Environ Sci Pollut Res DOI 10.1007/s11356-013-2288-3
RESEARCH ARTICLE
A short-term study to evaluate the uptake and accumulation of arsenic in Asian willow (Salix sp.) from arsenic-contaminated water Guangcai Chen & Xiaoli Zou & Yuan Zhou & Jianfeng Zhang & Gary Owens
Received: 27 June 2013 / Accepted: 24 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Five Asian willow species (Salix jiangsuensis J172, Salix matsudana, Salix integra Yizhibi, Salix integra Weishanhu, and Salix mongolica) were evaluated for their potential for phytofiltration of arsenic (As) from synthetically contaminated waters. Arsenic accumulation, tolerance, uptake influx, and phytofiltration ability of the five willow species were examined under hydroponic conditions in a glasshouse. Short-term exposure (2 weeks) to solutions containing 80 μmol L−1 arsenate (As(V)), resulted in significant accumulation of As in all willow species. Arsenic concentration in plant roots ranged from 322 mg kg−1 dry weight (DW) for S. matsudana to 604 mg kg−1 (DW) for S. integra Yizhibi. S. integra Yizhibi decreased As(V) concentration in water from 3.87 to 1.89 μmol L−1 (290 to 142 μg L−1) over 168 h, which is 50 % of the total As(V) in the solution. The results suggested that even though Asian willow was not a traditional aquatic species, it still had significant potential for phytofiltration of As from contaminated waters. Of the five willow species studied, S. integra Yizhibi had the greatest capacity to remove As from As-contaminated waters. Thus, Asian willow has significant potential for the phytofiltration of As and may also be suitable for practical phytoremediation of As in highly water-logged areas. Responsible editor: Elena Maestri G. Chen (*) : X. Zou : J. Zhang (*) Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, Zhejiang, China e-mail: [email protected] e-mail: [email protected] X. Zou : Y. Zhou Jiangxi University of Science and Technology, Ganzhou 341000, Jiangxi, China G. Owens Mawson Institute, University of South Australia, Mawson Lakes, SA 5095, Australia
Keywords Accumulation . Arsenic . Phytofiltration . Tolerance . Uptake . Willow
Introduction Arsenic (As) is a highly toxic element and is listed as a category 1 and group A human carcinogen by both the International Agency for Research on Cancer (IARC 2004) and the US Environmental Protection Agency (USEPA 1997), respectively. In the environment, As exists in a variety of inorganic and organic species with oxidation states varying between −3 and +5 (Moreno-Jiménez et al. 2012). The four major species commonly found in both aqueous and soil environments include the inorganic species, arsenate [As(V)] a n d a r s e n i t e [A s ( I I I) ] , a n d t h e o rg a n i c s p e c i e s monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA) (Akter et al. 2005). Worldwide, As contamination of water occurs mainly due to efflux of inorganic forms from both anthropogenic and geogenic sources (Cullen and Reimer 1989). Thermodynamically, As(V) is the predominant stable oxidation state in oxic waters, whereas in anoxic systems As(III) is the most stable oxidation state (Seyler and Martin 1989). In many places, discharge of agricultural and industrial effluents is the main cause of As contamination in water (Watts 1997; Schmöger et al. 2000). However, in Bangladesh and other parts of southeast Asia, As contamination of groundwater occurs primarily via a natural geologic mechanism, either through mineral dissolution from sedimentary rocks or from dilution of geothermal waters (Nickson et al. 1998; Chowdhury et al. 1999; Sun 2004). Regardless of the source, As contamination causes serious health problems globally via ingestion of potable water (Nordstrom 2002) or food (Zhu et al. 2008; Khan et al. 2009). Moreover, the World Health Organization (WHO 1999) has acknowledged
Environ Sci Pollut Res
As contamination as a “major public health issue” and adopted a drinking water standard of 10 μg L−1. Despite the ever-growing number of contaminated sites and the consequential risk to public health, limited remedial technologies are currently available for environmental remediation. Phytoremediation, where plants are used to clean contaminated soils and waters, has emerged as a “green” alternative to traditional engineered technologies (i.e., physical and chemical remediation) because of the advantages it offers in terms of environmental friendliness and costeffectiveness. Most of the recent phytoremediation studies have focused on the use of As-hyperaccumulator fern species to extract As from contaminated soils (Ma et al. 2001; Niazi et al. 2012). A field experiment lasting 27 months, suggested that Pityrogramma calomelanos var. austroamericana accumulated more As in its fronds (581±205 mg kg−1 DW) than Pteris vittata (401±174 mg kg−1 DW) under field conditions (Niazi et al. 2012). Using the As uptake of the two fern species, they calculated that it would take 55–125 years to decrease the total As to below 20 mg kg−1 in soil with P. calomelanos var. austroamericana compared to 143– 412 years with P. vittata (Niazi et al. 2012). However, while some herbaceous species such as P. calomelanos var. austroamericana are relatively fast growing, since most herbaceous species grow slowly and their biomass production is relatively small (Reichmann et al. 2004), it takes a long time and repeated cropping to achieve specific metal reduction targets, thus their applications in practical phytoremediation has been limited (Ebbs et al. 1997). Of course, plant growth would be heavily dependent on soil conditions and unfavorable environmental conditions, such as drought or nutrient deficiency would result in poor growth and