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Uptake and translocation of benzo[a]pyrene (B[a]P) in two ornamental plants and dissipation in soil
Ecotoxicology and Environmental Safety 124 (2016) 74–81
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Uptake and translocation of benzo[a]pyrene (B[a]P) in two ornamental plants and dissipation in soil Yuebing Sun a,c, Qixing Zhou b,c,n a
Innovation Team of Remediation for Heavy Metal Contaminated Farmlands, Agro-Environment Pollution Institute, Ministry of Agriculture, Tianjin 300191, China Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education), College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China c Key Laboratory of Terrestrial Ecological Process, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 June 2015 Received in revised form 21 September 2015 Accepted 22 September 2015
Pot experiments were conducted to evaluate the phytoremediation of B[a]P contaminated soil using two ornamental plants (Tagetes patula and Mirabilis jalapa). The results showed that the dry biomass of two plants was increased at low B[a]P contaminated soil and then inhibited with increasing B[a]P concentrations. It exhibited a significantly positive linear relationship between B[a]P absorption in roots, stems, leaves and shoots of the tested plants and the concentration of B[a]P in soils (Po 0.01). Meanwhile, the contents of B[a]P in different tissues of the plants increased with growing time. After planting T. patula and M. jalapa, plant-promoted biodegradation of B[a]P was account for 79.5–99.8% and 71.1– 99.9%, respectively, whereas the amount of B[a]P dissipation enhancement was only 0.2–20.5% and 0.1– 28.9%, respectively. Moreover, low bioaccumulation factor (BF) and translocation factor (TF) values indicated that T. patula and M. jalapa took up B[a]P from contaminated soil and transferred them to the aerial parts with low efficiency. The B[a]P removal rates in rhizosphere soils at different growing stages of T. patula and M. jalapa were 2.7–26.8% and 0.4%–33.9%, respectively, higher than those of non-rhizopshere soils. Therefore, the presence of T. patula and M. jalapa roots was effective in promoting the phytoremediation of B[a]P contaminated soils. & 2015 Elsevier Inc. All rights reserved.
Keywords: Phytoremediation Benzo[a]P (B[a]P) Uptake Distribution Dissipation
1. Introduction Benzo[a]Pyrene (B[a]P) is a popularly environmental pollutant emanating from industrial processes (e.g., exhaust fumes and combustion of all forms of fossil fuels, processing production and spillage of petroleum) and during household activities (e.g., cooking, barbequing, and smoking) (Sinha et al., 2005; Sun et al., 2013). B[a]P is a carcinogen known to induce tumors in a number of organs in animal models and humans. One of its metabolites is known to form DNA adducts which are responsible for mutagenesis and tumorigenesi (Uno et al. 2001; Gong et al., 2006). There are indications that B[a]P diol epoxide specifically targets the protective p53 gene, which is a transcription factor that regulates the cell cycle and hence functions as a tumor suppressor (Pfeifer et al., 2002). Immunocytochemistry and Western immunoblot analysis showed that B[a]P produced a marked loss of plasma n Corresponding author at: Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education), College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China. Fax: þ 86 22 23501117. E-mail addresses: [email protected] (Y. Sun), [email protected] (Q. Zhou).
http://dx.doi.org/10.1016/j.ecoenv.2015.09.037 0147-6513/& 2015 Elsevier Inc. All rights reserved.
membrane epidermal growth factor receptor (EGF-R) localized along intercellular boundaries and significantly decreased in βcatenin and cadherin protein levels (Mcgarry et al., 2002). Hence, B [a]P has been classified as a toxic and priority-controlled contaminant in the list of USEPA and European Union (Ko-Wasik et al., 2004; Dixit et al., 2011), and it is necessary to look for effective methods of remediation. However, the major limitations of remediation for B[a]P contaminated soil have been the requirement for long degradation periods due to its poor water-solubility and the difficulty in controlling the conditions, thus precluding the reclaiming of natural soil amendments (Singh and Jain, 2003; Dixit et al., 2011). As an option in certain situations, phytoremediation is gaining popularity for its low cost and environmental friendly alternative for decontamination of the contaminated soil (Zhou and Song, 2004; Zhang et al., 2012; Sun et al., 2013). Disappearance of polycyclic aromatic hydrocarbons (PAHs) and Polychlorinated biphenyls (PCBs) in those researches was found to be higher in planted soil (Johnson et al., 2004; Lu et al., 2010; Phillips et al., 2012; Wei et al., 2014). The mechanisms of phytoremediation for organic pollutants include biophysical and biochemical processes like adsorption, transport and translocation, as well as transformation and
Y. Sun, Q. Zhou / Ecotoxicology and Environmental Safety 124 (2016) 74–81
mineralization by plant enzymes (Perelo, 2010; Gao et al., 2010). It was assumed that plants enhanced biodegradation of organic matters by stimulating the growth of soil microbes due to the interactions between root exudates, microbes and contaminants that stimulated microbial activities, enzyme-catalyzed processes or cometabolic processes in rhizosphere (Gao et al., 2010). Lee et al. (2008) have reported 499% and 77–94% of phenanthrene (Phe) and pyrene (Pyr), respectively, had been degraded in planted soil, whereas 99% and 69% had been degraded in unplanted soil. 91.7–97.8% of Phe and 70.8–90.0% of Pyr had degraded in the planted soils, which were 1.9–3.2% and 8.9–20.7% larger than those in corresponding unplanted soils (Cheema et al., 2009). The objectives of the present work were: (1) to investigate the B[a]P uptake and translocation in two ornamental plants, and (2) to determine the mechanism of B[a]P phytodegradation and dissipation. Results obtained from this study are expected to provide some insight with regard to the feasibility of plant-enhanced phytoremediation for B[a]P contaminated soils. Tagetes patula and Mirabilis jalapa are selected for the experiment because of its extensive, widely branched root system providing a large root surface for the growth of microbial populations, which can strongly endure and effectively phytodegrade hydrocarbons (Peng et al., 2009; Sun et al., 2011; Zhou et al., 2012).
2. Materials and methods 2.1. Soil characterization The tested soil was collected from agricultural fields in the Shenyang Ecological Experiment Station, Chinese Academy of Sciences (Lat. 123°41′, and Lon. 41°31′). This soil is meadow burozem, and chemical analysis showed that total C, total N, total P, total K and pH were 1.6%, 0.11%, 0.04%, 0.60% and 6.50, respectively. The initial concentration of B[a]P was non-detectable. Surface (0–20 cm) soil samples which were ground to pass through a 4 mm mesh were used in the pot-culture experiment. 2.2. Experimental procedure According to the Environmental quality standard for soil environment in China (GB 4284-84), the soils should be remedied when B[a]P concentration was up to 3 mg kg 1. thus, in this study single B[a]P treatments were CK, 2, 5, 10 and 50 mg kg 1, respectively, as simulating non-, low-, medium- and serious-B[a]P contamination. acetone solution containing B[a]P was mixed with 200 g soil as a portion, then mixed with 800 g soil when acetone volatilized completely. B[a]P (purity 4 97%) was obtained from sigma chemical CO., USA. Seeds of T. patula and M. jalapa were sterilized in 2% (v/v) hydrogen peroxide for 15 min, washed with tap water and soaked in water 1 d. 3 seedlings of T. patula and M. jalapa with a similar size, about 4 wk old, and 6–8 cm height with 4–6 leaves were transplanted in each pot. The soil surface was covered with a layer of silica sand (75 g) to minimize B[a]P volatilization (Sun et al., 2011). The position of pots was changed randomly every week in order to eliminate the non-uniform illumination. Loss of water was made up using tap water to reach 75% of the field water-holding capacity and maintained this humidity by daily watering throughout the cultivation, and a petri dish was placed under each pot to collect potential leachate during the experiment. After growing for 30, 60 and 92 days, namely, the seedling phase, flowering and mature phases were used for evaluating characteristics of plant growth and B[a]P uptake and dissipation. The plant samples were washed with deionized water, and separated into roots, stems and leaves. After drying with filter paper, the sub-samples were freezed-dried
75
(at 530717 Pa and 5072 °C, BYK FD-1), and weighed to determine the biomass. The soil samples were air dried at room temperature and ground sufficiently to pass through a 100-mesh sieve. 2.3. Extraction and analysis of PAHs in soils and plants 2.0 g of dry soil samples were extracted using 20 mL of dichloromethane for 2 h in an ultrasonic bath (the system was o40 °C). 0.5 g of dried plant fragment was mixed into 10 mL solution of methanol, and extracted by ultrasonication for 30 min for 3 successive extractions (Fan et al., 2008). Then, 2 mL of the supernatant was filtered through 2 g of silica gel column and eluted consecutively with 10 mL mixture of 1:1 of hexane and dichloromethane (v/v). The extract was concentrated by evaporation of the dichloromethane under a stream of nitrogen and the residue was dissolved in hexane with a final volume of 1.0 mL for gas chromatography (GC) analysis. Samples extracts (1 μL) were analyzed by a Hewlett-Packard (HP) 6890 GC system equipped with a HP 5973 mass selective detector and a DB-5 capillary column (30 mm 0.25 mm 0.25 μm). The operation was following steps: (1) the initial oven temperature was 80 °C and held time for 1 min, (2) the oven temperature was increased to 275 °C at 15 min 1 and held for 1 min, (3) the oven was set to 285 °C at 10 min 1, and held for 1 min, and (4) the oven temperature was increased to 295 °C at 5 min 1, and held for 1 min. Nitrogen was used as the carrier gas (1.5 mL min 1) and makeup gas (35 mL min 1). A 1.0 μL aliquot of the extract was injected in the splitless mode. The injector was held at 250 °C and the detector at 300 °C. 2.4. Quality control Experiments on PAH recovery were carried out by spiking a known concentration of PAH standards (1 mg kg 1) in an uncontaminated soil and a plant. The results showed satisfactory recovery, and ranged from 94.5 77.1% to 101.978.7% for soils and plants, respectively. The limit of detection (LOD) of B[a]P in soil and plants was the same of 0.08 μg kg 1. 2.5. Statistical analysis All treatments were replicated three times in the experiments. The means and standard deviations (SD) were calculated by the Microsoft Office Excel 2003. One-way analysis of variance was carried out with SPSS10.0. When a significant (Po0.05 or Po 0.01) difference was observed between treatments, multiple comparisons were made by the LSD test.
3. Results 3.1. Plant biomass The shoot biomass of T. patula and M. jalapa on a dry weight basis is shown in Fig. 1. The application of B[a]P could enhance the growth and production of T. patula seedling under low B[a]P levels of 2 and 5 mg kg 1, resulting in 39.0% and 13.1% increase, respectively, in contrast with the control group. The similar trend of shoot biomass was found at flowing and mature stage, which increased with increasing of r10 B[a]P kg 1 in soil, and reached the maximum of 9.4 and 9.5 g pot 1, respectively, at B[a]P of 5 mg kg 1. However, the shoot biomass observed was significantly decreased when the B[a]P in soils was up to 50 mg kg 1 (Po0.05), suffering by 29.2%, 36.3% and 18.4% reduction, respectively, at the seedling, flowering and mature stage when compared with CK.
