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Bioaccumulation of Lanthanum and Its Effect on Growth of Maize Seedlings in a Red Loamy Soil1
Pedosphere 16(6): 79W305, 2006 ISSN 1002-0160/CN 32-1315/P @ 2006 Soil Science Society of China Published by Elsevier Limited and Science Press
PEDOSPHERE www.elsevier.com/lote/pedosphere
Bioaccumulation of Lanthanum and Its Effect on Growth of Maize Seedlings in a Red Loamy Soil*' HU Xin', WANG Xiao-Rong' and WANG Chao2
'
State Key Labomtory of Pollution Control and Resource Reuse, School of Environmental Science, Nanjing University, Nanjing 210093 (China). E-mail: huzinJu8yahoo. com.cn 2School of Environmental Science and Engineering, Hohai University, Nanjing 210098 (China) (Received February 27, 2006; revised July 6, 2006)
ABSTRACT Through a pot culture lanthanum nitrate was applied to maize seedlings grown in a red loamy soil t o investigate the physiological and toxic effects of added La on the growth of crop seedlings and La bioaccumulation to help understand the environmental chemistry behaviors of rare earth element as fertilizers in soils. Compared t o the control, La concentrations in shoots and especially in roots of maize seedlings increased with an increase of La in the soil. Also, with added concentrations of La 2 0.75 g La kg-' soil and 2 0.05 g La kg-' soil, the dry weight of shoots and roots of maize seedlings was significantly reduced (P 5 0.05), respectively, compared with the control. Additionally, La 2 0.5 g kg-' in the soil significantly inhibited (P 5 0.05) primary root elongation. Roots were more sensitive to La stress than shoots and thus could be used as a biomarker to La stress. Overall, in the red loamy soil studied, La had no significant beneficial effects on the growth of maize at the added La levels above 0.1 g kg-' soil. Key Words:
bioaccumulation, growth, lanthanum, maize seedlings, red loamy soil
INTRODUCTION Rare earth elements (REEs) as fertilizers have been widely used in China's agriculture over the last 20 years. It was believed that REEs could stimulate the growth of roots, improve the quantity and quality of crops, and enhance crop resistance (Ni, 1995; Pang et al., 2002a, b; Xie et al., 2002). Nevertheless, results from field trials, pot trials, and laboratory studies on the effect of REEs on crop growth and development have been inconsistent (Brown et al., 1990; He and Loh, 2000; Wahid et al., 2000; Xu et al., 2002; Hong et al., 2002; Hu et al., 2002; Tyler, 2004). For example, lanthanum (La) added to a medium at levels of below 40 mg L-I, 10 mg L-', and 30 mg L-l stimulated the growth of maize, rape, and rice, respectively (Xiong and Zhang, 1997). He and Loh (2000) also reported that the addition of cerium (Ce) nitrate (0.5-10 pmol L-I) or lanthanum nitrate (0.5-50 pmol L-') to a culture medium significantly increased the length of primary roots of Ambidopsis thaliana (L.) Heynh. However, Hu and Ye (1996) noted that 5 mg (REE),O, L-' inhibited the growth of rice roots. In addition, Diatloff et al. (1999) reported that concentrations of La and Ce in a nutrient solution from 0.1 to 2 mg L-' were toxic to root elongation of corn and mung beans; they also found that 0.03-0.7 mg La or Ce L-' reduced the uptake rate of all nutrients for mung beans, and that 0.7 mg La or Ce L-' had little effect on growth, but reduced the uptake of Ca for corn. In another case investigations of people living near La ore deposits revealed that the intake of low doses of rare earths through the food chain over time could cause nerve toxicity (Zhu et aL, 1997). Many questions concerning REEs remain unresolved, such as their bioavailability as well as the toxic and physiological effects of REE based fertilizers on plants and animals. Because of the increasing interest on the application of REEs in agricultural, more studies are required. *'Project supported by the Jiangsu Provincial Natural Science Foundation of China (No. BK99034) and the National Natural Science Foundation of China (No.29890280-1).
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It was reported that in agriculture over the last 20 years about 3.5 billion ha of cropland have used REEs-based fertilizers and about 11 000 t of rare-earth oxides have been applied in China (Pang, 2002a). La is one of the main components of REE-based fertilizers and the application of REE-based fertilizers has therefore caused large amounts of La to enter agricultural ecosystems. In fact, the mean background values of REEs and La in 61 kinds of major soils in China are 264 and 43.7 mg kg-l, respectively (Ran and Liu, 1994). Although La could affect the growth of crop seedlings, few studies concerning La have been undertaken, especially in the acidic red soils that occur widely in South China. In these soils mobility and phyto-toxicity of added metal elements were much higher than that in other types of soils because of the low soil pH (Zhang and Shan, 2001; Tyler and Olsson, 2001; Chen et al., 2004). In this study, lanthanum nitrate was applied to maize seedlings, an important crop in China, grown in a red loamy soil to investigate the physiological and toxic behaviors of added La and to help understand the effect of REEbased fertilizers on soil environment. MATERIALS AND METHODS The tested soil collected from Yingtan, Jiangxi Province in southern China was a red loamy soil derived from Quaternary red clay. The soil was air-dried at room temperature and sieved through a 2-mm sieve to remove large rocks, roots, and other large particles. The dry soil was then ground and passed through a l-mm sieve. Soil pH was measured in an aqueous slurry of a 1:l soil and water mixture. Organic matter in the soil was determined by the Wakley-Black method (Nelson and Sommers, 1982). Cation exchange capacities (CEC) of the soils were determined using the method described by Rhoades (1982). Selected properties of the soil sample were determined as: pH 4.68, CEC 19.7 cmol kg-', organic matter 12 g kg-', La 32.7 mg kg-', and REEs 231.5 mg kg-'. A completely random design (CRD) with nine La treatments including a control having no La in three replications was established. Treatments consisted of different levels of L a ( N 0 3 ) ~solution at 0, 0.10,0.25,0.50,0.75, 1.00, 1.50, 2.00, and 2.50 mg La kg-l soil. First, the soil was spiked with NH4N03 (at a rate of 0.1 g N-NO3 kg-' soil) and KHzP04 (at a rate of 0.08 g P and 0.1 g K kg-' soil). One week after fertilization, different levels of La(N03)3 solution were