oil and soil DHA both in the rhizosphere and the root-free soil. On the basis of the results from the present experiment, there is a synergistic inhibition on soil biota under Cd–PCP co-contamination especially at high pollutant levels. As
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reported by Maliszewska-Kordybach and Smreczak (2003) that inhibition of soil microbial parameters in many cases was stronger in soils contaminated with both groups of pollutants than in soils contaminated with PAH or metal only.
Conclusion Even though there was observed fast growth and easy harvesting, the emergent wetland species P. australis could not translocate Cd and PCP. P. australis had basic characteristics of Cd exclusion under Cd–PCP co-contaminated soil. So it is not suitable for phytoextraction of Cd under Cd–PCP co-contamination. The dissipation of PCP in both sole and Cd co-contaminated soil was enhanced by vegetation but the contribution of plant uptake was less than 0.3 %. So, it is conceivable that under Cd–PCP co-contamination, the remediation by emergent wetland plants is influenced by interactions of contaminants in the soil and the occurrence of high levels Cd and PCP had inhibitive effects on plant growth and PCP uptake and accumulation on the plant. For these reasons, additional strategies (e.g. exploring the partnership of plant–microbe and/or application of chelating compounds (citric acids)) are required to enhance the co-removal of Cd and PCP from co-contaminated soils. Acknowledgments The authors acknowledge all staff of Sol Direction and Biotechnology Centre in Tunisia for the technical support. We also would like to express gratitude to Professor Andrew Hursthouse and all staff of Physical Sciences SDG, School of Science, University of the West of Scotland for their great assistance in pollutant (PCP) extraction and quantification.
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RESEARCH ARTICLE
Evaluating the phytoremediation potential of Phragmites australis grown in pentachlorophenol and cadmium co-contaminated soils Nejla Hechmi & Nadhira Ben Aissa & Hassen Abdenaceur & Naceur Jedidi
Received: 4 February 2013 / Accepted: 10 July 2013 / Published online: 31 July 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Pot-culture experiments were conducted to evaluate the phytoremediation potential of a wetland plant species, Phragmites australis in cadmium (Cd) and pentachlorophenol (PCP) co-contaminated soil under glasshouse conditions for 70 days. The treatments included Cd (0, 5 and 50 mg kg−1) without or with PCP (50 and 250 mg kg−1). The results showed that growth of P. australis was significantly influenced by interaction of Cd and PCP, decreasing with either Cd or PCP additions. Plant biomass was inhibited and reduced by the rate of 89 and 92 % in the low and high Cd treatments and by 20 and 40 % in the low and high PCP treatments compared to the control. The mixture of low Cd and low PCP lessened Cd toxicity to plants, resulting in improved plant growth (by 144 %). Under the joint stress of the two contaminants, the ability of Cd uptake and translocation by P. australis was weak, and the BF and TF values were inferior to 1.0. A low proportion of the metal is found aboveground in comparison to roots, indicating a restriction on transport upwards and an excluding effect on Cd uptake. Thus, P. australis cannot be useful for phytoextraction. The removal rate of PCP increased significantly (70 %) in planted
Responsible editor: Philippe Garrigues N. Hechmi : H. Abdenaceur : N. Jedidi Laboratory of Wastewater Treatment, Water Research and Technologies Centre (CERTE), Technopole of Borj Cedria, BP 273, Soliman 8020, Tunisia N. Hechmi (*) : N. B. Aissa National Agronomic Institute of Tunisia, 43 Avenue Charles Nicolle, Mahrajene 1082, Tunisia e-mail: [email protected]
soil. Significant positive correlations were found between the DHA and the removal of PCP in planted soils which implied that plant root exudates promote the rhizosphere microorganisms and enzyme activity, thereby improving biodegradation of PCP. Based on results, P. australis cannot be effective for phytoremediation of soil co-contaminated with Cd and PCP. Further, high levels of pollutant hamper and eventually inhibit plant growth. Therefore, developing supplementary methods (e.g. exploring the partnership of plant–microbe) for either enhancing (phytoextraction) or reducing the bioavailability of contaminants in the rhizosphere (phytostabilization) as well as plant growth promoting could significantly improve the process of phytoremediation in co-contaminated soil. Keywords Phytoremediation . Wetland plant . Phragmites australis . Co-contamination . Pentachlorophenol . Cadmium
Introduction Soils have been submitted to several contaminants that vary in concentration and composition. Among all, organic chemicals such as persistent organic pollutants and heavy metals are recognized as two major chemical families that cause water and soil pollution (Sun et al. 2011). In many parts of the world, agricultural soil is slightly or moderately contaminated by cadmium (Cd) (Li et al. 2010). Cd is a heavy metal of great concern in agricultural ecosystems because of its high toxicity to animals and human health
