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The growth and uptake of Ga and In of rice (Oryza sative L.) seedlings as affected by Ga and In concentrations in hydroponic cultures
Ecotoxicology and Environmental Safety 135 (2017) 32–39
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
The growth and uptake of Ga and In of rice (Oryza sative L.) seedlings as affected by Ga and In concentrations in hydroponic cultures Chien-Hui Syu a,1, Po-Hsuan Chien b,1, Chia-Chen Huang b, Pei-Yu Jiang b, Kai-Wei Juang c, Dar-Yuan Lee b,n a
Division of Agricultural Chemistry, Taiwan Agricultural Research Institute, No.189, Zhongzheng Rd., Wufeng Dist., Taichung 41362, Taiwan Department of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan c Department of Agronomy, National Chiayi University, No.300 Syuefu Rd., Chiayi 60004, Taiwan b
art ic l e i nf o
a b s t r a c t
Article history: Received 11 July 2016 Received in revised form 9 September 2016 Accepted 19 September 2016
Limited information is available on the effects of gallium (Ga) and indium (In) on the growth of paddy rice. The Ga and In are emerging contaminants and widely used in high-tech industries nowadays. Understanding the toxicity and accumulation of Ga and In by rice plants is important for reducing the effect on rice production and exposure risk to human by rice consumption. Therefore, this study investigates the effect of Ga and In on the growth of rice seedlings and examines the accumulation and distribution of those elements in plant tissues. Hydroponic cultures were conducted in phytotron glasshouse with controlled temperature and relative humidity conditions, and the rice seedlings were treated with different levels of Ga and In in the nutrient solutions. The growth index and the concentrations of Ga and In in roots and shoots of rice seedlings were measured after harvesting. A significant increase in growth index with increasing Ga concentrations in culture solutions (o10 mg Ga L 1) was observed. In addition, the uptake of N, K, Mg, Ca, Mn by rice plants was also enhanced by Ga. However, the growth inhibition were observed while the In concentrations higher than 0.08 mg L 1, and the nutrients accumulated in rice plants were also significant decreased after In treatments. Based on the dose-response curve, we observed that the EC10 (effective concentration resulting in 10% growth inhibition) value for In treatment was 0.17 mg L 1. The results of plant analysis indicated that the roots were the dominant sink of Ga and In in rice seedlings, and it was also found that the capability of translocation of Ga from roots to shoots were higher than In. In addition, it was also found that the PT10 (threshold concentration of phytotoxicity resulting in 10% growth retardation) values based on shoot height and total biomass for In were 15.4 and 10.6 μg plant 1, respectively. The beneficial effects on the plant growth of rice seedlings were found by the addition of Ga in culture solutions. In contrast, the In treatments led to growth inhibition of rice seedlings. There were differences in the phytotoxicity, uptake, and translocation of the two emerging contaminants in rice seedlings. & 2016 Elsevier Inc. All rights reserved.
Keywords: Gallium Indium Emerging contaminants Paddy rice Hydroponic culture
1. Introduction Gallium (Ga) and indium (In) are regarded as toxic substances to humans based on previous reports (Fowler et al.. 1993; Ivanoff et al.. 2012; Kabata-Pendias and Mukherjee, 2007; Tanaka 2004). In general, Ga and In are produced as byproducts in the production of Al (bauxite), Pb (galena) and Zn (sphalerite), and these two elements are widely used in semiconductor manufacturing and the electro-optical and medical industries (Alfantazi and Moskalyk, 2003; Kabata-Pendias and Mukherjee, 2007; Kabata-Pendias 2011; Yu and Liao, 2011).
n
Corresponding author. E-mail address: [email protected] (D.-Y. Lee). 1 Dr. Chien-Hui Syu and Mr. Po-Hsuan Chien are equal contributors to this paper. http://dx.doi.org/10.1016/j.ecoenv.2016.09.016 0147-6513/& 2016 Elsevier Inc. All rights reserved.
Nowadays, due to progressively increasing usage of Ga and In, wastewater containing them may be discharged into farmland soils by the irrigation system, further raising the risk of human exposure to Ga and In through the food chain. Chen (2006) reported the high concentrations of Ga (up to 41 μg L 1) and In (up to 20 μg L 1) in groundwater contaminated by wastewater from semiconductor manufacturing area of Taiwan. To date, the information about the contents of Ga and In in soils contaminated by semiconductor manufacturing is limited. However, there were some studies indicated that the contents of Ga and In in soils near area of automobile and ZnPb smelting industries were 10.89–22.46 mg kg 1 and 0.11– 1.92 mg kg 1, respectively (Asami et al., 1990; Yu et al., 2015). Nevertheless, studies about the effects of Ga and In on the growth of edible crops are still scarce. Therefore, understanding the interaction between Ga and In and edible crops is necessary.
