Journal of Environmental Management 147 (2015) 73e80
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Recovering a copper mine soil using organic amendments and phytomanagement with Brassica juncea L. n Forja n, Vero nica Asensio 1 Alfonso Rodríguez-Vila*, Emma F. Covelo, Rube Department of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, Lagoas, Marcosende, 36310 Vigo, Pontevedra, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 May 2014 Received in revised form 31 July 2014 Accepted 9 September 2014 Available online
A 3-month greenhouse experiment was carried out for evaluating the effect of an amendment mixture and mustards on the chemical characteristics of a mine soil and the metal uptake by plants. A settling pond soil was amended with increasing percentages of a technosol and biochar mixture and vegetated with Brassica juncea L. Adding amendments and planting mustards increased the soil pH from 2.83 to 6.18 and the TSC from u.l to 131 g kg1 and generally reduced the CaCl2-extractable metal concentrations in the soil. However, the amendments increased the pseudo-total soil concentration of Ni from 9.27 to 31.9 mg kg1, Pb from 27.9 to 91.6 mg kg1 and Zn from 46.5 to 577 mg kg1. The technosol and biochar mixture increased the shoot biomass from 0.74 to 2.95 g and generally reduced the metal concentrations in plants, meaning B. juncea as a potential candidate for phytostabilization of mine soils.
Keywords: Settling pond Phytoremediation Technosol Biochar Brassica juncea
1. Introduction Mining activities negatively impacted the environment due to the deposition of a huge amount of waste material (Vega et al., 2005). Copper was extracted from a mine in Touro (Spain) between 1973 and 1988. Today, another company is extracting material from the mine for use in road building projects. In 1988, work began on recovering this mine by planting Pinus pinaster Aiton and Eucalyptus globulus Labill, and later by incorporating organic wastes into the soil, sewage sludges and paper mill residues, among others (Asensio et al., 2013a). The incorporation of organic amendments improves the quality of mine soils and makes it possible for vegetation to become established (Asensio et al., 2013b). Biochar has been described as a material that can possibly improve soil fertility (Sohi et al., 2010). Such biochar-amended soils can be phytomanaged (Paz-Ferreiro et al., 2014). Phytoextraction is based on using plants and associated microorganisms to remove contaminants from soils (Ali et al., 2013). Some plants, called hyperaccumulators, can uptake, accumulate and tolerate high metal concentrations in plant tissues (Evangelou et al., 2004). Indian mustard (Brassica juncea L.)
* Corresponding author. Tel.: þ34 986812630; fax: þ34 986812556. E-mail address: [email protected] (A. Rodríguez-Vila). 1 ~o Present address: Department of Plant Nutrition, CENA, Universidade de Sa rio 303, 13400-970 Piracicaba, SP, Brazil. Paulo (CENA-USP), Av. Centena http://dx.doi.org/10.1016/j.jenvman.2014.09.011 0301-4797/© 2014 Elsevier Ltd. All rights reserved.
© 2014 Elsevier Ltd. All rights reserved.
produces a large amount of shoot biomass and can be used for phytoextraction of metals in a reasonable period of time (Quartacci et al., 2006). Numerous studies have shown that adding organic amendments (sewage sludge and manure, among others) to soil promotes phytoextraction processes (Dede et al., 2012; Wei et al., 2011), but only a few of them have evaluated the combined effect of organic amendments and phytoextraction with B. juncea in copper mine soils (Novo et al., 2013). This study aimed at assessing the potential of B. juncea as a phytoextractor of metals from a mine soil polluted by copper and amended with wastes (sewage sludge, aluminium sludge, waste from food industries, sand and ashes from a paper mill) and biochar. 2. Materials and methods 2.1. Soil sampling The sampling area is located at a mine in Touro (Spain) (Lat/Lon) 0 00 0 00 (Datum ETRS89): 8 20 12.06 W 42 52 46.18 N. Copper extraction in this mine produced a settling pond covering an area of 71 ha which has been completely dry for several years. Some parts of the settling pond were colonized by native vegetation: gorse (Ulex sp.), heather (Erica sp.), Agrostis sp. and bryophytes. The mine soil (S) was collected from an untreated area of the settling pond without vegetation. The organic amendment used for the greenhouse experiment was a mixture of technosol (95%)
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gicos del Noroeste S.L. (Santiago de supplied by Tratamientos Ecolo Compostela, Spain) and biochar (5%) provided by PROININSO S.A. laga, Spain). The technosol (T) was made of municipal sewage (Ma sludge (60%), sludge from an aluminium company (10%), waste from food industries (canning and rabbit farms) (10%), sands from wastewater treatment plants (5%) and ashes from a paper mill (5%). This total does not account for 100% of the contents of the technosol, and the percentages are not completely accurate. The biochar (B) was made of holm oak wood treated at a pyrolysis temperature of 400 C for 8 h. The general characteristics of S, T and B are shown in Table 1.
Table 2 Labels for the soil samples in pots according to their mixture of soil and amendment. Soil mixture
Pots without plant
Pots with plant
Control: settling pond soil 80% soil þ 20% amendmenta 60% soil þ 40% amendmenta 20% soil þ 80% amendmenta 100% amendmenta
A0 A20 A40 A80 A100
A0P A20P A40P A80P A100P
a
Amendment mixture is made of 95% technosol þ 5% biochar.
65 ± 5% relative air humidity. The soil moisture was kept at 35 ± 5% according to field capacity.
2.2. Greenhouse experiment 2.3. Soil analyses The greenhouse experiment was carried out in 0.5 L pots with the settling pond soil (S) amended with the previously described mixture of technosol (T) and biochar (B) and vegetated with B. juncea L. seedlings previously germinated in other pots. The soil was amended with 20%, 40%, 80% and 100% of the amendment, always mixing T with B as follows: 19% T and 1% B (A20), 38% T and 2% B (A40), 76% T and 4% B (A80) and 95% T and 5% B (A100). Some pots were only filled with mine soil (A0, with 0% amendment) as the control treatment (Table 2). The plastic pots were filled with a dry weight of 200 g of the corresponding mixture. The control pots were supplemented with drinking straws cut into 1 cm pieces in order to minimize the difference in the substrate volume between the control soils and the amended soils (Puig et al., 2013). A total of 6 pots were filled with the different types of mixtures: three were used for planting mustard and the other three were left unplanted for evaluating the vegetation effect on soil physico-chemical properties. The samples planted with mustards were marked with the letter P (e.g. A20P). Three seedlings of B. juncea, previously germinated from seeds until they had two fully expanded leaves, were transferred to each pot. After 45 days, the plants were thinned to one per pot following uniform criteria. The plants, using 3 replicates per treatment, were harvested 90 days after sowing for subsequent analysis. Growth was allowed under greenhouse-controlled conditions: with a photoperiod of 11:13 light/dark, a temperature of 22 ± 2 C, and
Table 1 Selected characteristics and metal concentrations of the mine soil (S) and the two amendments (biochar B and technosol T) and the generic reference level (GRL) of metals (Macías and Calvo de Anta, 2009). S
B
T
GRL
pH H2O pH KCl TSC (g kg1) TN (g kg1) C/N
2.96 ± 0.07c 2.73 ± 0.02c u.l
9.93 ± 0.22a 9.64 ± 0.05a 667a
5.55 ± 0.07b 5.48 ± 0.05b 218b
e e e
u.l
5.8b
22.9a
e
e
115a
9b
e
Pseudo-total (mg kg1)
Co Cu Ni Pb Zn
31.9 ± 3.03a 452 ± 52.56b 18.9 ± 3.40b 20.9 ± 3.20b 65.4 ± 20.77b
u.l 28.7 ± 2.76c 27.9 ± 8.69b u.l 85.8 ± 11.38b
24.5 ± 2.53b 656 ± 47.55a 82.4 ± 17.84a 94 ± 2.95a 1446 ± 91.52a
40 50 75 80 200
CaCl2extractable (mg kg1)
Co Cu Ni Pb Zn
8.4 ± 0.07a 188 ± 10.88a 8.29 ± 0.07a u.l 2.64 ± 0.10c
0.25 ± 0.02b u.l 0.34 ± 0.02c u.l 1.28 ± 0.30b
0.71 ± 0.07b 4 ± 0.07b 4.25 ± 0.22b 0.43 ± 0.04 735 ± 3.83a
e e e e e
Mean ± CI values (n ¼ 3). Values followed by different letters differ significantly with P < 0.05. u.l: values below the detection limit; TSC: total soil carbon; TN: total soil nitrogen; GRL: generic reference level established for Galician soils (Macías and Calvo de Anta, 2009). Values in bold are over the GRL.