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(Huang et al. 2011). Pentachlorophenol (PCP) has been released into the environment as a wood preservative, pesticide, herbicide and antiseptic. This compound is a major soil pollutant due to its toxicity and recalcitrance (Miller and Dyer 2002), and it is regulated as one of the priority pollutants by the U.S. Environmental Protection Agency. Cocontamination of PCP and Cd often occurs in vegetable fields due to the conventional use of pesticides (Lin et al. 2007) as well as increased application of sewage sludge and wastewater irrigation (Li et al. 2010). Hence, it is critical to develop efficient and cost-effective approaches to simultaneously remove multiple contaminants from co-contaminated soils. Phytoremediation is the use of plants to remediate polluted soils, an eco-friendly and cost-effective technology that is currently receiving considerable global attention (Glick 2010). A large number of plant species are capable of hyperaccumulating heavy metals and organics in their tissues; however, phytoremediation in practice has several constraints at the level of sites as these are with a variety of different contaminants (Wu et al. 2006). Further, the success of phytoremediation depends upon a plant's capacity to tolerate and to accumulate high concentrations of the pollutant, while yielding a large plant biomass (Grčman et al. 2001). The mechanisms of phytoremediation systems comprise at least four pathways to reduce soil contaminants, such as abiotic losses (leachate, volatilization, photodegradation, irreversible sorption, chemical degradation and so on), indigenous microbial degradation, root tissues-enhanced dissipation and rootexudates-enhanced biodegradation (Sun et al. 2010). Up to now, there are numerous promising results indicating that the technique might become viable alternative to mechanical and chemical approaches in decontamination of metal polluted sites or a final polishing solution for organic contamination. However, little information was still available regarding the effectiveness and processes of phytoremediation of sites cocontaminated with organic and metal pollutants which have become more prevalent (Chigbo et al. 2013). The efficiency of phytoremediation of organic pollutants co-existing with heavy metals is complex and quite different from that in the singlepollutant system because different compounds may interact among themselves and/or with plants and their rhizosphere biota (Almeida et al. 2008). For instance, interaction of metals and organics with respect to degradation of organics and/or metal uptake can be negative or positive depending on type and concentration of both metals and organic pollutant (Zhang et al. 2012b). In a study of Cd and polycyclic aromatic hydrocarbons (PAHs) (phenanthrene (PHE) or pyrene (PYR)) remediation in co-contaminated soil planted with hyperaccumulator plant Sedum alfredii, removal rate of PYR decreased at the elevated Cd level (6.38 mg kg−1) in the soil. S. alfredii could effectively extract Cd from Cd-contaminated soils in the presence of PHE or PYR (Wang et al. 2012). Furthermore, planting of S. alfredii is an effective technique
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for phytoextraction of Cd and dichlorodiphenyltrichloroethane (DDT) in Cd–DDT co-contaminated soil with low Cd (0.895 mg kg−1) and high Cd ( 3.225 mg kg−1) concentrations, with 0.715 mg kg−1 DDT (Zhu et al. 2012). This uptake capability equaled that of the pot-grown crop Ricinus communis planted in spiked soils with DDT and Cd at the rate of 1.7 and 2.8 mg kg−1 soil. The total uptake of DDT and Cd varied from 83.1 to 267.8 and 66.0 to 155.1 μg pot−1, which indicates that R. communis (castor) has great potential for removing DDT and Cd from co-contaminated soils attributed to its strong absorption and accumulation for both DDT and Cd (Huang et al. 2011). The uptake and translocation of metals in various wetland plants have been studied (Liu et al. 2010; Zhang et al. 2010a), but few studies have focused on the uptake, translocation and/or metabolism of organic pollutants (e.g. PCP) in wetland species (Roy and Hänninen 1994) especially under co-contaminated conditions. However, the possible stimulatory, antagonistic (less than additive toxicity) and competitive uptake and metabolism of the combined PCP and Cd received little exploration (Hechmi et al. 2013). The growth and pollutant removal by emergent wetland plants may be influenced by Cd and PCP interaction. The knowledge about the pressure of these interactions between co-contaminants on the phytoremediation potential of wetland plants is relatively poor. Emergent aquatic macrophytes represent a diverse group of plants with an immense potential for removal/degradation of variety of contaminants (Dhir et al. 2009). Besides, macrophytes are more suitable for wastewater treatment than terrestrial plants due to their faster growth, production of more biomass and relative higher ability of pollutant uptake (Ali et al. 2013). Wetland plants can enhance metal removal and/or stabilization (Marchand et al. 2010) and may also relieve organic pollutant biodegradation (a) directly in the rhizosphere by the release of root exudates (Toyama et al. 2011) and (b) indirectly by improving soil biology via build up of organic carbon (Pilson-Smits 2005). The emergent wetland species such as Phragmites australis (Poaceae family) had numerous characteristics (e.g. cosmopolitan distribution, easy growth, inexpensive…) that make it a favoured test species. Previous studies reported that P. australis is one of the best plant organisms for detecting heavy metals and harmful compounds such as herbicides (Bonanno 2011). Furthermore, plant screening for phytoremediation of uranium, thorium, barium, nickel, strontium and lead contaminated soils showed that P. australis had the greatest removal capabilities for uranium (820 μg), thorium (103 μg) and lead (1,870 μg) (Li et al. (2011). Hence, effectiveness of P. australis in heavy metal filtration has prompted us to undertake this study. The main aims for the current investigation were (1) to examine the growing response of P. australis to single contamination of PCP and co-contamination of Cd–PCP, (2) to evaluate the plant uptake, accumulation, translocation and
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Material and methods
into shoots, roots and rhizomes. After drying with filter paper, the subsamples were freeze-dried and weighed to determine the biomass. The soil samples from the pots were separated into the rhizosphere and non-rhizosphere soils. Both types of soil samples were air-dried at room temperature (approximately 20–23 °C) and ground sufficiently to pass through a 100 mesh sieve.