C.-H. Syu et al. / Ecotoxicology and Environmental Safety 135 (2017) 32–39
Gallium and indium are grouped among the IIIA group of elements in the periodic table, and the content of Ga and In in worldwide soils ranges from 3 to 70 and 0.11–0.25 mg kg 1 respectively (Kabata-Pendias, 2011). In general, the oxidation states of Ga and In are þ3, but oxidation states of þ 1 and þ2 may also occur under anaerobic conditions. At 25 °C, the soluble species of Ga and In present in acidic conditions are Ga3 þ , Ga(OH)2 þ , Ga(OH)2 þ , and In3 þ , In(OH)2 þ , In(OH)2 þ ; the soluble species present in alkaline conditions are Ga(OH)4 and In(OH)4 , and the insoluble species Ga (OH)3 and In(OH)3 are present in conditions of pH 4–6 and pH 5–9 (Kabata-Pendias, 2011; Wood and Samson 2006). These solubility characteristics are similar to those of Al, which suggests that Ga and In are amphoteric elements. Orians and Bruland (1988) showed that the geochemistry and aquatic chemistry properties of Ga are similar to Al, but that the reactivity of Ga is less than that of Al. Rice is a dietary food for about half of the world's population, and for over 90% of the population in Asia (Meharg et al.. 2009). Because paddy fields may suffer from Ga- and In-associated wastewater contamination, study of the accumulation of Ga and In in rice plants and the effect of these two emerging contaminants on rice plant growth is merited. The chemical properties of Ga and In are similar to those of Al, and several studies have investigated Al toxicity and tolerance in rice plants (Roy and Bhadra, 2014; Silva 2012; Tanaka and Navasero, 1966). However, little information about the effects of Ga and In on the growth of rice plants exists to date. Yu et al.. (2015) reported an associated reduction in relative growth rate, transpiration rate and water use efficiency of rice seedlings grown for 2 days in solution culture of increasing Ga concentration. Yu and Zhang (2015) found that over-accumulation of Ga in plant tissue resulted in cell death and growth inhibition in rice seedlings. Some studies have also investigated the toxic effect and accumulation of Ga and In in other plant species (Berg and Steinnes, 1997; Fergusson 1990; Kopittke et al., 2009; Shacklette et al., 1978). Shacklette et al. (1978) reported that the concentration of Ga in a variety of native species ranged from 3 to 30 mg kg 1 in the United States. Berg and Steinnes (1997) indicated that atmospheric deposition may result in the elevation of Ga content by up to 16 mg kg 1 in moss growing wild in Norway. Fergusson (1990) found concentrations of In ranging from 80 to 300 μg/kg FW in beets, 0.64–1.8 μg/kg FW in leaves of fruit trees, and 30–710 μg/kg FW in vegetables. Kopittke et al. (2009) reported that soluble Ga and In reduced cowpea root growth and caused cell rupture in hydroponic experiments. To the best of our knowledge, the uptake and accumulation of Ga and In in rice plants and their effect on rice growth is still unclear. Therefore, our objective in this study was to investigate the effect of Ga and In on rice plant growth and how uptake of these two elements affects rice seedlings grown in solution cultures treated with different concentrations of Ga and In. In particular, Ga and In may impact the uptake of nutrients through competitive uptake by roots or through phytotoxicity; thus we also investigated the effect of Ga and In treatments on the uptake of nutrients by rice plants.