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Y. Sun, Q. Zhou / Ecotoxicology and Environmental Safety 124 (2016) 74–81
Dry weight (mg pot-1)
12 10
T. patula
a
a
a
a
seedling stage flowering stage mature stage a
ab
b
bc
8
c
6 4
Table 2 Relationships between B[a]P concentration in different tissues of tested plants and soil amended with B[a]P. Plants
Growth stage
T. patula
Seedling stage
b a
Flowering stage
ab
ab
ab b
2
Mature stage
Regression equation
R2
P
Root Shoot Root Shoot Root Shoot
Y ¼3.59X 5.81 Y ¼2.62X 4.16 Y ¼4.80X 7.39 Y ¼2.49X 2.96 Y ¼5.26X 7.75 Y ¼3.15X 3.92
0.83 0.78 0.84 0.98 0.83 0.97
0.01 0.01 0.01 0.01 0.01 0.01
Root Shoot Root Shoot Root Shoot
Y ¼1.70X 1.57 Y ¼1.18X 1.23 Y ¼8.43X 12.41 Y ¼3.05X 3.78 Y ¼9.66X 13.49 Y ¼3.97X 4.69
0.88 0.97 0.90 0.97 0.94 0.95
0.01 0.01 0.01 0.01 0.01 0.01
0
Dry weight (mg pot-1)
6
M. jalapa
a
5
3
M. jalap
Mature stage
b a
a
Seedling stage Flowering stage
a
b a
4
seedling stage flowering stage mature stage
a
c b
c a
c b a
2
3.2. B[a]P uptake and absorption in the plants
1
Table 1 exhibits the B[a]P uptake and partitioning in the different tissues of T. patula and M. jalapa. Across the 0–50 mg kg 1 B [a]P treatments, the concentrations of B[a]P in different tissues increased with increasing in soil B[a]P at the same growth stage, and according to these corresponding regression equations listed in Table 2. It displayed a markedly positive linear relationship between B[a]P uptake in roots, stems, leaves and shoots of the tested plants and the concentration of B[a]P in soils (P o0.01). Meanwhile, the contents of B[a]P in different tissues of the plants increased with growing time, and the B[a]P concentrations in roots, stems, leaves and shoots at mature stage were 1.08–1.41, 1.02–1.42, 0.36–1.49 and 0.60–1.38, respectively, times higher than those of flowering stage, and 1.79–5.49, 2.24–6.38, 0.89–3.06, and 1.29–3.46, respectively, times more than those of seedling stage for M. jalapa, and 1.08–1.31, 1.03–1.33, 1.11–1.43 and 1.10–1.27, respectively, times higher than those of flowering stage, and 1.47– 2.64, 1.14–4.91, 1.11–1.65, and 1.10–2.47, respectively, times more than those of seedling stage for T. patula. The concentration of B[a] P in tissues of the two plants on a whole was in the sequence of root4leaf 4stem and root4shoot, indicating the weak ability of B[a]P uptake and translocation by T. patula and M. jalapa. Fig. 2 shows the amount of B[a]P accumulated in the tissues of T. patula and M. jalapa. The concentrations of B[a]P in roots and
0
CK
2 5 10 B[a]P concentration (mg kg-1)
50
Fig. 1. Dry biomass of T. patula and M. jalapa grown in treatments with different B [a]P concentrations. Note: Letters above the bar diagram refer to the difference at significances level of 0.05 (LSD test).
The tolerance capability of M. jalapa to B[a]P contamination was slightly weaker than that of T. patula. Under the dose of 2 mg kg 1 B [a]P, the biomass of plants was enhanced at certain degree, resulting in 8.7%, 26.9% and 3.4% increase, respectively, at seedling, flowering and mature stage in comparison with unamended soil, and the significant increase in dry weight was got at flowering stage (Po0.05). However, it exhibited a inhibitory effect on plant biomass and decreased with increasing of the concentration of o2 mg kg 1 B[a]P in soils. In particular, at the flowering and mature stage, the dry weight of M. jalapa under the contamination of B[a]P (5–50 mg kg 1) was significantly lower than those of control group (Po0.05). Across the whole growing stage, the tolerant capability to B[a]P contamination was increased with time growing. Table 1 B[a]P concentrations in the different tissues of T. patula and M. jalapa. Growth stage
B[a]P treatments (mg kg 1)
T. patula Root
M. jalapa Stem
Leaf
Shoot
Root
Stem
Leaf
Shoot
Seedling stage
CK 2 5 10 50
ND 0.69 7 0.19 2.85 7 1.09 5.87 7 0.15 15.3 7 0.12
ND 0.497 0.70 1.22 7 0.26 3.09 7 2.02 10.3 7 1.58
ND 1.137 1.13 3.337 0.52 4.46 7 0.74 12.4 7 3.13
ND 0.83 7 0.92 2.277 0.01 3.69 7 0.75 11.7 7 1.20
ND 0.89 7 0.18 5.147 0.33 5.377 0.27 6.26 7 0.70
ND 0.54 7 0.08 1.177 0.20 1.477 0.16 2.53 7 0.18
ND 1.03 7 0.32 3.687 0.14 4.40 7 0.40 5.98 7 1.34
ND 0.84 70.16 2.7770.09 3.2470.11 4.69 70.95
Flower stage
CK 2 5 10 50
ND 1.39 7 0.05 5.157 0.90 7.727 0.17 20.9 7 0.81
ND 1.54 7 0.08 5.81 7 0.59 7.077 0.49 8.89 7 0.22
0.0027 0.001 1.487 0.12 3.63 7 0.28 5.62 7 0.08 11.17 0.23
0.0017 0.001 1.517 0.03 4.727 0.15 6.277 0.21 10.17 0.02
ND 3.95 7 0.26 6.53 7 0.03 19.5 7 9.92 34.4 7 3.79
ND 1.017 0.14 6.08 7 0.13 6.63 7 0.64 11.2 7 2.07
0.0017 0.001 2.54 7 0.42 7.58 7 0.12 9.247 0.01 13.0 7 1.96
0.001 70.001 1.79 70.17 6.87 70.02 8.12 70.29 12.1 72.30
Mature stage
CK 2 5 10 50
ND 1.83 7 0.17 6.46 7 0.45 9.247 1.04 22.6 7 1.98
0.0037 0.001 5.687 6.09 5.977 0.79 7.75 7 0.01 11.8 7 0.39
0.0027 0.002 4.647 4.51 5.197 0.75 7.34 7 0.41 13.8 7 1.47
0.0037 0.002 1.677 0.35 5.59 7 0.80 7.55 7 0.20 12.8 7 0.89
ND 4.417 0.74 9.187 0.49 26.717 3.65 37.2 7 2.51
0.0037 0.002 1.217 0.54 6.22 7 0.85 9.39 7 1.72 14.17 0.44
0.0017 0.001 0.92 7 0.31 11.3 7 1.68 13.17 0.22 15.2 7 0.23
0.002 70.001 1.08 70.14 8.98 70.41 11.2 70.91 14.8 70.39
6
0
0
60 18 40 12 20 6
0
Root B[a]P accumu
lation (µg pot -1 )
48 40
40 16 20
0
0 CK
2
5
10
200
36
150
27
100
18
50
9
0
0
200
60
8
45
80
24
50
-1
B[a]P treatment mg kg
0
250
250
32
2
3
100
Root B[a]P accumulation (µg kg -1 )
0
Root B[a]P accumulation (µg kg -1 )
80
24
4 6
0
Shoot B[a]P accumulation (µg pot -1 )
Root B[a]P accumulation (µg pot -1 )
30
6 9
Shoot B[a]P accumulation (µg kg -1 )
3
12
Shoot B[a]P accumulation (µg kg -1 )
12
8
60
)
6
15
-1
18
10
45 150 30 100 15 50
Shoot B[a]P accumulation (µg kg
9
77
18
Root B[a]P accumulation (µg kg -1 )
24
Shoot B[a]P accumulation (µg pot -1 )
12
Shoot B[a]P accumulation (µg pot -1 )
Root B[a] accumulation (µg pot -1 )
Y. Sun, Q. Zhou / Ecotoxicology and Environmental Safety 124 (2016) 74–81
0
0 CK
2
5
10
50
-1
B[a]P treatment (mg kg )
Fig. 2. B[a]P accumulation in T. patula and M. jalapa under different B[a]P treatments. Note: a–c refers seedling stage, flowering stage and mature stage of T. patula, respectively, and d–f refers seedling stage, flowering stage and mature stage of M. jalapa, respectively.
shoots of the plants steadily and strikingly increased with increasing soil B[a]P and the time of plant growth. However, the ability of B[a]P uptake and translocation by the two plants was weak, the highest B[a]P uptake by shoots of T. patula and M. jalapa was only 70.61 and 41.42 mg kg 1, respectively. Under B[a]P treated with 2–50 mg kg 1, most of B[a]P taken up was retained in the roots of M. jalapa, and 50.7–68.3%, 58.9–78.3% 75.8–79.0%, respectively, of B[a]P absorption by the plant was concentrated in its roots at seedling, flowering and mature stage. By contrast, the dominating B[a]P uptake by T. patula was in the shoots, up to 60.6– 78.9%, 71.0–86.6% and 70.1–86.7%, respectively, at seedling, flowering and mature stage. The amount of B[a]P accumulated in roots of M. jalapa, at seedling, flowering and mature stage was 1.49–3.07, 4.04–6.54 and 2.70–8.16, respectively, times higher than those of T. patula. In contrast, the accumulation of B[a]P in shoots of M. jalapa, at seedling, flowering and mature stage was 44.9–86.1%, 43.0– 71.1% and 32.5–60.4%, respectively, lower than those of T. patula. 3.3. B[a]P dissipation in rhizosphere and non-rhizosphere The dissipation rate of B[a]P in the soils of rhizosphere and non-rhizosphere were shown in Fig. 3. The residual B[a]P in the
soils were all significantly decreased after planting T. patula and M. jalapa, and the removal rate increased with growing time (P o0.05). The highest removal rates at seedling, flowering and mature stage was 74.3%, 90.2% and 92.4%, respectively, in rhizosphere soil, and 63.6%, 85.0% and 88.2%, respectively, in non-rhizosphere soil for T. patula, and 71.1%, 87.5% and 87.9%, respectively, in rhizosphere soil, and 67.8%, 87.5% and 83.1%, respectively, in non-rhizosphere soil for M. jalapa. The B[a]P removal rate in rhizosphere was higher than those of non-rhizosphere at seedling, flowering and mature stage, which was experienced by 10.8– 19.3%, 4.3–11.5% and 4.2–15.7%, respectively, higher than those of non-rhizosphere for T. patula, and 3.3–21.3%, 2.6–11.0% and 4.8– 20.2%, respectively, higher than those of non-rhizosphere for M. jalapa. However, the dissipation rate of B[a]P was generally inhibited with an increasing soil B[a]P content, which reached 37.9– 66.1% and 57.2–79.2%, and 41.2–52.4% and 21.7–38.4%, respectively, in rhizsphere and non-rhizosphere soils of different growth stage of T. patula and M. jalapa. The removal rates in rhizsphere soils at seedling, flowering and mature stage of T. patula were 3.2–16.0%, 2.7–25.6%, and 4.2–26.8%, respectively, higher than those of M. jalapa. By contrast, the decreases of 0.4–16.1%, 7.3–24.7%, and 14.2–33.9% in B[a]P dissipation rates in non-rhizosphere soils of T.
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Y. Sun, Q. Zhou / Ecotoxicology and Environmental Safety 124 (2016) 74–81
et al. (2009), the B[a]P content of rice grain, husk, and stem with leaf sampled from outdoor field was up to 7.33-, 9.21- and 27.10fold, respectively, higher than corresponding tissues, suggesting that polluted air was the main source of B[a]P in aboveground tissues (Li et al., 2009). Liu and Schnoor also found a small amount of PCBs (0.01–0.9 mg kg 1) were detected in the main stems of blank controls (Liu and Schnoor, 2008). This is, in some way, consistent with the observations on phenanthrene (Phe) and pyrene (Pyr) absorption by plant roots by Gao and Zhu (2004) and Xu et al. (2005). Despite B[a]P uptake and accumulation in the shoots might come from air, the translocation of B[a]P from roots to shoots was significant (Sun et al., 2011). Several literatures have pointed out that the most of PAH accumulation in the shoots was transferred from the roots (Gao and Zhu, 2004; Xu et al., 2005). It was found that an average 90.2% of Pyr and 74.3% of Phe in the shoots of ryegrass derived from the roots, and only 9.9% of Pyr and 25.7% of Phe in shoots was from the atmosphere (Xu et al., 2005). Schroll et al. (1994) demonstrated that about 22–95% of Phe and 32–96% of Pyr in the shoots were translocated from the roots. Translocation factor (TF), the quotient of contaminant concentration in shoots to roots (Zhou and Song, 2004; Gao and Zhu, 2004), and bioaccumulation factor (BF), the ratio of chemical concentration in a plant to soil (Sun et al., 2011), was used to evaluate the effectiveness of a plant in transferring a chemical from soil to roots and from roots to aerial part (Sun et al., 2008). As listed in Table 3, the BF values in T. patula and M. jalapa fist increased and then decreased with the increase of soil B[a]P concentrations, and its exhibited mature stage4flowering stage4 seedling stage, showing a significant B[a]P uptake in shoots of the two plants with growing time. In contrast, TFs in T. patula decreased with increasing of B[a]P applied to soil. In other words, the relationship between the TF values and soil B[a]P contents was negative and logarithmic linear, indicating a diminishing efficiency of B[a]P accumulation with increasing soil B[a]P concentrations (Zhou et al., 2006). However, there were no regular trends in TFs in M. jalapa under different treatments. The BF and TF value in most treatments was o1.0, low BFs and TFs indicated that T. patula and M. jalapa took up B[a]P from soil and transferred them to the aboveground parts with low efficiency (Sun et al., 2011). It was observed that concentrations of B[a]P in roots of two plants were higher than those in aerial parts (stems, leaves and shoots) (Table 1). Numerous studies have suggested that lipophilic organic contaminant entering plant's root from soil rely on the Kow (Simonich and Hites, 1995; Xu et al., 2005; Gao et al., 2008; Su, Zhu, 2005). In general, the higher the Kow of a pollutant, the greater would be the expectancy to partition from the soil organic matter, and thus the lower would be transported to plant aerial tissue. It was reported that the uptake of PAHs into plant roots from contaminated soils was directly proportional to Kow (Gao et al., 2008; Su, Zhu, 2005). Gao et al. (2008) found that the root concentration factors of Pye were generally a little larger or equal to those of Phe, which was about consistent with their log Kow.