Soil characterization
Extraction and analysis of PCP in soils and plants
The tested soil without detectable PCP and Cd was collected from the top layer (0–20 cm) of an agricultural field in NE of Tunisia. The soil was sandy loam containing 60 % sand, 30 % silt and 10 % clay. Selected soil properties were as follows: pH 6.4(1:2 soil/water), EC 0.012 dSm−1, total organic carbon 3.02 g kg−1, total nitrogen 0.22 g kg−1 and total phosphorus 0.12 g kg−1 . The soil was air-dried and sieved through a 2-mm sieve prior to spike with PCP and Cd.
PCP is extracted according to a slightly modified method previously described by Sharma et al. (2009). Soil (2 g) was ultrasonically extracted (45 kHz, 300 W) in 5 ml of methanol for 30 min followed by 4,000 rpm centrifugation for 10 min. As for plant tissues analysis, about 0.5 g of dried plant fragment was weighed into a 25-ml centrifuge tube and extracted by ultrasonication for 30 min with a 10-ml solution of methanol for three successive extractions. The extraction was centrifuged at 4,000 rpm for 5 min to separate the supernatant from the soil or plant. The solvent was removed gently by blowing under a stream of N2. The residue was derivatized in 400 μl of ethyl acetate and analyzed immediately on a gas chromatography–mass spectrometry (GC– MS). The GC–MS analyses were performed in electron ionization (EI) mode (70 eV) with an Agilent 6,890 N gas chromatograph, equipped with 5973 MSD (Agilent Technologies, Palo Alto, CA, USA). An HP-5MS (Agilent, USA) column was used at a temperature programme of 45 °C for 1.5 min, increased to 100 °C at 10 °C/min, increased to 180 °C at 4 °C/min and finally increased to 300 °C at 40 °C/min and held at 290 °C for 5 min. Helium was used as the carrier gas at a constant flow of 1.2 ml/min. The samples were analyzed in split mode (1:10) at an injection temperature of 250 °C, an EI source temperature of 230 °C and a quadrupole analyzer temperature of 150 °C, unit mass resolution, scan range m/z 35–500, with a scan cycle of three scans. The injected volume was 1 μl. The recovery rate of PCP was 89 %.
dissipation behaviours of Cd and PCP and (3) the interactive effects of Cd, PCP and plant on soil dehydrogenase activity (DHA) which is good indicators of soil microbial activity.
Experimental procedure Experiments were set up by artificially co-contaminating the soil sample with PCP and Cd. PCP>97 % purity (Sigma– Aldrich) is dissolved in acetone and added to 25 % by weight of the required amount of soil at concentrations of 0, 50 or 250 mg kg−1 (P0, P1 and P2) PCP. Cd (as CdCl2 ×21/2H2O, analytical grade, AJAX Chemicals) was dissolved in Milli-Q water and added to the PCP spiked soils at concentrations of 0, 5 or 50 mg kg−1 (C0, C1 and C2), resulting in a total of nine treatments including C0P0 (T1), C0P1(T2), C0P2(T3), C1P0 (T4), C1P1(T5), C1P2(T6), C2P0 (T7), C2P1(T8) and C2P2(T9). A modified Hoagland nutrient solution contained (mg kg−1 soil) N 30, P 20, K 89, S 30, Ca 40, Cl 73, Mg 4, Mn 3.26, Zn 2, Cu 0.50, B 0.11, Co 0.10 and Mo 0.08 was added to all treatments at the same rates and was mixed uniformly to the soils. The modified Hoagland nutrient solution was prepared as described by Dordio et al. (2009). The spiked soils were then sieved again through a 2-mm sieve to ensure homogeneity and stored for analysis. A cylindrical water-permeable polyethylene rhizobags (7.5 cm in diameter and 13 cm in length) were used to separate rhizosphere from non-rhizosphere soil. Six hundred fifty grams of soil was added to each rhizobag, and the space outside the bag was filled with 350 g of the same soil. In this system, the surfaces of the soil inside and outside the rhizobag were in the same level. The seedlings of P.australis (Cav.) were collected from an uncontaminated field site in NE of Tunisia and transplanted (with initial plant fresh weight 5.0±0.5 g per pot) into each rhizobag. All treatments were irrigated with the running water every day, and the depth of water was kept at 2– 3 cm high at the surface of the soil. The exposure experiment was carried out in a glasshouse with controlled temperature of 30–35/20–25 °C (day/night) under natural light conditions from early July to mid-September 2010). The plants were harvested after 70 days and washed with deionized water and separated
Cd content determination The soil samples and plant were digested with a solution of 3:1 HNO3:HClO4 (v/v). The concentrations of Cd were determined using the atomic absorption spectrophotometry. DHA Soil DHA was measured by the reduction of 2,3,5-triphenyl tetrazolium chloride (TTC) to 1,3,5-triphenyl formazan (TPF) using a modification of the methods of (Mills et al. 2006). Five gram soil samples, collected from each pot, were individually placed in 30 ml plastic vials with 0.1 g of CaCO3 and 3 ml of TTC solution (5 g L−1 in 0.2 M Tris–HCl buffer, pH 7.4) and were incubated for 24 h at 37 °C. After incubation, 20 ml of
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methanol was added, the sample shaken and then filtered through Whatman 41 filter paper to extract the TPF. The absorbance of the filtered solutions was measured at 485 nm using a spectrophotometer (Beckman DU 640).