2. Material and methods 2.1. Hydroponic cultures The hydroponic cultures were performed in a phytotron with a controlled temperature (25/20 °C, day/night) and relative humidity (70–95%) under sunlight. The cultivar of paddy rice (Oryza sative L., cv Taikeng 9) was used in this study because it is commonly planted in Taiwan and considered to be of high quality. Rice seeds were sterilized in a solution containing 1% sodium hydrochloride solution and one drop of Tween 20 for 30 min, and then washed with distilled water for 30 min. The seeds were then germinated in Petri dishes containing tissue paper under moist conditions at 37 °C for
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2 days. After germination, thirty seedlings were then transferred to an iron mesh set on the surface of the culture solution, contained in a 0.6-L beaker. Seedlings were then raised in half-strength modified Kimura B nutrient solution (pH was adjusted to 4.8–5.0 and the solution was renewed every three days) for 14 days until they reached the three-leaf age. Afterward, the solution was replaced with full-strength nutrient solution and treated with the indicated amount of Ga or In stock solution. The stock solutions of Ga and In were prepared using GaCl3 (99.999%, ultra dry, Alfa Aesar) and InCl3 (99.999%, anhydrous, Alfa Aesar) respectively. The Ga treatment concentrations were 0, 1, 3, 5, 10, 15 mg L 1, and the exposure time was 40 days (growth period: Nov.–Dec., 2014) The In treatments were separated into two parts: (a) For the high-In treatment (preliminary experiment), the In treatment concentrations were 0, 0.1, 1, 3, 5, 10 mg L 1, and the exposure time was 25 days (growth period: Jul.–Aug., 2014); (b) for the low-In treatment, the In treatment concentrations were 0, 0.04, 0.08, 0.1, 0.15, 1, 2 mg L 1, and the exposure time was 40 days (growth period: Nov.–Dec., 2014). Despite the concentrations of Ga and In in wastewater higher than 10 mg L 1 were very rare, the concentration ranges of Ga and In used in this study were intended to clarify the toxicity concentrations to rice seedlings such as EC10 (effective concentration resulting in 10% growth inhibition) and PT10 (threshold concentration of phytotoxicity resulting in 10% growth retardation) values. In order to avoid Ga and In precipitation, the culture solutions were made just before use and renewed every day. We verified that no significant changes in Ga and In concentrations in the culture solutions had occurred at 24 h. Three replicates (pots) for each of the Ga and In treatments. The half-strength modified Kimura B nutrient solution was used in this study, which contains the following compositions: 0.18 mM (NH4)2SO4, 0.09 mM KNO3, 0.27 mM MgSO4‧7H2O, 0.09 mM KH2PO4, 30.6 μM Fe-citrate, 183 μM Ca(NO3)2‧4H20, 25.1 μM H3BO3, 2.01 μM MnSO4‧4H2O, 2.02 μM ZnSO4‧7H2O, 1.19 μM CuSO4‧5H2O and 0.49 μM MoO3. The concentrations of micro-elements (B, Mn, Zn, Cu and Mo) in full-strength modified nutrient solution were identical with the half-strength modified Kimura B nutrient solution, and the concentrations of other elements in full-strength modified nutrient solution were two folds higher than half-strength modified Kimura B nutrient solution. After harvesting, the rice seedlings were separated into root and shoot and rinsed first with tap water and then with deionized water. The biomass and lengths of each root and shoot were measured. In addition, their chlorophyll content was also measured with a chlorophyll meter (SPAD-502, Spectrum Technologies). Briefly, 10 plants per pot were selected and the chlorophyll meter was used to measure three points on the least expanded leaf of each plant (the interval between two points was 5 cm). Iron plaque on the roots was removed using modified cold DCB (dithionite-citratebicarbonate) solution (Liu et al., 2004) before performing plant digestion. The procedure of DCB extraction was the same as that described in our previous research (Lee et al., 2013). One gram of fresh roots was extracted for 1 h at ambient temperature (20–25 °C) in a 40 mL solution containing 0.03 M sodium citrate (Z99.0%, J.T. Baker) and 0.125 M sodium bicarbonate (Z99.7%, J.T. Baker), with the addition of 0.6 g sodium dithionite powder (Z82%, Sigma-Aldrich), and then the DCB extracts were discarded. The roots were then washed three times with deionized water, removing the residues of DCB extracts. Afterward, the roots without iron plaque and the shoots were oven dried at 70 °C for 72 h, and then ground to a fine powder and stored in a desiccator cabinet. 2.2. Plant digestion and analysis The dried root and shoot samples (0.1 g) were digested separately with concentrated HNO3 (69–70%, J.T. Baker)/H2O2 (30%(w/w) in H2O, Sigma-Aldrich) in heating blocks (Meharg and Rahman,
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2003). The volume of the digests was diluted by deionized water to 50 mL, filtered through a 0.45 μm filter and stored in plastic bottles for subsequent element analysis. The concentrations of Ca, In, Ca, Mg, K, Fe and P in the digests were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES, Optima 8000 DV, Perkin Elmer), and the concentrations of Mn and Zn in the digests were determined by inductively coupled plasma-mass spectrometry (ICP-MS 7700, Agilent Technologies). Total nitrogen (N) content in rice plants was determined by the Kjeldahl method (Bremner and Mulvaney, 1982). 2.3. Data analysis The statistical analysis was carried out using ANOVA (analysis of variance) to test the effect of Ga and In treatment on plant growth (biomass, root length and shoot height) and on the uptake of Ga, In, and essential nutrient elements in the rice seedlings. To test for differences between the treatments, we used the least significant difference (LSD) test at the level of P¼0.05. ANOVA and LSD tests were performed using the SAS 9.2 software package. Data presented in this study are means (n¼3) plus or minus standard deviations (SD).