added, mixed thoroughly, and flooded with deionized water for 3 weeks. Soil pH was maintained with Ca(0H)z and 1 mol HC1 L-l. The soil was then air-dried at room temperature with all treatments being ground and passed through a 2-mm sieve again before pot-culture use. Maize (Zea mays L.) was chosen as the test plant. The seeds were surface sterilized with 2% NaClO for 30 min and washed thoroughly with distilled water. They were then placed in an illuminated incubator at 25 f 1 OC for germination, which occurred after 3 days. Next, five seedlings were planted in a 500-mL pot with 300 g test soil and cultivated in a greenhouse with 14 h sunshine each day. The temperature was kept at 22-25 "C during the day and 17-18 "C at night; soil moisture was maintained at about 15% (W/W), and, the soil was watered every day by weighing. The maize seedlings were harvested after 14 days of growth, washed with distilled water and measured for the root length. Plant samples were dried at 85 OC in an oven and the dry weight of the plant, root, and shoot was measured. The dry samples of shoots and roots were then placed in 25-mL beakers, soaked with concentrated HNOs (4.0 mL) and HC104 (1.0 mL) for 24 h, and heated. When the solution had evaporated to near dryness, the beakers were removed from the heater; after cooling, the residues were dissolved in 7% HN03. The resultant solutions were subsequently used for analysis by inductively coupled plasma-atomic emission spectrometry (ICP-AES) to determine La (detection limits of La in ICP-AES was 0.0015 mg La L-l) and selected mineral elements (Ca, Mg, K, Fe, Cu, Zn, and Mn). After the maize was harvested, the soils were collected and air-dried at room temperature. The dry soil was ground and passed through a l-mm plastic sieve for use. Then, 4 g dry soil samples from each treatment were added to 50 mL polypropylene centrifuge tubes with 40 mL of 1 mol NH4N03 L-' at room temperature and oscillated for 2 h. The mixture was then centrifuged at 10000 x g for 10 min, and the supernatant solutions were separated for analysis by ICP-AES. All extractions were completed
LANTHANUM AND MAIZE SEEDLING
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in duplicate. Bioaccumulation and distribution of La in seedlings for the 9 treatments was calculated with a linear regression model. Correlation of La content in shoots and roots with the added La levels in the soil was analyzed. After the maize seedlings were harvested the available fractions of La for the 9 La-treatments were determined and shown with a linear regression model. An ANOVA was used to determine the effects of La on primary root elongation of seedlings as well as dry weights of roots and shoots to test whether La could have beneficial effects on the growth of roots of maize. RESULTS AND DISCUSSION
Bioaccumulation and distribution of La in seedlings The background level of La in seedlings of the control was 2.22 mg kg-' in shoots and 16.25 mg kg-' in roots. Fig. 1 showed that La concentrations in shoots and roots increased with an increase of La in the soil. Changes of La concentrations in shoots of maize seedlings (Fig. la) were small except at 2.5 g La kg-', where the La concentration in the shoot was much higher than that at other concentrations. A regression of La concentration in the shoots to the concentration of La in the red soil revealed r2 = 0.788. However, La in the roots increased greatly and a linear regression showed a strong relationship (r2 = 0.980) between La in the roots and the La concentrations in the red soil (Fig. lb). Correlation analysis using Pearson's correlation (2-tailed) showed that the correlation coefficient for shoots and roots was 0.89 and 0.99, respectively, so correlation was significant at the 0.01 level for La content in shoots and roots with the added La. Fig. 1 also showed that the La concentrations in roots were much higher than those in shoots, and the ratio of La content in roots to that in shoots ranged from 20 to 302 for the 9 La-treatments. So La was mainly deposited in the roots of the seedlings. Results from other experiments have also shown that REEs were mainly distributed in the roots of crops (Cao et al., 2000; Wang et al., 2001; Zhang and Shan, 2001; Hu et al., 2002; Xu et al., 2002). This was also consistent with the results from heavy metal studies where roots were considered a barrier for translocation of metals to shoots. Thus, the compartmentalization of La at the whole plant level may be an important physiological aspect of metal tolerance. 35 h
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Added La (g kg-') Fig. 1 Regression analysis of the lanthanum (La) added to the red loamy soil and La accumulation in shoots (a) and roots (b) of maize seedlings.
In order to better understand the environmental behaviors of exogenous La, 1 mol NHINOB L-' was used to extract the available fraction of La in the red soil after the maize seedlings were harvested. Fig.2 showed that when La concentrations in the soil increased from 0 to 2.5 g kg-', the available fraction of La increased with the regression line of the variation for the added La concentrations in the red soil (r2 = 0.993). So after a short-term crop culture, plant growth did not have a strong effect on the fractionation of La.
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Effects of La on primary mot elongation of seedlings Compared with the control, La at low levels (from 0.10 to 0.25 g kg-') had no significant effects on promoting the elongation of maize roots (Table I). However, with increasing La concentrations added to the soil the primary root length became significantly shorter (P 5 0.05) (Table I). As La additions increased the seedlings also became smaller and the roots became thicker. At the concentrations of La in the soil above 1.5 to 2.5 g kg-l the root tips became stiff and dark. Therefore, at the concentrations of 0.10 to 0.25 g kg-' La had no significant beneficial effects on root elongation, and above 0.25 g kg-' root elongation was inhibited. TABLE I Primary root length of maize seedlings grown in the red loamy soil in treatments with different La concentrations neatment
Root length ~
~
Range g La kg-' 0 0.10 0.25 0.50 0.75 1.00 1.50 2.00 2.50
Mean cm
12.3-17.2 11.8-19.8 9.5-15.5 7.5-11.5 6.0-8.5 4.0-7.2 2.5-5.0 2.5-4.8 1.5-3.5 ~
')Means followed by the same letter are not significantly different at P
14.2ab') 15.0a 12.5b 9.5c 7.2d 5.4d 3.2e 3.h 2.4f ~~
5 0.05 by the Tukey's HSD test (n = 12).