B[aP]=2
B[a]P removal rate (%)
B[aP]=5 B[a]P=10 B[a]P=50
non-rhizosphere rhizosphere
non-rhizosphere
non-rhizosphere rhizosphere
rhizosphere
re
Seedling stage
Flower stage
Mature stage
B[a]P=2 B[a]P=5
B[a]P removal rate (%)
B[a]P=10 B[a]P=50
non-rhizosphere rhizosphere
Seedling stage
non-rhizosphere
non-rhizosphere rhizosphere
rhizosphere
Flower stage
Mature stage
Fig. 3. Removal rate of B[a]P in rhizosphere and non-rhizosphere. Note: (a) and (b) refers T. patula M. jalapa, respectively.
patula were exhibited than those of M. jalapa.
4. Discussion 4.1. B[a]P absorption and translocation in plants As listed in Table 1, it is notable that under the corresponding experiments the concentrations of B[a]P in different parts of T. patula and M. jalapa at seedling stage were under the detect limit, whereas it was found in aerial parts of two tested plants with the time growing. At flowering stage, B[a]P were found in leaves and shoots, which were 0.002 and 0.001 mg kg 1, respectively, for T. patula and 0.001 and 0.001 mg kg 1, respectively for M. jalapa. When growing to mature stage, B[a]P was detected in aerial parts (stems, leaves and shoots) of T. patula and M. jalapa under unspiked soils from 0.002 to 0.003 mg kg 1 and from 0.001 to 0.003 mg kg 1, respectively. It indicated that the absorption of B [a]P in above-ground parts of plants from ambient air probably through the retention of vapor phase of B[a]P on the waxy leaf cuticle (Simonich and Hites, 1994; Gao and Zhu, 2004). An airquality controlled greenhouse experiments were conducted by Li
Table 3 The bioaccumulation factor and transfer factor of B[a]P in T. patula and M. jalapa under different B[a]P treatments. B[a]P concentration (mg kg 1)
2 5 10 50
T. patula
M. jalapa
Seedling stage
Flower stage
Mature stage
Seedling stage
Flower stage
Mature stage
BF
TF
BF
TF
BF
TF
BF
TF
BF
TF
BF
TF
0.42 0.45 0.37 0.23
1.20 0.79 0.63 0.76
0.76 0.94 0.63 0.20
1.09 0.92 0.81 0.48
0.83 1.12 0.75 0.26
0.91 0.86 0.82 0.57
0.42 0.55 0.32 0.09
0.94 0.54 0.60 0.75
0.90 1.37 0.81 0.24
0.45 1.05 0.42 0.35
0.54 1.80 1.12 0.30
0.24 0.98 0.42 0.40
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79
Table 4 Contribution of T. patula and M. jalapa to the removal of B[a]P in soils. Growth stage
Treatments (mg kg 1)
T. patula
M. jalapa
Td (mg pot 1)
Pac (mg pot 1)
Seedling stage
2 5 10 50
0.22 0.52 1.33 9.69
2.46 6.15 7.90 19.6
Flower stage
2 5 10 50
0.20 0.26 1.01 5.82
Mature stage
2 5 10 50
0.28 0.26 1.62 6.65
Pac/Td (%)
Td (mg pot 1t)
Pac (mg pot 1)
Pac/Td (%)
1.12 1.18 0.59 0.20
0.07 0.34 2.14 9.76
1.96 5.05 6.81 8.82
2.80 1.49 0.32 0.09
12.6 44.2 52.6 49.2
6.28 17.0 5.21 0.84
0.06 0.31 1.13 5.37
8.47 19.3 22.6 35.0
14.1 6.23 2.00 0.65
13.6 53.2 55.8 70.6
4.86 20.5 3.44 1.06
0.10 0.09 1.29 10.2
4.43 26.0 33.7 41.4
4.43 28.9 2.61 0.41
Moreover, these phenomena showed that the accumulation of PAHs in the roots was influenced by hydrophobic compositions of plant roots, such as specific surface area and lipid (Li et al., 2005; Su, Zhu, 2005; Yin et al., 2014). It was also found in this study that there was a significant positive linear correlation between B[a]P concentrations in roots and shoots of T. patula and M. jalapa under different growing stage and B[a]P spiked in soils (P o0.001) (Table 2), which was consistent with Borneff et al. (1973), who pointed out that the concentrations of B[a]P in carrot epidermis was correlated with soil B[a]P. Based on the results of Gao and Zhu (2004), the accumulation of Phe and Pye in the roots or shoots of 12 plant species was elevated with the increase of their soil concentrations. In the present study, the B[a]P concentrations and BFs in most case in M. jalapa were higher than those of T. patula, and while it was just the opposite to that of the TF values. The ability of B[a]P uptake and accumulation would be distinctive different among species due to their different activities of H þ -ATPase and affinities of transporter (Yin et al., 2014). It has been observed that PAHs are actively transported into the cells via H þ -coupled symporters and transporters in various plants have different affinities to PAHs (Zhang et al., 2012). The higher the depolarization of membrane potential, the stronger the capacity of PAHs uptake were (Yin et al., 2014). 4.2. Plant-enhanced phytoremediation of soil B[a]P The mechanisms of the dissipation of PAHs in rhizosphere soils involve volatilization, leaching, biodegradation, plant uptake and accumulation, and incorporation of the pollutant into soil organic material (Gao and Zhu, 2004; Sun et al., 2011). Whereas, the dissipation of B[a]P in non-rhizosphere soils was the sum of leaching, abiotic dissipation, biodegradation, and incorporation of the pollutant into soil organic material. In contrast, Thus the loss of B[a]P in vegetated soils could be calculated as follows (Gao and Zhu, 2004):
Trs = Tl + Ta + Tb + Pac
(1)
Tnrs = Tl + Ta + Tb ’
(2)
where Trs and Tnrs were the dissipation of B[a]P in rhizosphere and non-rhizosphere soils (mg pot 1). Tl and Ta denoted the dissipation by leaching and abiotic dissipation. Tb and Tb′ were the loss by biodegradation in rhizosphere and non-rhizosphere soils, respectively. Pac refers B[a]P uptake and accumulation in plants. Reilley
et al. (1996) has noticed that 4–5 rings of PAHs in leachate were undetectable from soils with or without plants. Moreover, the phytovolatilization of such organic compounds was negligible, and plant metabolism was not significant (Trapp et al., 1990). So the dissipation of PAHs would overwhelmingly derive of plant accumulation and promoted biodegradation. The corresponding equations can be expressed as
Trs = Tb + Pac
(3)
Tnrs = Tb ’
(4)
Therefore, the dissipation enhancement (Td) of B[a]P in rhizosphere versus non-rhizosphere soils was
Td = Trs –Tnrs = Pac + Tb –Tb ’
(5)
Tbp = Tb –Tb ’
(6)
In Eq. (6), Tpb refers the loss of B[a]P by the plant-promoted biodegradation. As listed in Table 4, there was a significant increasing of the B[a]P amounts of plant uptake and plant-promoted biodegradation under gradient B[a]P concentration in soil at different growing stages (Po0.05) and the accumulation of B[a]P in T. patula and M. jalapa increased with the increasing growth time, showing mature stage4flowering stage4seedling stage. However, at the same treatments of B[a]P, there exhibited a slight difference in plantenhanced biodegradation of B[a]P at different growing stages. Furthermore, MacLeod and Semple (2000) revealed that the extent of plant assisted PAH removal from soil decreased with soil incubation time. Aged PAHs are found to less microbially bioavailable due to an increased binding to soil organic matter (Nam and Kim, 2002). The dominant of B[a]P dissipation was contributed to plant-enhanced biodegradation, accounting for 79.54–99.78% and 71.1–99.9%, respectively, after planting T. patula and M. jalapa. By contrast, the amount of Tac was 0.2–20.5% and 0.1–28.9% of dissipation enhancement for B[a]P after planting T. patula and M. jalapa. The results above were consistent with Gao and Zhu (2004), who found that larger than 99.7% dissipation enhancement of Phe and Pyr in planted soils came from plant-promoted biodegradation. 4.3. Rhizosphere effects on B[a]P removal Microbial degradation in the rhizosphere might be the most significant mechanism for removal of organic contaminants in vegetated contaminated soil (Gao and Zhu, 2004, Zhou and Song,
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2004). Deposition of plant-derived carbon and nitrogen sources through root exudation, and/or root turnover provides rhizosphere bacteria with numerous organic substrates (Kamath et al., 2007). Therefore, the rhizosphere of plants constitutes a heterogeneous environment with numerous fluctuating parameters that can contribute significantly to the biodegradation of PAHs during phytoremediation processes such as an increase in microbial community structure and functional diversity (Davey and O’toole, 2000; Liu et al., 2014), a promotion in catabolic enzymes (Huang et al. 2005; Cai et al., 2010; Liu et al., 2014), a modification in bioavailability (Kamath et al., 2007), and an induction of co-oxidation of contaminants (Trapp and Karlson, 2001; Li et al., 2008). The removal ratio of B[a]P ranged from 41.2–71.1%, 49.0–87.5% and 52.4–87.9%, respectively, in rhizospher soils, and 21.7–67.8%, 38.4– 85.0% and 32.2–83.1%, respectively, in non-rhizosphere soils at seedling, flowering and mature stage of M. jalapa. T. patula has hairy roots which can provide a very good environment for detoxification of xenobiotics. The B[a]P removal percentages at different growing stages of T. patula were 3.2–16.0%, 2.7–25.6% and 4.1–26.8%, respectively, under 2–50 mg kg 1 B[a]P contaminated rhizosphere soils, and 0.4–16.2%, 7.2–24.7% and 14.2–33.9%, respectively, under 5–50 mg kg 1 B[a]P treated non-rhizopshere soils higher than those of M. jalapa. Under seedling, flowering and mature stage, the removal ratio values of B[a]P in rhizosphere were 3.3–21.3%, 2.5–11.0% and 1.3–20.2%, respectively, for M. jalapa, and 10.3–19.3%, 4.3–11.5% and 4.2–15.7%, respectively, for T. patula higher than those of in non-rhizosphere, indicating that plants play an important role in removing B[a]P from contaminated soils. It has been found that microbial populations in rhizosphere soils can be 10–100 times higher than those in the surrounding bulk soils and have greater xenobiotic degrading capabilities (Fan et al., 2008). Lee et al. (2008) have reported 499% and 77–94% of Phe and Pyr, respectively, had been degraded in planted soil, whereas 99% and 69% had been degraded in unplanted soil. 91.7–97.8% of Phe and 70.8–90.0% of Pyr had degraded in the planted soils, which were 1.9–3.2% and 8.9–20.7% larger than those in corresponding unplanted soils (Cheema et al., 2009). However, the removal ratio of B[a]P in rhizosphere and non-rhizosphere soils at different growing stages of M. jalapa and T. patula decreased with the increasing of soil B[a]P concentrations. Similar trend on Pye has also been reported by Fan et al. (2008), and the possible occurrence might be due to either inactivation of soil enzymatic activities or on account of limiting microbial communities under B[a]P stress (Phllips et al., 2012).
