Environ Sci Pollut Res DOI 10.1007/s11356-015-4344-7
RESEARCH ARTICLE
Effects of selenite and selenate application on growth and shoot selenium accumulation of pak choi (Brassica chinensis L.) during successive planting conditions Jun Li 1 & Dongli Liang 1 & Siyue Qin 1 & Puyang Feng 1 & Xiongping Wu 1
Received: 24 October 2014 / Accepted: 9 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Selenate and selenite are two main kinds of inorganic selenium (Se) sources in soil, but these substances can pose threats to the environment. Phytoextraction is an emerging technology to remove Se from polluted soils by using a hyper-accumulator. In this study, a pot experiment was conducted to investigate Se phytoextraction potential of pak choi (Brassica chinensis L.) and to determine the effects of Se on growth and Se accumulation of pak choi under successive planting conditions (four crops). Results showed that Se concentration in pak choi shoots significantly increased as selenate and selenite rates increased. Se concentration increased in successive crops on soil treated with selenite; by contrast, Se concentration decreased in crops on soil treated with selenate. Se concentrations of pak choi on soil treated with selenate were higher than those on soil treated with selenite. The maximum Se accumulations amount in crops on selenite- and selenate-treated soil were 7818 and 8828 μg·pot−1, respectively. High bioconcentration factor (BCF) values indicated that pak choi could accumulate more Se from Secontaminated soil. The Se phytoextraction efficiency of pak choi increased under successive planting conditions in selenite and selenate treatments; the maximum Se phytoextraction efficiencies of four successive crops of pak choi on selenite- and selenate-treated soil were 4.91 and 31.90 %, respectively. These differences between selenate and selenite treatments were Responsible editor: Elena Maestri * Dongli Liang [email protected] 1
College of Natural Resources and Environment, Northwest A&F University, Key Laboratory of Plant Nutrition and the Agri-environment in Northwest China, Ministry of Agriculture, Yangling, Shaanxi 712100, China
attributed to the differences in Se forms in soil. Total and available Se contents in soil decreased significantly during repeated planting crops on soil treated with selenate; conversely, total and available Se contents decreased slightly in crops on soil treated with selenite. These results suggested that pak choi could highly tolerate and accumulate Se. Thus, pak choi may remove Se from contaminated soil; indeed, pak choi can be used in the phytoextraction of Se in polluted soil. Keywords Se . Accumulation . Phytoremediation . Successive planting . Pak choi
Introduction Selenium (Se) is an essential trace element for humans and animals (Sharma et al. 2010); however, deficient and excessive Se in the environment can cause adverse effects on organisms. As such, Se is considered as a Bdouble-edged sword^ (Boyd 2011; Fordyce 2013). Considering the narrow range of Se among deficient (400 μg day−1) levels (Hegarty 1997), researchers intensively investigated Se in different fields (Carey et al. 2012; Kikkert and Berkelaar 2013; Terry et al. 2000). Hawkesford and Zhao (2007) reported that total Se in soil of >3 mg kg−1 can be considered excessive for human nutrition. With the efflux of Se from anthropogenic activities, such as increasing Se application in metal smelting, mining, glass production, and ceramic industries, Se pollution in the environment has exacerbated (Lim and Goh 2005). Contamination of Se in the environment can cause serious environmental problems that can affect the quality of water and soil and directly threaten human health. In humans, Se intoxication events, such as selenosis in America, Canada, and Mexico and poisoning in Enshi and Ziyang, China, have occurred occasionally because
Environ Sci Pollut Res
Se has entered the food chain; these events are caused by excessive Se in soil and water (Fordyce et al. 2000; Lemly 2004; Skorupa 1998; Tan et al. 2002). Se is difficult to remove once this element pollutes the soil because of low mobility. Several treatment technologies, including physical, chemical, and biological methods, have been proposed to reduce Se load in Se-contaminated soil (Esringü and Turan 2012; Hasan et al. 2010). However, physical and chemical treatment technologies have limited practical applications because of complicated procedures, prohibitive costs, and secondary contamination (Ali et al. 2013). Phytoremediation is a simple, environmentally friendly, and cost-effective Bgreen^ biological remediation method. Phytoextraction can be applied to remove adequate Se from contaminated soils to achieve safe levels