In the preliminary experiment (high-In treatments), it was found that In treatment inhibited plant growth across the whole range of concentrations tested (0.1–10 mg L 1). Therefore, experiments with lower concentrations of In (low-In treatments) were performed to further understand the toxicity and accumulation of In in rice seedlings under low In conditions (0.04– 2 mg L 1). Table 1 shows the effect of In treatments on the growth of rice seedlings under both high-In and low-In treatments. Significant differences in the root length, shoot height, biomass of root and shoot, and chlorophyll SPAD value of rice seedlings among various In concentrations were found in the One-Way ANOVA analysis, and the values of these growth indices all decreased as In concentration increased, except for the chlorophyll SPAD value under the low-In treatments. The extent of decrease of these growth indices was 9–25%, 9–25%, 37–55%, 22–42% and 13– 29% for root length, shoot height, biomass of root and shoot, and chlorophyll SPAD value, respectively, under the high In treatments. Under the low-In treatments, the extent of decrease of these growth indices was 7–11%, 6–13%, 0.2–16%, and 4–13% for root length, shoot height, biomass of root and shoot, respectively. 3.2. Concentration of Ga and In in rice seedlings
3. Results 3.1. The effect of Ga and In treatment on rice seedling growth Table 1 shows the root length, shoot height, plant biomass, and chlorophyll SPAD value of rice seedlings grown in culture solutions with different Ga treatments. ANOVA analysis indicated that there were significant differences in the root length (P o0.001), shoot height (P o0.01), biomass of root (Po 0.001) and shoot (P o0.001), and chlorophyll SPAD value (Po 0.01) of rice seedlings among the Ga treatments, and these growth indices all increased as Ga concentration was increased in the culture solution. The extent of increase of these growth indices was 10–20%, 2–12%, 2–34%, 1–30% and 3–13% for root length, shoot height, biomass of root and shoot, and chlorophyll SPAD value of rice seedlings, respectively.
Fig. 1 shows the concentration of Ga and In measured in the roots and shoots of rice seedlings grown under different Ga and In treatment conditions. Ga and In concentration in the roots and shoots increased as the Ga and the In concentration in the culture solution was increased, and there were significant differences among the various Ga and In treatments according to the ANOVA and the LSD analysis. The concentration of Ga and In in the control treatments were below the detection limit (N.D.: o10 mg kg 1). The concentration of Ga in the roots and shoots of rice seedlings under different Ga treatments were N.D. 774 and N.D. 74 mg kg 1, respectively. The concentration of In in the roots and shoots of rice seedlings under high-In treatments (root: N.D. 1320 mg kg 1, shoot: N.D. 151 mg kg 1) was higher than that under low-In treatments (root: N.D. 566 mg kg 1, shoot: N.D. 33 mg kg 1). It was found that the Ga and In concentration in the roots was about
Table 1 The root length, shoot height, biomass of root and shoot (dry weight), and chlorophyll SPAD value of rice seedlings grown in culture solutions with different Ga and In treatments.