5. Conclusions The present study showed that the tolerant capability at flowering and mature stage was greater than that of seedling stage, the dry weight of M. jalapa and T. patula increased firstly and then gradually inhibited with increasing B[a]P. Under B[a]P contamination of 0–50 mg kg 1, the concentration and accumulation of B[a]P in all parts of plants were elevated with the increment of their soil concentrations and plant growing time, and significantly positive correlations were exhibited between B[a]P contents in roots, stems, leaves and shoots and soil B[a]P concentrations (P o0.05). The dominant of B[a]P dissipation was contributed to plant-enhanced biodegradation, accounting for 79.5–99.8% and 71.1–99.9%, respectively, after planting T. patula and M. jalapa. Furthermore, the B[a]P removal percentages in rhizosphere soils at different growing stages of T. patula and M. jalapa were 2.7–26.8% and 0.4–33.9%, respectively, higher than those of non-rhizopshere soils. Therefore, it supports the notion that T. patula and M. jalapa can be using for phytoremediation of B[a]P-contaminated soil.
Acknowledgments This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201203045) and the National Natural Science Foundation of China (21177068; 41401362).
References Borneff, J., Farkazdi, G., Glathe, H., Kunte, H., 1973. The fate of polycyclic aromatic hydrocarbons in experiments using sewage sludge–garbage composts as fertilizers. Zentralbl Bakteriol Mikrobiol Hyg [B] 157, 151–164. Cai, Z., Zhou, Q.X., Peng, S.W., Li, K.N., 2010. Promoted biodegradation and microbiological effects of petroleum hydrocarbons by Impatiens balsamina L. with strong endurance. J. Hazard. Mater. 183, 731–737. Cheema, S.A., Khan, M.I., Tang, X.J., Zhang, C.K., Shen, C.F., Malik, Z., Shen, K.L., Chen, X.C., Chen, Y.X., 2009. Enhancement of phenanthrene and pyrene degradation in rhizosphere of Tall Fescue (Festuca arundinacea). J. Hazard. Mater. 166, 1226–1231. Davey, M.E., O’toole, G.A., 2000. Microbial Biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64, 847–867. Dixit, P., Mukherjee, P.K., Sherkhane, P.D., Kale, S.P., Eapen, S., 2011. Enhanced tolerance and remediation of anthracene by transgenic tobacco plants expressing a fungal glutathione transferase gene. J. Hazard. Mater. 192, 270–276. Fan, S.X., Li, P.J., Gong, Z.Q., Ren, W.X., He, N., 2008. Promotion of pyrene degradation in rhizosphere of Alfalfa (Medicago sativa L.). Chemosphere 71, 1593–1598. Gao, Y., Wu, S.C., Yu, X.Z., Wong, M.H., 2010. Dissipation gradients of phenanthrene and pyrene in the rice rhizosphere. Environ. Pollut. 158, 2596–2603. Gao, Y.Z., Shen, Q., Ling, W.T., Ren, L.L., 2008. Uptake of polycyclic aromatic hydrocarbons by Trifolium pretense L. from water in the presence of a nonionic surfactant. Chemosphere 72, 636–643. Gao, Y.Z., Zhu, L.Z., 2004. Plant uptake, accumulation and translocation of phenanthrene and pyrene in soils. Chemosphere 55, 1169–1178. Gong, Z., Yang, J., Yang, M., Wang, F.M., Wei, Q., Tanguay, R.M., Wu, T., 2006. Benzo (a)pyrene inhibits expression of inducible heat shock protein 70 in vascular endothelial cells. Toxicol. Lett. 166, 229–236. Huang, X.D., Ei–Alawi, Y., Gurska, J., Glick, B.R., Greenberg, B.M., 2005. A multiprocess phytoremediation system for decontamination of persistent total petroleum hydrocarbons (TPHs) from soils. Microchem. J. 81, 139–147. Johnson, D.L., Maguire, K.L., Anderson, D.R., McGragh, S.P., 2004. Enhanced dissipation of chrysene in planted soil: the impact of a rhizobial inoculum. Soil Biol. Biochem. 36, 33–38. Kamath, R., Rentz, J.A., Schnoor, J.L., Alvarez, P.J.J., 2007. Phytoremediation of Hydrocarboncontaminated Soils; Principles and Applications. University of Iowa, Iowa City, Iowa, USA, p. 52242. Ko-Wasik, A., Da˛browska, D., Namiesnik, J., 2004. Photodegradation and biodegradation study of benzo(a)pyrene in different liquid media. J. Photochem. Photobiol. A: Chem. 168, 109–115. Li, H., Sheng, G., Chiou, C.T., Xu, O.Y., 2005. Relation of organic contaminant equilibrium sorption and kinetic uptake in plants. Environ. Sci. Technol. 39, 4864–4870. Li, P.J., Li, X.J., Stagnitti, F., Zhang, H.R., Lin, X., Zhang, S.Y., Zhuo, J.C., Xiong, X.Z., 2009. Studies on the sources of benzo[a]pyrene in grain and aboveground tissues of rice plants. J. Hazard. Mater. 162, 463–468. Li, X.J., Li, P.J., Lin, X., Zhang, C.H., Li, Q., Gong, Z.Q., 2008. Biodegradation of aged polycyclic aromatic hydrocarbons (PAHs) by microbial consortia in soil and slurry phases. J. Hazard. Mater. 150, 21–26. Liu, J.Y., Schnoor, J.L., 2008. Uptake and translocation of lesser-chlorinated polychlorinated biphenyls (PCBs) in whole hybrid poplar plants after hydroponic exposure. Chemosphere 73, 1608–1616. Liu, R., Xiao, N., Wei, S.H., Zhao, L.X., An, J., 2014. Rhizosphere effects of PAH–contaminated soil phytoremediation using a special plant named Fire Phoenix. Sci. Total Environ. 473-474, 350–358. Lu, S.J., Teng, Y.G., Wang, J.S., Sun, Z.J., 2010. Enhancement of pyrene removed from contaminated soils by Bidens maximowicziana. Chemosphere 81, 645–650. MacLeod, C., Semple, K., 2000. Influence of contact time on extractability and degradation of pyrene in soils. Environ. Sci. Technol. 34, 4952–4957. Mcgarry, M.A., Charles, G.D., Medrano, T., Bubb, M.R., Grant, M.B., Campbell– Thompson, M., Shiverick, K.T., 2002. Benzo(a)pyrene, but not 2,3,7,8-tetrachlorodibenzo-p-dioxin, alters cell adhesion proteins in human uterine RL95-2 cells. Biochem. Biophys. Res. Commun. 294, 101–107. Nam, K., Kim, J.Y., 2002. Role of loosely bound humic substances and humin in the bioavailability of phenanthrene aged in soil. Environ. Pollut. 118, 427–433. Peng, S.W., Zhou, Q.X., Cai, Z., Zhang, Z.N., 2009. Phytoremediation of petroleum contaminated soils by Mirabilis jalapa L. in a field plot experiment. J. Hazard. Mater. 168, 1490–1496. Perelo, L.W., 2010. Review: In situ and bioremediation of organic pollutants in aquatic sediments. J. Hazard. Mater. 177, 81–89. Pfeifer, G.P., Denissenko, M.F., Olivier, M., Tretyakova, N., Hecht, S.S., Hainaut, P., 2002. Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking–associated cancers. Oncogene 21, 7435–7451. Phillips, L.A., Greer, C.W., Farrell, R.E., Germida, J.J., 2012. Plant root exudates impact
Y. Sun, Q. Zhou / Ecotoxicology and Environmental Safety 124 (2016) 74–81
the hydrocarbon degradation potential of a weathered–hydrocarbon contaminated soil. Appl. Soil Ecol. 52, 56–64. Reilley, K.A., Banks, M.K., Schwab, A.P., 1996. Dissipation of polycyclic aromatic hydrocarbons in the rhizosphere. J. Environ. Qual. 25, 212–219. Schroll, R., Bierling, B., Cao, G., Dorfler, U., Lahaniati, M., Langenbach, T., Scheunert, I., Winkler, R., 1994. Uptake pathways of organic chemicals from soil by agricultural plants. Chemosphere 28, 297–303. Simonich, S.L., Hites, R.A., 1994. Vegetation-atmosphere partitioning of polycyclic aromatic hydrocarbons. Environ. Sci. Technol. 28, 939–943. Simonich, S.L., Hites, R.A., 1995. Organic pollutant accumulation in vegetation. Environ. Sci. Technol. 29, 2095–2103. Singh, O.V., Jain, R.K., 2003. Phytoremediation of toxic aromatic pollutants from soil. Appl. Microbiol. Biotechnol. 63, 128–135. Sinha, R., Kulldorff, M., Gunter, M.J., Strickland, P., Rothman, N., 2005. Dietary Benzo [a]Pyrene intake and risk of colorectal adenoma. Cancer Epidemiol. Biomak. Prev. 14, 2030–2034. Su, Y.H., Zhu, Y.G., 2005. Uptake of selected PAHs from contaminated soils by rice seedlings (Oryza sativa) and influence of rhizosphere on PAH distribution. Environ. Pollut. 155, 359–365. Sun, Y.B., Zhou, Q.X., Diao, C.Y., 2008. Effects of cadmium and arsenic on growth and metal accumulation of Cd–hyperaccumulator Solanum nigrum L. Bioresour. Technol. 99, 1103–1110. Sun, Y.B., Zhou, Q.X., Xu, Y.M., Wang, L., Liang, X.F., 2011. Phytoremediation for cocontaminated soils of benzo[a]pyrene (B[a]P) and heavy metals using ornamental plant Tagetes patula. J. Hazard. Mater. 186, 2075–2082. Sun, Y.B., Zhou, Q.X., Xu, Y.M., Wang, L., Lin, D.S., Liang, X.F., 2013. The potential of gibberellic acid 3 (GA3) and Tween-80 induced phytoremediation of co–contamination of Cd and B[a]P using Tagetes patula. J. Environ. Manag. 114, 202–208.
81
Trapp, S., Karlson, U., 2001. Aspects of phytoremediation of organic pollutants. J. Soil. Sediment. 1, 1–7. Trapp, S., Matthies, M., Scheunert, I., Topp, E.M., 1990. Modeling the bioconcentration of organic chemicals in plants. Environ. Sci. Technol. 24, 1246–1252. Uno, S., Dalton, T.P., Shertzer, H.G., Genter, M.B., Warshawsky, D., Talaska, G., Nebert, D.W., 2001. Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1 ( / ) knockout mice having increased hepatic BaP-DNA adduct levels. Biochem. Biophys. Res. Commun. 289, 1049–1056. Wei, J., Liu, X.Y., Wang, Q., Wang, C.H., Chen, X.P., Li, H.B., 2014. Effect of rhizodeposition on pyrene bioaccessibility and microbial structure in pyrene and pyrene–lead polluted soil. Chemosphere 97, 92–97. Xu, S.Y., Chen, Y.X., Lin, Q., Wu, W.X., Xue, S.G., Shen, C.F., 2005. Uptake and accumulation of phenanthrene and pyrene in spiked soils by Ryegrass (Lolium perenne L.). J. Environ. Sci. 17, 817–822. Yin, X.M., Liang., X., Xu, G.H., Zhan, X.H., 2014. Effect of phenanthrene uptake on membrane potential in roots of soybean, wheat and carrot. Environ. Exp. Bot. 99, 53–58. Zhang, Z.H., Rengel, Z., Chang, H., Meney, K., Pantelic, L., Tomanovic, R., 2012. Phytoremediation potential of Juncus subsecundus in soils contaminated with cadmium and polynuclear aromatic hydrocarbons (PAHs). Geoderma 175–176, 1–8. Zhou, Q.X., Diao, C.Y., Sun, Y.B., Zhou, J.L., 2012. Tolerance, uptake and removal of nitrobenzene by a newly-found remediation species Mirabilis jalapa L. Chemosphere 86, 994–1000. Zhou, Q.X., Song, Y.F., 2004. Principles and Methods of Contaminated Soil Remediation. Science Press, Beijing, China. Zhou, Q.X., Wei, S.H., Zhang, Q.R., 2006. Ecological Remediation. Environmental Science Press, Beijing, China.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Uptake and translocation of benzo[a]pyrene (B[a]P) in two ornamental plants and dissipation in soil Yuebing Sun a,c, Qixing Zhou b,c,n a
Innovation Team of Remediation for Heavy Metal Contaminated Farmlands, Agro-Environment Pollution Institute, Ministry of Agriculture, Tianjin 300191, China Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education), College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China c Key Laboratory of Terrestrial Ecological Process, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 June 2015 Received in revised form 21 September 2015 Accepted 22 September 2015
Pot experiments were conducted to evaluate the phytoremediation of B[a]P contaminated soil using two ornamental plants (Tagetes patula and Mirabilis jalapa). The results showed that the dry biomass of two plants was increased at low B[a]P contaminated soil and then inhibited with increasing B[a]P concentrations. It exhibited a significantly positive linear relationship between B[a]P absorption in roots, stems, leaves and shoots of the tested plants and the concentration of B[a]P in soils (Po 0.01). Meanwhile, the contents of B[a]P in different tissues of the plants increased with growing time. After planting T. patula and M. jalapa, plant-promoted biodegradation of B[a]P was account for 79.5–99.8% and 71.1– 99.9%, respectively, whereas the amount of B[a]P dissipation enhancement was only 0.2–20.5% and 0.1– 28.9%, respectively. Moreover, low bioaccumulation factor (BF) and translocation factor (TF) values indicated that T. patula and M. jalapa took up B[a]P from contaminated soil and transferred them to the aerial parts with low efficiency. The B[a]P removal rates in rhizosphere soils at different growing stages of T. patula and M. jalapa were 2.7–26.8% and 0.4%–33.9%, respectively, higher than those of non-rhizopshere soils. Therefore, the presence of T. patula and M. jalapa roots was effective in promoting the phytoremediation of B[a]P contaminated soils. & 2015 Elsevier Inc. All rights reserved.