without causing soil alteration by repeated cropping (Li et al. 2014). Optimal alternative plants (hyper-accumulators) should exhibit rapid growth, reasonable biomass production, and high metal tolerance and accumulation to facilitate phytoremediation practically; hyper-accumulators should also allow easy translocation of pollutants in contaminated soil from underground to aboveground (Ali et al. 2013). Hamilton and Beath (1964) analyzed Se amount and chemical form in 18 common garden vegetables planted in soils that contain variable levels of Se; among these vegetables, Brassicaceae cabbage contains the highest Se concentration. Dhillon and Dhillon (2009) analyzed 11 vegetable crops raised in selenate-rich alkaline clay loam soil and revealed that Brassica radish yields the greatest Se concentration. Yawata et al. (2010) found that Indian mustard, nozawana (Brassica rapa var. hakabura), and komatsuna (B. rapa var. peruviridis) grown in selenate-contaminated soil in Japan exhibit similar Se-accumulating abilities. These findings indicate that Brassicaceae plants can accumulate and tolerate Se. For instance, pak choi is a vegetable species of Brassicaceae commonly found in China. In a previous study, pak choi [B. chinensis Jus l. var. parachinensis (Bailey) Tsen & Lee] was compared with two plants, namely kale (B. alboglabra L. H. Bailey) and broccoli (B. oleracea L. var. italica); the results reveal that pak choi undergoes the same biotransformation of selenate as that of the two other Brassicaceae plants and accumulates higher Se than kale (Thosaikham et al. 2014). However, no other in-depth studies regarding Se uptake of pak choi have been conducted. Although Se exists in soil at different valence states, particularly Se (IV), Se (VI), Se (II), and Se (0), Se can be absorbed by plants in the form of selenite and selenate (Hawrylak-Nowak 2013). Studies on the difference in selenite and selenate absorption and translocation in plants have applied hydroponic conditions or soil culture with only one crop (short-time) condition (Arvy 1993; Lavu et al. 2012; Li et al. 2008, 2010). However, a knowledge gap remains in studies regarding
selenite and selenate uptake and accumulation in Brassicaceae pak choi during sequential planting in Se-contaminated soil conditions; no systematic study has been conducted on the potential of pak choi as Se accumulator for phytoremediation. In the present study, four sequential crops of pak choi (Brassica chinensis L.) were planted on soils with different concentrations of selenite and selenate. This study aimed to (1) explore the effects of two mineral Se (selenite and selenate) on growth and shoot accumulation of pak choi, (2) elucidate the potential of pak choi to treat Se-contaminated soil and the remediation efficiency of pak choi as Se accumulator, and (3) provide further insights into Se transport in a soil– plant system.
Material and methods Experimental materials Non-contaminated soil classified as cinnamon soil was collected at a depth of 0 to 20 cm in the experimental field of Northwest A&F University in Shaanxi Province, China. Basic physicochemical properties of the soil were analyzed according to the procedure described by Bao (2000). In brief, soil pH was determined in water extracts at a soil-to-water ratio of 1:2.5 by using a pH meter; cation exchange capacity (CEC) was determined using NH4OAC method. Clay and carbonate contents were determined by laser particle size analysis and gas volumetric method, respectively. Organic matter content was measured by hot K2CrO4 oxidation and FeSO4 titration. Total nitrogen was determined using Kjeldahl method. Total Se in soil was identified using an atomic fluorescence spectrophotometer after soil was digested with HNO3 and HClO4 (3:2, v/v). The results are shown in Table 1. Selenite (Na2SeO3) and selenate (Na2SeO4) used in this study were of analytical grade and produced by a chemical reagent factory in Tianjing, China. Pak choi (B. chinensis L.) seeds were provided by Northwest A&F University Seeds Co., Ltd., Shaanxi, China. Experimental design There were 11 treatments, including control (CK, uncontaminated soil) and five different selenate and selenite treatments (2.5, 5.0, 10.0, 20.0, and 40.0 mg Se kg−1). These concentrations were selected according to our previous studies (Wu et al. 2009). Each treatment was prepared in four replicates. The soil was air-dried at room temperature, homogenized, and allowed to pass through a 5 mm sieve. Chemical fertilizers, including 100 mg kg−1 N (urea), 75 mg kg−1 P2O5 (calcium superphosphate), and 75 mg kg−1 K2O (potassium chlorine), were mixed thoroughly with 14.0 kg of air-dried soil
Environ Sci Pollut Res Table 1
Soil physical and chemical properties