mg L 1
Root length cm
Shoot height cm
Root biomass mg plant 1
Shoot biomass mg plant 1
Chlorophyll SPAD value
Ga treatments 0 1 3 5 10 15
17.7 70.1 cn 17.2 70.9 c 17.6 71.0c 19.5 7 0.8 b 20.9 7 0.3 a 21.3 7 0.4 a
61.9 7 1.3 d 63.4 73.9 dc 67.17 0.6 abc 69.17 0.7 a 67.9 71.6 ab 64.37 2.7 bcd
51.5 72.2c 49.57 3.5c 52.7 75.3 bc 60.5 73.7 ab 69.17 4.5 a 68.57 8.2 a
282 7 12.0c 284 7 12.7c 3137 27.4b c 3517 5.6 a 366 721.3 a 3367 24.5 ab
31.2 7 1.1c 32.2 71.0 bc 33.9 7 1.2 ab 33.3 7 1.1 ab 34.2 71.1 a 35.2 7 0.7 a
High-In treatments 0 0.1 1 3 5 10
18.3 7 0.3 a 14.7 7 1.8 b 13.7 7 1.1 b 13.8 7 0.8 b 14.17 0.2 b 13.9 7 0.8 b
43.8 70.9 a 39.9 70.7 b 35.9 7 1.2c 36.1 7 1.9c 33.9 7 2.1 cd 32.7 70.6 d
63.4 78.7 a 39.9 717.4 b 33.0 7 5.9 b 36.7 76.1 b 29.4 72.2 b 28.4 71.7 b
1827 15.1 a 1427 25.5 b 1237 15.9 bc 1317 20.9 bc 1057 14.3c 1107 5.3c
30.3 70.7 a 26.4 75.1 ab 22.4 74.6 b 23.2 72.4 b 22.4 74.5 b 21.6 7 1.2 b
Low-In treatments 0 0.04 0.08 0.10 0.15 1 2
17.7 70.1 a 16.6 7 0.8 b 16.6 7 0.4 b 16.3 7 0.9 b 15.7 7 0.1 b 16.0 7 0.2 b 15.8 7 0.2 b
61.9 7 1.3 a 58.2 70.5 b 56.9 70.8 bc 56.17 1.5c 57.0 7 0.3 bc 55.6 71.6 cd 54.17 1.2 d
51.5 72.2 a 51.4 71.8 a 46.17 1.6 b 44.6 7 3.2 b 43.5 73.5 b 43.3 71.2 b 48.37 5.5 ab
282 7 12.0 a 2717 14.8 ab 254 7 5.9 bc 254 7 8.9 bc 249 7 20.8 bc 244 710.8 c 2477 17.0 c
31.2 7 1.1 d 32.9 72.0 dc 34.5 70.8 bc 34.7 70.9 bc 34.6 71.0 bc 36.2 70.4 ab 36.7 70.1 a
n
Different letters indicated the differences in the value among the Ga/In treatments based on the LSD test (P o0.05).
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Fig. 1. The concentrations of Ga and In in (a–c) root and (d–f) shoot of rice seedlings grown in culture solutions with different Ga and In treatments. Data are means 7SD (n ¼3). Different letters above the bars indicate significant difference in the value among the Ga or In treatments based on the LSD test (P o 0.05). The treatment without bar shown on the diagram represents the concentrations of Ga/In in the treatment below the detection limit.
ten times higher than in the shoots. For the low-In treatments, the concentration of In in the shoots was below the detection limit when rice seedlings were grown in culture solutions with In concentration below 0.15 mg L 1 (Fig. 1f). 3.3. The content and distribution of Ga and In in rice seedlings Fig. 2 shows the content (total uptake) and distribution of Ga and In in rice seedlings grown in culture solutions of different Ga and In concentration. The content of Ga and In in plant tissues was calculated as the concentration of Ga and In in plant tissue the
biomass of plant tissue. The results indicate that the content of Ga and In in roots and shoots increased as the concentration in the culture solution increased, and the content in roots was higher than in shoots under all Ga treatment conditions (root: 68–100%, shoot: 0–32%) and all In treatment conditions (root: 70–100%, shoot: 0–31%). No significant difference in the Ga content in the shoots was found between the Ga 10 treatment and the Ga 15 treatment. For high-In treatments, there was no significant difference in In content in roots when the rice seedlings were grown in culture solutions with In concentrations higher than 3 mg L 1, and for low-In treatments, there was no significant difference in In
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Fig. 3 shows the relationship between Ga (In) content in rice plants and the Ga (In) concentration in culture solutions, respectively, which clearly indicates a significantly positive correlation in both cases. The relationship between Ga content in roots and Ga concentration in culture solution was best described by a linear model (R2 ¼0.9953, Po0.001), but the Ga content in shoots was best described by a logarithmic model (R2 ¼ 0.9649, Po0.01). These results indicate that the rice seedling shoot and root accumulation kinetics of Ga were different. For high-In and low-In treatments, the relationship between In content in roots (high-In: R2 ¼9463, Po0.01; low-In: R2 ¼0.9627, Po0.001) and shoots (high-In: R2 ¼ 0.9497, Po0.01) and In concentration in culture solution were best described by logarithmic models, except for the shoot content under low-In treatment (linear model, R2 ¼ 0.9980, Po0.001). These results indicate that the accumulation kinetics of In by the roots and by the shoots under highIn treatments and that by the roots under low-In treatments followed a saturation curve, but the accumulation behavior of In by the shoots under low-In treatments exhibited a linear tendency. 3.4. The effect of Ga and In treatment on accumulation of nutrients in rice seedlings
Fig. 2. The contents and distribution of (a) Ga and (b–c) In in rice seedlings grown in culture solutions with different Ga and In treatments. Different small and capital letters above the bars indicate significant difference in the Ga or In concentrations in root and shoot, respectively among the Ga or In treatments based on the LSD test (P o0.05). Content (μg Ga or In plant 1): Ga or In concentration (μg Ga or In g 1) biomass (g plant 1). The treatment without bar shown on the diagram represents the concentrations of Ga/In in the treatment below the detection limit, thus the contents of Ga/In are regarded as 0.