Keywords: Phytoremediation Benzo[a]P (B[a]P) Uptake Distribution Dissipation
1. Introduction Benzo[a]Pyrene (B[a]P) is a popularly environmental pollutant emanating from industrial processes (e.g., exhaust fumes and combustion of all forms of fossil fuels, processing production and spillage of petroleum) and during household activities (e.g., cooking, barbequing, and smoking) (Sinha et al., 2005; Sun et al., 2013). B[a]P is a carcinogen known to induce tumors in a number of organs in animal models and humans. One of its metabolites is known to form DNA adducts which are responsible for mutagenesis and tumorigenesi (Uno et al. 2001; Gong et al., 2006). There are indications that B[a]P diol epoxide specifically targets the protective p53 gene, which is a transcription factor that regulates the cell cycle and hence functions as a tumor suppressor (Pfeifer et al., 2002). Immunocytochemistry and Western immunoblot analysis showed that B[a]P produced a marked loss of plasma n Corresponding author at: Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education), College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China. Fax: þ 86 22 23501117. E-mail addresses: [email protected] (Y. Sun), [email protected] (Q. Zhou).
http://dx.doi.org/10.1016/j.ecoenv.2015.09.037 0147-6513/& 2015 Elsevier Inc. All rights reserved.
membrane epidermal growth factor receptor (EGF-R) localized along intercellular boundaries and significantly decreased in βcatenin and cadherin protein levels (Mcgarry et al., 2002). Hence, B [a]P has been classified as a toxic and priority-controlled contaminant in the list of USEPA and European Union (Ko-Wasik et al., 2004; Dixit et al., 2011), and it is necessary to look for effective methods of remediation. However, the major limitations of remediation for B[a]P contaminated soil have been the requirement for long degradation periods due to its poor water-solubility and the difficulty in controlling the conditions, thus precluding the reclaiming of natural soil amendments (Singh and Jain, 2003; Dixit et al., 2011). As an option in certain situations, phytoremediation is gaining popularity for its low cost and environmental friendly alternative for decontamination of the contaminated soil (Zhou and Song, 2004; Zhang et al., 2012; Sun et al., 2013). Disappearance of polycyclic aromatic hydrocarbons (PAHs) and Polychlorinated biphenyls (PCBs) in those researches was found to be higher in planted soil (Johnson et al., 2004; Lu et al., 2010; Phillips et al., 2012; Wei et al., 2014). The mechanisms of phytoremediation for organic pollutants include biophysical and biochemical processes like adsorption, transport and translocation, as well as transformation and
Y. Sun, Q. Zhou / Ecotoxicology and Environmental Safety 124 (2016) 74–81
mineralization by plant enzymes (Perelo, 2010; Gao et al., 2010). It was assumed that plants enhanced biodegradation of organic matters by stimulating the growth of soil microbes due to the interactions between root exudates, microbes and contaminants that stimulated microbial activities, enzyme-catalyzed processes or cometabolic processes in rhizosphere (Gao et al., 2010). Lee et al. (2008) have reported 499% and 77–94% of phenanthrene (Phe) and pyrene (Pyr), respectively, had been degraded in planted soil, whereas 99% and 69% had been degraded in unplanted soil. 91.7–97.8% of Phe and 70.8–90.0% of Pyr had degraded in the planted soils, which were 1.9–3.2% and 8.9–20.7% larger than those in corresponding unplanted soils (Cheema et al., 2009). The objectives of the present work were: (1) to investigate the B[a]P uptake and translocation in two ornamental plants, and (2) to determine the mechanism of B[a]P phytodegradation and dissipation. Results obtained from this study are expected to provide some insight with regard to the feasibility of plant-enhanced phytoremediation for B[a]P contaminated soils. Tagetes patula and Mirabilis jalapa are selected for the experiment because of its extensive, widely branched root system providing a large root surface for the growth of microbial populations, which can strongly endure and effectively phytodegrade hydrocarbons (Peng et al., 2009; Sun et al., 2011; Zhou et al., 2012).
2. Materials and methods 2.1. Soil characterization The tested soil was collected from agricultural fields in the Shenyang Ecological Experiment Station, Chinese Academy of Sciences (Lat. 123°41′, and Lon. 41°31′). This soil is meadow burozem, and chemical analysis showed that total C, total N, total P, total K and pH were 1.6%, 0.11%, 0.04%, 0.60% and 6.50, respectively. The initial concentration of B[a]P was non-detectable. Surface (0–20 cm) soil samples which were ground to pass through a 4 mm mesh were used in the pot-culture experiment. 2.2. Experimental procedure According to the Environmental quality standard for soil environment in China (GB 4284-84), the soils should be remedied when B[a]P concentration was up to 3 mg kg 1. thus, in this study single B[a]P treatments were CK, 2, 5, 10 and 50 mg kg 1, respectively, as simulating non-, low-, medium- and serious-B[a]P contamination. acetone solution containing B[a]P was mixed with 200 g soil as a portion, then mixed with 800 g soil when acetone volatilized completely. B[a]P (purity 4 97%) was obtained from sigma chemical CO., USA. Seeds of T. patula and M. jalapa were sterilized in 2% (v/v) hydrogen peroxide for 15 min, washed with tap water and soaked in water 1 d. 3 seedlings of T. patula and M. jalapa with a similar size, about 4 wk old, and 6–8 cm height with 4–6 leaves were transplanted in each pot. The soil surface was covered with a layer of silica sand (75 g) to minimize B[a]P volatilization (Sun et al., 2011). The position of pots was changed randomly every week in order to eliminate the non-uniform illumination. Loss of water was made up using tap water to reach 75% of the field water-holding capacity and maintained this humidity by daily watering throughout the cultivation, and a petri dish was placed under each pot to collect potential leachate during the experiment. After growing for 30, 60 and 92 days, namely, the seedling phase, flowering and mature phases were used for evaluating characteristics of plant growth and B[a]P uptake and dissipation. The plant samples were washed with deionized water, and separated into roots, stems and leaves. After drying with filter paper, the sub-samples were freezed-dried
75
(at 530717 Pa and 5072 °C, BYK FD-1), and weighed to determine the biomass. The soil samples were air dried at room temperature and ground sufficiently to pass through a 100-mesh sieve. 2.3. Extraction and analysis of PAHs in soils and plants 2.0 g of dry soil samples were extracted using 20 mL of dichloromethane for 2 h in an ultrasonic bath (the system was o40 °C). 0.5 g of dried plant fragment was mixed into 10 mL solution of methanol, and extracted by ultrasonication for 30 min for 3 successive extractions (Fan et al., 2008). Then, 2 mL of the supernatant was filtered through 2 g of silica gel column and eluted consecutively with 10 mL mixture of 1:1 of hexane and dichloromethane (v/v). The extract was concentrated by evaporation of the dichloromethane under a stream of nitrogen and the residue was dissolved in hexane with a final volume of 1.0 mL for gas chromatography (GC) analysis. Samples extracts (1 μL) were analyzed by a Hewlett-Packard (HP) 6890 GC system equipped with a HP 5973 mass selective detector and a DB-5 capillary column (30 mm 0.25 mm 0.25 μm). The operation was following steps: (1) the initial oven temperature was 80 °C and held time for 1 min, (2) the oven temperature was increased to 275 °C at 15 min 1 and held for 1 min, (3) the oven was set to 285 °C at 10 min 1, and held for 1 min, and (4) the oven temperature was increased to 295 °C at 5 min 1, and held for 1 min. Nitrogen was used as the carrier gas (1.5 mL min 1) and makeup gas (35 mL min 1). A 1.0 μL aliquot of the extract was injected in the splitless mode. The injector was held at 250 °C and the detector at 300 °C. 2.4. Quality control Experiments on PAH recovery were carried out by spiking a known concentration of PAH standards (1 mg kg 1) in an uncontaminated soil and a plant. The results showed satisfactory recovery, and ranged from 94.5 77.1% to 101.978.7% for soils and plants, respectively. The limit of detection (LOD) of B[a]P in soil and plants was the same of 0.08 μg kg 1. 2.5. Statistical analysis All treatments were replicated three times in the experiments. The means and standard deviations (SD) were calculated by the Microsoft Office Excel 2003. One-way analysis of variance was carried out with SPSS10.0. When a significant (Po0.05 or Po 0.01) difference was observed between treatments, multiple comparisons were made by the LSD test.
3. Results 3.1. Plant biomass The shoot biomass of T. patula and M. jalapa on a dry weight basis is shown in Fig. 1. The application of B[a]P could enhance the growth and production of T. patula seedling under low B[a]P levels of 2 and 5 mg kg 1, resulting in 39.0% and 13.1% increase, respectively, in contrast with the control group. The similar trend of shoot biomass was found at flowing and mature stage, which increased with increasing of r10 B[a]P kg 1 in soil, and reached the maximum of 9.4 and 9.5 g pot 1, respectively, at B[a]P of 5 mg kg 1. However, the shoot biomass observed was significantly decreased when the B[a]P in soils was up to 50 mg kg 1 (Po0.05), suffering by 29.2%, 36.3% and 18.4% reduction, respectively, at the seedling, flowering and mature stage when compared with CK.