would also lead to a relatively small biomass production (Reichmann et al. 2004). Additionally, most of the terrestrial metal hyperaccumulating plants are not generally suitable for water-logged conditions or for phytofiltration in general where native halophytes are preferred. Duckweed halophytes have previously shown high As removal from water via phytofiltration. Duckweed, Spirodela polyrhiza L., accumulated 999±95 mg As kg−1 DW when grown in a solution containing 320 μM L−1 As(V) for 7 days (Zhang et al. 2011). Similarly, Lemna gibba accumulated 1,022±250 mg As kg−1 DW when grown in a solution containing As(V) 100 μg L −1 for 21 days (Mkandawire et al. 2004). Other halophyte species, such as Eleocharis macrostachya , have been successfully used to increase the As retention capacity by up to 90 % in constructed wetlands compared to only 27 % in the absence of plants (Olmos-Márquez et al. 2012), suggesting higher As removal from water via phytofiltration. To more practically facilitate phytofiltration an ideal plant species would be a fast growing, high biomass species exhibiting a large tolerance and/or accumulation of metals
and if not an aquatic species at least a species significantly resistant to water-logging with an extensive root system. Species such as willow (Salix sp.) and poplar (Populus) which are fast-growing high-biomass species address many of these criteria. When used for the phytoremediation of As-, Cd-, Pb-, and Zn-contaminated soils, the remediation effectiveness of Salix dasyclados for As, assessed by the remediation factor (RF), corresponding to the annual percentage of element removed from a specific volume of soil, of 0.033, was similar to that obtained using two hyperaccumulators, Arabidopsis halleri and Thlaspi caerulescens, with RFs of 0.067 and 0.028, respectively (Fischerová et al. 2006). Since willow exhibited large biomass production and was not directly associated with the food chain, willow could remove pollutants via short-rotation cultivation and periodic harvesting (Mirck et al. 2005). However, one of the main disadvantages of soil phytoremediation is that it is generally relatively slow, with clean-up generally occurring over many years depending on the degree of soil contamination and the physiochemical properties of the target metals (French et al. 2006). Recovery of metals from the roots is also possible after final harvest of the plants (Kuzovkina et al. 2004). Landberg and Greger (1994) proposed recovery of root biomass from soil as part of a biofuel production cycle, but as this would occur only every 25 to 30 years this would not be suitable for rapid remediation of a contaminated site. Thus while it is not desirable to harvest willow root from soil, fast remediation of waters may potentially be obtained when willow is alternatively used for phytofiltration, where plants are used to remove contaminants from water by harvesting plant biomass (Dushenkov and Kapulnik 2000). Indeed, Kuzovkina and Volk (2009) proposed that since willow exhibited high resistance to salt, alkali, drought, and water-logging stress, and grew well in a variety of waters and soils, willow might have potential for use in phytofiltration. However, to date, only a few studies on the phytoremediation capacity of willow to remediate As from contaminated waters exist in the literature (French et al. 2006; Purdy and Smart 2008; Puckett et al. 2012). While four willow clones grown in nutrient solution accumulated As in the leaves (2 to 329 μg As g−1 DW), and in the stems (2 to 201 μg As g−1 DW), As was predominantly accumulated in willow roots (800 to 5,800 μg As g−1 DW), suggesting that willow could tolerate As toxicity by preferentially partitioning As in the roots without significantly affecting phosphate transport to the aboveground plant parts (Purdy and Smart 2008). In short-term (3 weeks) growth experiments conducted in a hydroponic solution containing 250 μM As(V), As accumulation of an As-tolerant willow (Salix viminalis ×Salix miyabeana ) reached 66.8, 34.2, and 3, 170 mg kg−1 and an As-sensitive willow (Salix eriocephala) reached 20.3, 16.8, and 2,380 mg kg−1 DW for leaf, stem, and roots, respectively (Puckett et al. 2012). They also found that, for both genotypes, the addition of phosphate resulted in
Environ Sci Pollut Res
greater As accumulation than in treatments without phosphate, a result that contrasts with previous studies with various plant species which had shown that increased phosphate concentrations generally decreased plant accumulation and translocation of As(V) in hydroponic solution (Meharg and Macnair 1991; Zhao et al. 2009; Moreno-Jiménez et al. 2012). Since As(V) is a phosphate analogue, uptake and translocation of As(V) in plants is mainly through phosphate transporters (Meharg and Macnair 1992; Esteban et al. 2003). The phosphate/As(V) mechanism of absorption involves the cotransport of the anion with protons, in a stoichiometry of 2H+ for each anion (Zhao et al. 2009). Restricting As(V) influx by constitutive suppression of high-affinity phosphate/As(V) transport might be an important mechanism to avoid toxicity and to improve As(V) tolerance in plants (Meharg and Macnair 1992; Bleeker et al. 2003). While As tolerance has been identified for a wide variety of species, both in hyperaccumulating and nonhyperaccumulating plant species, the exact mechanism for tolerance, uptake, and accumulation is still a topic for significant research. Since there has been no systematic study of Asian willows for phytoremediation, the potential of five Asian willow species to remediate As-contaminated waters was conducted to provide baseline information on their potential for phytofiltration.