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Total pollutant accumulation ¼ ðpollutant concentration in shoots shoot DWÞ þ ðpollutant concentration in roots root DWÞ
ð1Þ
Data analysis Total accumulation of Cd or PCP in plants, expressed as microgram pot−1 and was calculated as:
The percentage removal of Cd or PCP by plants was calculated as:
Removal of Cd or PCP by plantsð%Þ . ¼ total Cd or PCP accumulation in plants total added Cd or total measured PCP in soil 100
ð2Þ
Statistical analyses were carried out using SPSS13.0. A one-way analysis of variance followed by a Tukey's multiple comparison test (at p ≤0.05) was employed.
high PCP treatments compared to the control. Compared to the low Cd treatment alone, the total plant biomass was significantly (p ≤0.05) increased (by 144 %) in the mixture of low Cd and low PCP.
Results
Cd uptake in P. australis under joint stress of Cd and PCP
Plant growth
The amount and distribution of Cd absorption in the tissues of P. australis are shown in Table 1. The concentrations of Cd increased with Cd additions and decreased with PCP additions. The total Cd accumulation in plant tissues increased by 165 % in the mixture of low Cd and low PCP compared to the low Cd treatment alone. Although the accumulation of Cd decreased by 22 and 14 %, respectively, no significant difference was observed in the mixtures of either low or high Cd or high PCP
Figure 1 shows the dry matter yield of P. australis under different soil PCP and Cd rates. The growth of P. australis was significantly (p ≤0.05) influenced by Cd, PCP and their interaction. The influence of Cd on biomass was much stronger compared to PCP revealed that the occurrence of Cd had prohibitive effects on the growth of P. australis. Plant biomass was inhibited by the rate of 89 and 92 % in the low and high Cd treatments and by 20 and 40 % in the low and
Table 1 The concentrations of Cd in plant tissues, BF and TFs under combined contamination of Cd and PCP after 70 days of plant growth Treatment
T4 T5 T6 T7 T8 T9
Fig. 1 Plant biomass influenced by Cd and PCP treatments after 70 days of growth. Bars (means ± SE, n =3). Treatment: C0P0 (T1), C0 P1(T2), C0P2(T3), C1P0 (T4), C1P1(T5), C1P2(T6), C2 P0 (T7), C2P1(T8) and C2P2(T9). C0, C1 and C2 represent 0, 5 and 50 mg Cd kg−1; P0, P1 and P2 represent 0, 50 and 250 mg PCP kg−1
Concentration of Cd (mg kg−1) Root
Rhizomes
Shoot
456±61d 436±105d 135±54e 1,278±18a 1,099±81b 824±25c
287±14e 386±39e 231±0.04b 1,109±154a 857±152e 611±88e
114±21c 89±12c 70±2c 545±137a 363±25b 280±41b
BF
TF
0. 43 0.77 0.63 0.56 0.43 0.13
0.31 0.20 0.52 0.42 0.34 0.33
Treatment: C1P0 (T4), C1P1(T5), C1P2(T6), C2 P0 (T7), C2P1(T8) and C2P2(T9). C0 and C2 represent 0, 5 and 50 mg Cd kg−1 ; P1, P1 and P2 represent 0, 50 and 250 mg PCP kg−1 . Means (±SE, n =3) followed by the same lowercase letter within columns are not significantly different according to LSD (p ≤0.05). Bioaccumulation factor (BF) is defined as the ratio of the metal concentration in shoots to that in the soil, and transfer factor (TF) is defined as the ratio of the metal concentration in shoots to that in roots
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in plants was significantly (p ≤0.05) influenced by Cd additions (Table 2).