pH
CEC cmol kg−1
Clay %
CaCO3 g kg−1
Orangic matter g kg−1
Total N g kg−1
Total Se mg kg−1
7.75
23.34
39.5
55.00
16.33
1.1126
0.113
(bulk soil) in plastic pots (30 cm in height and 30 cm in diameter) before the plants were cultivated (repeated for each crop). Different concentrations of selenite and selenate spiked solutions were aspirated onto dry soil by using a plastic nebulizer. After equilibrating was performed for 14 days, the first crop of pak choi seeds was sown in the pots. Ten seedlings were retained in each pot after these plants emerged and grew under natural lighting conditions. During the planting period, the soil moisture was maintained at approximately 70 % water holding capacity by weighing the pots at an interval of 2 to 3 days. Once the first crop was harvested, the second crop was planted. A total of four successive crops were planted during the planting periods of April 7 to May 10 (33 days), May 13 to June 14 (32 days), June 20 to July 22 (32 days), and July 24 to August 28 (35 days) in 2008, which were 33, 66, 99, and 132 days after selenite and selenate application, respectively. After pak choi was harvested for each growth period, shoot biomass (root biomass was excluded because of incomplete collection), Se concentration in shoots, total Se, and available Se content in soil were determined.
became clear. After acid digestion was completed, sample solutions were cooled and diluted with deionized water in the glass tube. Available Se in soil was divided into soluble Se and exchangeable Se fractions, which were analyzed using a twostep sequential extraction method (Wang et al. 2012). Soil samples (1.0000 g) were extracted with 0.25 M KCl (soil/extractant ratio 1:10) by shaking at 25 °C for 1 h, centrifuged at 4000 r/min for 10 min, and filtered. The supernatant was collected for soluble Se analysis. The residue from the soluble Se fraction was continuously extracted with 0.7 M KH2PO4 by shaking at 25 °C for 4 h, centrifuged at 4000 r/ min for 10 min, and filtered. The supernatant was collected for exchangeable Se analysis. The total of soluble Se and exchangeable Se fractions was operationally defined as available Se in soil. Se in all of the samples was analyzed using an atomic fluorescence spectrophotometer with hydride generation (AFS-930; Beijing Titan Instruments Co., Ltd.). Calcareous soil (GBW07404, 0.64±0.18 mg kg−1) and cabbage sample (GBW10014, 0.21±0.01 mg kg−1) as certified reference materials were included in each analytical batches for quality control in all of the samples with recoveries of 95 to 98 % and 94 to 99 %, respectively.
Sample collection and chemical analysis Approximately 100 g of soil samples was collected from each pot and subjected to chemical analysis after each crop was harvested. Pak choi with roots harvested from each pot was initially washed with tap water and then with deionized water; afterward, the washed pak choi was dried with blotting paper. Shoots and roots were separated. The samples were oven-dried at 95 °C for 30 min and then at 50 °C in constant weight. Dried plant samples were ground in an electrical blender and sieved using a 2 mm sieve except the roots because only few materials were harvested. Total Se concentrations in soil and plants were determined using a previously described method (Wang et al. 2012) with slight modifications. Soil samples were aciddigested using 3:2 (v/v) HNO3-HClO4; plant samples were digested using 4:1 (v/v) HNO3-HClO4. In brief, 0.5000 g of each sample was weighed in 100 mL glass tube. Concentrated HNO3 and concentrated HClO4 with a combined volume of 10 mL were added; the digestion tubes were stored overnight at room temperature. Acid digestion was conducted at 170 °C in an automatic temperature control furnace until the digestion solution
Statistical analysis Data were analyzed by ANOVA and Duncan’s multiple comparison test in SPSS 20.0. The following concepts were applied in this study: Total shoot Se accumulation amount ¼ C shoot M shoot Bioconcentration factor ðBC F Þ ¼ C shoot =C soil
ð1Þ ð2Þ
Where Cshoot (mg kg−1 DW) is the average Se concentration in pak choi shoots, Mshoot (g·pot−1 DW) is the corresponding average biomass weight, and Csoil (mg kg−1 DW) is the average total Se concentration in corresponding soil. Shoot phytoextraction efficiency ð%Þ ¼ C shoot M shoot =ðC soil M soil Þ 100%
ð3Þ
where Cshoot (mg kg−1 DW) is the average Se concentration in pak choi shoots of each harvested crop, Mshoot (g·pot−1 DW) is the corresponding average biomass weight, Csoil (mg kg−1 DW) is the average soil Se concentration before each crop of
Environ Sci Pollut Res 18
pak choi was planted, and Msoil is the soil mass (14, 13.9, 13.8, and 13.7 kg·pot−1 DW).