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Y. Sun, Q. Zhou / Ecotoxicology and Environmental Safety 124 (2016) 74–81
Dry weight (mg pot-1)
12 10
T. patula
a
a
a
a
seedling stage flowering stage mature stage a
ab
b
bc
8
c
6 4
Table 2 Relationships between B[a]P concentration in different tissues of tested plants and soil amended with B[a]P. Plants
Growth stage
T. patula
Seedling stage
b a
Flowering stage
ab
ab
ab b
2
Mature stage
Regression equation
R2
P
Root Shoot Root Shoot Root Shoot
Y ¼3.59X 5.81 Y ¼2.62X 4.16 Y ¼4.80X 7.39 Y ¼2.49X 2.96 Y ¼5.26X 7.75 Y ¼3.15X 3.92
0.83 0.78 0.84 0.98 0.83 0.97
0.01 0.01 0.01 0.01 0.01 0.01
Root Shoot Root Shoot Root Shoot
Y ¼1.70X 1.57 Y ¼1.18X 1.23 Y ¼8.43X 12.41 Y ¼3.05X 3.78 Y ¼9.66X 13.49 Y ¼3.97X 4.69
0.88 0.97 0.90 0.97 0.94 0.95
0.01 0.01 0.01 0.01 0.01 0.01
0
Dry weight (mg pot-1)
6
M. jalapa
a
5
3
M. jalap
Mature stage
b a
a
Seedling stage Flowering stage
a
b a
4
seedling stage flowering stage mature stage
a
c b
c a
c b a
2
3.2. B[a]P uptake and absorption in the plants
1
Table 1 exhibits the B[a]P uptake and partitioning in the different tissues of T. patula and M. jalapa. Across the 0–50 mg kg 1 B [a]P treatments, the concentrations of B[a]P in different tissues increased with increasing in soil B[a]P at the same growth stage, and according to these corresponding regression equations listed in Table 2. It displayed a markedly positive linear relationship between B[a]P uptake in roots, stems, leaves and shoots of the tested plants and the concentration of B[a]P in soils (P o0.01). Meanwhile, the contents of B[a]P in different tissues of the plants increased with growing time, and the B[a]P concentrations in roots, stems, leaves and shoots at mature stage were 1.08–1.41, 1.02–1.42, 0.36–1.49 and 0.60–1.38, respectively, times higher than those of flowering stage, and 1.79–5.49, 2.24–6.38, 0.89–3.06, and 1.29–3.46, respectively, times more than those of seedling stage for M. jalapa, and 1.08–1.31, 1.03–1.33, 1.11–1.43 and 1.10–1.27, respectively, times higher than those of flowering stage, and 1.47– 2.64, 1.14–4.91, 1.11–1.65, and 1.10–2.47, respectively, times more than those of seedling stage for T. patula. The concentration of B[a] P in tissues of the two plants on a whole was in the sequence of root4leaf 4stem and root4shoot, indicating the weak ability of B[a]P uptake and translocation by T. patula and M. jalapa. Fig. 2 shows the amount of B[a]P accumulated in the tissues of T. patula and M. jalapa. The concentrations of B[a]P in roots and
0
CK
2 5 10 B[a]P concentration (mg kg-1)
50
Fig. 1. Dry biomass of T. patula and M. jalapa grown in treatments with different B [a]P concentrations. Note: Letters above the bar diagram refer to the difference at significances level of 0.05 (LSD test).
The tolerance capability of M. jalapa to B[a]P contamination was slightly weaker than that of T. patula. Under the dose of 2 mg kg 1 B [a]P, the biomass of plants was enhanced at certain degree, resulting in 8.7%, 26.9% and 3.4% increase, respectively, at seedling, flowering and mature stage in comparison with unamended soil, and the significant increase in dry weight was got at flowering stage (Po0.05). However, it exhibited a inhibitory effect on plant biomass and decreased with increasing of the concentration of o2 mg kg 1 B[a]P in soils. In particular, at the flowering and mature stage, the dry weight of M. jalapa under the contamination of B[a]P (5–50 mg kg 1) was significantly lower than those of control group (Po0.05). Across the whole growing stage, the tolerant capability to B[a]P contamination was increased with time growing. Table 1 B[a]P concentrations in the different tissues of T. patula and M. jalapa. Growth stage
B[a]P treatments (mg kg 1)
T. patula Root
M. jalapa Stem
Leaf
Shoot
Root
Stem
Leaf
Shoot
Seedling stage
CK 2 5 10 50
ND 0.69 7 0.19 2.85 7 1.09 5.87 7 0.15 15.3 7 0.12
ND 0.497 0.70 1.22 7 0.26 3.09 7 2.02 10.3 7 1.58
ND 1.137 1.13 3.337 0.52 4.46 7 0.74 12.4 7 3.13
ND 0.83 7 0.92 2.277 0.01 3.69 7 0.75 11.7 7 1.20
ND 0.89 7 0.18 5.147 0.33 5.377 0.27 6.26 7 0.70
ND 0.54 7 0.08 1.177 0.20 1.477 0.16 2.53 7 0.18
ND 1.03 7 0.32 3.687 0.14 4.40 7 0.40 5.98 7 1.34
ND 0.84 70.16 2.7770.09 3.2470.11 4.69 70.95
Flower stage
CK 2 5 10 50
ND 1.39 7 0.05 5.157 0.90 7.727 0.17 20.9 7 0.81
ND 1.54 7 0.08 5.81 7 0.59 7.077 0.49 8.89 7 0.22
0.0027 0.001 1.487 0.12 3.63 7 0.28 5.62 7 0.08 11.17 0.23
0.0017 0.001 1.517 0.03 4.727 0.15 6.277 0.21 10.17 0.02
ND 3.95 7 0.26 6.53 7 0.03 19.5 7 9.92 34.4 7 3.79
ND 1.017 0.14 6.08 7 0.13 6.63 7 0.64 11.2 7 2.07
0.0017 0.001 2.54 7 0.42 7.58 7 0.12 9.247 0.01 13.0 7 1.96
0.001 70.001 1.79 70.17 6.87 70.02 8.12 70.29 12.1 72.30
Mature stage
CK 2 5 10 50
ND 1.83 7 0.17 6.46 7 0.45 9.247 1.04 22.6 7 1.98
0.0037 0.001 5.687 6.09 5.977 0.79 7.75 7 0.01 11.8 7 0.39
0.0027 0.002 4.647 4.51 5.197 0.75 7.34 7 0.41 13.8 7 1.47
0.0037 0.002 1.677 0.35 5.59 7 0.80 7.55 7 0.20 12.8 7 0.89
ND 4.417 0.74 9.187 0.49 26.717 3.65 37.2 7 2.51
0.0037 0.002 1.217 0.54 6.22 7 0.85 9.39 7 1.72 14.17 0.44
0.0017 0.001 0.92 7 0.31 11.3 7 1.68 13.17 0.22 15.2 7 0.23
0.002 70.001 1.08 70.14 8.98 70.41 11.2 70.91 14.8 70.39
6
0
0
60 18 40 12 20 6
0
Root B[a]P accumu
lation (µg pot -1 )
48 40
40 16 20
0
0 CK
2
5
10
200
36
150
27
100
18
50
9
0
0
200
60
8
45
80
24
50
-1
B[a]P treatment mg kg
0
250
250
32
2
3
100
Root B[a]P accumulation (µg kg -1 )
0
Root B[a]P accumulation (µg kg -1 )
80
24
4 6
0
Shoot B[a]P accumulation (µg pot -1 )
Root B[a]P accumulation (µg pot -1 )
30
6 9
Shoot B[a]P accumulation (µg kg -1 )
3
12
Shoot B[a]P accumulation (µg kg -1 )
12
8
60
)
6
15
-1
18
10
45 150 30 100 15 50
Shoot B[a]P accumulation (µg kg
9
77
18
Root B[a]P accumulation (µg kg -1 )
24
Shoot B[a]P accumulation (µg pot -1 )
12
Shoot B[a]P accumulation (µg pot -1 )
Root B[a] accumulation (µg pot -1 )
Y. Sun, Q. Zhou / Ecotoxicology and Environmental Safety 124 (2016) 74–81
0
0 CK
2
5
10
50
-1
B[a]P treatment (mg kg )
Fig. 2. B[a]P accumulation in T. patula and M. jalapa under different B[a]P treatments. Note: a–c refers seedling stage, flowering stage and mature stage of T. patula, respectively, and d–f refers seedling stage, flowering stage and mature stage of M. jalapa, respectively.
shoots of the plants steadily and strikingly increased with increasing soil B[a]P and the time of plant growth. However, the ability of B[a]P uptake and translocation by the two plants was weak, the highest B[a]P uptake by shoots of T. patula and M. jalapa was only 70.61 and 41.42 mg kg 1, respectively. Under B[a]P treated with 2–50 mg kg 1, most of B[a]P taken up was retained in the roots of M. jalapa, and 50.7–68.3%, 58.9–78.3% 75.8–79.0%, respectively, of B[a]P absorption by the plant was concentrated in its roots at seedling, flowering and mature stage. By contrast, the dominating B[a]P uptake by T. patula was in the shoots, up to 60.6– 78.9%, 71.0–86.6% and 70.1–86.7%, respectively, at seedling, flowering and mature stage. The amount of B[a]P accumulated in roots of M. jalapa, at seedling, flowering and mature stage was 1.49–3.07, 4.04–6.54 and 2.70–8.16, respectively, times higher than those of T. patula. In contrast, the accumulation of B[a]P in shoots of M. jalapa, at seedling, flowering and mature stage was 44.9–86.1%, 43.0– 71.1% and 32.5–60.4%, respectively, lower than those of T. patula. 3.3. B[a]P dissipation in rhizosphere and non-rhizosphere The dissipation rate of B[a]P in the soils of rhizosphere and non-rhizosphere were shown in Fig. 3. The residual B[a]P in the
soils were all significantly decreased after planting T. patula and M. jalapa, and the removal rate increased with growing time (P o0.05). The highest removal rates at seedling, flowering and mature stage was 74.3%, 90.2% and 92.4%, respectively, in rhizosphere soil, and 63.6%, 85.0% and 88.2%, respectively, in non-rhizosphere soil for T. patula, and 71.1%, 87.5% and 87.9%, respectively, in rhizosphere soil, and 67.8%, 87.5% and 83.1%, respectively, in non-rhizosphere soil for M. jalapa. The B[a]P removal rate in rhizosphere was higher than those of non-rhizosphere at seedling, flowering and mature stage, which was experienced by 10.8– 19.3%, 4.3–11.5% and 4.2–15.7%, respectively, higher than those of non-rhizosphere for T. patula, and 3.3–21.3%, 2.6–11.0% and 4.8– 20.2%, respectively, higher than those of non-rhizosphere for M. jalapa. However, the dissipation rate of B[a]P was generally inhibited with an increasing soil B[a]P content, which reached 37.9– 66.1% and 57.2–79.2%, and 41.2–52.4% and 21.7–38.4%, respectively, in rhizsphere and non-rhizosphere soils of different growth stage of T. patula and M. jalapa. The removal rates in rhizsphere soils at seedling, flowering and mature stage of T. patula were 3.2–16.0%, 2.7–25.6%, and 4.2–26.8%, respectively, higher than those of M. jalapa. By contrast, the decreases of 0.4–16.1%, 7.3–24.7%, and 14.2–33.9% in B[a]P dissipation rates in non-rhizosphere soils of T.
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Y. Sun, Q. Zhou / Ecotoxicology and Environmental Safety 124 (2016) 74–81
et al. (2009), the B[a]P content of rice grain, husk, and stem with leaf sampled from outdoor field was up to 7.33-, 9.21- and 27.10fold, respectively, higher than corresponding tissues, suggesting that polluted air was the main source of B[a]P in aboveground tissues (Li et al., 2009). Liu and Schnoor also found a small amount of PCBs (0.01–0.9 mg kg 1) were detected in the main stems of blank controls (Liu and Schnoor, 2008). This is, in some way, consistent with the observations on phenanthrene (Phe) and pyrene (Pyr) absorption by plant roots by Gao and Zhu (2004) and Xu et al. (2005). Despite B[a]P uptake and accumulation in the shoots might come from air, the translocation of B[a]P from roots to shoots was significant (Sun et al., 2011). Several literatures have pointed out that the most of PAH accumulation in the shoots was transferred from the roots (Gao and Zhu, 2004; Xu et al., 2005). It was found that an average 90.2% of Pyr and 74.3% of Phe in the shoots of ryegrass derived from the roots, and only 9.9% of Pyr and 25.7% of Phe in shoots was from the atmosphere (Xu et al., 2005). Schroll et al. (1994) demonstrated that about 22–95% of Phe and 32–96% of Pyr in the shoots were translocated from the roots. Translocation factor (TF), the quotient of contaminant concentration in shoots to roots (Zhou and Song, 2004; Gao and Zhu, 2004), and bioaccumulation factor (BF), the ratio of chemical concentration in a plant to soil (Sun et al., 2011), was used to evaluate the effectiveness of a plant in transferring a chemical from soil to roots and from roots to aerial part (Sun et al., 2008). As listed in Table 3, the BF values in T. patula and M. jalapa fist increased and then decreased with the increase of soil B[a]P concentrations, and its exhibited mature stage4flowering stage4 seedling stage, showing a significant B[a]P uptake in shoots of the two plants with growing time. In contrast, TFs in T. patula decreased with increasing of B[a]P applied to soil. In other words, the relationship between the TF values and soil B[a]P contents was negative and logarithmic linear, indicating a diminishing efficiency of B[a]P accumulation with increasing soil B[a]P concentrations (Zhou et al., 2006). However, there were no regular trends in TFs in M. jalapa under different treatments. The BF and TF value in most treatments was o1.0, low BFs and TFs indicated that T. patula and M. jalapa took up B[a]P from soil and transferred them to the aboveground parts with low efficiency (Sun et al., 2011). It was observed that concentrations of B[a]P in roots of two plants were higher than those in aerial parts (stems, leaves and shoots) (Table 1). Numerous studies have suggested that lipophilic organic contaminant entering plant's root from soil rely on the Kow (Simonich and Hites, 1995; Xu et al., 2005; Gao et al., 2008; Su, Zhu, 2005). In general, the higher the Kow of a pollutant, the greater would be the expectancy to partition from the soil organic matter, and thus the lower would be transported to plant aerial tissue. It was reported that the uptake of PAHs into plant roots from contaminated soils was directly proportional to Kow (Gao et al., 2008; Su, Zhu, 2005). Gao et al. (2008) found that the root concentration factors of Pye were generally a little larger or equal to those of Phe, which was about consistent with their log Kow.