PCP removal from soil
Fig. 2 Cd accumulation in plant tissues and percentage of Cd removal from soils under stress of Cd and PCP after 70 days of growth. Bars, means ± SE, n =3 with different letters are significantly different based on LSD (p ≤0.05). Treatment: C0P0 (T1), C0P1(T2), C0P2(T3), C1P0 (T4), C1P1(T5), C1P2(T6), C2 P0 (T7), C2P1(T8) and C2P2(T9). C0, C1 and C2 represent 0, 5 and 50 mg Cd kg−1; P0 P1 and P2 represent 0, 50 and 250 mg PCP kg−1
compared to either low or high Cd treatment alone (Fig. 2). Cd accumulation in different plant tissues was in the sequence root > rhizome > shoot among the treatments with added Cd. The highest percent Cd removal from soils by plants was in the mixture of low Cd and low PCP, which increased significantly (p ≤0.05) (2.2-fold) compared to the low Cd treatment alone. PCP concentration and partitioning in P. australis The concentration of PCP in the tested plants could strikingly increase with an increase in the content of PCP in soil. The highest amount of PCP concentrated in roots was 3.10–7.6 times higher than those in shoots. The concentration of PCP
The percentage of PCP removal from soils was significantly influenced by the interaction of Cd, PCP and planting/nonplanting treatments (Fig. 3). The percentage of PCP removal was significantly higher (p ≤0.05) in the low than high PCP treatments. The removal of PCP decreased with Cd additions, which was more obvious in the non-planted treatments. Compared to the non-planted treatments, PCP removal significantly increased (p ≤0.05) by 16, 17 and 19 %, respectively, in the planted mixtures of low Cd and either low or high PCP and mixture of high Cd and low PCP.
Desydrogenase activity in soil The DHA in unspiked, and unplanted soil was taken as the control value of 100 %. As shown in Fig. 4a, DHA in unplanted soils co-contaminated by Cd and PCP was significantly lower (p ≤0.05) than the control value, decreasing to an average of 64 % of the control value. It tended to decline along with increase in soil PCP and Cd concentration in soil. It was observed that a low concentration of PCP alone contamination can increase DHA slightly. However, with an increase in the Cd level, DHA decreased to 64 % of the control value in 5 mg kg−1 Cd treatment and 55 % of the control value in 50 mg kg−1 Cd treatment, respectively. DHA in planted soils was increased significantly (p ≤0.05) by 11 % than in unplanted soil.
Table 2 Concentrations of PCP in plant tissues, BF and the root-toshoot TFs influenced by Cd treatments after 70 days of plant growth Treatment
T2 T3 T5 T6 T8 T9
Concentration of PCP (mg kg−1) Root
Rhizomes
Shoot
40±2.61d* 45±1.75d 47±0.54e 48±1.28a 39±0.61b 37±2.05b
2.6±14a 4.06±39e 3.11±0.11d 1.2±0.15a 6.5±0.2b 6.11±0.3d
7.2±0.11a 11±0.11c 10±0.2c 21±0.13b 6.9±0.15b 6±0.31b
BF
TF
0.36 0.44 0.56 0.63 0.76 0.72
0.18 0.24 0.22 0.43 0.18 0.16
Treatment: C0 P1(T2), C0P2(T3), C1P1(T5), C1P2(T6), C2P1(T8) and C2P2(T9). C0 and C2 represent 0, 5 and 50 mg Cd kg−1 ; P1, P1 and P2 represent 0, 50 and 250 mg PCP kg−1 . Bioaccumulation factor (BF) is defined as the ratio of the metal concentration in shoots to that in the soil, and transfer factor (TF) is defined as the ratio of the metal concentration in shoots to that in roots *p ≤0.05 (means (±SE, n =3) followed by the same lowercase letter within columns are not significantly different according to LSD)
Fig. 3 Percentage of PCP removal from soils. Bars, means ± SE, n =3. Treatment: C0P0 (T1), C0P1(T2), C0P2(T3), C1P0 (T4), C1P1(T5), C1P2(T6), C2 P0 (T7), C2P1(T8) and C2P2(T9). C0, C1 and C2 represent 0, 5 and 50 mg Cd kg−1; P0, P1 and P2 represent 0, 50 and 250 mg PCP kg−1
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Fig. 4 Changes in dehydrogenase activity in unplanted and planted soils with different treatments (a). Correlation between the dehydrogenase activity and removal ratio of pentachlorophenol in unplanted and planted soils (b). Bars, means ± SE, n =3 (p ≤0.05). Treatments: C0P0
(T1), C0P1(T2), C0P2(T3), C1P0 (T4), C1P1(T5), C1P2(T6), C2P0 (T7), C2P1(T8) and C2P2(T9). C0, C1 and C2 represent 0, 5 and 50 mg Cd kg−1; P0, P1 and P2 represent 0, 50 and 250 mg PCP kg−1
Discussion
high treatment). In the same way, Zhang et al. (2012a) revealed that the growth of Scirpus triqueter tends to decrease (including height, diameter, shoot number and biomass) in the relatively high concentration of 80 and 160 mg kg−1 PYR. These results were different to those of Hechmi et al. (2013), who reported that with the initial concentration of 50 mg kg−1 PCP, no outward sign of phytotoxicity or even better growth for the crop Z. mays was shown with the increment of soil PCP level. An interactive effect of Cd and PCP on plant growth was observed in the present study. In the mixture of low Cd and low PCP, plants grew better than in the low Cd treatment alone (Fig. 1), implying that PCP could at least partly alleviate Cd toxicity effects. The available study investigating the combined toxicity of metals and organics have indicated that toxicity may be independent, additive, synergistic (greater than additive toxicity), or antagonistic (less than additive toxicity) (Zhang et al. 2012b). For instance, PCP present in a 50 mg kg−1 alleviated also Cu toxicity to ryegrass (Lolium perenne) and radish (Raphanus sativus), while high