−1
where Ci (mg kg DW) is the shoot Se concentration in ith crop of pak choi (i=1, 2, 3, 4), Mi (g·pot−1 DW) is the shoot biomass weight in ith crop of pak choi, Csoil (mg kg−1 DW) is the initial Se application concentration, and Msoil is the soil mass (14 kg·pot−1).
15
shoot biomass/g.pot-1
Total phytoextraction efficiency f or four crops ð%Þ Xn ¼ C M i =ðC soil M soil Þ 100% ð4Þ 1 i
CK 10
2.5 20
5 40
(a) a
a
12 bb
a
a
bb
a
9 6
a
cc
b
b
b
aa aaa
c d
c
3 b
0
1st
2nd
3rd
4th
Crops
Results
Pak choi biomass directly indicated the effects of selenite and selenate rates on plant growth. Shoot biomass showed different patterns as selenite and selenate applications were increased in the same crop; these different patterns were also observed in the four successive crops (Fig. 1). At 5 mg Se kg−1 selenite, the highest shoot biomass of pak choi was found in second and third crops on selenite-treated soil; shoot biomass respectively increased by 16.3 and 22.3 % compared with the control treatment. At 40 mg Se kg−1, the shoot biomass of pak choi significantly decreased for all four crops. At 10 and 20 mg Se kg−1, the shoot biomass of the third and fourth harvests of pak choi significantly decreased, respectively (Fig. 1a). At 2.5 mg Se kg−1, no significant difference was observed in the shoot biomass of pak choi in selenate application and control treatments of the third and fourth crops; by contrast, shoot biomass declined significantly as selenate application rates increased (P
RESEARCH ARTICLE
Effects of selenite and selenate application on growth and shoot selenium accumulation of pak choi (Brassica chinensis L.) during successive planting conditions Jun Li 1 & Dongli Liang 1 & Siyue Qin 1 & Puyang Feng 1 & Xiongping Wu 1
Received: 24 October 2014 / Accepted: 9 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Selenate and selenite are two main kinds of inorganic selenium (Se) sources in soil, but these substances can pose threats to the environment. Phytoextraction is an emerging technology to remove Se from polluted soils by using a hyper-accumulator. In this study, a pot experiment was conducted to investigate Se phytoextraction potential of pak choi (Brassica chinensis L.) and to determine the effects of Se on growth and Se accumulation of pak choi under successive planting conditions (four crops). Results showed that Se concentration in pak choi shoots significantly increased as selenate and selenite rates increased. Se concentration increased in successive crops on soil treated with selenite; by contrast, Se concentration decreased in crops on soil treated with selenate. Se concentrations of pak choi on soil treated with selenate were higher than those on soil treated with selenite. The maximum Se accumulations amount in crops on selenite- and selenate-treated soil were 7818 and 8828 μg·pot−1, respectively. High bioconcentration factor (BCF) values indicated that pak choi could accumulate more Se from Secontaminated soil. The Se phytoextraction efficiency of pak choi increased under successive planting conditions in selenite and selenate treatments; the maximum Se phytoextraction efficiencies of four successive crops of pak choi on selenite- and selenate-treated soil were 4.91 and 31.90 %, respectively. These differences between selenate and selenite treatments were Responsible editor: Elena Maestri * Dongli Liang [email protected] 1
College of Natural Resources and Environment, Northwest A&F University, Key Laboratory of Plant Nutrition and the Agri-environment in Northwest China, Ministry of Agriculture, Yangling, Shaanxi 712100, China
attributed to the differences in Se forms in soil. Total and available Se contents in soil decreased significantly during repeated planting crops on soil treated with selenate; conversely, total and available Se contents decreased slightly in crops on soil treated with selenite. These results suggested that pak choi could highly tolerate and accumulate Se. Thus, pak choi may remove Se from contaminated soil; indeed, pak choi can be used in the phytoextraction of Se in polluted soil. Keywords Se . Accumulation . Phytoremediation . Successive planting . Pak choi