The soil samples collected from the field and from the pots were air dried, passed through a 2 mm sieve and homogenized prior to analysis. A series of soil characteristics were determined (Table 3). Soil pH was determined using a pH electrode in 1:2.5 water or KCl to soil extracts (Porta, 1986). Total soil carbon (TSC) and total nitrogen (TN) were determined in a LECO CN-2000 module using solid samples. Co, Cu, Ni, Pb and Zn were extracted with acidified 0.01 M CaCl2, which is operationally defined as phytoavailable (Houba et al., 2000). The pseudo-total concentrations of Co, Cu, Ni, Pb and Zn were extracted with aqua regia in a microwave oven (Milestone ETHOS 1). The certified soil reference material CRM026 was analysed in parallel with samples to check the effectiveness and precision of the extraction analysis. Metal concentrations were determined by ICPeAES (Optima 4300 DV; PerkineElmer) and were compared with the generic reference level established for Galician soils (Macías and Calvo de Anta, 2009) (Table 4). 2.4. Pore water analysis One week before harvesting the mustards, a “Rhizon” soil pore water sampler (Eijkelkamp Agrisearch Equipment, The Netherlands) was carefully inserted into the soil of each treatment replicate pot at an angle of approximately 45 . Vacuum tubes (10 mL) were attached through a Luer lock system with hypodermic needles to extract pore water. Pore water samples were analysed for metal concentrations by ICPeAES, carried out in triplicate. 2.5. Plant growth and determination of metals in plant tissues After 90 days, the roots and shoots were sampled and carefully washed with deionized water. Fresh biomass was immediately weighed and dry mass was assessed after oven drying for 48 h at 80 C and cooling down to room temperature. Table 3 Selected properties in soil samples at 3 months of experiment. Soil sample
pH H2O
A0 A20 A40 A80 A100 A0P A20P A40P A80P A100P
2.83 4.56 5.16 5.97 6.02 2.70 4.58 5.56 6.18 6.07
± ± ± ± ± ± ± ± ± ±
0.08g 0.11f 0.13e 0.28c 0.1bc 0.17g 0.05f 0.26d 0.24a 0.1b
pH KCl 2.72 4.49 5.12 5.94 5.98 2.60 4.54 5.52 6.17 6.06
± ± ± ± ± ± ± ± ± ±
0.04g 0.11f 0.15e 0.26c 0.1c 0.18h 0.07f 0.21d 0.23a 0.07b
TSC (g kg1)
TN (g kg1)
C/N
u.l 26.23e 56.53d 131b 238a u.l 21e 59.1d 110c 236a
u.l 2.6e 5.5d 11.4b 20.23a u.l 1.97e 5.03d 9.5c 20.5a
e 9.67b 10b 11a 11a e 10b 11a 11a 11a
Mean ± CI values (n ¼ 9). Values followed by different letters in each row of each treatment differ significantly with P < 0.05. u.l: values below the detection limit; TSC: total soil carbon; TN: total soil nitrogen.
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Table 4 Pseudo-total and CaCl2-extractable (phytoavailable) Co, Cu, Ni, Pb and Zn concentrations in soil samples at 3 months of experiment; concentrations of the reference soil CRM026 and generic reference level (GRL) of metals (Macías and Calvo de Anta, 2009). Pseudo-total (mg kg1)
Soil sample A0 A20 A40 A80 A100 A0P A20P A40P A80P A100P GRL a
Co
Cu
32.5 ± 6.72a 33 ± 3.29a 30.8 ± 6.72 ab 22.2 ± 7.67c 16.7 ± 5.52d 32.6 ± 6.75a 30.1 ± 6.41b 28.7 ± 3.43b 22.3 ± 4.11c 13.9 ± 2.81d
341 419 408 352 349 344 408 350 300 315
40
50 23.1 ± 3.33 18.8 ± 0.87 3.01
CRM026 12.2 ± 0.98 CRM026b 6.8 ± 0.67 SD 3.85
CaCl2-extractable (mg kg1) Ni
± ± ± ± ± ± ± ± ± ±
53.74bc 40.65a 98.15a 84.51b 27.08b 88.12b 134a 60.86b 63.56d 41.94cd
Pb
Zn
Co
29.9 ± 11.17gh 42.8 ± 14.43f 52.4 ± 10.33e 91.6 ± 24.67c 125 ± 20.54a 27.9 ± 9.16h 37.6 ± 9.37 fg 52 ± 16.89e 60.8 ± 19.52d 105 ± 18.91b
46.5 ± 9.73h 127 ± 17.39g 246 ± 59.36e 577 ± 200c 876 ± 187a 49.3 ± 12.9h 175 ± 52.72 fg 244 ± 84.26ef 408 ± 128d 773 ± 72.72b
7.15 3.49 3.28 0.74 0.36 6.88 4.09 1.75 0.78 0.35
75
80
200
19.5 ± 1.97 14.4 ± 1.54 3.63
45.3 ± 6.28 25.6 ± 1.88 13.92
174.5 ± 17.68 140 ± 7.09 24.38
11.9 14.8 22.1 31.9 46.4 9.27 19.4 23.2 26.1 42.1
± ± ± ± ± ± ± ± ± ±
3.75 fg 2.84f 6.03de 8.56c 8.57a 2.9g 1.24e 5.64de 5.76d 4.81b
Cu
Ni
152 ± 34.87a 98.15 ± 13.6b 54.01 ± 16.94c 3.43 ± 0.67e 2.03 ± 0.5e 149 ± 38.49a 110 ± 20.13b 30.54 ± 6.01d 3.27 ± 0.89e 1.67 ± 0.21e
7.22 5.11 6.08 3.16 2.12 6.88 5.97 4.32 3.07 1.98
e
e
e
e
e
e e e
e e e
e e e
e e e
e e e
± ± ± ± ± ± ± ± ± ±
2.64a 0.95bc 1.06c 0.14e 0.06e 2.17a 1.39b 0.39d 0.08e 0.05e
Pb ± ± ± ± ± ± ± ± ± ±
2.68a 1.37c 1.77b 0.18d 0.13e 2.19a 1.92b 0.63c 0.32d 0.22e
u.l 4.83 3.14 0.58 0.68 u.l 5.27 2.82 0.63 0.58
Zn ± ± ± ±
0.78b 0.78c 0.04d 0.16d
± ± ± ±
0.65a 0.85c 0.11d 0.19d
2.78 ± 0.12g 69.43 ± 18.12f 168 ± 48.84c 299 ± 8.03a 291 ± 11.96a 3.08 ± 0.43g 86.61 ± 31.17e 156 ± 29.63d 281 ± 8.57b 271 ± 26.88b
Mean ± CI values (n ¼ 9). Values followed by different letters in each row of each treatment differ significantly with P < 0.05. u.l: values below the detection limit; GRL: generic reference level established for Galician soils (Macías and Calvo de Anta, 2009). Values in bold are over the GRL. a Values obtained from this study. b Certified values.
The plant tissues were air dried and ground. The total concentrations of Co, Cu, Ni, Pb and Zn in plants were extracted by acid digestion using a mixture of H2O2 and HNO3 (1:6 v/v) in a microwave oven. The certified reference plant material ERM-CD281 was analysed in parallel with the samples. The transfer coefficient (TC) for the plants was here calculated using the following equation:
TC ¼
Cp Cso
(1)
where Cp is the metal concentration in shoots (mg kg1) and Cso is the metal content of soil (mg kg1) (Peijnenburg and Jager, 2003). A TC with high value indicates a high shoot concentration of the element compared to the pseudo-total concentration in soil (Karami et al., 2011). A plant species is considered as an accumulator or hyperaccumulator biosystem when TC > 1 (Busuioc et al., 2011). The translocation factor (TF) for the plants is expressed by the following equation:
Cs TF ¼ Cr
(2)
where Cs and Cr are metal concentrations (mg kg1) in shoots and roots, respectively. A TF with a high value indicates a relatively high shoot metal concentration compared to its root concentration. A plant species translocates metals effectively from the roots to shoots when TF > 1 (Baker and Brooks, 1989). The bioconcentration factor (BF) for plants is expressed by the following equation:
BF ¼
Cp Cpw
(3)
where Cp is the shoot metal concentration (mg kg1) and Cpw is the metal concentration in the soil pore water (mg L1) (Mahon and Mathewes, 1983). The BF describes the ratio of metal in pore water concentration that is taken up into shoots. High BF values indicate high shoot element concentration compared to the element concentration in the soil pore water (Karami et al., 2011).