Effects of La on seedling dry yields Compared with the control, the dry weight of seedlings was significantly reduced (P 5 0.05) (Table 11) with the added concentrations of La 2 0.75 g kg-l for shoots and 2 0.05 g kg-' for roots. This was consistent with other results in pot cultural experiments where growth and yield of rape were inhibited when the dose of La was more than 300 mg kg-' and killed after 600 mg kg-'. Also, in a solution cultural experiment with rape, the critical concentration of La was determined as 30 mg kg-' (Zeng et al., 2001a, b). Meanwhile, Diatleff et al. (1999) reported that La or Ce in a culture medium from 0.1 to 2 mg L-' was toxic to root elongation of corn and mung beans and that Ce at 0.03 mg L-' reduced the total dry weight of mung beans by 44%. Table I1 also showed that La caused more serious damage to roots than to shoots. This was consistent with the results of root elongation mentioned above. Thus,
LANTHANUM AND MAIZE SEEDLING
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roots were more sensitive to La in the red soil and could be considered as a sensitive biomarker of La stress. In addition, La may change some metabolic functions of plants when taken up. Thus, the effects of La on plant growth could resuIt from its bioaccumulation in plants. TABLE I1 Dry weights of roots and shoots of maize seedlings in treatments with different La concentrations Treatment
Shoot
Root ~
g La kg-' 0 0.10 0.25 0.50 0.75 1.00 1.50 2.00 2.50 ~
Range
Mean
37.9-53.7 41.2-57.1 36.6-52.0 37.2-60.3 28.6-49.7 20.8-45.2 14.5-31.7 18.0-25.0 11.4-18.5
47.9aa) 49.8a 47.5a 46.8a 36.9b 28.5~ 20.6d 22.5d 15.4e
Range
Mean
39.3-68.5 36.0-70.2 34.8-57.3 25.7-56.6 30.3-47.9 18.7-39.2 16.6-24.8 14.4-21.5 13.5-19.5
50.2a 52.2a 47.8a 41.0b 35.6~ 27.0d 20.k 18.k 15.9f
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P 5 0.05 by the Tukey's HSD test (n = 12).
Effects of La on major mineral elements in maize seedlings
Mineral elements were analyzed to test whether La could have beneficial effects on nutrient uptake and accumulation. Table I11 showed that in shoots, except for one case, and most of the time in roots, except for Ca and Mn, the main mineral elements of seedlings with La treatments, such as Ca, K, Mg, Cu, Mn, Fe and Zn, were less than the control. This meant that La generally interfered with essential nutrients and thereby disturbed the mineral nutrition of plants. This was consistent with the results of Diatloff's studies (1999) mentioned in the introduction. However, Qun et al. (1992) reported that in maize 5 mg La and Ce L-' in solution promoted the uptake of micronutrients, such as Zn, Mn, and TABLE I11 Effect of lanthanum (La) on mineral elements in shoots and roots of maize seedlings in treatments with different La concentrations ~~
Treatment
Ca
K
g La kg-' 0
0.10 0.25 0.50 0.75 1.00 1.50 2.00 2.50
Mg
Fe
cu
Mn
Zn
8.20 7.47 7.34 6.68 7.79 6.36 6.01 4.75 5.25
80.2 78.2 72.5 63.9 53.6 69.1 58.3 61.4 52.0
73.7 66.4 69.6 63.7 62.5 61.0 60.6 59.4 59.2
11.1 10.0 9.88 8.90 8.70 8.06 7.72 7.94 7.15
54.9 57.9 50.2 52.4 58.8 55.4 61.3 65.7 57.8
63.5 61.6 61.9 56.5 46.2 48.5 44.8 42.9 51.7
mg g-' 6.05 5.81 5.44 5.24 4.17 4.02 3.21 3.71 3.14
25.8 23.6 22.9 25.4 23.4 22.2 24.6 24.8 22.3
2.78 2.31 2.62 2.13 2.21 2.09 2.26 2.37
Shoot 0.158 0.151 0.149 0.132 0.121 0.115 0.106 0.093 0.095
5.60 5.58 5.67 6.44 5.99 6.11 6.94 6.21 6.31
27.0 28.4 26.0 21.3 19.8 17.5 19.0 16.8 13.1
4.03 4.05 3.75 3.66 3.55 3.14 3.50 3.40 2.96
2.47 2.47 2.48 2.39 2.15 2.25 2.14 2.02 1.85
2.80
Root 0
0.10 0.25 0.50 0.75 1.00 1.50 2.00 2.50
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Mo. It could be concluded that low doses of La or Ce had some beneficial effects on nutrient uptake. In fact, no evidence has indicated that REEs are essential for plant growth. However, because of a similar ionic radius tQCa2+ and a higher valence than Ca2+, La3+ could bind to superficially located Ca2+ absorption sites in a less reversible manner than Ca2+. La3+ could then block calcium ionic channels (Lewis and Spalding, 1998; Pineros and Tester, 1997) and disturb the uptake of nutrient ions through calcium channels. Therefore, nutrient disturbances caused the decrease of the dry yields of seedlings and inhibited the elongation of roots. CONCLUSIONS Maize seedlings could accumulate La in their tissues from their grown-soil and La was mainly distributed in roots. The ratio of La content in roots to that in shoots ranged from 20 to 302 for the 9 La-treatments. Compared with the control, the dry weight of shoots and roots, respectively, of maize seedlings was significantly reduced (P 5 0.05) with additions of La 2 0.75 g La kg-' soil and 2 0.05 g La kg-' soil. And La 2 0.5 g kg-' in the soil significantly inhibited (P 5 0.05) primary root elongation. Added La also influenced the uptake of main mineral elements of seedlings, such as Ca, K, Mg, Cu, Mn, and Zn. This study implies that La could not have beneficial effects on the growth of maize at the added La levels above 0.1 g kg-' red soil. ACKNOWLEDGEMENT Thanks are due to Pro. Les Evans from Guelph University, Canada for his help in modifying this article. REFERENCES Brown, P. H., Rathjen, A. H., Graham, R. D. and 'Ikibe, D. E. 1990. Rare earth elements in biological systems. In Gschneidner Jr., K. A. and Eyring, L. (eds.) Handbook on the Physics and Chemistry of Rare Earths. Vol. 13. Elsevier Science Publishers, Amsterdam. pp. 423-453. Cao, X. D., Wang, X. R. and Zhao, G. W. 2000. Assessment of the bioavailability of rare earth elements in soils by chemical fractionation and multiple regression analysis. Chemoaphere. 