Materials and methods Preparation of plant material Five willow species, Salix jiangsuensis J172, Salix matsudana, Salix integra Yizhibi, Salix integra Weishanhu, and Salix mongolica, were collected from a nursery in the Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang Province, China, an area where the soil was free of As or any other metal contamination. For each willow species, all cuttings (clones) were taken from one mother plant, being 1-year-old branches that were cut into uniform 10 cm lengths, and healthy cuttings were cultivated in a basal nutrient solution contained in a 20-L plastic box. The composition of the basal Hoagland nutrient solution was: 0.51 g L −1 KNO 3 , 0.82 g L −1 Ca(NO 3 ) 2 , 0.49 g L −1 MgSO4·7H2O, 0.136 g L−1 KH2PO4, 2.86 mg L−1 H3BO4, 1.81 mg L−1 MnCl2·4H2O, 0.22 mg L−1 ZnSO4·7H2O, 0.45 mg L −1 (NH 4) 6Mo7 O 24 , 0.6 mg L −1 FeSO 4, and 0.744 mg L−1 ethylenediaminetetraacetic acid disodium salt (Na2EDTA). The nutrient solution was renewed every 2 weeks and the pH adjusted daily to 6.0±0.1 with 0.1 mol L−1 NaOH or HCl. The plants were grown under glasshouse conditions with natural light, day/night temperature of 26/20 °C, and day/ night humidity of 70/85 %. Depending on the willow species,
the height of the plants reached 0.8 to 1.5 m after 8 weeks of cultivation. Screening experiment Screening and tolerance experiments were conducted in a glasshouse to determine which species had the greatest potential for phytofiltration. For each species, 8-week-old willows of uniform size were selected and transferred to 5-L plastic containers filled with full-strength Hoagland solution which also contained 80 μmol L−1 As(V) which was prepared by dissolving 0.161 g of Na2HAsO4·12H2O in 5 L of nutrient solution. Exposure to As(V) was chosen because this is the dominant species in the surficial layer of natural oxic waters and the bioavailable form for aquatic plants (Seyler and Martin 1989; Sizova et al. 2002). The nutrient solution was renewed weekly and the pH was adjusted daily to 6.0±0.1 with 0.1 mol L−1 NaOH or HCl. Each treatment was conducted in triplicate, and each replicate was applied to four willow plants. Willows were harvested 2 weeks after exposure to As and the roots were washed first with tap water, then with deionized water, and finally blotted dry with paper towel. The roots were rinsed (desorbed) in an ice-cold phosphate buffer solution (1 mmol L−1 K2HPO4, 5 mmol L−1 2-(N morpholin) ethansulfonic acid (MES), and 0.5 mmol L−1 Ca(NO 3 ) 2 ) for 20 min to remove apoplastic As(V) (Abedin et al. 2002). After desorption, the roots were again washed with deionized water and blotted dry. The plants were divided into roots, stems, and leaves and were oven-dried for 72 h at 70 °C. Subsamples of dried plant material were digested to determine the total As concentration in the plant biomass. S. integra Yizhibi which had the highest As accumulation among the five species was selected for further testing of As(V) concentration-dependent uptake kinetics, accumulation, tolerance, and phytofiltration efficiency. Uptake kinetics of As in willow plants Since the speed of uptake is an important parameter in determining effective phytoremediation, the kinetics of As uptake was examined in response to different As concentrations. Eight-week-old willow plants (S. integra Yizhibi) of uniform size were washed with tap water followed by deionized water prior to commencing uptake kinetic experiments. A single plant was incubated in each test solution (400 mL), which contained 5.0 mmol L−1 MES and 0.5 mmol L−1 Ca(NO3)2 (pH 5) (Chen et al. 2005; Zhang et al. 2009), at seven different concentrations of As(V) (0, 10, 20, 40, 80, 160, and 320 μmol L−1). Each test solution was placed in a 500-mL opaque conical flask covered with a black plastic bag, and the roots were submerged completely in the solution. After an hour, the willows were collected, washed, and the roots were
Environ Sci Pollut Res
then desorbed following the method described in the screening experiment. The plants were subsequently washed with tap water, deionized water, blotted dry, divided into roots and shoots, and oven-dried for 72 h at 70 °C and the total As concentrations in plants were determined. Uptake kinetic experiments of As(V) were conducted in a controlledenvironment growth chamber with a photoperiod of 16 h light/8 h dark cycles, where temperature was maintained at 25 °C (day) and 20 °C (night) and light intensity and relative humidity were maintained at 260 μmol(m2 s)−1 and 60 %, respectively. Each treatment corresponded to a set of four replicates. Arsenic accumulation and tolerance Since the effectiveness of phytoremediation depends on the ability of plants to accumulate As, experiments were conducted to evaluate the uptake of As by S. integra Yizhibi in response to variable As exposure. Uniform willow plants (8 weeks old) were washed with tap water, deionized water, and placed in 5-L plastic containers filled with full strength Hoagland solution containing different concentrations of As(V) (0, 10, 20, 40, 80, 160, and 320 μmol L−1). The nutrient solution was renewed weekly and the pH controlled daily following the experimental design and protocol previously described in the screening