Introduction Selenium (Se) is an essential trace element for humans and animals (Sharma et al. 2010); however, deficient and excessive Se in the environment can cause adverse effects on organisms. As such, Se is considered as a Bdouble-edged sword^ (Boyd 2011; Fordyce 2013). Considering the narrow range of Se among deficient (400 μg day−1) levels (Hegarty 1997), researchers intensively investigated Se in different fields (Carey et al. 2012; Kikkert and Berkelaar 2013; Terry et al. 2000). Hawkesford and Zhao (2007) reported that total Se in soil of >3 mg kg−1 can be considered excessive for human nutrition. With the efflux of Se from anthropogenic activities, such as increasing Se application in metal smelting, mining, glass production, and ceramic industries, Se pollution in the environment has exacerbated (Lim and Goh 2005). Contamination of Se in the environment can cause serious environmental problems that can affect the quality of water and soil and directly threaten human health. In humans, Se intoxication events, such as selenosis in America, Canada, and Mexico and poisoning in Enshi and Ziyang, China, have occurred occasionally because
Environ Sci Pollut Res
Se has entered the food chain; these events are caused by excessive Se in soil and water (Fordyce et al. 2000; Lemly 2004; Skorupa 1998; Tan et al. 2002). Se is difficult to remove once this element pollutes the soil because of low mobility. Several treatment technologies, including physical, chemical, and biological methods, have been proposed to reduce Se load in Se-contaminated soil (Esringü and Turan 2012; Hasan et al. 2010). However, physical and chemical treatment technologies have limited practical applications because of complicated procedures, prohibitive costs, and secondary contamination (Ali et al. 2013). Phytoremediation is a simple, environmentally friendly, and cost-effective Bgreen^ biological remediation method. Phytoextraction can be applied to remove adequate Se from contaminated soils to achieve safe levels without causing soil alteration by repeated cropping (Li et al. 2014). Optimal alternative plants (hyper-accumulators) should exhibit rapid growth, reasonable biomass production, and high metal tolerance and accumulation to facilitate phytoremediation practically; hyper-accumulators should also allow easy translocation of pollutants in contaminated soil from underground to aboveground (Ali et al. 2013). Hamilton and Beath (1964) analyzed Se amount and chemical form in 18 common garden vegetables planted in soils that contain variable levels of Se; among these vegetables, Brassicaceae cabbage contains the highest Se concentration. Dhillon and Dhillon (2009) analyzed 11 vegetable crops raised in selenate-rich alkaline clay loam soil and revealed that Brassica radish yields the greatest Se concentration. Yawata et al. (2010) found that Indian mustard, nozawana (Brassica rapa var. hakabura), and komatsuna (B. rapa var. peruviridis) grown in selenate-contaminated soil in Japan exhibit similar Se-accumulating abilities. These findings indicate that Brassicaceae plants can accumulate and tolerate Se. For instance, pak choi is a vegetable species of Brassicaceae commonly found in China. In a previous study, pak choi [B. chinensis Jus l. var. parachinensis (Bailey) Tsen & Lee] was compared with two plants, namely kale (B. alboglabra L. H. Bailey) and broccoli (B. oleracea L. var. italica); the results reveal that pak choi undergoes the same biotransformation of selenate as that of the two other Brassicaceae plants and accumulates higher Se than kale (Thosaikham et al. 2014). However, no other in-depth studies regarding Se uptake of pak choi have been conducted. Although Se exists in soil at different valence states, particularly Se (IV), Se (VI), Se (II), and Se (0), Se can be absorbed by plants in the form of selenite and selenate (Hawrylak-Nowak 2013). Studies on the difference in selenite and selenate absorption and translocation in plants have applied hydroponic conditions or soil culture with only one crop (short-time) condition (Arvy 1993; Lavu et al. 2012; Li et al. 2008, 2010). However, a knowledge gap remains in studies regarding
selenite and selenate uptake and accumulation in Brassicaceae pak choi during sequential planting in Se-contaminated soil conditions; no systematic study has been conducted on the potential of pak choi as Se accumulator for phytoremediation. In the present study, four sequential crops of pak choi (Brassica chinensis L.) were planted on soils with different concentrations of selenite and selenate. This study aimed to (1) explore the effects of two mineral Se (selenite and selenate) on growth and shoot accumulation of pak choi, (2) elucidate the potential of pak choi to treat Se-contaminated soil and the remediation efficiency of pak choi as Se accumulator, and (3) provide further insights into Se transport in a soil– plant system.