B[aP]=2
B[a]P removal rate (%)
B[aP]=5 B[a]P=10 B[a]P=50
non-rhizosphere rhizosphere
non-rhizosphere
non-rhizosphere rhizosphere
rhizosphere
re
Seedling stage
Flower stage
Mature stage
B[a]P=2 B[a]P=5
B[a]P removal rate (%)
B[a]P=10 B[a]P=50
non-rhizosphere rhizosphere
Seedling stage
non-rhizosphere
non-rhizosphere rhizosphere
rhizosphere
Flower stage
Mature stage
Fig. 3. Removal rate of B[a]P in rhizosphere and non-rhizosphere. Note: (a) and (b) refers T. patula M. jalapa, respectively.
patula were exhibited than those of M. jalapa.
4. Discussion 4.1. B[a]P absorption and translocation in plants As listed in Table 1, it is notable that under the corresponding experiments the concentrations of B[a]P in different parts of T. patula and M. jalapa at seedling stage were under the detect limit, whereas it was found in aerial parts of two tested plants with the time growing. At flowering stage, B[a]P were found in leaves and shoots, which were 0.002 and 0.001 mg kg 1, respectively, for T. patula and 0.001 and 0.001 mg kg 1, respectively for M. jalapa. When growing to mature stage, B[a]P was detected in aerial parts (stems, leaves and shoots) of T. patula and M. jalapa under unspiked soils from 0.002 to 0.003 mg kg 1 and from 0.001 to 0.003 mg kg 1, respectively. It indicated that the absorption of B [a]P in above-ground parts of plants from ambient air probably through the retention of vapor phase of B[a]P on the waxy leaf cuticle (Simonich and Hites, 1994; Gao and Zhu, 2004). An airquality controlled greenhouse experiments were conducted by Li
Table 3 The bioaccumulation factor and transfer factor of B[a]P in T. patula and M. jalapa under different B[a]P treatments. B[a]P concentration (mg kg 1)
2 5 10 50
T. patula
M. jalapa
Seedling stage
Flower stage
Mature stage
Seedling stage
Flower stage
Mature stage
BF
TF
BF
TF
BF
TF
BF
TF
BF
TF
BF
TF
0.42 0.45 0.37 0.23
1.20 0.79 0.63 0.76
0.76 0.94 0.63 0.20
1.09 0.92 0.81 0.48
0.83 1.12 0.75 0.26
0.91 0.86 0.82 0.57
0.42 0.55 0.32 0.09
0.94 0.54 0.60 0.75
0.90 1.37 0.81 0.24
0.45 1.05 0.42 0.35
0.54 1.80 1.12 0.30
0.24 0.98 0.42 0.40
Y. Sun, Q. Zhou / Ecotoxicology and Environmental Safety 124 (2016) 74–81
79
Table 4 Contribution of T. patula and M. jalapa to the removal of B[a]P in soils. Growth stage
Treatments (mg kg 1)
T. patula
M. jalapa
Td (mg pot 1)
Pac (mg pot 1)
Seedling stage
2 5 10 50
0.22 0.52 1.33 9.69
2.46 6.15 7.90 19.6
Flower stage
2 5 10 50
0.20 0.26 1.01 5.82
Mature stage
2 5 10 50
0.28 0.26 1.62 6.65
Pac/Td (%)
Td (mg pot 1t)
Pac (mg pot 1)
Pac/Td (%)
1.12 1.18 0.59 0.20
0.07 0.34 2.14 9.76
1.96 5.05 6.81 8.82
2.80 1.49 0.32 0.09
12.6 44.2 52.6 49.2
6.28 17.0 5.21 0.84
0.06 0.31 1.13 5.37
8.47 19.3 22.6 35.0
14.1 6.23 2.00 0.65
13.6 53.2 55.8 70.6
4.86 20.5 3.44 1.06
0.10 0.09 1.29 10.2
4.43 26.0 33.7 41.4
4.43 28.9 2.61 0.41
Moreover, these phenomena showed that the accumulation of PAHs in the roots was influenced by hydrophobic compositions of plant roots, such as specific surface area and lipid (Li et al., 2005; Su, Zhu, 2005; Yin et al., 2014). It was also found in this study that there was a significant positive linear correlation between B[a]P concentrations in roots and shoots of T. patula and M. jalapa under different growing stage and B[a]P spiked in soils (P o0.001) (Table 2), which was consistent with Borneff et al. (1973), who pointed out that the concentrations of B[a]P in carrot epidermis was correlated with soil B[a]P. Based on the results of Gao and Zhu (2004), the accumulation of Phe and Pye in the roots or shoots of 12 plant species was elevated with the increase of their soil concentrations. In the present study, the B[a]P concentrations and BFs in most case in M. jalapa were higher than those of T. patula, and while it was just the opposite to that of the TF values. The ability of B[a]P uptake and accumulation would be distinctive different among species due to their different activities of H þ -ATPase and affinities of transporter (Yin et al., 2014). It has been observed that PAHs are actively transported into the cells via H þ -coupled symporters and transporters in various plants have different affinities to PAHs (Zhang et al., 2012). The higher the depolarization of membrane potential, the stronger the capacity of PAHs uptake were (Yin et al., 2014). 4.2. Plant-enhanced phytoremediation of soil B[a]P The mechanisms of the dissipation of PAHs in rhizosphere soils involve volatilization, leaching, biodegradation, plant uptake and accumulation, and incorporation of the pollutant into soil organic material (Gao and Zhu, 2004; Sun et al., 2011). Whereas, the dissipation of B[a]P in non-rhizosphere soils was the sum of leaching, abiotic dissipation, biodegradation, and incorporation of the pollutant into soil organic material. In contrast, Thus the loss of B[a]P in vegetated soils could be calculated as follows (Gao and Zhu, 2004):
Trs = Tl + Ta + Tb + Pac
(1)
Tnrs = Tl + Ta + Tb ’
(2)
where Trs and Tnrs were the dissipation of B[a]P in rhizosphere and non-rhizosphere soils (mg pot 1). Tl and Ta denoted the dissipation by leaching and abiotic dissipation. Tb and Tb′ were the loss by biodegradation in rhizosphere and non-rhizosphere soils, respectively. Pac refers B[a]P uptake and accumulation in plants. Reilley
et al. (1996) has noticed that 4–5 rings of PAHs in leachate were undetectable from soils with or without plants. Moreover, the phytovolatilization of such organic compounds was negligible, and plant metabolism was not significant (Trapp et al., 1990). So the dissipation of PAHs would overwhelmingly derive of plant accumulation and promoted biodegradation. The corresponding equations can be expressed as
Trs = Tb + Pac
(3)
Tnrs = Tb ’
(4)
Therefore, the dissipation enhancement (Td) of B[a]P in rhizosphere versus non-rhizosphere soils was
Td = Trs –Tnrs = Pac + Tb –Tb ’
(5)
Tbp = Tb –Tb ’
(6)
In Eq. (6), Tpb refers the loss of B[a]P by the plant-promoted biodegradation. As listed in Table 4, there was a significant increasing of the B[a]P amounts of plant uptake and plant-promoted biodegradation under gradient B[a]P concentration in soil at different growing stages (Po0.05) and the accumulation of B[a]P in T. patula and M. jalapa increased with the increasing growth time, showing mature stage4flowering stage4seedling stage. However, at the same treatments of B[a]P, there exhibited a slight difference in plantenhanced biodegradation of B[a]P at different growing stages. Furthermore, MacLeod and Semple (2000) revealed that the extent of plant assisted PAH removal from soil decreased with soil incubation time. Aged PAHs are found to less microbially bioavailable due to an increased binding to soil organic matter (Nam and Kim, 2002). The dominant of B[a]P dissipation was contributed to plant-enhanced biodegradation, accounting for 79.54–99.78% and 71.1–99.9%, respectively, after planting T. patula and M. jalapa. By contrast, the amount of Tac was 0.2–20.5% and 0.1–28.9% of dissipation enhancement for B[a]P after planting T. patula and M. jalapa. The results above were consistent with Gao and Zhu (2004), who found that larger than 99.7% dissipation enhancement of Phe and Pyr in planted soils came from plant-promoted biodegradation. 4.3. Rhizosphere effects on B[a]P removal Microbial degradation in the rhizosphere might be the most significant mechanism for removal of organic contaminants in vegetated contaminated soil (Gao and Zhu, 2004, Zhou and Song,
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2004). Deposition of plant-derived carbon and nitrogen sources through root exudation, and/or root turnover provides rhizosphere bacteria with numerous organic substrates (Kamath et al., 2007). Therefore, the rhizosphere of plants constitutes a heterogeneous environment with numerous fluctuating parameters that can contribute significantly to the biodegradation of PAHs during phytoremediation processes such as an increase in microbial community structure and functional diversity (Davey and O’toole, 2000; Liu et al., 2014), a promotion in catabolic enzymes (Huang et al. 2005; Cai et al., 2010; Liu et al., 2014), a modification in bioavailability (Kamath et al., 2007), and an induction of co-oxidation of contaminants (Trapp and Karlson, 2001; Li et al., 2008). The removal ratio of B[a]P ranged from 41.2–71.1%, 49.0–87.5% and 52.4–87.9%, respectively, in rhizospher soils, and 21.7–67.8%, 38.4– 85.0% and 32.2–83.1%, respectively, in non-rhizosphere soils at seedling, flowering and mature stage of M. jalapa. T. patula has hairy roots which can provide a very good environment for detoxification of xenobiotics. The B[a]P removal percentages at different growing stages of T. patula were 3.2–16.0%, 2.7–25.6% and 4.1–26.8%, respectively, under 2–50 mg kg 1 B[a]P contaminated rhizosphere soils, and 0.4–16.2%, 7.2–24.7% and 14.2–33.9%, respectively, under 5–50 mg kg 1 B[a]P treated non-rhizopshere soils higher than those of M. jalapa. Under seedling, flowering and mature stage, the removal ratio values of B[a]P in rhizosphere were 3.3–21.3%, 2.5–11.0% and 1.3–20.2%, respectively, for M. jalapa, and 10.3–19.3%, 4.3–11.5% and 4.2–15.7%, respectively, for T. patula higher than those of in non-rhizosphere, indicating that plants play an important role in removing B[a]P from contaminated soils. It has been found that microbial populations in rhizosphere soils can be 10–100 times higher than those in the surrounding bulk soils and have greater xenobiotic degrading capabilities (Fan et al., 2008). Lee et al. (2008) have reported 499% and 77–94% of Phe and Pyr, respectively, had been degraded in planted soil, whereas 99% and 69% had been degraded in unplanted soil. 91.7–97.8% of Phe and 70.8–90.0% of Pyr had degraded in the planted soils, which were 1.9–3.2% and 8.9–20.7% larger than those in corresponding unplanted soils (Cheema et al., 2009). However, the removal ratio of B[a]P in rhizosphere and non-rhizosphere soils at different growing stages of M. jalapa and T. patula decreased with the increasing of soil B[a]P concentrations. Similar trend on Pye has also been reported by Fan et al. (2008), and the possible occurrence might be due to either inactivation of soil enzymatic activities or on account of limiting microbial communities under B[a]P stress (Phllips et al., 2012).