experiment. Plants were harvested 28 days after As exposure, washed, desorbed, separated, and dried as described in Section 2.2 prior to being homogenized and the total As and P concentrations in root, stem, and leaf determined. Arsenic phytofiltration potential of willow species Since the time required to remove As to below a specific threshold value is an important consideration, phytofiltration experiments were conducted to determine the rate of As removal from solution. Willow plants (8 weeks old) of uniform size were washed with deionized water and pretreated with 0.1 mmol L−1 CaCl2 solution (400 mL) for 24 h. Willow plants were then exposed to 0.1 mmol L−1 CaCl2 solution (400 mL) containing 3.87 μmol L−1 As(V) (corresponding to 290 μg L−1). A 0.1 mmol L−1 CaCl2 solution containing 3.87 μM As(V) without willow plants was used as a control. Each treatment had one plant and each treatment had four replicates. Subsequently during the experiment deionized water was added through the weighing method to maintain the volume near 400 mL and to replace the volume of nutrient solution lost due to uptake and evaporation. Periodically, at 0, 0.5, 1, 2, 8, 24, 48, 72, 96, 120, 144, and 168 h, 3 mL of solution was sampled and the total As concentration in the solution was determined at each sampling time. In determining the total amount of As removed by the willow plants, the amount of As removed during sampling was allowed for by
subtraction of the small amount of As removed at each sampling time from the total amount of As removed by the willow. Analysis of arsenic and phosphorous Dried plant material (0.1 g) was digested in a mixture of highpurity nitric (5 mL) and perchloric acids (1 mL) with heating on an electric hot plate. Briefly, the temperature was gently raised in the sequence 70, 150, and 200 °C with a holding time of 20 min at each step. Finally, the digests were heated at 250 °C for 60 min before removing them from the hotplate and allowing them to cool to room temperature. A reagent blank and a certified reference material (bush twigs and leaves, GBW07603 from the National Research Center for Standard Materials in China) were included for quality assurance. The digests, or an aliquot (1 mL) of the sampled solutions, were mixed with hydrochloric acid (5 mL) and a mixture (10 mL) of thiourea (5 %, 5 g/100 mL) and ascorbic acid (5 %, 5 g/100 mL), and made up to 50 mL with ultrapure water. Total As was subsequently determined using a hydride generation atomic fluorescence spectrometer (AFS-9130, Beijing Titan Instruments Co., Ltd.). The concentrations of phosphorous in the digests were determined using an ultraviolet and visible spectrophotometer (UV-1800 PC, Shanghai Mapada Instruments Co., Ltd.) at 700 nm. Data analysis All statistical analysis was performed using SPSS v.13.0 (SPSS, Chicago, IL, USA) and plotted with Microcal Origin 7.0 (Originlab Corporation, Northampton, MA, USA). Differences among As content in five willow species, biomass production, As content, and P concentration among different As treatments for S. integra Yizhibi, and As removal from solution by S. integra Yizhibi were tested with a one-way analysis of variance using the general linear model. All statistical tests were conducted using least significant difference (LSD) tests at a significance level of 0.05. Uptake kinetics was fit to the Michaelis−Menten equation (Eq. 1) using Origin7.5 software to estimate V max and K m . V ¼ aC þ
V max C Km þ C
ð1Þ
where V was the uptake rate (nmol kg−1 DW min−1), C was the As(V) concentration in the uptake solution (μmol L−1), a was a parameter characterizing the linear part of the uptake rate, V max was the maximum uptake rate, reflecting the inherent potential of uptake by plant roots, the higher V max value (nmol kg−1 DW min−1) indicates a higher inherent potential;
Environ Sci Pollut Res
Σ Cm Σm n 1
i
n 1
i
ð4Þ
i
where, C p (mg kg−1 DW) was the average As content in the whole plant and C s (mg L−1) was the As concentration in corresponding solution. The translocation factor (TF), which is indicative of the ability of willow to translocate As from roots to shoots, was defined as the ratio of As concentration in the shoots to that in the roots. As TF ¼ Ar
ð5Þ
where, A s (mg kg−1 DW) was the total amount of As accumulated in the shoots and A r (mg kg−1 DW) was the total amount of As accumulated in the roots. In general, if the TF and BF values are
RESEARCH ARTICLE
A short-term study to evaluate the uptake and accumulation of arsenic in Asian willow (Salix sp.) from arsenic-contaminated water Guangcai Chen & Xiaoli Zou & Yuan Zhou & Jianfeng Zhang & Gary Owens