For calculating the TC and BF, we used the shoot metal concentrations of the mustard plants, as under field conditions this is the plant part that would be removed by a combine harvester. Moreover, the analysed roots had low harvestable mineral mass (root DW yield root concentration for each metal). 2.6. Statistical analysis All analytical determinations were performed in triplicate. The data obtained were statistically treated using SPSS version 19.0 for Windows. An analysis of variance and homogeneity of variance test were carried out. In case of homogeneity, a post-hoc least significant difference test was carried out. If there was no homogeneity, Dunnett's T3 test was performed. We also carried out a correlated bivariate analysis. 3. Results 3.1. Characteristics of the mine soil and amendments The untreated soil (S) had an extremely acid pH and high concentrations of pseudo-total Cu (Table 1). The total soil carbon (TSC) and total nitrogen (TN) contents were below the detection limit for the mine soil (Table 1). The addition of amendments can help to correct these TSC and TN lacks because of their high content in organic matter and inorganic carbon. The technosol is however contaminated by Cu, Ni, Pb and Zn (Table 1). These metals can be provided by technosol components such as sewage and aluminium sludges (Asensio et al., 2013c). 3.2. Chemical properties of the greenhouse soil samples The chemical properties of the mine soil (pots A0 and A0P) were significantly changed 3 months after the soil amendment and cultivation (Tables 3 and 4), while the pH of untreated soil remained extremely acid according to the USDA (1998). In the cultivated untreated soil (A0P), all plants died one week after planting. The amended soils had significantly higher pH values than the untreated soils. The highest increase in soil pH was found in A80P. In the amended soils, the pH rose to 4.6e6.2, probably due to their high concentration in basic cations and the buffer action of the
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organic matter which increases the CEC, according to previous studies (Asensio et al., 2013b). The TSC and TN were below the detection limits in both A0 and A0P soils. Both TSC and TN concentrations increased significantly with the amendment incorporation. The pseudo-total Co values were lower than the generic reference level (GRL) for Galician soils in all studied soil samples (Table 4) and their concentration was generally diluted by adding technosol and biochar to the mine soil. The pseudo-total Cu concentrations were higher than the GRL in all soil samples (Table 4) and the highest ones were detected in A20, A20P and A40, but the highest CaCl2-extractable Cu was observed in A0 and A0P. A significant negative correlation was obtained between the CaCl2extractable Cu concentration in soils with plants and this concentration in soils without plants (P < 0.01, r ¼ 0.325). The pseudototal and CaCl2-extractable Ni concentrations were lower than the GRL in all soil samples (Table 4), but the higher the amendment rate, the higher the Ni pseudo-total concentration. The highest pseudo-total Pb concentration was found in the A100 and A100P soils, with values over the GRL. The pseudo-total Zn exceeded the GRL in the soil samples with the highest amendment rate. 3.3. Plant biomass and uptake of metals The shoot DW yield peaked for plants grown in the A80P soil (Table 5). The TC and the TF values for Co could only be calculated for the sample A20P, as shoot and root Co concentration were below the Co detection limit (0.005 mg L1). The TC and TF values of Co for plants cultivated in the A20P soil were 0.33 and 0.2, respectively. For Cu, Ni, Pb and Zn, the higher the amendment rate, the lower the TC value (Fig. 1). The TF showed the opposite pattern than the TC for Cu and Zn, but the same in the case of Ni. It was not possible to calculate the TC or TF for Ni in A80P as the shoot Ni concentration was below the Ni detection limit. The TF for Pb could not be calculated because the root Pb concentration was below the detection limit for all root samples. The TC for Pb was significantly the highest in A20P and A80P and the lowest in A100P. It was not possible to calculate it in A40P because the Pb concentration in the plants was below the detection limit. Amendment incorporation into the S soil significantly increased the BF for Cu only for A20P compared to A80P (Fig. 2a). The highest BF for Ni was observed in the treatments A20P and A100P (Fig. 2b). For Pb, the highest BF was found in A80P and A100P (Fig. 2c). For Zn, the application of amendments significantly increased the BF for A20P compared to A40P and A80P (Fig. 2d). The shoot metal removal (shoot DW yield shoot metal concentration) of plant in each pot was computed to account for changes in shoot DW yield (Fig. 3). For Cu, the higher amendment rate, the higher the shoot Cu removal (Fig. 3a). The A20P plants had the highest shoot Ni removal (Fig. 3b). The highest shoot Pb
Table 5 Biomass produced and concentration of Co, Cu, Ni, Pb and Zn in B. juncea plants grown in the different soil treatments at 3 months of experiment. A20P Biomass (g) Co (mg kg1) Cu (mg kg1) Ni (mg kg1) Pb (mg kg1) Zn (mg kg1)
0.74 64.32 223 55.19 6.67 1113
± ± ± ± ± ±
0.12c 8.28 8.69a 8.12a 0.87b 33.36a
A40P
A80P
A100P
2.4 ± 0.23b u.l 67.08 ± 8.92b 25.01 ± 0.9b u.l 716 ± 9.41b
2.95 ± 0.25a u.l 47.05 ± 2.67c 4.26 ± 0.02d 11.78 ± 2.59a 729 ± 23.77b
2.55 ± 0.67 ab u.l 34.45 ± 3.55d 19.07 ± 4.28c 11.72 ± 2.48a 922 ± 44.78a
Mean ± CI values (n ¼ 3 for biomass and n ¼ 9 for metals). Values followed by different letters in each row of each treatment differ significantly with P < 0.05. u.l: values below the detection limit.
removal was obtained for the A80P plants, followed by the A100P plants (Fig. 3c). For Zn, a similar behaviour to Cu was observed, as the higher the amendment rate, the higher the shoot Zn removal (Fig. 3d). 4. Discussion 4.1. Metal concentrations in soils The low Co concentration in the amended soils (Table 4) was likely due to the dilution effect of mixing the amendments with the S soil. In addition, all soil samples had high pseudo-total Cu concentrations above the GRL, as both the untreated mine soil and the technosol have a high total Cu concentration (Table 1). For the same amendment rate, the CaCl2-extractable Cu concentration was generally lower in the cultivated amended soils and technosols, than in the barren ones (Table 4). This indicates that the mustards extracted Cu from the soils. In fact, a significant negative correlation was obtained between the CaCl2-extractable Cu concentration in soils with plants and this concentration in soils without plants (P < 0.01, r ¼ 0.325). The unamended soils had the highest CaCl2-extractable Cu concentrations (Table 4), which may be mainly due to the low TSC to retain this metal and to the extremely acid pH (Table 3). This suggests that the high pH and TSC provided by the organic amendments reduced Cu mobility in soil. This metal can be strongly bound to organic matter and forms both rezsoluble and insoluble complexes with organic compounds (Pe Esteban et al., 2014). The higher pseudo-total Ni concentration in the amended soils than in the untreated ones (Table 4) was likely due to high Ni concentration in the technosol (Table 1). Nevertheless, the highest CaCl2-extractable Ni concentrations were observed in the unamended soils (Table 4). As for Cu, this may reflect the low TSC and soil pH in these soils samples. Higher pH values increase the surface rezcharges of soil particles and therefore metal retention in soil (Pe Esteban et al., 2014). Adding amendments to the S soil increased the concentrations of both pseudo-total Pb and Zn and CaCl2-extractable Zn in the soil, which exceeded the GRL in the samples with the highest amendment rate (Table 4). This may be due to the Pb and Zn provided by the wastes used (Table 1) (Kumar and Chopra, 2014). 4.2. Uptake and transfer of metals to mustards 4.2.1. Cobalt The total Co concentration in B. juncea plants was higher than the Co detection limit only for the soil A20P. The TC for A20P was lower than 1, which indicates that the mustards did not extract this metal from soil efficiently (Busuioc et al., 2011). The TF of less than 1 for Co for A20P indicates low root to shoot translocation (Baker and Brooks, 1989). Also, it was only possible to calculate the BF and the shoot Co removal for this sample. 4.2.2. Copper The application of technosol and biochar as a soil amendment resulted in lower Cu concentrations in plant shoots (Table 5). The TC for Cu was lower than 1 for all soil samples (Fig. 1a). Mustards did not extract Cu from soil efficiently due to their low shoot Cu concentrations (Table 5), according to recent studies (van der Ent et al., 2013). The TF for Cu was also lower than 1 for all of the samples (Fig. 1a) indicating low root to shoot translocation (Baker and Brooks, 1989). The highest BF value for Cu was found in the A100P, A40P and A20P treatments and the lowest in A80P (Fig. 2a). This demonstrates that the BF on its own is not enough to assess the efficiency of plants to extract metals from soils and
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Fig. 1. Transfer of Cu (a), Ni (b), Pb (c) and Zn (d) to and within mustards according to transfer coefficient (TC) (shoots concentration/pseudo-total soil concentration) and translocation factor (TF) (shoots concentration/roots concentration). The solid line is the median, the box represents the upper and lower quartiles and whiskers are the 10th and 90th percentiles (n ¼ 9). Different letters indicate significant differences in TC or TF between treatments (P < 0.05). The TF and TC for Co were not represented because they only could be calculated for the soil A20P. The TF for Pb could not be calculated because Pb concentration in mustard roots was below the detection limit.