5. Conclusions The present study showed that the tolerant capability at flowering and mature stage was greater than that of seedling stage, the dry weight of M. jalapa and T. patula increased firstly and then gradually inhibited with increasing B[a]P. Under B[a]P contamination of 0–50 mg kg 1, the concentration and accumulation of B[a]P in all parts of plants were elevated with the increment of their soil concentrations and plant growing time, and significantly positive correlations were exhibited between B[a]P contents in roots, stems, leaves and shoots and soil B[a]P concentrations (P o0.05). The dominant of B[a]P dissipation was contributed to plant-enhanced biodegradation, accounting for 79.5–99.8% and 71.1–99.9%, respectively, after planting T. patula and M. jalapa. Furthermore, the B[a]P removal percentages in rhizosphere soils at different growing stages of T. patula and M. jalapa were 2.7–26.8% and 0.4–33.9%, respectively, higher than those of non-rhizopshere soils. Therefore, it supports the notion that T. patula and M. jalapa can be using for phytoremediation of B[a]P-contaminated soil.
Acknowledgments This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201203045) and the National Natural Science Foundation of China (21177068; 41401362).
References Borneff, J., Farkazdi, G., Glathe, H., Kunte, H., 1973. The fate of polycyclic aromatic hydrocarbons in experiments using sewage sludge–garbage composts as fertilizers. Zentralbl Bakteriol Mikrobiol Hyg [B] 157, 151–164. Cai, Z., Zhou, Q.X., Peng, S.W., Li, K.N., 2010. Promoted biodegradation and microbiological effects of petroleum hydrocarbons by Impatiens balsamina L. with strong endurance. J. Hazard. Mater. 183, 731–737. Cheema, S.A., Khan, M.I., Tang, X.J., Zhang, C.K., Shen, C.F., Malik, Z., Shen, K.L., Chen, X.C., Chen, Y.X., 2009. Enhancement of phenanthrene and pyrene degradation in rhizosphere of Tall Fescue (Festuca arundinacea). J. Hazard. Mater. 166, 1226–1231. Davey, M.E., O’toole, G.A., 2000. Microbial Biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64, 847–867. Dixit, P., Mukherjee, P.K., Sherkhane, P.D., Kale, S.P., Eapen, S., 2011. Enhanced tolerance and remediation of anthracene by transgenic tobacco plants expressing a fungal glutathione transferase gene. J. Hazard. Mater. 192, 270–276. Fan, S.X., Li, P.J., Gong, Z.Q., Ren, W.X., He, N., 2008. Promotion of pyrene degradation in rhizosphere of Alfalfa (Medicago sativa L.). Chemosphere 71, 1593–1598. Gao, Y., Wu, S.C., Yu, X.Z., Wong, M.H., 2010. Dissipation gradients of phenanthrene and pyrene in the rice rhizosphere. Environ. Pollut. 158, 2596–2603. Gao, Y.Z., Shen, Q., Ling, W.T., Ren, L.L., 2008. Uptake of polycyclic aromatic hydrocarbons by Trifolium pretense L. from water in the presence of a nonionic surfactant. Chemosphere 72, 636–643. Gao, Y.Z., Zhu, L.Z., 2004. Plant uptake, accumulation and translocation of phenanthrene and pyrene in soils. Chemosphere 55, 1169–1178. Gong, Z., Yang, J., Yang, M., Wang, F.M., Wei, Q., Tanguay, R.M., Wu, T., 2006. Benzo (a)pyrene inhibits expression of inducible heat shock protein 70 in vascular endothelial cells. Toxicol. Lett. 166, 229–236. Huang, X.D., Ei–Alawi, Y., Gurska, J., Glick, B.R., Greenberg, B.M., 2005. A multiprocess phytoremediation system for decontamination of persistent total petroleum hydrocarbons (TPHs) from soils. Microchem. J. 81, 139–147. Johnson, D.L., Maguire, K.L., Anderson, D.R., McGragh, S.P., 2004. Enhanced dissipation of chrysene in planted soil: the impact of a rhizobial inoculum. Soil Biol. Biochem. 36, 33–38. Kamath, R., Rentz, J.A., Schnoor, J.L., Alvarez, P.J.J., 2007. Phytoremediation of Hydrocarboncontaminated Soils; Principles and Applications. University of Iowa, Iowa City, Iowa, USA, p. 52242. Ko-Wasik, A., Da˛browska, D., Namiesnik, J., 2004. Photodegradation and biodegradation study of benzo(a)pyrene in different liquid media. J. Photochem. Photobiol. A: Chem. 168, 109–115. Li, H., Sheng, G., Chiou, C.T., Xu, O.Y., 2005. Relation of organic contaminant equilibrium sorption and kinetic uptake in plants. Environ. Sci. Technol. 39, 4864–4870. Li, P.J., Li, X.J., Stagnitti, F., Zhang, H.R., Lin, X., Zhang, S.Y., Zhuo, J.C., Xiong, X.Z., 2009. Studies on the sources of benzo[a]pyrene in grain and aboveground tissues of rice plants. J. Hazard. Mater. 162, 463–468. Li, X.J., Li, P.J., Lin, X., Zhang, C.H., Li, Q., Gong, Z.Q., 2008. Biodegradation of aged polycyclic aromatic hydrocarbons (PAHs) by microbial consortia in soil and slurry phases. J. Hazard. Mater. 150, 21–26. Liu, J.Y., Schnoor, J.L., 2008. Uptake and translocation of lesser-chlorinated polychlorinated biphenyls (PCBs) in whole hybrid poplar plants after hydroponic exposure. Chemosphere 73, 1608–1616. Liu, R., Xiao, N., Wei, S.H., Zhao, L.X., An, J., 2014. Rhizosphere effects of PAH–contaminated soil phytoremediation using a special plant named Fire Phoenix. Sci. Total Environ. 473-474, 350–358. Lu, S.J., Teng, Y.G., Wang, J.S., Sun, Z.J., 2010. Enhancement of pyrene removed from contaminated soils by Bidens maximowicziana. Chemosphere 81, 645–650. MacLeod, C., Semple, K., 2000. Influence of contact time on extractability and degradation of pyrene in soils. Environ. Sci. Technol. 34, 4952–4957. Mcgarry, M.A., Charles, G.D., Medrano, T., Bubb, M.R., Grant, M.B., Campbell– Thompson, M., Shiverick, K.T., 2002. Benzo(a)pyrene, but not 2,3,7,8-tetrachlorodibenzo-p-dioxin, alters cell adhesion proteins in human uterine RL95-2 cells. Biochem. Biophys. Res. Commun. 294, 101–107. Nam, K., Kim, J.Y., 2002. Role of loosely bound humic substances and humin in the bioavailability of phenanthrene aged in soil. Environ. Pollut. 118, 427–433. Peng, S.W., Zhou, Q.X., Cai, Z., Zhang, Z.N., 2009. Phytoremediation of petroleum contaminated soils by Mirabilis jalapa L. in a field plot experiment. J. Hazard. Mater. 168, 1490–1496. Perelo, L.W., 2010. Review: In situ and bioremediation of organic pollutants in aquatic sediments. J. Hazard. Mater. 177, 81–89. Pfeifer, G.P., Denissenko, M.F., Olivier, M., Tretyakova, N., Hecht, S.S., Hainaut, P., 2002. Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking–associated cancers. Oncogene 21, 7435–7451. Phillips, L.A., Greer, C.W., Farrell, R.E., Germida, J.J., 2012. Plant root exudates impact
Y. Sun, Q. Zhou / Ecotoxicology and Environmental Safety 124 (2016) 74–81
the hydrocarbon degradation potential of a weathered–hydrocarbon contaminated soil. Appl. Soil Ecol. 52, 56–64. Reilley, K.A., Banks, M.K., Schwab, A.P., 1996. Dissipation of polycyclic aromatic hydrocarbons in the rhizosphere. J. Environ. Qual. 25, 212–219. Schroll, R., Bierling, B., Cao, G., Dorfler, U., Lahaniati, M., Langenbach, T., Scheunert, I., Winkler, R., 1994. Uptake pathways of organic chemicals from soil by agricultural plants. Chemosphere 28, 297–303. Simonich, S.L., Hites, R.A., 1994. Vegetation-atmosphere partitioning of polycyclic aromatic hydrocarbons. Environ. Sci. Technol. 28, 939–943. Simonich, S.L., Hites, R.A., 1995. Organic pollutant accumulation in vegetation. Environ. Sci. Technol. 29, 2095–2103. Singh, O.V., Jain, R.K., 2003. Phytoremediation of toxic aromatic pollutants from soil. Appl. Microbiol. Biotechnol. 63, 128–135. Sinha, R., Kulldorff, M., Gunter, M.J., Strickland, P., Rothman, N., 2005. Dietary Benzo [a]Pyrene intake and risk of colorectal adenoma. Cancer Epidemiol. Biomak. Prev. 14, 2030–2034. Su, Y.H., Zhu, Y.G., 2005. Uptake of selected PAHs from contaminated soils by rice seedlings (Oryza sativa) and influence of rhizosphere on PAH distribution. Environ. Pollut. 155, 359–365. Sun, Y.B., Zhou, Q.X., Diao, C.Y., 2008. Effects of cadmium and arsenic on growth and metal accumulation of Cd–hyperaccumulator Solanum nigrum L. Bioresour. Technol. 99, 1103–1110. Sun, Y.B., Zhou, Q.X., Xu, Y.M., Wang, L., Liang, X.F., 2011. Phytoremediation for cocontaminated soils of benzo[a]pyrene (B[a]P) and heavy metals using ornamental plant Tagetes patula. J. Hazard. Mater. 186, 2075–2082. Sun, Y.B., Zhou, Q.X., Xu, Y.M., Wang, L., Lin, D.S., Liang, X.F., 2013. The potential of gibberellic acid 3 (GA3) and Tween-80 induced phytoremediation of co–contamination of Cd and B[a]P using Tagetes patula. J. Environ. Manag. 114, 202–208.
81
Trapp, S., Karlson, U., 2001. Aspects of phytoremediation of organic pollutants. J. Soil. Sediment. 1, 1–7. Trapp, S., Matthies, M., Scheunert, I., Topp, E.M., 1990. Modeling the bioconcentration of organic chemicals in plants. Environ. Sci. Technol. 24, 1246–1252. Uno, S., Dalton, T.P., Shertzer, H.G., Genter, M.B., Warshawsky, D., Talaska, G., Nebert, D.W., 2001. Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1 ( / ) knockout mice having increased hepatic BaP-DNA adduct levels. Biochem. Biophys. Res. Commun. 289, 1049–1056. Wei, J., Liu, X.Y., Wang, Q., Wang, C.H., Chen, X.P., Li, H.B., 2014. Effect of rhizodeposition on pyrene bioaccessibility and microbial structure in pyrene and pyrene–lead polluted soil. Chemosphere 97, 92–97. Xu, S.Y., Chen, Y.X., Lin, Q., Wu, W.X., Xue, S.G., Shen, C.F., 2005. Uptake and accumulation of phenanthrene and pyrene in spiked soils by Ryegrass (Lolium perenne L.). J. Environ. Sci. 17, 817–822. Yin, X.M., Liang., X., Xu, G.H., Zhan, X.H., 2014. Effect of phenanthrene uptake on membrane potential in roots of soybean, wheat and carrot. Environ. Exp. Bot. 99, 53–58. Zhang, Z.H., Rengel, Z., Chang, H., Meney, K., Pantelic, L., Tomanovic, R., 2012. Phytoremediation potential of Juncus subsecundus in soils contaminated with cadmium and polynuclear aromatic hydrocarbons (PAHs). Geoderma 175–176, 1–8. Zhou, Q.X., Diao, C.Y., Sun, Y.B., Zhou, J.L., 2012. Tolerance, uptake and removal of nitrobenzene by a newly-found remediation species Mirabilis jalapa L. Chemosphere 86, 994–1000. Zhou, Q.X., Song, Y.F., 2004. Principles and Methods of Contaminated Soil Remediation. Science Press, Beijing, China. Zhou, Q.X., Wei, S.H., Zhang, Q.R., 2006. Ecological Remediation. Environmental Science Press, Beijing, China.