Received: 27 June 2013 / Accepted: 24 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Five Asian willow species (Salix jiangsuensis J172, Salix matsudana, Salix integra Yizhibi, Salix integra Weishanhu, and Salix mongolica) were evaluated for their potential for phytofiltration of arsenic (As) from synthetically contaminated waters. Arsenic accumulation, tolerance, uptake influx, and phytofiltration ability of the five willow species were examined under hydroponic conditions in a glasshouse. Short-term exposure (2 weeks) to solutions containing 80 μmol L−1 arsenate (As(V)), resulted in significant accumulation of As in all willow species. Arsenic concentration in plant roots ranged from 322 mg kg−1 dry weight (DW) for S. matsudana to 604 mg kg−1 (DW) for S. integra Yizhibi. S. integra Yizhibi decreased As(V) concentration in water from 3.87 to 1.89 μmol L−1 (290 to 142 μg L−1) over 168 h, which is 50 % of the total As(V) in the solution. The results suggested that even though Asian willow was not a traditional aquatic species, it still had significant potential for phytofiltration of As from contaminated waters. Of the five willow species studied, S. integra Yizhibi had the greatest capacity to remove As from As-contaminated waters. Thus, Asian willow has significant potential for the phytofiltration of As and may also be suitable for practical phytoremediation of As in highly water-logged areas. Responsible editor: Elena Maestri G. Chen (*) : X. Zou : J. Zhang (*) Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, Zhejiang, China e-mail: [email protected] e-mail: [email protected] X. Zou : Y. Zhou Jiangxi University of Science and Technology, Ganzhou 341000, Jiangxi, China G. Owens Mawson Institute, University of South Australia, Mawson Lakes, SA 5095, Australia
Keywords Accumulation . Arsenic . Phytofiltration . Tolerance . Uptake . Willow
Introduction Arsenic (As) is a highly toxic element and is listed as a category 1 and group A human carcinogen by both the International Agency for Research on Cancer (IARC 2004) and the US Environmental Protection Agency (USEPA 1997), respectively. In the environment, As exists in a variety of inorganic and organic species with oxidation states varying between −3 and +5 (Moreno-Jiménez et al. 2012). The four major species commonly found in both aqueous and soil environments include the inorganic species, arsenate [As(V)] a n d a r s e n i t e [A s ( I I I) ] , a n d t h e o rg a n i c s p e c i e s monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA) (Akter et al. 2005). Worldwide, As contamination of water occurs mainly due to efflux of inorganic forms from both anthropogenic and geogenic sources (Cullen and Reimer 1989). Thermodynamically, As(V) is the predominant stable oxidation state in oxic waters, whereas in anoxic systems As(III) is the most stable oxidation state (Seyler and Martin 1989). In many places, discharge of agricultural and industrial effluents is the main cause of As contamination in water (Watts 1997; Schmöger et al. 2000). However, in Bangladesh and other parts of southeast Asia, As contamination of groundwater occurs primarily via a natural geologic mechanism, either through mineral dissolution from sedimentary rocks or from dilution of geothermal waters (Nickson et al. 1998; Chowdhury et al. 1999; Sun 2004). Regardless of the source, As contamination causes serious health problems globally via ingestion of potable water (Nordstrom 2002) or food (Zhu et al. 2008; Khan et al. 2009). Moreover, the World Health Organization (WHO 1999) has acknowledged
Environ Sci Pollut Res
As contamination as a “major public health issue” and adopted a drinking water standard of 10 μg L−1. Despite the ever-growing number of contaminated sites and the consequential risk to public health, limited remedial technologies are currently available for environmental remediation. Phytoremediation, where plants are used to clean contaminated soils and waters, has emerged as a “green” alternative to traditional engineered technologies (i.e., physical and chemical remediation) because of the advantages it offers in terms of environmental friendliness and costeffectiveness. Most of the recent phytoremediation studies have focused on the use of As-hyperaccumulator fern species to extract As from contaminated soils (Ma et al. 2001; Niazi et al. 2012). A field experiment lasting 27 months, suggested that Pityrogramma calomelanos var. austroamericana accumulated more As in its fronds (581±205 mg kg−1 DW) than Pteris vittata (401±174 mg kg−1 DW) under field conditions (Niazi et al. 2012). Using the As uptake of the two fern species, they calculated that it would take 55–125 years to decrease the total As to below 20 mg kg−1 in soil with P. calomelanos var. austroamericana compared to 143– 412 years with P. vittata (Niazi et al. 2012). However, while some herbaceous species such as P. calomelanos var. austroamericana are relatively fast growing, since most herbaceous species grow slowly and their biomass production is relatively small (Reichmann et al. 2004), it takes a long time and repeated cropping to achieve specific metal reduction targets, thus their applications in practical phytoremediation has been limited (Ebbs et al. 1997). Of course, plant growth would be heavily dependent on soil conditions and unfavorable environmental conditions, such as drought or nutrient deficiency would result in poor growth and would also lead to a relatively small biomass production (Reichmann et al. 2004). Additionally, most of the terrestrial metal hyperaccumulating plants are not generally suitable for water-logged conditions or for phytofiltration in general where native halophytes are preferred. Duckweed halophytes have previously shown high As removal from water via phytofiltration. Duckweed, Spirodela polyrhiza L., accumulated 999±95 mg As kg−1 DW when grown in a solution containing 320 μM L−1 As(V) for 7 days (Zhang et al. 2011). Similarly, Lemna gibba accumulated 1,022±250 mg As kg−1 DW when grown in a solution containing As(V) 100 μg L −1 for 21 days (Mkandawire et al. 2004). Other halophyte species, such as Eleocharis macrostachya , have been successfully used to increase the As retention capacity by up to 90 % in constructed wetlands compared to only 27 % in the absence of plants (Olmos-Márquez et al. 2012), suggesting higher As removal from water via phytofiltration. To more practically facilitate phytofiltration an ideal plant species would be a fast growing, high biomass species exhibiting a large tolerance and/or accumulation of metals