that it is also necessary to take the shoot metal removal into account. The values for shoot Cu removal are statistically similar for all treatments except for A20P, and above a 40% amendment rate, mustard plants are not capable of taking up more Cu from the soil (Fig. 3). 4.2.3. Nickel Nickel concentrations in plants decreased significantly with the application of technosol and biochar as a soil amendment (Table 5). Both the TC and TF for Ni were lower than 1 in all of the studied samples (Fig. 1b), indicating that mustards do not extract Ni efficiently from these soils (Busuioc et al., 2011) with a very low root to shoot translocation of this metal (Baker and Brooks, 1989). Both the TC and TF significantly decrease with the addition of amendments to the mine soil (Fig. 1b). Amendment incorporation into the S soil significantly decreased the values of shoot Ni removal for A40P compared to A20P (Fig. 3b). A significantly positive correlation was obtained between CaCl2-ext. Ni and Ni in lixiviates and the total plant concentration (P < 0.05). Therefore, the data obtained indicates that the higher the concentration of Ni
in the soil solution, the higher the concentration taken up by the mustard shoots. 4.2.4. Lead The incorporation of organic amendments into the soil significantly increased the total Pb concentration in plant tissues for A80P compared to A20P (Table 5). The TC and TF have values lower than 1 for Pb in all of the samples (Fig. 1c), indicating that mustard plants were not capable of efficiently extracting this metal from soils (Busuioc et al., 2011) or translocating it from the roots to shoots (Baker and Brooks, 1989). The BF also had low values (Fig. 2c). The application of technosol and biochar as a soil amendment significantly increased the values for shoot Pb removal for A80P compared to A20P (Fig. 3c). Due to the low concentration of Pb in phytoavailable form, it was not possible to assess the phytoextraction potential of Pb by mustards. 4.2.5. Zinc Zinc concentrations in plants decreased significantly with the application of technosol and biochar as a soil amendment for A40P
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Fig. 2. Transfer of Cu (a), Ni (b), Pb (c) and Zn (d) to mustards according to bioconcentration factor (BF) (shoots concentration/soil pore water concentration). The solid line is the median, the box represents the upper and lower quartiles and whiskers are the 10th and 90th percentiles (n ¼ 9). Different letters indicate significant differences in BF between treatments (P < 0.05). The BF for Co was not represented because it only could be calculated for the soil A20P, since the Co total concentration in plants was below the detection limit in the others.
and A80P compared to A20P (Table 5). The TC for Zn was equal or higher than 1 for all of the samples, indicating that mustards are efficient in the uptake of this metal from soils and are also hyperaccumulators of it (Busuioc et al., 2011) (Fig. 1d). The data showed that the higher the amendment rate, the lower the TC for Zn. This is probably because the higher the percentage of amendment, the lower the concentration of Zn in soil pore water (data not shown). In fact, the highest BF for Zn is in the samples with the highest percentage of amendment (Fig. 2d). Moreover, the samples with the highest percentage of amendment had the highest values for shoot metal removal and the highest plant biomass. The TF for Zn (Fig. 1d) increases by adding organic amendment to the soil, probably because it promotes the plant biomass produced. The TF was higher than 1 for all of the samples (Fig. 1d), indicating that mustards efficiently uptake Zn from roots to shoots (Baker and Brooks, 1989). Many studies have shown that these plants are suitable for the phytoextraction of Zn in soils polluted by this metal (e.g. Novo et al., 2013). 4.3. Viability of technosol and biochar to reclaim polluted soils Organic amendments such as wastes and biochar have previously been used to improve the quality of metal polluted mine soils (Asensio et al., 2013b; Fellet et al., 2014). Technosols made of wastes have been successfully used to increase the growth and the uptake
of Zn by mustards in mine soils (Novo et al., 2013). The combined use of biochar with other organic amendments could be an effective way to improve the quality of soils, as the combined application of these two materials could have synergistic effects on improving soil condition (Karami et al., 2011). Mendez and Maier (2008) reported that plants which show a TC > 1 and a TF > 1 could be suitable for phytoextraction, while plants with TC < 1 and TF < 1 should be used for phytostabilization. The results of this study showed that B. juncea plants could be useful for phytostabilization purposes rather than phytoextraction ones in mine soils contaminated with copper. 5. Conclusions The incorporation of amendments made of wastes and biochar to a Cu-contaminated soil improves the soil conditions for the establishment of mustards by reducing the extreme acidity of the soil, increasing the concentrations of TSC and TN and generally reducing the CaCl2-extractable concentration of metals. However, the used technosol increased the Zn concentrations in the amended soils. It was not possible to grow mustards without amendment in the settling pond soil due to its high Cu concentration. The combined use of technosol and biochar could have a synergistic effect that is highly effective in reducing metal concentrations in pore water. The incorporation of technosol and biochar as a soil
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Fig. 3. Harvestable amounts of total Cu, Ni, Pb and Zn in mustards shoots of the different soil treatments [A20P ¼ 20% amendment (19% technosol þ 1% biochar); A40P ¼ 40% amendment (38% technosol þ 2% biochar); A80P ¼ 80% amendment (76% technosol þ 4% biochar); and A100P ¼ 100% amendment (92% technosol þ 8% biochar)]. Bars with the same letters are not significantly different at P < 0.05. Means of three replicates are reported with confidence interval (CI). The harvestable amount of total Co in mustards was not represented because it was detected only for the soil A20P, since the Co total concentration in plants was below the detection limit for the others.
amendment generally reduced the metal concentrations in plant tissues and enhanced the phytostabilization ability of B. juncea plants in this mine soil. Acknowledgements This study was supported by the Spanish Ministry of Education and Science through the project CGL2009-07843. We also thank the anonymous reviewers for their comments, which helped to improve the quality of this article. References Ali, H., Khan, E., Sajad, M.A., 2013. Phytoremediation of heavy metals e concepts and applications. Chemosphere 91, 869e881. Asensio, V., Vega, F.A., Andrade, M.L., Covelo, E.F., 2013a. Tree vegetation and waste amendments to improve the physical condition of copper mine soils. Chemosphere 90, 603e610. Asensio, V., Vega, F.A., Andrade, M.L., Covelo, E.F., 2013b. Technosols made of wastes to improve physico-chemical characteristics of a copper mine soil. Pedosphere 23, 1e9. Asensio, V., Vega, F.A., Singh, B.R., Covelo, E.F., 2013c. Effects of tree vegetation and waste amendments on the fractionation of Cr, Cu, Ni, Pb and Zn in polluted mine soils. Sci. Total Environ. 443, 446e453.
Baker, A.J.M., Brooks, R.R., 1989. Terrestrial higher plants which hyperaccumulate metallic elements e a review of their distribution, ecology and phytochemistry. Biorecovery 1, 81e126. Busuioc, G., Elekes, C.C., Stihi, C., Iordache, S., Ciulei, S.C., 2011. The bioaccumulation and translocation of Fe, Zn, and Cu in species of mushrooms from Russula genus. Environ. Sci. Pollut. Res. Int. 18, 890e896. Dede, G., Ozdemir, S., Hulusi Dede, O., 2012. Effect of soil amendments on phytoextraction potential of Brassica juncea growing on sewage sludge. Int. J. Environ. Sci. Technol. 9, 559e564. Evangelou, M.W.H., Daghan, H., Schaeffer, A., 2004. The influence of humic acids on the phytoextraction of cadmium from soil. Chemosphere 57, 207e213. Fellet, G., Marmiroli, M., Marchiol, L., 2014. Elements uptake by metal accumulator species grown on mine tailings amended with three types of biochar. Sci. Total Environ. 468e469, 598e608. Houba, V.J.G., Temminghoff, E.J.M., Gaikhorst, G.A., Van Vark, W., 2000. Soil analysis procedures using 0.01 M calcium chloride as extraction reagent. Commun. Soil Sci. Plant Anal. 31, 1299e1396. nez, E., Lepp, N.W., Beesley, L., 2011. Efficiency Karami, N., Clemente, R., Moreno-Jime of green waste compost and biochar soil amendments for reducing lead and copper mobility and uptake to ryegrass. J. Hazard. Mater. 191, 41e48. Kumar, V., Chopra, A.K., 2014. Accumulation and translocation of metals in soil and different parts of French bean (Phaseolus vulgaris L.) amended with sewage sludge. Bull. Environ. Contam. Toxicol. 92, 103e108. ricos de referencia de metales Macías, F., Calvo de Anta, R., 2009. Niveles gene pesados y otros elementos traza en los suelos de Galicia, Xunta de Galicia. Xunta de Galicia, Spain. Mahon, D.C., Mathewes, R.W., 1983. Seasonal variation in the accumulation of radionuclides of the uranium series by yellow pond-lily (Nuphar lutea). Bull. Environ. Contam. Toxicol. 30, 575e581.