40: 23-28. Chen, G. C., He, Z. L. and Wang, Y. J. 2004. Impact of pH on microbial biomass carbon and microbial biomass phosphorus in red soils. Pedoaphere. 14(1): 9-15. Diatloff, E., Asher, C. J. and Smith, F. W. 1999. The effects of rare earth elements on the growth and nutrition of plants. Rare Earths' 98. Materiab Science Forum. 315(3): 354-360. He, Y. W. and Loh, C. S. 2000. Cerium and lanthanum promote floral initiation and reproductive growth of Ambidopaia thaliana. Plant Science. 159: 117-124. Hong, F. S.,Wang, L., Meng, X.X., Wei, Z. and Zhao, G. W. 2002. The effect of cerium (111) on the chlorophyll formation in spinach. Biological l h c e Element Research. 89: 263-276. Hu, Q. H. and Ye, Z. J. 1996. Physiological effects of rare-earth elements on plants. Plant Phyaiology Communications (in Chinese). 32: 296-300. Hu, X., Ding, Z. H., Chen, Y. J., Wang, X. R. and Dai, L. M. 2002. Bioaccumulation of lanthanum and cerium and their effects on the growth of wheat ( %ticum aestivum L.) seedlings. Chemosphere. 48: 621-629. Lewis, B. D. and Spalding, E. P. 1998. Nonselective block by La3+ of Ambidopaia ion channels involved in signal transduction. J. Membmne Biology. 162: 81-90. Nelson, D. W. and Sommers, L. E. 1982. Total carbon, organic carbon, and organic matter. In Page, A. L., Miller, R.H. and Keeney, D. R. (eds.) Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties. 2nd Edition. American Society of Agronomy, Madison, WI. pp. 539-579. Ni, J. Z. 1995. Bioinorganic Chemistry of Rare Earth Elements (in Chinese). Science Press, Beijing. 450pp. Pang, X.,Li, D. C. and Peng, A. 2002a. Application of rare-earth elements in the agriculture of China and its environmental behavior in soil. Environmental Science and Pollution Reaeurch. @(2): 143-148. Pang, X.,Wang, D. H., Xing, X.Y., Peng, A., Zhang, F. S. and Li, C. J. 2002b. Effect of La3+ on the activities of antioxidant enzymes in wheat seedlings under lead stress in solution culture. Chemosphere. 47( 10): 1033-1 039. Pineros, M. and Tester, M. 1997. Calcium channels in higher plant cells: Selectivity, regulation and pharmacology. J. Ezperimental Botany. 48: 551-577. Qun, B. Z., Gao, W. J., Yang, X. L. and Wu, L. N. 1992. Effects of rare earth elements on the uptake of Zn, Cu, Mn, Mo, Ca on corn seedlings. In He, M. Y. and Xiao, J. M. (eds.) Theoretical and Application Researches on Rare Earths in
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China (in Chinese). High Education Press, Beijing. pp. 386-390. Ran, Y.and Liu, Z. 1994. Content and distribution of rare earth elements in main types of soil in China. Acta Chinese Rare Earth (in Chinese). 12: 248-252. Rhoades, J. D. 1982. Cation-exchange capacity. In Page, A. L., Miller, R. H. and Keeney, D. R. (eds.) Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties. 2nd Edition. America Society of Agronomy, Madison, WI. pp. 149-158. Tyler, G. 2004. Rare earth elements in soil and plant systems-A review. Plant and Soil. 267: 191-206. Tyler, G.and Olsson, T. 2001. Plant uptake of major and minor mineral elements as influenced by soil acidity and liming. Plant and Soil. 230: 307-321. Wahid, P. A., Valiathan, M. S.,Kamalam, N. V., Eapen, J. T., Vijayalakshmi, S., Prabhu, R. K. and Mahalingam, T. R. 2000. Effect of rare earth elements on growth and nutrition of coconut palm and root competition for these elements between the palm and Calotropis gigantea. J . Plant Nutr. 23: 329-338. Wang, Z. J., Liu, D. F., Lu, P. and Wang, C. X. 2001. Accumulation of rare earth elements in corn after agricultural application. J. Enuimn. Qual. 30: 37-45. Xie, Z. B., Zhu, J. G., Chu, H. Y.,Zhang, Y.L., Zeng, Q., Ma, H. L. and Cao, Z. H. 2002. Effect of lanthanum on rice production, nutrient uptake, and distribution. J. Plant Nutr. 25: 2 315-2 331. Xiong, J. L.and Zhang, Z. L. 1997. Maximum threshold of the rare earth concentration and crop growth. Chinese Journal of Ecology (in Chinese). 16: 1-7. Xu, X. K.,Zhu, W. Z., Wang, Z. J. and Witkamp, G. J. 2002. Distributions of rare earths and heavy metals in field-grown maize after application of rare earth-containing fertilizer. Sci. Total Environ. 293: 97-105. Zeng, Q., Zhu, J. G., Xie, Z. B., Chu, H. Y.and Cao, Z. H. 2001a. Does response of rape to La and critical concentration of La in red earth. Environmental Science (in Chinese). 22(4): 77-80. Zeng, Q., Zhu, J. G., Xie, Z. B., Chu, H. Y.and Cao, Z. H. 2001b. Effects of rare earth La on some physiological indices of rape and its critical concentration. Rum1 Em-Environment (in Chinese). 17(3): 26-29. Zhang, S. Z. and Shan, X. Q. 2001. Speciation of rare earth elements in soil and accumulation by wheat with rare earth fertilizer application. Environmental Pollution. 112: 395-405. Zhu, W. F.,Xu, S.,Shao, P. P. and Zhang, H. 1997, Investigation on intake allowance of rare earth-A study on bio-effect of rare earth in south Jiangxi. Chinese Environmental Science (in Chinese). 17: 6246.