content both in the roots and in the shoots when the rice seedlings were grown in culture solutions with In concentrations ranging from 0.04 to 0.15 mg L 1. In addition, both for Ga and for In, the translocation factor from roots to shoots (content in shoots/content in roots) decreased as the concentration increased in the culture solution. The average translocation factor of Ga (0.69 70.18) was higher than that of In (0.33 70.10).
Table S1 (Supporting information) shows the content (total uptake) of nutrients in rice seedlings grown in culture solution under different Ga treatments. No significant effect on the accumulation of P, K, Zn, and Mn in roots under different Ga treatments was observed. The content of Mg in roots significantly increased with increased Ga concentration, whereas the content of Ca and Fe in roots significantly decreased. In shoots, the content of N, K, Mg, Ca, Mn significantly increased as Ga concentration increased, and the extent of increase of those nutrients was 5–25%, 7–21%, 2–25%, 3–27% and 12–51%, respectively. The content of P and Fe in shoots significantly decreased as Ga concentration in culture solution increased, but no significant difference in Zn content among different Ga treatments was found. The nutrient content in rice seedlings as effected by In treatment is presented in Table S2 (Supporting Information). Under high-In treatment, no significant difference in the content of Ca in roots and in the content of N and K in shoots was observed among different In treatments, whereas the content of the other nutrients tested significantly decreased as In concentration in the culture solution increased. The extent of decrease of P, Mg, Ca, Fe, Zn, Mn content in shoots was 13–17%, 36–64%, 43–62%, 14–29, 59–77% and 32–58%, respectively. Under low-In treatment, no significant decrease in the content of P, K and Mg was observed in the roots as In concentration increased, and in the shoots no significant decrease in the content of P and K were observed. Mg content in the shoots did decrease significantly with increased concentration of In as did the content of the other nutrients considered, both in the roots and the shoots. The extent of decrease of N, Mg, Ca, Fe, Zn, Mn content in the shoots was 5–13%, 8–36%, 18–39%, 16–49, 10–20% and 7–12%, respectively. In addition, it was found that the percentage of decrease of nutrient content in shoots under high-In treatment was higher than under low-In treatment for all nutrients tested except for Fe. 3.5. Phytotoxicity of In to rice seedlings grown in culture solutions No growth inhibition to rice seedlings grown in Ga-containing culture solution was observed; instead, a significant increase of plant growth was found as the concentration of Ga increased (Table 1). However, under In treatment, a significant decrease in plant growth was observed as the amount of In in culture solution increased (Table 1). Thus, the relationship between plant growth response and In uptake by plant could be used to assess the phytotoxicity of In. Fig. 4 shows the shoot height and total biomass of rice seedlings grown in In-containing cultural solutions as a function of plant (rootþshoot) In content for illustrating the phytotoxicity of In. The threshold of In phytotoxicity is
C.-H. Syu et al. / Ecotoxicology and Environmental Safety 135 (2017) 32–39
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Fig. 3. Relationship between the contents of Ga and In in (a–c) root and (d–f) shoot of rice seedlings and the concentrations of Ga and In in culture solutions.