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Mendez, M.O., Maier, R.M., 2008. Phytoremediation of mine tailings in temperate and arid environments. Rev. Environ. Sci. Biotechnol. 7, 47e59. Novo, L.A.B., Covelo, E.F., Gonz alez, L., 2013. Phytoremediation of amended copper mine tailings with Brassica juncea. Int. J. Min. Reclam. Environ. 27, 215e226. ndez, A., Gasco , G., 2014. Use of phytoremediation Paz-Ferreiro, J., Lu, H., Fu, S., Me and biochar to remediate heavy metal polluted soils: a review. Solid Earth 5, 65e75. Peijnenburg, W.J.G., Jager, T., 2003. Monitoring approaches to assess bioaccessibility and bioavailability of metals: matrix issues. Ecotoxicol. Environ. Saf. 56, 63e77. rez-Esteban, J., Escola stico, C., Moliner, A., Masaguer, A., Ruiz-Ferna ndez, J., 2014. Pe Phytostabilization of metals in mine soils using Brassica juncea in combination with organic amendments. Plant Soil 377, 97e109. cnicas y experimentos en Edafología. Collegi Oficial D'Enginyers Porta, J., 1986. Te Agronoms de Catalunya, Barcelona, Spain. Puig, C.G., Alvarez-Iglesias, L., Reigosa, M.J., Pedrol, N., 2013. Eucalyptus globulus leaves incorporated as green manure for weed control in maize. Weed Sci. 61, 154e161.
Quartacci, M.F., Argilla, A., Baker, A.J.M., Navari-Izzo, F., 2006. Phytoextraction of metals from a multiply contaminated soil by Indian mustard. Chemosphere 63, 918e925. Sohi, S.P., Krull, E., Lopez-Capel, E., Bol, R., 2010. A review of biochar and its use and function in soil. In: Sparks, D.L. (Ed.), Advances in Agronomy. Academic Press, pp. 47e82 (Chapter 2). USDA, 1998. Soil Quality Indicators: pH 2. van der Ent, A., Baker, A.J.M., Reeves, R.D., Pollard, A.J., Schat, H., 2013. Hyperaccumulators of metal and metalloid trace elements: facts and fiction. Plant Soil 362, 319e334. Vega, F.A., Covelo, E.F., Andrade, M.L., 2005. Limiting factors for reforestation of mine spoils from Galicia (Spain). Land Degrad. Dev. 16, 27e36. Wei, S., Zhu, J., Zhou, Q.X., Zhan, J., 2011. Fertilizer amendment for improving the phytoextraction of cadmium by a hyperaccumulator Rorippa globosa (Turcz.) Thell. J. Soils Sedim. 11, 915e922.
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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Recovering a copper mine soil using organic amendments and phytomanagement with Brassica juncea L. n Forja n, Vero nica Asensio 1 Alfonso Rodríguez-Vila*, Emma F. Covelo, Rube Department of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, Lagoas, Marcosende, 36310 Vigo, Pontevedra, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 May 2014 Received in revised form 31 July 2014 Accepted 9 September 2014 Available online
A 3-month greenhouse experiment was carried out for evaluating the effect of an amendment mixture and mustards on the chemical characteristics of a mine soil and the metal uptake by plants. A settling pond soil was amended with increasing percentages of a technosol and biochar mixture and vegetated with Brassica juncea L. Adding amendments and planting mustards increased the soil pH from 2.83 to 6.18 and the TSC from u.l to 131 g kg1 and generally reduced the CaCl2-extractable metal concentrations in the soil. However, the amendments increased the pseudo-total soil concentration of Ni from 9.27 to 31.9 mg kg1, Pb from 27.9 to 91.6 mg kg1 and Zn from 46.5 to 577 mg kg1. The technosol and biochar mixture increased the shoot biomass from 0.74 to 2.95 g and generally reduced the metal concentrations in plants, meaning B. juncea as a potential candidate for phytostabilization of mine soils.
Keywords: Settling pond Phytoremediation Technosol Biochar Brassica juncea
1. Introduction Mining activities negatively impacted the environment due to the deposition of a huge amount of waste material (Vega et al., 2005). Copper was extracted from a mine in Touro (Spain) between 1973 and 1988. Today, another company is extracting material from the mine for use in road building projects. In 1988, work began on recovering this mine by planting Pinus pinaster Aiton and Eucalyptus globulus Labill, and later by incorporating organic wastes into the soil, sewage sludges and paper mill residues, among others (Asensio et al., 2013a). The incorporation of organic amendments improves the quality of mine soils and makes it possible for vegetation to become established (Asensio et al., 2013b). Biochar has been described as a material that can possibly improve soil fertility (Sohi et al., 2010). Such biochar-amended soils can be phytomanaged (Paz-Ferreiro et al., 2014). Phytoextraction is based on using plants and associated microorganisms to remove contaminants from soils (Ali et al., 2013). Some plants, called hyperaccumulators, can uptake, accumulate and tolerate high metal concentrations in plant tissues (Evangelou et al., 2004). Indian mustard (Brassica juncea L.)
* Corresponding author. Tel.: þ34 986812630; fax: þ34 986812556. E-mail address: [email protected] (A. Rodríguez-Vila). 1 ~o Present address: Department of Plant Nutrition, CENA, Universidade de Sa rio 303, 13400-970 Piracicaba, SP, Brazil. Paulo (CENA-USP), Av. Centena http://dx.doi.org/10.1016/j.jenvman.2014.09.011 0301-4797/© 2014 Elsevier Ltd. All rights reserved.
© 2014 Elsevier Ltd. All rights reserved.
produces a large amount of shoot biomass and can be used for phytoextraction of metals in a reasonable period of time (Quartacci et al., 2006). Numerous studies have shown that adding organic amendments (sewage sludge and manure, among others) to soil promotes phytoextraction processes (Dede et al., 2012; Wei et al., 2011), but only a few of them have evaluated the combined effect of organic amendments and phytoextraction with B. juncea in copper mine soils (Novo et al., 2013). This study aimed at assessing the potential of B. juncea as a phytoextractor of metals from a mine soil polluted by copper and amended with wastes (sewage sludge, aluminium sludge, waste from food industries, sand and ashes from a paper mill) and biochar. 2. Materials and methods 2.1. Soil sampling The sampling area is located at a mine in Touro (Spain) (Lat/Lon) 0 00 0 00 (Datum ETRS89): 8 20 12.06 W 42 52 46.18 N. Copper extraction in this mine produced a settling pond covering an area of 71 ha which has been completely dry for several years. Some parts of the settling pond were colonized by native vegetation: gorse (Ulex sp.), heather (Erica sp.), Agrostis sp. and bryophytes. The mine soil (S) was collected from an untreated area of the settling pond without vegetation. The organic amendment used for the greenhouse experiment was a mixture of technosol (95%)
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gicos del Noroeste S.L. (Santiago de supplied by Tratamientos Ecolo Compostela, Spain) and biochar (5%) provided by PROININSO S.A. laga, Spain). The technosol (T) was made of municipal sewage (Ma sludge (60%), sludge from an aluminium company (10%), waste from food industries (canning and rabbit farms) (10%), sands from wastewater treatment plants (5%) and ashes from a paper mill (5%). This total does not account for 100% of the contents of the technosol, and the percentages are not completely accurate. The biochar (B) was made of holm oak wood treated at a pyrolysis temperature of 400 C for 8 h. The general characteristics of S, T and B are shown in Table 1.
Table 2 Labels for the soil samples in pots according to their mixture of soil and amendment. Soil mixture
Pots without plant
Pots with plant
Control: settling pond soil 80% soil þ 20% amendmenta 60% soil þ 40% amendmenta 20% soil þ 80% amendmenta 100% amendmenta
A0 A20 A40 A80 A100
A0P A20P A40P A80P A100P
a
Amendment mixture is made of 95% technosol þ 5% biochar.
65 ± 5% relative air humidity. The soil moisture was kept at 35 ± 5% according to field capacity.