PEDOSPHERE www.elsevier.com/lote/pedosphere
Bioaccumulation of Lanthanum and Its Effect on Growth of Maize Seedlings in a Red Loamy Soil*' HU Xin', WANG Xiao-Rong' and WANG Chao2
'
State Key Labomtory of Pollution Control and Resource Reuse, School of Environmental Science, Nanjing University, Nanjing 210093 (China). E-mail: huzinJu8yahoo. com.cn 2School of Environmental Science and Engineering, Hohai University, Nanjing 210098 (China) (Received February 27, 2006; revised July 6, 2006)
ABSTRACT Through a pot culture lanthanum nitrate was applied to maize seedlings grown in a red loamy soil t o investigate the physiological and toxic effects of added La on the growth of crop seedlings and La bioaccumulation to help understand the environmental chemistry behaviors of rare earth element as fertilizers in soils. Compared t o the control, La concentrations in shoots and especially in roots of maize seedlings increased with an increase of La in the soil. Also, with added concentrations of La 2 0.75 g La kg-' soil and 2 0.05 g La kg-' soil, the dry weight of shoots and roots of maize seedlings was significantly reduced (P 5 0.05), respectively, compared with the control. Additionally, La 2 0.5 g kg-' in the soil significantly inhibited (P 5 0.05) primary root elongation. Roots were more sensitive to La stress than shoots and thus could be used as a biomarker to La stress. Overall, in the red loamy soil studied, La had no significant beneficial effects on the growth of maize at the added La levels above 0.1 g kg-' soil. Key Words:
bioaccumulation, growth, lanthanum, maize seedlings, red loamy soil
INTRODUCTION Rare earth elements (REEs) as fertilizers have been widely used in China's agriculture over the last 20 years. It was believed that REEs could stimulate the growth of roots, improve the quantity and quality of crops, and enhance crop resistance (Ni, 1995; Pang et al., 2002a, b; Xie et al., 2002). Nevertheless, results from field trials, pot trials, and laboratory studies on the effect of REEs on crop growth and development have been inconsistent (Brown et al., 1990; He and Loh, 2000; Wahid et al., 2000; Xu et al., 2002; Hong et al., 2002; Hu et al., 2002; Tyler, 2004). For example, lanthanum (La) added to a medium at levels of below 40 mg L-I, 10 mg L-', and 30 mg L-l stimulated the growth of maize, rape, and rice, respectively (Xiong and Zhang, 1997). He and Loh (2000) also reported that the addition of cerium (Ce) nitrate (0.5-10 pmol L-I) or lanthanum nitrate (0.5-50 pmol L-') to a culture medium significantly increased the length of primary roots of Ambidopsis thaliana (L.) Heynh. However, Hu and Ye (1996) noted that 5 mg (REE),O, L-' inhibited the growth of rice roots. In addition, Diatloff et al. (1999) reported that concentrations of La and Ce in a nutrient solution from 0.1 to 2 mg L-' were toxic to root elongation of corn and mung beans; they also found that 0.03-0.7 mg La or Ce L-' reduced the uptake rate of all nutrients for mung beans, and that 0.7 mg La or Ce L-' had little effect on growth, but reduced the uptake of Ca for corn. In another case investigations of people living near La ore deposits revealed that the intake of low doses of rare earths through the food chain over time could cause nerve toxicity (Zhu et aL, 1997). Many questions concerning REEs remain unresolved, such as their bioavailability as well as the toxic and physiological effects of REE based fertilizers on plants and animals. Because of the increasing interest on the application of REEs in agricultural, more studies are required. *'Project supported by the Jiangsu Provincial Natural Science Foundation of China (No. BK99034) and the National Natural Science Foundation of China (No.29890280-1).
800
X. HU
et at.
It was reported that in agriculture over the last 20 years about 3.5 billion ha of cropland have used REEs-based fertilizers and about 11 000 t of rare-earth oxides have been applied in China (Pang, 2002a). La is one of the main components of REE-based fertilizers and the application of REE-based fertilizers has therefore caused large amounts of La to enter agricultural ecosystems. In fact, the mean background values of REEs and La in 61 kinds of major soils in China are 264 and 43.7 mg kg-l, respectively (Ran and Liu, 1994). Although La could affect the growth of crop seedlings, few studies concerning La have been undertaken, especially in the acidic red soils that occur widely in South China. In these soils mobility and phyto-toxicity of added metal elements were much higher than that in other types of soils because of the low soil pH (Zhang and Shan, 2001; Tyler and Olsson, 2001; Chen et al., 2004). In this study, lanthanum nitrate was applied to maize seedlings, an important crop in China, grown in a red loamy soil to investigate the physiological and toxic behaviors of added La and to help understand the effect of REEbased fertilizers on soil environment. MATERIALS AND METHODS The tested soil collected from Yingtan, Jiangxi Province in southern China was a red loamy soil derived from Quaternary red clay. The soil was air-dried at room temperature and sieved through a 2-mm sieve to remove large rocks, roots, and other large particles. The dry soil was then ground and passed through a l-mm sieve. Soil pH was measured in an aqueous slurry of a 1:l soil and water mixture. Organic matter in the soil was determined by the Wakley-Black method (Nelson and Sommers, 1982). Cation exchange capacities (CEC) of the soils were determined using the method described by Rhoades (1982). Selected properties of the soil sample were determined as: pH 4.68, CEC 19.7 cmol kg-', organic matter 12 g kg-', La 32.7 mg kg-', and REEs 231.5 mg kg-'. A completely random design (CRD) with nine La treatments including a control having no La in three replications was established. Treatments consisted of different levels of L a ( N 0 3 ) ~solution at 0, 0.10,0.25,0.50,0.75, 1.00, 1.50, 2.00, and 2.50 mg La kg-l soil. First, the soil was spiked with NH4N03 (at a rate of 0.1 g N-NO3 kg-' soil) and KHzP04 (at a rate of 0.08 g P and 0.1 g K kg-' soil). One week after fertilization, different levels of La(N03)3 solution were added, mixed thoroughly, and flooded with deionized water for 3 weeks. Soil pH was maintained with Ca(0H)z and 1 mol HC1 L-l. The soil was