concentrations intensify that toxicity (Lin et al. 2006). Similarly, Hechmi et al. (2013) reported that in Cd co-contaminated soil with the initial PCP concentration of 50 and 100 mg kg−1, Z. mays grew better with the increment of soil Cd level (0, 2 and 6 mg kg−1), implying that combinations of Cd and PCP exerted antagonistic toxic effects on plant growth. Besides, Lin et al. (2008) observed an increase in shoot yield of Z. mays with copper and PYR co-contamination, whereas our results showed that the yield of both root and shoot of P. australis decreased significantly (p ≤0.05) with the increase of soil PCP and Cd concentrations. This suggests a synergistic effect of metals and organics in co-contaminated soil and is supported by Zhang et al. (2009) which showed that PYR did not alleviate the toxicity Cd to Z. mays. These diverse results suggest that
Interactive effects of Cd and PCP on plant growth Several studies have illustrated symptoms and responses of wetland plants upon toxic metal exposure (Bonanno and Giudice 2010). The inhibiting effects of Cd on biomass accumulation, height, root length and other biometric parameters of plants were reported in most investigations. For instance, Zhang et al. (2010a) observed an adverse effect of Cd on the relative growth rates of four wetland species. The relative growth rates of the three species (Baumea articulata, Schoenoplectus validus and Juncus subsecundus) changed little in various Cd treatments, but was severely inhibited for Baumea juncea at 20 mg l−1of Cd, whereas an increase of growth for S. validus was observed at 5 mg l−1. In the present study, dry weight of P. australis was significantly reduced by Cd additions after 70 days of growth in soils (Fig. 1). The effects of Cd toxicity have been studied for other plants. Hechmi et al. (2013) reported nonsignificant inhibitory effects of Cd (up to 6.00 mg kg−1) on shoot growth of Zea mays. However, significant growth enhancement of S. alfredii was observed at level 6 mg kg−1 of Cd (Wang et al. 2012). This different response to Cd in the degree of expressed phytotoxicity could be due to various Cd levels as well as to different plant species and its tolerance. The effect of organic pollutants on wetland plant growth could be species specific regardless of pollutant types and media (Zhang et al. 2010b). For instance, exposure to low doses of PCP can stimulate growth of Phragmites communis Trin, Typha orientalis Pres and Scirpus validus Vahl, but high doses of PCP (>2 μg kg−1 dry weight) hamper and eventually inhibit plant growth (Zhao et al. 2011). In the present study, biomass of P. australis was significantly reduced (p ≤0.05) by PCP additions (40 % in PCP
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growth response of plants to joint toxicity of metal and organic pollutants are dependent on certain factors including plant species, age, type and concentration of contaminants (Zhang et al. 2012b; Chigbo et al. 2013). Cd/ PCP uptake and partitioning in P. australis Our results showed that whether soil Cd was present in single or mixed contamination, higher Cd concentrations were always observed in the root. The Cd removal efficiency by P. australis was both relative to biomass production and tissue concentration. The observed increase in total Cd accumulation with increasing soil Cd concentration (Table. 1) could be as a result of increased plant biomass. The ability of plants to tolerate and accumulate heavy metals is useful for phytoextraction and phytostabilization purpose measured using translocation factor (TF) and bioconcentration factor (BF). BF and TF are usually >1 (or≫1) in hyperaccumulators and leaf > stem > root and all the parameters (BF and TF reached 3.28 and 1.51, respectively) were very low (Table 1). Hence, P. australis was inappropriate for Cd phytoextraction. An added experiments carried out by Zhang et al. (2012b) showed an order of Cd accumulation shoot > root > rhizome among the treatments with added Cd (5, 10 and 20 mg kg−1) and TF between 144 and 307 %, suggesting the phytoextraction potential of wetland plant J. subsecundus. This inconsistency may be due to differences in the levels, durations of exposure to Cd and the different growth media as well as plant species which can differ in rates of metal uptake, allocation and excretion in metal dynamics. Plant uptake of hydrophobic organics, such as PCP, could be described as partitioning between the soil aqueous solution and plant roots, with translocation from root to shoot being highly restricted (Gao et al. 2011). Obviously, PCP uptake is not a significant pathway of dissipation for PCP in this experiment. PCP was undetectable in the aboveground part of the P. australis and BF and TF values were 0.36–0.76 and 0.16–0.34, respectively (Table 2). Therefore, the mechanisms of dissipation evaluated were biodegradation and incorporation into soil. In the present investigation, P. australis plants were able to accumulate xenobiotics such as PCP only in their root which corroborate with the previous research on aquatic macrophyte and rice demonstrating that plant could remediate the PCP-contaminated soil by uptake PCP into root tissue. The maximum BF of PCP in the roots of