Material and methods Experimental materials Non-contaminated soil classified as cinnamon soil was collected at a depth of 0 to 20 cm in the experimental field of Northwest A&F University in Shaanxi Province, China. Basic physicochemical properties of the soil were analyzed according to the procedure described by Bao (2000). In brief, soil pH was determined in water extracts at a soil-to-water ratio of 1:2.5 by using a pH meter; cation exchange capacity (CEC) was determined using NH4OAC method. Clay and carbonate contents were determined by laser particle size analysis and gas volumetric method, respectively. Organic matter content was measured by hot K2CrO4 oxidation and FeSO4 titration. Total nitrogen was determined using Kjeldahl method. Total Se in soil was identified using an atomic fluorescence spectrophotometer after soil was digested with HNO3 and HClO4 (3:2, v/v). The results are shown in Table 1. Selenite (Na2SeO3) and selenate (Na2SeO4) used in this study were of analytical grade and produced by a chemical reagent factory in Tianjing, China. Pak choi (B. chinensis L.) seeds were provided by Northwest A&F University Seeds Co., Ltd., Shaanxi, China. Experimental design There were 11 treatments, including control (CK, uncontaminated soil) and five different selenate and selenite treatments (2.5, 5.0, 10.0, 20.0, and 40.0 mg Se kg−1). These concentrations were selected according to our previous studies (Wu et al. 2009). Each treatment was prepared in four replicates. The soil was air-dried at room temperature, homogenized, and allowed to pass through a 5 mm sieve. Chemical fertilizers, including 100 mg kg−1 N (urea), 75 mg kg−1 P2O5 (calcium superphosphate), and 75 mg kg−1 K2O (potassium chlorine), were mixed thoroughly with 14.0 kg of air-dried soil
Environ Sci Pollut Res Table 1
Soil physical and chemical properties
pH
CEC cmol kg−1
Clay %
CaCO3 g kg−1
Orangic matter g kg−1
Total N g kg−1
Total Se mg kg−1
7.75
23.34
39.5
55.00
16.33
1.1126
0.113
(bulk soil) in plastic pots (30 cm in height and 30 cm in diameter) before the plants were cultivated (repeated for each crop). Different concentrations of selenite and selenate spiked solutions were aspirated onto dry soil by using a plastic nebulizer. After equilibrating was performed for 14 days, the first crop of pak choi seeds was sown in the pots. Ten seedlings were retained in each pot after these plants emerged and grew under natural lighting conditions. During the planting period, the soil moisture was maintained at approximately 70 % water holding capacity by weighing the pots at an interval of 2 to 3 days. Once the first crop was harvested, the second crop was planted. A total of four successive crops were planted during the planting periods of April 7 to May 10 (33 days), May 13 to June 14 (32 days), June 20 to July 22 (32 days), and July 24 to August 28 (35 days) in 2008, which were 33, 66, 99, and 132 days after selenite and selenate application, respectively. After pak choi was harvested for each growth period, shoot biomass (root biomass was excluded because of incomplete collection), Se concentration in shoots, total Se, and available Se content in soil were determined.
became clear. After acid digestion was completed, sample solutions were cooled and diluted with deionized water in the glass tube. Available Se in soil was divided into soluble Se and exchangeable Se fractions, which were analyzed using a twostep sequential extraction method (Wang et al. 2012). Soil samples (1.0000 g) were extracted with 0.25 M KCl (soil/extractant ratio 1:10) by shaking at 25 °C for 1 h, centrifuged at 4000 r/min for 10 min, and filtered. The supernatant was collected for soluble Se analysis. The residue from the soluble Se fraction was continuously extracted with 0.7 M KH2PO4 by shaking at 25 °C for 4 h, centrifuged at 4000 r/ min for 10 min, and filtered. The supernatant was collected for exchangeable Se analysis. The total of soluble Se and exchangeable Se fractions was operationally defined as available Se in soil. Se in all of the samples was analyzed using an atomic fluorescence spectrophotometer with hydride generation (AFS-930; Beijing Titan Instruments Co., Ltd.). Calcareous soil (GBW07404, 0.64±0.18 mg kg−1) and cabbage sample (GBW10014, 0.21±0.01 mg kg−1) as certified reference materials were included in each analytical batches for quality control in all of the samples with recoveries of 95 to 98 % and 94 to 99 %, respectively.