then air-dried at room temperature with all treatments being ground and passed through a 2-mm sieve again before pot-culture use. Maize (Zea mays L.) was chosen as the test plant. The seeds were surface sterilized with 2% NaClO for 30 min and washed thoroughly with distilled water. They were then placed in an illuminated incubator at 25 f 1 OC for germination, which occurred after 3 days. Next, five seedlings were planted in a 500-mL pot with 300 g test soil and cultivated in a greenhouse with 14 h sunshine each day. The temperature was kept at 22-25 "C during the day and 17-18 "C at night; soil moisture was maintained at about 15% (W/W), and, the soil was watered every day by weighing. The maize seedlings were harvested after 14 days of growth, washed with distilled water and measured for the root length. Plant samples were dried at 85 OC in an oven and the dry weight of the plant, root, and shoot was measured. The dry samples of shoots and roots were then placed in 25-mL beakers, soaked with concentrated HNOs (4.0 mL) and HC104 (1.0 mL) for 24 h, and heated. When the solution had evaporated to near dryness, the beakers were removed from the heater; after cooling, the residues were dissolved in 7% HN03. The resultant solutions were subsequently used for analysis by inductively coupled plasma-atomic emission spectrometry (ICP-AES) to determine La (detection limits of La in ICP-AES was 0.0015 mg La L-l) and selected mineral elements (Ca, Mg, K, Fe, Cu, Zn, and Mn). After the maize was harvested, the soils were collected and air-dried at room temperature. The dry soil was ground and passed through a l-mm plastic sieve for use. Then, 4 g dry soil samples from each treatment were added to 50 mL polypropylene centrifuge tubes with 40 mL of 1 mol NH4N03 L-' at room temperature and oscillated for 2 h. The mixture was then centrifuged at 10000 x g for 10 min, and the supernatant solutions were separated for analysis by ICP-AES. All extractions were completed
LANTHANUM AND MAIZE SEEDLING
801
in duplicate. Bioaccumulation and distribution of La in seedlings for the 9 treatments was calculated with a linear regression model. Correlation of La content in shoots and roots with the added La levels in the soil was analyzed. After the maize seedlings were harvested the available fractions of La for the 9 La-treatments were determined and shown with a linear regression model. An ANOVA was used to determine the effects of La on primary root elongation of seedlings as well as dry weights of roots and shoots to test whether La could have beneficial effects on the growth of roots of maize. RESULTS AND DISCUSSION
Bioaccumulation and distribution of La in seedlings The background level of La in seedlings of the control was 2.22 mg kg-' in shoots and 16.25 mg kg-' in roots. Fig. 1 showed that La concentrations in shoots and roots increased with an increase of La in the soil. Changes of La concentrations in shoots of maize seedlings (Fig. la) were small except at 2.5 g La kg-', where the La concentration in the shoot was much higher than that at other concentrations. A regression of La concentration in the shoots to the concentration of La in the red soil revealed r2 = 0.788. However, La in the roots increased greatly and a linear regression showed a strong relationship (r2 = 0.980) between La in the roots and the La concentrations in the red soil (Fig. lb). Correlation analysis using Pearson's correlation (2-tailed) showed that the correlation coefficient for shoots and roots was 0.89 and 0.99, respectively, so correlation was significant at the 0.01 level for La content in shoots and roots with the added La. Fig. 1 also showed that the La concentrations in roots were much higher than those in shoots, and the ratio of La content in roots to that in shoots ranged from 20 to 302 for the 9 La-treatments. So La was mainly deposited in the roots of the seedlings. Results from other experiments have also shown that REEs were mainly distributed in the roots of crops (Cao et al., 2000; Wang et al., 2001; Zhang and Shan, 2001; Hu et al., 2002; Xu et al., 2002). This was also consistent with the results from heavy metal studies where roots were considered a barrier for translocation of metals to shoots. Thus, the compartmentalization of La at the whole plant level may be an important physiological aspect of metal tolerance. 35 h
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Added La (g kg-') Fig. 1 Regression analysis of the lanthanum (La) added to the red loamy soil and La accumulation in shoots (a) and roots (b) of maize seedlings.
In order to better understand the environmental behaviors of exogenous La, 1 mol NHINOB L-' was used to extract the available fraction of La in the red soil after the maize seedlings were harvested. Fig.2 showed that when La concentrations in the soil increased from 0 to 2.5 g kg-', the available fraction of La increased with the regression line of the variation for the added La concentrations in the red soil (r2 = 0.993). So after a short-term crop culture, plant growth did not have a strong effect on the fractionation of La.
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Effects of La on primary mot elongation of seedlings Compared with the control, La at low levels (from 0.10 to 0.25 g kg-') had no significant effects on promoting the elongation of maize roots (Table I). However, with increasing La concentrations added to the soil the primary root length became significantly shorter (P 5 0.05) (Table I). As La additions increased the seedlings also became smaller and the roots became thicker. At the concentrations of La in the soil above 1.5 to 2.5 g kg-l the root tips became stiff and dark. Therefore, at the concentrations of 0.10 to 0.25 g kg-' La had no significant beneficial effects on root elongation, and above 0.25 g kg-' root elongation was inhibited. TABLE I Primary root length of maize seedlings grown in the red loamy soil in treatments with different La concentrations neatment
Root length ~
~
Range g La kg-' 0 0.10 0.25 0.50 0.75 1.00 1.50 2.00 2.50
Mean cm
12.3-17.2 11.8-19.8 9.5-15.5 7.5-11.5 6.0-8.5 4.0-7.2 2.5-5.0 2.5-4.8 1.5-3.5 ~
')Means followed by the same letter are not significantly different at P
14.2ab') 15.0a 12.5b 9.5c 7.2d 5.4d 3.2e 3.h 2.4f ~~
5 0.05 by the Tukey's HSD test (n = 12).