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the macrophyte P. communis Trin was 0.71 at sediment PCP concentration of 2 μg mg−1 (Zhao et al. 2011). The root fraction of the rice treatment had the highest PCP concentration, but accounted for only 0.015 μg of the added PCP of 75 μg (Terry and Elisa 2005). The main reason of this substantiation might be the physicochemical characteristics (the octanol–water partition coefficient K ow) of PCP. Since the log Kow of PCP was 5.01, which might make it difficult to transport from root tissue to upper part tissue (Schröder et al. 2008). Since uptake of PCP in shoots was negligible, PCP may be catabolized by plant enzymes either mineralized completely to inorganic compounds (e.g. CO2, H2O) or degraded partially to a stable intermediate that is stored in the plants (Pilson-Smits 2005). There is a trend that antagonistic and synergistic responses of the plant reflected bioaccumulation patterns in the cocontamination soil. For instance, Sun et al. (2011) reported that under the co-contamination of heavy metals and PAH, the accumulation of Cd in shoots of T. patula was favourably influenced by the presence of PAH in soil. Nevertheless, adding PAH with two levels (25 and 150 mg kg−1) of PHE or PYR could restrain Cd translocation in S. alfredii (Wang et al. 2012). On the other hand, in the present investigation, whether soil Cd was present in single or mixed contamination, higher Cd concentrations were always observed in the root and PCP addition increased significantly (p ≤0.05) the uptake of Cd by the roots of P. australis. This could be as a result of the influence on plant growth as observed in the present study where co-contamination reduced shoot dry weight significantly (p ≤0.05). Chigbo et al. (2013) observed that PYR significantly affected Cu uptake as a result of reduction in growth of B. juncea. As a potential remediation plant, the basic characteristic is high tolerant capability and exhibits a variety of responses to mechanical stresses that enable them to tolerate and evolve resistance to adverse conditions that are toxic to most other plants (Sun et al. 2008). Based on the results in this investigation and in the previous one, we can point out that under the coexistence of Cd and PCP in contaminated soil, the interaction between metal and organic pollutant could influence plant growth, metal uptake and accumulation by plants. The phytoextraction of Cd by P. australis is very restricted, and it could be that PCP altered the way P. australis influenced Cd sorption and solubility. Z. mays unlikely has higher capability to phytoextract Cd and could be useful for remediation of Cd even at co-contamination of PCP–Cd, but the ability of Cd phytoextraction would be inhibited under co-contamination of high level of PCP (PCP>100 mg kg−1 DW) in highly Cd polluted soil (Cd>6 mg kg−1 DW) (Hechmi et al. 2013). Z. mays as well can normally grow in the co-contaminated soil with high level Cd and PCP and can effectively remedy sites co-contaminated with these two types of pollutant.
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PCP removal PCP removal in planted soil was greater than in no planted soil except in co-contaminated soils which showed similar rates of removal (Fig. 3). The removal rate of PCP in soils excelled 70 % for all the planted soils after 70 days of remediation, which was far more than that in the control. Likewise, Zhao et al. (2011) reported that the removal rates of PCP in the sediments planted with three macrophytes P. communis Trin, T. orientalis Pres and S.validus Vahl were 90.35, 99.23 and 99.33 %, respectively, while the rate in the control sediment was only 29.87 %. The removal rates are also more than that in previous reports of PCP phytoremediation using terrestrial , such as Chinese chive (40 %, 28 days) and rice (63 %, 82 days) (Takashi et al. 2004; Terry and Elisa 2005). During phytoremediation, organic contaminants could be removed by plant uptake, plant accumulation and plant enhanced dissipation; however, plant enhanced dissipation was the main way (Cheema et al. 2010). In the current investigation, enhanced dissipation of PCP in planted versus unplanted soil would overwhelmingly derive from P. australis promoted biodegradation which is the dominant contribution in this present study. Similarly, Liste and Alexander (2000) investigated the capability of nine plant species to promote the degradation of PYR in soil and the results showed that more PYR was degraded in the presence of roots of all nine species than in unplanted soil. The enhanced dissipation of PCP in planted soil could be attributed to the increased microbial activity in the rhizosphere due to presence of plant root exudates leading to increased microbial transformation and/or mineralization of contaminants (Bolan et al. 2011). This explanation can be supported by the results of He et al. (2005) reported that the largest microbial biomass carbon was found at each PCP concentration treatment in the near-rhizosphere soils and the degradation gradient followed the order: near rhizosphere > root compartment > far-rhizosphere soil zones. Wide investigations have demonstrated the soil environment influenced by plant roots or rhizosphere represents an interesting potential to stimulate biodegradation of organic contaminants, including PYR (Xie et al. 2012). In the present investigation, the removal rate of PCP decreased significantly (p ≤0.05) with the addition of Cd. A negative effect of Cd on dissipation of PCP was observed mainly in higher Cd level (50 mg kg−1) suggesting the change of the microbial numbers and activity and/or the root physiology under Cd stress which was unfavourable to PCP dissipation. Hechmi et al. (2013) reported also that with the increment of Cd level, residual PCP in the planted soil with Z. mays tended to increase. The interactive effect on pollutant removal was also observed for other organic pollutants with metals. For instance, Wang et al. (2012) revealed that removal rate of PYR decreased at the elevated Cd level