and if not an aquatic species at least a species significantly resistant to water-logging with an extensive root system. Species such as willow (Salix sp.) and poplar (Populus) which are fast-growing high-biomass species address many of these criteria. When used for the phytoremediation of As-, Cd-, Pb-, and Zn-contaminated soils, the remediation effectiveness of Salix dasyclados for As, assessed by the remediation factor (RF), corresponding to the annual percentage of element removed from a specific volume of soil, of 0.033, was similar to that obtained using two hyperaccumulators, Arabidopsis halleri and Thlaspi caerulescens, with RFs of 0.067 and 0.028, respectively (Fischerová et al. 2006). Since willow exhibited large biomass production and was not directly associated with the food chain, willow could remove pollutants via short-rotation cultivation and periodic harvesting (Mirck et al. 2005). However, one of the main disadvantages of soil phytoremediation is that it is generally relatively slow, with clean-up generally occurring over many years depending on the degree of soil contamination and the physiochemical properties of the target metals (French et al. 2006). Recovery of metals from the roots is also possible after final harvest of the plants (Kuzovkina et al. 2004). Landberg and Greger (1994) proposed recovery of root biomass from soil as part of a biofuel production cycle, but as this would occur only every 25 to 30 years this would not be suitable for rapid remediation of a contaminated site. Thus while it is not desirable to harvest willow root from soil, fast remediation of waters may potentially be obtained when willow is alternatively used for phytofiltration, where plants are used to remove contaminants from water by harvesting plant biomass (Dushenkov and Kapulnik 2000). Indeed, Kuzovkina and Volk (2009) proposed that since willow exhibited high resistance to salt, alkali, drought, and water-logging stress, and grew well in a variety of waters and soils, willow might have potential for use in phytofiltration. However, to date, only a few studies on the phytoremediation capacity of willow to remediate As from contaminated waters exist in the literature (French et al. 2006; Purdy and Smart 2008; Puckett et al. 2012). While four willow clones grown in nutrient solution accumulated As in the leaves (2 to 329 μg As g−1 DW), and in the stems (2 to 201 μg As g−1 DW), As was predominantly accumulated in willow roots (800 to 5,800 μg As g−1 DW), suggesting that willow could tolerate As toxicity by preferentially partitioning As in the roots without significantly affecting phosphate transport to the aboveground plant parts (Purdy and Smart 2008). In short-term (3 weeks) growth experiments conducted in a hydroponic solution containing 250 μM As(V), As accumulation of an As-tolerant willow (Salix viminalis ×Salix miyabeana ) reached 66.8, 34.2, and 3, 170 mg kg−1 and an As-sensitive willow (Salix eriocephala) reached 20.3, 16.8, and 2,380 mg kg−1 DW for leaf, stem, and roots, respectively (Puckett et al. 2012). They also found that, for both genotypes, the addition of phosphate resulted in
Environ Sci Pollut Res
greater As accumulation than in treatments without phosphate, a result that contrasts with previous studies with various plant species which had shown that increased phosphate concentrations generally decreased plant accumulation and translocation of As(V) in hydroponic solution (Meharg and Macnair 1991; Zhao et al. 2009; Moreno-Jiménez et al. 2012). Since As(V) is a phosphate analogue, uptake and translocation of As(V) in plants is mainly through phosphate transporters (Meharg and Macnair 1992; Esteban et al. 2003). The phosphate/As(V) mechanism of absorption involves the cotransport of the anion with protons, in a stoichiometry of 2H+ for each anion (Zhao et al. 2009). Restricting As(V) influx by constitutive suppression of high-affinity phosphate/As(V) transport might be an important mechanism to avoid toxicity and to improve As(V) tolerance in plants (Meharg and Macnair 1992; Bleeker et al. 2003). While As tolerance has been identified for a wide variety of species, both in hyperaccumulating and nonhyperaccumulating plant species, the exact mechanism for tolerance, uptake, and accumulation is still a topic for significant research. Since there has been no systematic study of Asian willows for phytoremediation, the potential of five Asian willow species to remediate As-contaminated waters was conducted to provide baseline information on their potential for phytofiltration.