Effects of La on seedling dry yields Compared with the control, the dry weight of seedlings was significantly reduced (P 5 0.05) (Table 11) with the added concentrations of La 2 0.75 g kg-l for shoots and 2 0.05 g kg-' for roots. This was consistent with other results in pot cultural experiments where growth and yield of rape were inhibited when the dose of La was more than 300 mg kg-' and killed after 600 mg kg-'. Also, in a solution cultural experiment with rape, the critical concentration of La was determined as 30 mg kg-' (Zeng et al., 2001a, b). Meanwhile, Diatleff et al. (1999) reported that La or Ce in a culture medium from 0.1 to 2 mg L-' was toxic to root elongation of corn and mung beans and that Ce at 0.03 mg L-' reduced the total dry weight of mung beans by 44%. Table I1 also showed that La caused more serious damage to roots than to shoots. This was consistent with the results of root elongation mentioned above. Thus,
LANTHANUM AND MAIZE SEEDLING
803
roots were more sensitive to La in the red soil and could be considered as a sensitive biomarker of La stress. In addition, La may change some metabolic functions of plants when taken up. Thus, the effects of La on plant growth could resuIt from its bioaccumulation in plants. TABLE I1 Dry weights of roots and shoots of maize seedlings in treatments with different La concentrations Treatment
Shoot
Root ~
g La kg-' 0 0.10 0.25 0.50 0.75 1.00 1.50 2.00 2.50 ~
Range
Mean
37.9-53.7 41.2-57.1 36.6-52.0 37.2-60.3 28.6-49.7 20.8-45.2 14.5-31.7 18.0-25.0 11.4-18.5
47.9aa) 49.8a 47.5a 46.8a 36.9b 28.5~ 20.6d 22.5d 15.4e
Range
Mean
39.3-68.5 36.0-70.2 34.8-57.3 25.7-56.6 30.3-47.9 18.7-39.2 16.6-24.8 14.4-21.5 13.5-19.5
50.2a 52.2a 47.8a 41.0b 35.6~ 27.0d 20.k 18.k 15.9f
mg
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')Means followed by the same letter are not significantly different at
~~
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P 5 0.05 by the Tukey's HSD test (n = 12).
Effects of La on major mineral elements in maize seedlings
Mineral elements were analyzed to test whether La could have beneficial effects on nutrient uptake and accumulation. Table I11 showed that in shoots, except for one case, and most of the time in roots, except for Ca and Mn, the main mineral elements of seedlings with La treatments, such as Ca, K, Mg, Cu, Mn, Fe and Zn, were less than the control. This meant that La generally interfered with essential nutrients and thereby disturbed the mineral nutrition of plants. This was consistent with the results of Diatloff's studies (1999) mentioned in the introduction. However, Qun et al. (1992) reported that in maize 5 mg La and Ce L-' in solution promoted the uptake of micronutrients, such as Zn, Mn, and TABLE I11 Effect of lanthanum (La) on mineral elements in shoots and roots of maize seedlings in treatments with different La concentrations ~~
Treatment
Ca
K
g La kg-' 0
0.10 0.25 0.50 0.75 1.00 1.50 2.00 2.50
Mg
Fe
cu
Mn
Zn
8.20 7.47 7.34 6.68 7.79 6.36 6.01 4.75 5.25
80.2 78.2 72.5 63.9 53.6 69.1 58.3 61.4 52.0
73.7 66.4 69.6 63.7 62.5 61.0 60.6 59.4 59.2
11.1 10.0 9.88 8.90 8.70 8.06 7.72 7.94 7.15
54.9 57.9 50.2 52.4 58.8 55.4 61.3 65.7 57.8
63.5 61.6 61.9 56.5 46.2 48.5 44.8 42.9 51.7
mg g-' 6.05 5.81 5.44 5.24 4.17 4.02 3.21 3.71 3.14
25.8 23.6 22.9 25.4 23.4 22.2 24.6 24.8 22.3
2.78 2.31 2.62 2.13 2.21 2.09 2.26 2.37
Shoot 0.158 0.151 0.149 0.132 0.121 0.115 0.106 0.093 0.095
5.60 5.58 5.67 6.44 5.99 6.11 6.94 6.21 6.31
27.0 28.4 26.0 21.3 19.8 17.5 19.0 16.8 13.1
4.03 4.05 3.75 3.66 3.55 3.14 3.50 3.40 2.96
2.47 2.47 2.48 2.39 2.15 2.25 2.14 2.02 1.85
2.80
Root 0
0.10 0.25 0.50 0.75 1.00 1.50 2.00 2.50
804
X.HU et al.