2.2. Greenhouse experiment 2.3. Soil analyses The greenhouse experiment was carried out in 0.5 L pots with the settling pond soil (S) amended with the previously described mixture of technosol (T) and biochar (B) and vegetated with B. juncea L. seedlings previously germinated in other pots. The soil was amended with 20%, 40%, 80% and 100% of the amendment, always mixing T with B as follows: 19% T and 1% B (A20), 38% T and 2% B (A40), 76% T and 4% B (A80) and 95% T and 5% B (A100). Some pots were only filled with mine soil (A0, with 0% amendment) as the control treatment (Table 2). The plastic pots were filled with a dry weight of 200 g of the corresponding mixture. The control pots were supplemented with drinking straws cut into 1 cm pieces in order to minimize the difference in the substrate volume between the control soils and the amended soils (Puig et al., 2013). A total of 6 pots were filled with the different types of mixtures: three were used for planting mustard and the other three were left unplanted for evaluating the vegetation effect on soil physico-chemical properties. The samples planted with mustards were marked with the letter P (e.g. A20P). Three seedlings of B. juncea, previously germinated from seeds until they had two fully expanded leaves, were transferred to each pot. After 45 days, the plants were thinned to one per pot following uniform criteria. The plants, using 3 replicates per treatment, were harvested 90 days after sowing for subsequent analysis. Growth was allowed under greenhouse-controlled conditions: with a photoperiod of 11:13 light/dark, a temperature of 22 ± 2 C, and
Table 1 Selected characteristics and metal concentrations of the mine soil (S) and the two amendments (biochar B and technosol T) and the generic reference level (GRL) of metals (Macías and Calvo de Anta, 2009). S
B
T
GRL
pH H2O pH KCl TSC (g kg1) TN (g kg1) C/N
2.96 ± 0.07c 2.73 ± 0.02c u.l
9.93 ± 0.22a 9.64 ± 0.05a 667a
5.55 ± 0.07b 5.48 ± 0.05b 218b
e e e
u.l
5.8b
22.9a
e
e
115a
9b
e
Pseudo-total (mg kg1)
Co Cu Ni Pb Zn
31.9 ± 3.03a 452 ± 52.56b 18.9 ± 3.40b 20.9 ± 3.20b 65.4 ± 20.77b
u.l 28.7 ± 2.76c 27.9 ± 8.69b u.l 85.8 ± 11.38b
24.5 ± 2.53b 656 ± 47.55a 82.4 ± 17.84a 94 ± 2.95a 1446 ± 91.52a
40 50 75 80 200
CaCl2extractable (mg kg1)
Co Cu Ni Pb Zn
8.4 ± 0.07a 188 ± 10.88a 8.29 ± 0.07a u.l 2.64 ± 0.10c
0.25 ± 0.02b u.l 0.34 ± 0.02c u.l 1.28 ± 0.30b
0.71 ± 0.07b 4 ± 0.07b 4.25 ± 0.22b 0.43 ± 0.04 735 ± 3.83a
e e e e e
Mean ± CI values (n ¼ 3). Values followed by different letters differ significantly with P < 0.05. u.l: values below the detection limit; TSC: total soil carbon; TN: total soil nitrogen; GRL: generic reference level established for Galician soils (Macías and Calvo de Anta, 2009). Values in bold are over the GRL.
The soil samples collected from the field and from the pots were air dried, passed through a 2 mm sieve and homogenized prior to analysis. A series of soil characteristics were determined (Table 3). Soil pH was determined using a pH electrode in 1:2.5 water or KCl to soil extracts (Porta, 1986). Total soil carbon (TSC) and total nitrogen (TN) were determined in a LECO CN-2000 module using solid samples. Co, Cu, Ni, Pb and Zn were extracted with acidified 0.01 M CaCl2, which is operationally defined as phytoavailable (Houba et al., 2000). The pseudo-total concentrations of Co, Cu, Ni, Pb and Zn were extracted with aqua regia in a microwave oven (Milestone ETHOS 1). The certified soil reference material CRM026 was analysed in parallel with samples to check the effectiveness and precision of the extraction analysis. Metal concentrations were determined by ICPeAES (Optima 4300 DV; PerkineElmer) and were compared with the generic reference level established for Galician soils (Macías and Calvo de Anta, 2009) (Table 4). 2.4. Pore water analysis One week before harvesting the mustards, a “Rhizon” soil pore water sampler (Eijkelkamp Agrisearch Equipment, The Netherlands) was carefully inserted into the soil of each treatment replicate pot at an angle of approximately 45 . Vacuum tubes (10 mL) were attached through a Luer lock system with hypodermic needles to extract pore water. Pore water samples were analysed for metal concentrations by ICPeAES, carried out in triplicate. 2.5. Plant growth and determination of metals in plant tissues After 90 days, the roots and shoots were sampled and carefully washed with deionized water. Fresh biomass was immediately weighed and dry mass was assessed after oven drying for 48 h at 80 C and cooling down to room temperature. Table 3 Selected properties in soil samples at 3 months of experiment. Soil sample
pH H2O
A0 A20 A40 A80 A100 A0P A20P A40P A80P A100P
2.83 4.56 5.16 5.97 6.02 2.70 4.58 5.56 6.18 6.07
± ± ± ± ± ± ± ± ± ±
0.08g 0.11f 0.13e 0.28c 0.1bc 0.17g 0.05f 0.26d 0.24a 0.1b
pH KCl 2.72 4.49 5.12 5.94 5.98 2.60 4.54 5.52 6.17 6.06
± ± ± ± ± ± ± ± ± ±
0.04g 0.11f 0.15e 0.26c 0.1c 0.18h 0.07f 0.21d 0.23a 0.07b
TSC (g kg1)
TN (g kg1)
C/N
u.l 26.23e 56.53d 131b 238a u.l 21e 59.1d 110c 236a
u.l 2.6e 5.5d 11.4b 20.23a u.l 1.97e 5.03d 9.5c 20.5a
e 9.67b 10b 11a 11a e 10b 11a 11a 11a
Mean ± CI values (n ¼ 9). Values followed by different letters in each row of each treatment differ significantly with P < 0.05. u.l: values below the detection limit; TSC: total soil carbon; TN: total soil nitrogen.
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Table 4 Pseudo-total and CaCl2-extractable (phytoavailable) Co, Cu, Ni, Pb and Zn concentrations in soil samples at 3 months of experiment; concentrations of the reference soil CRM026 and generic reference level (GRL) of metals (Macías and Calvo de Anta, 2009). Pseudo-total (mg kg1)
Soil sample A0 A20 A40 A80 A100 A0P A20P A40P A80P A100P GRL a
Co
Cu
32.5 ± 6.72a 33 ± 3.29a 30.8 ± 6.72 ab 22.2 ± 7.67c 16.7 ± 5.52d 32.6 ± 6.75a 30.1 ± 6.41b 28.7 ± 3.43b 22.3 ± 4.11c 13.9 ± 2.81d
341 419 408 352 349 344 408 350 300 315
40
50 23.1 ± 3.33 18.8 ± 0.87 3.01
CRM026 12.2 ± 0.98 CRM026b 6.8 ± 0.67 SD 3.85
CaCl2-extractable (mg kg1) Ni
± ± ± ± ± ± ± ± ± ±
53.74bc 40.65a 98.15a 84.51b 27.08b 88.12b 134a 60.86b 63.56d 41.94cd
Pb
Zn
Co
29.9 ± 11.17gh 42.8 ± 14.43f 52.4 ± 10.33e 91.6 ± 24.67c 125 ± 20.54a 27.9 ± 9.16h 37.6 ± 9.37 fg 52 ± 16.89e 60.8 ± 19.52d 105 ± 18.91b
46.5 ± 9.73h 127 ± 17.39g 246 ± 59.36e 577 ± 200c 876 ± 187a 49.3 ± 12.9h 175 ± 52.72 fg 244 ± 84.26ef 408 ± 128d 773 ± 72.72b
7.15 3.49 3.28 0.74 0.36 6.88 4.09 1.75 0.78 0.35
75
80
200
19.5 ± 1.97 14.4 ± 1.54 3.63
45.3 ± 6.28 25.6 ± 1.88 13.92
174.5 ± 17.68 140 ± 7.09 24.38
11.9 14.8 22.1 31.9 46.4 9.27 19.4 23.2 26.1 42.1
± ± ± ± ± ± ± ± ± ±
3.75 fg 2.84f 6.03de 8.56c 8.57a 2.9g 1.24e 5.64de 5.76d 4.81b
Cu
Ni
152 ± 34.87a 98.15 ± 13.6b 54.01 ± 16.94c 3.43 ± 0.67e 2.03 ± 0.5e 149 ± 38.49a 110 ± 20.13b 30.54 ± 6.01d 3.27 ± 0.89e 1.67 ± 0.21e
7.22 5.11 6.08 3.16 2.12 6.88 5.97 4.32 3.07 1.98
e
e
e
e
e
e e e
e e e
e e e
e e e
e e e
± ± ± ± ± ± ± ± ± ±
2.64a 0.95bc 1.06c 0.14e 0.06e 2.17a 1.39b 0.39d 0.08e 0.05e
Pb ± ± ± ± ± ± ± ± ± ±
2.68a 1.37c 1.77b 0.18d 0.13e 2.19a 1.92b 0.63c 0.32d 0.22e
u.l 4.83 3.14 0.58 0.68 u.l 5.27 2.82 0.63 0.58
Zn ± ± ± ±
0.78b 0.78c 0.04d 0.16d
± ± ± ±
0.65a 0.85c 0.11d 0.19d
2.78 ± 0.12g 69.43 ± 18.12f 168 ± 48.84c 299 ± 8.03a 291 ± 11.96a 3.08 ± 0.43g 86.61 ± 31.17e 156 ± 29.63d 281 ± 8.57b 271 ± 26.88b
Mean ± CI values (n ¼ 9). Values followed by different letters in each row of each treatment differ significantly with P < 0.05. u.l: values below the detection limit; GRL: generic reference level established for Galician soils (Macías and Calvo de Anta, 2009). Values in bold are over the GRL. a Values obtained from this study. b Certified values.