Materials and methods Preparation of plant material Five willow species, Salix jiangsuensis J172, Salix matsudana, Salix integra Yizhibi, Salix integra Weishanhu, and Salix mongolica, were collected from a nursery in the Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang Province, China, an area where the soil was free of As or any other metal contamination. For each willow species, all cuttings (clones) were taken from one mother plant, being 1-year-old branches that were cut into uniform 10 cm lengths, and healthy cuttings were cultivated in a basal nutrient solution contained in a 20-L plastic box. The composition of the basal Hoagland nutrient solution was: 0.51 g L −1 KNO 3 , 0.82 g L −1 Ca(NO 3 ) 2 , 0.49 g L −1 MgSO4·7H2O, 0.136 g L−1 KH2PO4, 2.86 mg L−1 H3BO4, 1.81 mg L−1 MnCl2·4H2O, 0.22 mg L−1 ZnSO4·7H2O, 0.45 mg L −1 (NH 4) 6Mo7 O 24 , 0.6 mg L −1 FeSO 4, and 0.744 mg L−1 ethylenediaminetetraacetic acid disodium salt (Na2EDTA). The nutrient solution was renewed every 2 weeks and the pH adjusted daily to 6.0±0.1 with 0.1 mol L−1 NaOH or HCl. The plants were grown under glasshouse conditions with natural light, day/night temperature of 26/20 °C, and day/ night humidity of 70/85 %. Depending on the willow species,
the height of the plants reached 0.8 to 1.5 m after 8 weeks of cultivation. Screening experiment Screening and tolerance experiments were conducted in a glasshouse to determine which species had the greatest potential for phytofiltration. For each species, 8-week-old willows of uniform size were selected and transferred to 5-L plastic containers filled with full-strength Hoagland solution which also contained 80 μmol L−1 As(V) which was prepared by dissolving 0.161 g of Na2HAsO4·12H2O in 5 L of nutrient solution. Exposure to As(V) was chosen because this is the dominant species in the surficial layer of natural oxic waters and the bioavailable form for aquatic plants (Seyler and Martin 1989; Sizova et al. 2002). The nutrient solution was renewed weekly and the pH was adjusted daily to 6.0±0.1 with 0.1 mol L−1 NaOH or HCl. Each treatment was conducted in triplicate, and each replicate was applied to four willow plants. Willows were harvested 2 weeks after exposure to As and the roots were washed first with tap water, then with deionized water, and finally blotted dry with paper towel. The roots were rinsed (desorbed) in an ice-cold phosphate buffer solution (1 mmol L−1 K2HPO4, 5 mmol L−1 2-(N morpholin) ethansulfonic acid (MES), and 0.5 mmol L−1 Ca(NO 3 ) 2 ) for 20 min to remove apoplastic As(V) (Abedin et al. 2002). After desorption, the roots were again washed with deionized water and blotted dry. The plants were divided into roots, stems, and leaves and were oven-dried for 72 h at 70 °C. Subsamples of dried plant material were digested to determine the total As concentration in the plant biomass. S. integra Yizhibi which had the highest As accumulation among the five species was selected for further testing of As(V) concentration-dependent uptake kinetics, accumulation, tolerance, and phytofiltration efficiency. Uptake kinetics of As in willow plants Since the speed of uptake is an important parameter in determining effective phytoremediation, the kinetics of As uptake was examined in response to different As concentrations. Eight-week-old willow plants (S. integra Yizhibi) of uniform size were washed with tap water followed by deionized water prior to commencing uptake kinetic experiments. A single plant was incubated in each test solution (400 mL), which contained 5.0 mmol L−1 MES and 0.5 mmol L−1 Ca(NO3)2 (pH 5) (Chen et al. 2005; Zhang et al. 2009), at seven different concentrations of As(V) (0, 10, 20, 40, 80, 160, and 320 μmol L−1). Each test solution was placed in a 500-mL opaque conical flask covered with a black plastic bag, and the roots were submerged completely in the solution. After an hour, the willows were collected, washed, and the roots were
Environ Sci Pollut Res
then desorbed following the method described in the screening experiment. The plants were subsequently washed with tap water, deionized water, blotted dry, divided into roots and shoots, and oven-dried for 72 h at 70 °C and the total As concentrations in plants were determined. Uptake kinetic experiments of As(V) were conducted in a controlledenvironment growth chamber with a photoperiod of 16 h light/8 h dark cycles, where temperature was maintained at 25 °C (day) and 20 °C (night) and light intensity and relative humidity were maintained at 260 μmol(m2 s)−1 and 60 %, respectively. Each treatment corresponded to a set of four replicates. Arsenic accumulation and tolerance Since the effectiveness of phytoremediation depends on the ability of plants to accumulate As, experiments were conducted to evaluate the uptake of As by S. integra Yizhibi in response to variable As exposure. Uniform