Mo. It could be concluded that low doses of La or Ce had some beneficial effects on nutrient uptake. In fact, no evidence has indicated that REEs are essential for plant growth. However, because of a similar ionic radius tQCa2+ and a higher valence than Ca2+, La3+ could bind to superficially located Ca2+ absorption sites in a less reversible manner than Ca2+. La3+ could then block calcium ionic channels (Lewis and Spalding, 1998; Pineros and Tester, 1997) and disturb the uptake of nutrient ions through calcium channels. Therefore, nutrient disturbances caused the decrease of the dry yields of seedlings and inhibited the elongation of roots. CONCLUSIONS Maize seedlings could accumulate La in their tissues from their grown-soil and La was mainly distributed in roots. The ratio of La content in roots to that in shoots ranged from 20 to 302 for the 9 La-treatments. Compared with the control, the dry weight of shoots and roots, respectively, of maize seedlings was significantly reduced (P 5 0.05) with additions of La 2 0.75 g La kg-' soil and 2 0.05 g La kg-' soil. And La 2 0.5 g kg-' in the soil significantly inhibited (P 5 0.05) primary root elongation. Added La also influenced the uptake of main mineral elements of seedlings, such as Ca, K, Mg, Cu, Mn, and Zn. This study implies that La could not have beneficial effects on the growth of maize at the added La levels above 0.1 g kg-' red soil. ACKNOWLEDGEMENT Thanks are due to Pro. Les Evans from Guelph University, Canada for his help in modifying this article. REFERENCES Brown, P. H., Rathjen, A. H., Graham, R. D. and 'Ikibe, D. E. 1990. Rare earth elements in biological systems. In Gschneidner Jr., K. A. and Eyring, L. (eds.) Handbook on the Physics and Chemistry of Rare Earths. Vol. 13. Elsevier Science Publishers, Amsterdam. pp. 423-453. Cao, X. D., Wang, X. R. and Zhao, G. W. 2000. Assessment of the bioavailability of rare earth elements in soils by chemical fractionation and multiple regression analysis. Chemoaphere. 40: 23-28. Chen, G. C., He, Z. L. and Wang, Y. J. 2004. Impact of pH on microbial biomass carbon and microbial biomass phosphorus in red soils. Pedoaphere. 14(1): 9-15. Diatloff, E., Asher, C. J. and Smith, F. W. 1999. The effects of rare earth elements on the growth and nutrition of plants. Rare Earths' 98. Materiab Science Forum. 315(3): 354-360. He, Y. W. and Loh, C. S. 2000. Cerium and lanthanum promote floral initiation and reproductive growth of Ambidopaia thaliana. Plant Science. 159: 117-124. Hong, F. S.,Wang, L., Meng, X.X., Wei, Z. and Zhao, G. W. 2002. The effect of cerium (111) on the chlorophyll formation in spinach. Biological l h c e Element Research. 89: 263-276. Hu, Q. H. and Ye, Z. J. 1996. Physiological effects of rare-earth elements on plants. Plant Phyaiology Communications (in Chinese). 32: 296-300. Hu, X., Ding, Z. H., Chen, Y. J., Wang, X. R. and Dai, L. M. 2002. Bioaccumulation of lanthanum and cerium and their effects on the growth of wheat ( %ticum aestivum L.) seedlings. Chemosphere. 48: 621-629. Lewis, B. D. and Spalding, E. P. 1998. Nonselective block by La3+ of Ambidopaia ion channels involved in signal transduction. J. Membmne Biology. 162: 81-90. Nelson, D. W. and Sommers, L. E. 1982. Total carbon, organic carbon, and organic matter. In Page, A. L., Miller, R.H. and Keeney, D. R. (eds.) Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties. 2nd Edition. American Society of Agronomy, Madison, WI. pp. 539-579. Ni, J. Z. 1995. Bioinorganic Chemistry of Rare Earth Elements (in Chinese). Science Press, Beijing. 450pp. Pang, X.,Li, D. C. and Peng, A. 2002a. Application of rare-earth elements in the agriculture of China and its environmental behavior in soil. Environmental Science and Pollution Reaeurch. @(2): 143-148. Pang, X.,Wang, D. H., Xing, X.Y., Peng, A., Zhang, F. S. and Li, C. J. 2002b. Effect of La3+ on the activities of antioxidant enzymes in wheat seedlings under lead stress in solution culture. Chemosphere. 47( 10): 1033-1 039. Pineros, M. and Tester, M. 1997. Calcium channels in higher plant cells: Selectivity, regulation and pharmacology. J. Ezperimental Botany. 48: 551-577. Qun, B. Z., Gao, W. J., Yang, X. L. and Wu, L. N. 1992. Effects of rare earth elements on the uptake of Zn, Cu, Mn, Mo, Ca on corn seedlings. In He, M. Y. and Xiao, J. M. (eds.) Theoretical and Application Researches on Rare Earths in
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China (in Chinese). High Education Press, Beijing. pp. 386-390. Ran, Y.and Liu, Z. 1994. Content and distribution of rare earth elements in main types of soil in China. Acta Chinese Rare Earth (in Chinese). 12: 248-252. Rhoades, J. D. 1982. Cation-exchange capacity. In Page, A. L., Miller, R. H. and Keeney, D. R. (eds.) Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties. 2nd Edition. America Society of Agronomy, Madison, WI. pp. 149-158. Tyler, G. 2004. Rare earth elements in soil and plant systems-A review. Plant and Soil. 267: 191-206. Tyler, G.and Olsson, T. 2001. Plant uptake of major and minor mineral elements as influenced by soil acidity and liming. Plant and Soil. 230: 307-321. Wahid, P. A., Valiathan, M. S.,Kamalam, N. V., Eapen, J. T., Vijayalakshmi, S., Prabhu, R. K. and Mahalingam, T. R. 2000. Effect of rare earth elements on growth and nutrition of coconut palm and root competition for these elements between the palm and Calotropis gigantea. J . Plant Nutr. 23: 329-338. Wang, Z. J., Liu, D. F., Lu, P. and Wang, C. X. 2001. Accumulation of rare earth elements in corn after agricultural application. J. Enuimn. Qual. 30: 37-45. Xie, Z. B., Zhu, J. G., Chu, H. Y.,Zhang, Y.L., Zeng, Q., Ma, H. L. and Cao, Z. H. 2002. Effect of lanthanum on rice production, nutrient uptake, and distribution. J. Plant Nutr. 25: 2 315-2 331. Xiong, J. L.and Zhang, Z. L. 1997. Maximum threshold of the rare earth concentration and crop growth. Chinese Journal of Ecology (in Chinese). 16: 1-7. Xu, X. K.,Zhu, W. Z., Wang, Z. J. and Witkamp, G. J. 2002. Distributions of rare earths and heavy metals in field-grown maize after application of rare earth-containing fertilizer. Sci. Total Environ. 293: 97-105. Zeng, Q., Zhu, J. G., Xie, Z. B., Chu, H. Y.and Cao, Z. H. 2001a. Does response of rape to La and critical concentration of La in red earth. Environmental Science (in Chinese). 22(4): 77-80. Zeng, Q., Zhu, J. G., Xie, Z. B., Chu, H. Y.and Cao, Z. H. 2001b. Effects of rare earth La on some physiological indices of rape and its critical concentration. Rum1 Em-Environment (in Chinese). 17(3): 26-29. Zhang, S. Z. and Shan, X. Q. 2001. Speciation of rare earth elements in soil and accumulation by wheat with rare earth fertilizer application. Environmental Pollution. 112: 395-405. Zhu, W. F.,Xu, S.,Shao, P. P. and Zhang, H. 1997, Investigation on intake allowance of rare earth-A study on bio-effect of rare earth in south Jiangxi. Chinese Environmental Science (in Chinese). 17: 6246.