The plant tissues were air dried and ground. The total concentrations of Co, Cu, Ni, Pb and Zn in plants were extracted by acid digestion using a mixture of H2O2 and HNO3 (1:6 v/v) in a microwave oven. The certified reference plant material ERM-CD281 was analysed in parallel with the samples. The transfer coefficient (TC) for the plants was here calculated using the following equation:
TC ¼
Cp Cso
(1)
where Cp is the metal concentration in shoots (mg kg1) and Cso is the metal content of soil (mg kg1) (Peijnenburg and Jager, 2003). A TC with high value indicates a high shoot concentration of the element compared to the pseudo-total concentration in soil (Karami et al., 2011). A plant species is considered as an accumulator or hyperaccumulator biosystem when TC > 1 (Busuioc et al., 2011). The translocation factor (TF) for the plants is expressed by the following equation:
Cs TF ¼ Cr
(2)
where Cs and Cr are metal concentrations (mg kg1) in shoots and roots, respectively. A TF with a high value indicates a relatively high shoot metal concentration compared to its root concentration. A plant species translocates metals effectively from the roots to shoots when TF > 1 (Baker and Brooks, 1989). The bioconcentration factor (BF) for plants is expressed by the following equation:
BF ¼
Cp Cpw
(3)
where Cp is the shoot metal concentration (mg kg1) and Cpw is the metal concentration in the soil pore water (mg L1) (Mahon and Mathewes, 1983). The BF describes the ratio of metal in pore water concentration that is taken up into shoots. High BF values indicate high shoot element concentration compared to the element concentration in the soil pore water (Karami et al., 2011).
For calculating the TC and BF, we used the shoot metal concentrations of the mustard plants, as under field conditions this is the plant part that would be removed by a combine harvester. Moreover, the analysed roots had low harvestable mineral mass (root DW yield root concentration for each metal). 2.6. Statistical analysis All analytical determinations were performed in triplicate. The data obtained were statistically treated using SPSS version 19.0 for Windows. An analysis of variance and homogeneity of variance test were carried out. In case of homogeneity, a post-hoc least significant difference test was carried out. If there was no homogeneity, Dunnett's T3 test was performed. We also carried out a correlated bivariate analysis. 3. Results 3.1. Characteristics of the mine soil and amendments The untreated soil (S) had an extremely acid pH and high concentrations of pseudo-total Cu (Table 1). The total soil carbon (TSC) and total nitrogen (TN) contents were below the detection limit for the mine soil (Table 1). The addition of amendments can help to correct these TSC and TN lacks because of their high content in organic matter and inorganic carbon. The technosol is however contaminated by Cu, Ni, Pb and Zn (Table 1). These metals can be provided by technosol components such as sewage and aluminium sludges (Asensio et al., 2013c). 3.2. Chemical properties of the greenhouse soil samples The chemical properties of the mine soil (pots A0 and A0P) were significantly changed 3 months after the soil amendment and cultivation (Tables 3 and 4), while the pH of untreated soil remained extremely acid according to the USDA (1998). In the cultivated untreated soil (A0P), all plants died one week after planting. The amended soils had significantly higher pH values than the untreated soils. The highest increase in soil pH was found in A80P. In the amended soils, the pH rose to 4.6e6.2, probably due to their high concentration in basic cations and the buffer action of the
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organic matter which increases the CEC, according to previous studies (Asensio et al., 2013b). The TSC and TN were below the detection limits in both A0 and A0P soils. Both TSC and TN concentrations increased significantly with the amendment incorporation. The pseudo-total Co values were lower than the generic reference level (GRL) for Galician soils in all studied soil samples (Table 4) and their concentration was generally diluted by adding technosol and biochar to the mine soil. The pseudo-total Cu concentrations were higher than the GRL in all soil samples (Table 4) and the highest ones were detected in A20, A20P and A40, but the highest CaCl2-extractable Cu was observed in A0 and A0P. A significant negative correlation was obtained between the CaCl2extractable Cu concentration in soils with plants and this concentration in soils without plants (P < 0.01, r ¼ 0.325). The pseudototal and CaCl2-extractable Ni concentrations were lower than the GRL in all soil samples (Table 4), but the higher the amendment rate, the higher the Ni pseudo-total concentration. The highest pseudo-total Pb concentration was found in the A100 and A100P soils, with values over the GRL. The pseudo-total Zn exceeded the GRL in the soil samples with the highest amendment rate. 3.3. Plant biomass and uptake of metals The shoot DW yield peaked for plants grown in the A80P soil (Table 5). The TC and the TF values for Co could only be calculated for the sample A20P, as shoot and root Co concentration were below the Co detection limit (0.005 mg L1). The TC and TF values of Co for plants cultivated in the A20P soil were 0.33 and 0.2, respectively. For Cu, Ni, Pb and Zn, the higher the amendment rate, the lower the TC value (Fig. 1). The TF showed the opposite pattern than the TC for Cu and Zn, but the same in the case of Ni. It was not possible to calculate the TC or TF for Ni in A80P as the shoot Ni concentration was below the Ni detection limit. The TF for Pb could not be calculated because the root Pb concentration was below the detection limit for all root samples. The TC for Pb was significantly the highest in A20P and A80P and the lowest in A100P. It was not possible to calculate it in A40P because the Pb concentration in the plants was below the detection limit. Amendment incorporation into the S soil significantly increased the BF for Cu only for A20P compared to A80P (Fig. 2a). The highest BF for Ni was observed in the treatments A20P and A100P (Fig. 2b). For Pb, the highest BF was found in A80P and A100P (Fig. 2c). For Zn, the application of amendments significantly increased the BF for A20P compared to A40P and A80P (Fig. 2d). The shoot metal removal (shoot DW yield shoot metal concentration) of plant in each pot was computed to account for changes in shoot DW yield (Fig. 3). For Cu, the higher amendment rate, the higher the shoot Cu removal (Fig. 3a). The A20P plants had the highest shoot Ni removal (Fig. 3b). The highest shoot Pb
Table 5 Biomass produced and concentration of Co, Cu, Ni, Pb and Zn in B. juncea plants grown in the different soil treatments at 3 months of experiment. A20P Biomass (g) Co (mg kg1) Cu (mg kg1) Ni (mg kg1) Pb (mg kg1) Zn (mg kg1)
0.74 64.32 223 55.19 6.67 1113
± ± ± ± ± ±
0.12c 8.28 8.69a 8.12a 0.87b 33.36a
A40P
A80P
A100P
2.4 ± 0.23b u.l 67.08 ± 8.92b 25.01 ± 0.9b u.l 716 ± 9.41b
2.95 ± 0.25a u.l 47.05 ± 2.67c 4.26 ± 0.02d 11.78 ± 2.59a 729 ± 23.77b
2.55 ± 0.67 ab u.l 34.45 ± 3.55d 19.07 ± 4.28c 11.72 ± 2.48a 922 ± 44.78a
Mean ± CI values (n ¼ 3 for biomass and n ¼ 9 for metals). Values followed by different letters in each row of each treatment differ significantly with P < 0.05. u.l: values below the detection limit.