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(6.38 mg kg−1) in the soil planted with hyperaccumulator plant S. alfredii. Furthermore, Olaniran et al. (2011) reported that heavy metals have different impacts on the degree of 1,2-dichloroethane degradation and suggested that the degree of inhibition is metal specific. Therefore, an interactive effect of heavy metals and organic pollutants on contaminants removal from co-contaminated soil can either cause a negative or positive effect depending on the type and concentration of both PAHs and metals (Zhang et al. 2012b). Microbial activity in rhizosphere of P. australis As a vital constituent part, soil enzymes are direct participants of biochemical reactions and the activities of which designate the degree of biochemical processes such as material transformation, energy metabolism and pollutant degradation (Xie et al. 2012). Organic pollutants can be catabolized by enzymes either mineralized completely to inorganic compounds (e.g. carbon dioxide and water) or degraded partially to a stable intermediate (Zhu and Zhang 2008 ). Among all enzymes, the dehydrogenase is an important oxidoreductase in soils, which is the catalyst for important metabolic processes, including the decomposition of organic inputs and the detoxification of xenobiotics (Margesin et al. 2000). In the study presented here, a stimulation of the metabolic soil processes (increase in DHA) was observed in planted soils (11 %) compared to unplanted soil (Fig. 4a). By comparison, it was found that DHA depressed by pollutant addition and increased (p ≤0.05) remarkably with the plantation of P. australis, which might be attributed to the increased microbial activity as a result of root exudation of plants (Cheema et al. 2009). The results suggested that this P. australis can effectively alleviate the inhibitory effect of pollutants on soil microbial activity, and that cultivation could improve the activities of some enzymes (DHA). Similar reports by Yang et al. (2011) during phytoremediation of butachlor contaminated soil by different wetland plants: P. australis, Zizania aquatica and Acorus calamus which indicated that throughout the incubation periods (35 days), the rhizosphere soil of A. calamus showed higher enzymatic activities followed by P. australis and Z. aquatica. Significant positive correlations were found between the DHA and the removal ratio of PCP in soils (Fig. 4b). R2 values for linear regressions were 0.8828 and 0.9658 for DHA in unplanted and planted soil, respectively. Such results indicated that the dehydrogenase was a dominating enzyme involved in PCP biochemical transformation. Similarly, Kaimi et al. (2006) reported that there was a significant correlation between the dissipation rate of diesel oil and soil DHA both in the rhizosphere and the root-free soil. On the basis of the results from the present experiment, there is a synergistic inhibition on soil biota under Cd–PCP co-contamination especially at high pollutant levels. As
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reported by Maliszewska-Kordybach and Smreczak (2003) that inhibition of soil microbial parameters in many cases was stronger in soils contaminated with both groups of pollutants than in soils contaminated with PAH or metal only.
Conclusion Even though there was observed fast growth and easy harvesting, the emergent wetland species P. australis could not translocate Cd and PCP. P. australis had basic characteristics of Cd exclusion under Cd–PCP co-contaminated soil. So it is not suitable for phytoextraction of Cd under Cd–PCP co-contamination. The dissipation of PCP in both sole and Cd co-contaminated soil was enhanced by vegetation but the contribution of plant uptake was less than 0.3 %. So, it is conceivable that under Cd–PCP co-contamination, the remediation by emergent wetland plants is influenced by interactions of contaminants in the soil and the occurrence of high levels Cd and PCP had inhibitive effects on plant growth and PCP uptake and accumulation on the plant. For these reasons, additional strategies (e.g. exploring the partnership of plant–microbe and/or application of chelating compounds (citric acids)) are required to enhance the co-removal of Cd and PCP from co-contaminated soils. Acknowledgments The authors acknowledge all staff of Sol Direction and Biotechnology Centre in Tunisia for the technical support. We also would like to express gratitude to Professor Andrew Hursthouse and all staff of Physical Sciences SDG, School of Science, University of the West of Scotland for their great assistance in pollutant (PCP) extraction and quantification.
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