willow plants (8 weeks old) were washed with tap water, deionized water, and placed in 5-L plastic containers filled with full strength Hoagland solution containing different concentrations of As(V) (0, 10, 20, 40, 80, 160, and 320 μmol L−1). The nutrient solution was renewed weekly and the pH controlled daily following the experimental design and protocol previously described in the screening experiment. Plants were harvested 28 days after As exposure, washed, desorbed, separated, and dried as described in Section 2.2 prior to being homogenized and the total As and P concentrations in root, stem, and leaf determined. Arsenic phytofiltration potential of willow species Since the time required to remove As to below a specific threshold value is an important consideration, phytofiltration experiments were conducted to determine the rate of As removal from solution. Willow plants (8 weeks old) of uniform size were washed with deionized water and pretreated with 0.1 mmol L−1 CaCl2 solution (400 mL) for 24 h. Willow plants were then exposed to 0.1 mmol L−1 CaCl2 solution (400 mL) containing 3.87 μmol L−1 As(V) (corresponding to 290 μg L−1). A 0.1 mmol L−1 CaCl2 solution containing 3.87 μM As(V) without willow plants was used as a control. Each treatment had one plant and each treatment had four replicates. Subsequently during the experiment deionized water was added through the weighing method to maintain the volume near 400 mL and to replace the volume of nutrient solution lost due to uptake and evaporation. Periodically, at 0, 0.5, 1, 2, 8, 24, 48, 72, 96, 120, 144, and 168 h, 3 mL of solution was sampled and the total As concentration in the solution was determined at each sampling time. In determining the total amount of As removed by the willow plants, the amount of As removed during sampling was allowed for by
subtraction of the small amount of As removed at each sampling time from the total amount of As removed by the willow. Analysis of arsenic and phosphorous Dried plant material (0.1 g) was digested in a mixture of highpurity nitric (5 mL) and perchloric acids (1 mL) with heating on an electric hot plate. Briefly, the temperature was gently raised in the sequence 70, 150, and 200 °C with a holding time of 20 min at each step. Finally, the digests were heated at 250 °C for 60 min before removing them from the hotplate and allowing them to cool to room temperature. A reagent blank and a certified reference material (bush twigs and leaves, GBW07603 from the National Research Center for Standard Materials in China) were included for quality assurance. The digests, or an aliquot (1 mL) of the sampled solutions, were mixed with hydrochloric acid (5 mL) and a mixture (10 mL) of thiourea (5 %, 5 g/100 mL) and ascorbic acid (5 %, 5 g/100 mL), and made up to 50 mL with ultrapure water. Total As was subsequently determined using a hydride generation atomic fluorescence spectrometer (AFS-9130, Beijing Titan Instruments Co., Ltd.). The concentrations of phosphorous in the digests were determined using an ultraviolet and visible spectrophotometer (UV-1800 PC, Shanghai Mapada Instruments Co., Ltd.) at 700 nm. Data analysis All statistical analysis was performed using SPSS v.13.0 (SPSS, Chicago, IL, USA) and plotted with Microcal Origin 7.0 (Originlab Corporation, Northampton, MA, USA). Differences among As content in five willow species, biomass production, As content, and P concentration among different As treatments for S. integra Yizhibi, and As removal from solution by S. integra Yizhibi were tested with a one-way analysis of variance using the general linear model. All statistical tests were conducted using least significant difference (LSD) tests at a significance level of 0.05. Uptake kinetics was fit to the Michaelis−Menten equation (Eq. 1) using Origin7.5 software to estimate V max and K m . V ¼ aC þ
V max C Km þ C
ð1Þ
where V was the uptake rate (nmol kg−1 DW min−1), C was the As(V) concentration in the uptake solution (μmol L−1), a was a parameter characterizing the linear part of the uptake rate, V max was the maximum uptake rate, reflecting the inherent potential of uptake by plant roots, the higher V max value (nmol kg−1 DW min−1) indicates a higher inherent potential;
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Σ Cm Σm n 1
i
n 1
i
ð4Þ
i
where, C p (mg kg−1 DW) was the average As content in the whole plant and C s (mg L−1) was the As concentration in corresponding solution. The translocation factor (TF), which is indicative of the ability of willow to translocate As from roots to shoots, was defined as the ratio of As concentration in the shoots to that in the roots. As TF ¼ Ar
ð5Þ
where, A s (mg kg−1 DW) was the total amount of As accumulated in the shoots and A r (mg kg−1 DW) was the total amount of As accumulated in the roots. In general, if the TF and BF values are