removal was obtained for the A80P plants, followed by the A100P plants (Fig. 3c). For Zn, a similar behaviour to Cu was observed, as the higher the amendment rate, the higher the shoot Zn removal (Fig. 3d). 4. Discussion 4.1. Metal concentrations in soils The low Co concentration in the amended soils (Table 4) was likely due to the dilution effect of mixing the amendments with the S soil. In addition, all soil samples had high pseudo-total Cu concentrations above the GRL, as both the untreated mine soil and the technosol have a high total Cu concentration (Table 1). For the same amendment rate, the CaCl2-extractable Cu concentration was generally lower in the cultivated amended soils and technosols, than in the barren ones (Table 4). This indicates that the mustards extracted Cu from the soils. In fact, a significant negative correlation was obtained between the CaCl2-extractable Cu concentration in soils with plants and this concentration in soils without plants (P < 0.01, r ¼ 0.325). The unamended soils had the highest CaCl2-extractable Cu concentrations (Table 4), which may be mainly due to the low TSC to retain this metal and to the extremely acid pH (Table 3). This suggests that the high pH and TSC provided by the organic amendments reduced Cu mobility in soil. This metal can be strongly bound to organic matter and forms both rezsoluble and insoluble complexes with organic compounds (Pe Esteban et al., 2014). The higher pseudo-total Ni concentration in the amended soils than in the untreated ones (Table 4) was likely due to high Ni concentration in the technosol (Table 1). Nevertheless, the highest CaCl2-extractable Ni concentrations were observed in the unamended soils (Table 4). As for Cu, this may reflect the low TSC and soil pH in these soils samples. Higher pH values increase the surface rezcharges of soil particles and therefore metal retention in soil (Pe Esteban et al., 2014). Adding amendments to the S soil increased the concentrations of both pseudo-total Pb and Zn and CaCl2-extractable Zn in the soil, which exceeded the GRL in the samples with the highest amendment rate (Table 4). This may be due to the Pb and Zn provided by the wastes used (Table 1) (Kumar and Chopra, 2014). 4.2. Uptake and transfer of metals to mustards 4.2.1. Cobalt The total Co concentration in B. juncea plants was higher than the Co detection limit only for the soil A20P. The TC for A20P was lower than 1, which indicates that the mustards did not extract this metal from soil efficiently (Busuioc et al., 2011). The TF of less than 1 for Co for A20P indicates low root to shoot translocation (Baker and Brooks, 1989). Also, it was only possible to calculate the BF and the shoot Co removal for this sample. 4.2.2. Copper The application of technosol and biochar as a soil amendment resulted in lower Cu concentrations in plant shoots (Table 5). The TC for Cu was lower than 1 for all soil samples (Fig. 1a). Mustards did not extract Cu from soil efficiently due to their low shoot Cu concentrations (Table 5), according to recent studies (van der Ent et al., 2013). The TF for Cu was also lower than 1 for all of the samples (Fig. 1a) indicating low root to shoot translocation (Baker and Brooks, 1989). The highest BF value for Cu was found in the A100P, A40P and A20P treatments and the lowest in A80P (Fig. 2a). This demonstrates that the BF on its own is not enough to assess the efficiency of plants to extract metals from soils and
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Fig. 1. Transfer of Cu (a), Ni (b), Pb (c) and Zn (d) to and within mustards according to transfer coefficient (TC) (shoots concentration/pseudo-total soil concentration) and translocation factor (TF) (shoots concentration/roots concentration). The solid line is the median, the box represents the upper and lower quartiles and whiskers are the 10th and 90th percentiles (n ¼ 9). Different letters indicate significant differences in TC or TF between treatments (P < 0.05). The TF and TC for Co were not represented because they only could be calculated for the soil A20P. The TF for Pb could not be calculated because Pb concentration in mustard roots was below the detection limit.
that it is also necessary to take the shoot metal removal into account. The values for shoot Cu removal are statistically similar for all treatments except for A20P, and above a 40% amendment rate, mustard plants are not capable of taking up more Cu from the soil (Fig. 3). 4.2.3. Nickel Nickel concentrations in plants decreased significantly with the application of technosol and biochar as a soil amendment (Table 5). Both the TC and TF for Ni were lower than 1 in all of the studied samples (Fig. 1b), indicating that mustards do not extract Ni efficiently from these soils (Busuioc et al., 2011) with a very low root to shoot translocation of this metal (Baker and Brooks, 1989). Both the TC and TF significantly decrease with the addition of amendments to the mine soil (Fig. 1b). Amendment incorporation into the S soil significantly decreased the values of shoot Ni removal for A40P compared to A20P (Fig. 3b). A significantly positive correlation was obtained between CaCl2-ext. Ni and Ni in lixiviates and the total plant concentration (P < 0.05). Therefore, the data obtained indicates that the higher the concentration of Ni
in the soil solution, the higher the concentration taken up by the mustard shoots. 4.2.4. Lead The incorporation of organic amendments into the soil significantly increased the total Pb concentration in plant tissues for A80P compared to A20P (Table 5). The TC and TF have values lower than 1 for Pb in all of the samples (Fig. 1c), indicating that mustard plants were not capable of efficiently extracting this metal from soils (Busuioc et al., 2011) or translocating it from the roots to shoots (Baker and Brooks, 1989). The BF also had low values (Fig. 2c). The application of technosol and biochar as a soil amendment significantly increased the values for shoot Pb removal for A80P compared to A20P (Fig. 3c). Due to the low concentration of Pb in phytoavailable form, it was not possible to assess the phytoextraction potential of Pb by mustards. 4.2.5. Zinc Zinc concentrations in plants decreased significantly with the application of technosol and biochar as a soil amendment for A40P
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Fig. 2. Transfer of Cu (a), Ni (b), Pb (c) and Zn (d) to mustards according to bioconcentration factor (BF) (shoots concentration/soil pore water concentration). The solid line is the median, the box represents the upper and lower quartiles and whiskers are the 10th and 90th percentiles (n ¼ 9). Different letters indicate significant differences in BF between treatments (P < 0.05). The BF for Co was not represented because it only could be calculated for the soil A20P, since the Co total concentration in plants was below the detection limit in the others.
and A80P compared to A20P (Table 5). The TC for Zn was equal or higher than 1 for all of the samples, indicating that mustards are efficient in the uptake of this metal from soils and are also hyperaccumulators of it (Busuioc et al., 2011) (Fig. 1d). The data showed that the higher the amendment rate, the lower the TC for Zn. This is probably because the higher the percentage of amendment, the lower the concentration of Zn in soil pore water (data not shown). In fact, the highest BF for Zn is in the samples with the highest percentage of amendment (Fig. 2d). Moreover, the samples with the highest percentage of amendment had the highest values for shoot metal removal and the highest plant biomass. The TF for Zn (Fig. 1d) increases by adding organic amendment to the soil, probably because it promotes the plant biomass produced. The TF was higher than 1 for all of the samples (Fig. 1d), indicating that mustards efficiently uptake Zn from roots to shoots (Baker and Brooks, 1989). Many studies have shown that these plants are suitable for the phytoextraction of Zn in soils polluted by this metal (e.g. Novo et al., 2013). 4.3. Viability of technosol and biochar to reclaim polluted soils Organic amendments such as wastes and biochar have previously been used to improve the quality of metal polluted mine soils (Asensio et al., 2013b; Fellet et al., 2014). Technosols made of wastes have been successfully used to increase the growth and the uptake
of Zn by mustards in mine soils (Novo et al., 2013). The combined use of biochar with other organic amendments could be an effective way to improve the quality of soils, as the combined application of these two materials could have synergistic effects on improving soil condition (Karami et al., 2011). Mendez and Maier (2008) reported that plants which show a TC > 1 and a TF > 1 could be suitable for phytoextraction, while plants with TC < 1 and TF < 1 should be used for phytostabilization. The results of this study showed that B. juncea plants could be useful for phytostabilization purposes rather than phytoextraction ones in mine soils contaminated with copper. 5. Conclusions The incorporation of amendments made of wastes and biochar to a Cu-contaminated soil improves the soil conditions for the establishment of mustards by reducing the extreme acidity of the soil, increasing the concentrations of TSC and TN and generally reducing the CaCl2-extractable concentration of metals. However, the used technosol increased the Zn concentrations in the amended soils. It was not possible to grow mustards without amendment in the settling pond soil due to its high Cu concentration. The combined use of technosol and biochar could have a synergistic effect that is highly effective in reducing metal concentrations in pore water. The incorporation of technosol and biochar as a soil
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Fig. 3. Harvestable amounts of total Cu, Ni, Pb and Zn in mustards shoots of the different soil treatments [A20P ¼ 20% amendment (19% technosol þ 1% biochar); A40P ¼ 40% amendment (38% technosol þ 2% biochar); A80P ¼ 80% amendment (76% technosol þ 4% biochar); and A100P ¼ 100% amendment (92% technosol þ 8% biochar)]. Bars with the same letters are not significantly different at P < 0.05. Means of three replicates are reported with confidence interval (CI). The harvestable amount of total Co in mustards was not represented because it was detected only for the soil A20P, since the Co total concentration in plants was below the detection limit for the others.
amendment generally reduced the metal concentrations in plant tissues and enhanced the phytostabilization ability of B. juncea plants in this mine soil. Acknowledgements This study was supported by the Spanish Ministry of Education and Science through the project CGL2009-07843. We also thank the anonymous reviewers for their comments, which helped to improve the quality of this article. References Ali, H., Khan, E., Sajad, M.A., 2013. Phytoremediation of heavy metals e concepts and applications. Chemosphere 91, 869e881. Asensio, V., Vega, F.A., Andrade, M.L., Covelo, E.F., 2013a. Tree vegetation and waste amendments to improve the physical condition of copper mine soils. Chemosphere 90, 603e610. Asensio, V., Vega, F.A., Andrade, M.L., Covelo, E.F., 2013b. Technosols made of wastes to improve physico-chemical characteristics of a copper mine soil. Pedosphere 23, 1e9. Asensio, V., Vega, F.A., Singh, B.R., Covelo, E.F., 2013c. Effects of tree vegetation and waste amendments on the fractionation of Cr, Cu, Ni, Pb and Zn in polluted mine soils. Sci. Total Environ. 443, 446e453.
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