Sample collection and chemical analysis Approximately 100 g of soil samples was collected from each pot and subjected to chemical analysis after each crop was harvested. Pak choi with roots harvested from each pot was initially washed with tap water and then with deionized water; afterward, the washed pak choi was dried with blotting paper. Shoots and roots were separated. The samples were oven-dried at 95 °C for 30 min and then at 50 °C in constant weight. Dried plant samples were ground in an electrical blender and sieved using a 2 mm sieve except the roots because only few materials were harvested. Total Se concentrations in soil and plants were determined using a previously described method (Wang et al. 2012) with slight modifications. Soil samples were aciddigested using 3:2 (v/v) HNO3-HClO4; plant samples were digested using 4:1 (v/v) HNO3-HClO4. In brief, 0.5000 g of each sample was weighed in 100 mL glass tube. Concentrated HNO3 and concentrated HClO4 with a combined volume of 10 mL were added; the digestion tubes were stored overnight at room temperature. Acid digestion was conducted at 170 °C in an automatic temperature control furnace until the digestion solution
Statistical analysis Data were analyzed by ANOVA and Duncan’s multiple comparison test in SPSS 20.0. The following concepts were applied in this study: Total shoot Se accumulation amount ¼ C shoot M shoot Bioconcentration factor ðBC F Þ ¼ C shoot =C soil
ð1Þ ð2Þ
Where Cshoot (mg kg−1 DW) is the average Se concentration in pak choi shoots, Mshoot (g·pot−1 DW) is the corresponding average biomass weight, and Csoil (mg kg−1 DW) is the average total Se concentration in corresponding soil. Shoot phytoextraction efficiency ð%Þ ¼ C shoot M shoot =ðC soil M soil Þ 100%
ð3Þ
where Cshoot (mg kg−1 DW) is the average Se concentration in pak choi shoots of each harvested crop, Mshoot (g·pot−1 DW) is the corresponding average biomass weight, Csoil (mg kg−1 DW) is the average soil Se concentration before each crop of
Environ Sci Pollut Res 18
pak choi was planted, and Msoil is the soil mass (14, 13.9, 13.8, and 13.7 kg·pot−1 DW).
−1
where Ci (mg kg DW) is the shoot Se concentration in ith crop of pak choi (i=1, 2, 3, 4), Mi (g·pot−1 DW) is the shoot biomass weight in ith crop of pak choi, Csoil (mg kg−1 DW) is the initial Se application concentration, and Msoil is the soil mass (14 kg·pot−1).
15
shoot biomass/g.pot-1
Total phytoextraction efficiency f or four crops ð%Þ Xn ¼ C M i =ðC soil M soil Þ 100% ð4Þ 1 i
CK 10
2.5 20
5 40
(a) a
a
12 bb
a
a
bb
a
9 6
a
cc
b
b
b
aa aaa
c d
c
3 b
0
1st
2nd
3rd
4th
Crops
Results
Pak choi biomass directly indicated the effects of selenite and selenate rates on plant growth. Shoot biomass showed different patterns as selenite and selenate applications were increased in the same crop; these different patterns were also observed in the four successive crops (Fig. 1). At 5 mg Se kg−1 selenite, the highest shoot biomass of pak choi was found in second and third crops on selenite-treated soil; shoot biomass respectively increased by 16.3 and 22.3 % compared with the control treatment. At 40 mg Se kg−1, the shoot biomass of pak choi significantly decreased for all four crops. At 10 and 20 mg Se kg−1, the shoot biomass of the third and fourth harvests of pak choi significantly decreased, respectively (Fig. 1a). At 2.5 mg Se kg−1, no significant difference was observed in the shoot biomass of pak choi in selenate application and control treatments of the third and fourth crops; by contrast, shoot biomass declined significantly as selenate application rates increased (P