defined by the content of In in rice seedlings corresponding to 10% growth retardation (PT10). The PT10 values based on shoot height and total biomass for In were 15.4 and 10.6 μg plant 1, respectively. The dose-response curves for In treatment, shown in Fig. 5, indicates a significant reduction in shoot height as In concentration increased. The relationship between shoot height and In concentration was well described by a logarithmic model (R2 ¼0.8354, Po0.001). The EC10 value (effective concentration resulting in 10% growth inhibition) calculated by the dose-response curve of In treatment was 0.17 mg L 1. 4. Discussion In this study, we found differences between Ga and In with regard to their phytotoxicity, translocation, and accumulation in rice
plants, despite their being classified into the same group (IIIA) of the periodic table. Analysis of the various growth indices showed that the addition of Ga in culture solution enhanced the growth of rice seedlings grown in culture solution with Ga concentrations from 0 to 15 mg L 1 (Table 1). The significant increase in nutrient content (N, K, Mg, Ca, Mn) in shoots after Ga treatment (Table S1, Supporting information) might be one reason for the enhancement of plant growth associated with Ga treatment. These results differ from reports on the effect of Ga treatment in previous studies. Yu et al. (2015) reported a reduction of relative growth rate, transpiration rate and water use efficiency of rice seedlings while the Ga concentrations in the culture solution studied were higher 2.14, 12.83 and 4.28 mg L 1, respectively (exposure periods were 2 days), and found that the inhibition rate increased with Ga concentration.
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Fig. 4. Phytotoxicity thresholds (PT10) defined as the contents of In in rice seedling corresponding to 10% growth inhibition, respectively, for shoot height (a) and total biomass (b) of rice seedlings.
Fig. 5. Dose-response relation model as a function of the concentration of In for the relative shoot height of rice seedlings grown in culture solutions under In treatments.
The results of Yu and Zhang (2015) also indicated that over-accumulation of Ga in plants tissue caused the formation of DNA-protein cross-links (DPCs) in roots, which led to cell death and growth inhibition of rice seedlings (Ga treatments: 1.06–15.63 mg L 1, 4-d exposure period). This discrepant finding regarding the effect of Ga on rice plants may be attributed to differences in the Ga exposure time and rice cultivars considered. Given that we observed that the extent of increase of shoot height and biomass of root and shoot diminished when Ga concentration exceeded 10 mg L 1, further study is needed to investigate whether phytotoxicity to rice seedlings of the cultivar we used would occur at Ga concentrations higher than 15 mg L 1. Kopittke et al. (2009) reported that the average elongation rate of cowpea root was reduced by 90% when
grown in culture solution of 0.59 μM (0.041 mg Ga L 1) Ga concentration. This Ga concentration is much lower than those used in the present study, which indicates that cowpeas are more sensitive to Ga phytotoxicity than paddy rice. The physiological mechanisms resulting in the positive effect on the plant growth and nutrients accumulation of rice seedlings under Ga treatments were still unclear. It needs further investigation. The growth responses of rice seedlings under In treatments were different from those under Ga treatments, which resulted in a significant reduction in all the growth indices studied, namely root length, shoot height, and plant biomass (Table 1). Obvious toxicity symptoms of chlorosis appeared on the leaf of rice shoots grown under high-In treatment conditions. In addition, the nutrient content of P, Mg, Ca, Fe, Zn, Mn measured in shoots significantly decreased under high-In treatment (Table S2, Supporting information), which may have resulted in the observed growth inhibition of the rice seedlings. Whether this reduction in nutrient uptake by the rice seedlings resulted from In toxicity or from competitive uptake between In and nutrients merits further investigation. For the lowIn treatments, although the extent of decrease of the growth indices was lower than that of the high-In treatments, growth inhibition and decrease of nutrients in the rice seedlings still occurred at In concentrations higher than 0.08 mg L 1. Kopittke et al. (2008) suggested that the toxicity to plants of trace elements, including In, Ga, Al, Cu, Gd, Hg, La, Ru and Sc, is due to their strong binding to the cell walls of roots, which leads to increased rigidity of the cell walls located in the zone of elongation, thereby reducing root growth and forming cell ruptures. Therefore, in this study, it predicted that the rice root suffered from In toxicity under In treatments, and induced the nutritional imbalances and reduction of nutrients (P, Mg, Ca, Fe, Zn, Mn) uptake and accumulated in rice seedlings (Table S2, Supporting information), thus leading to the plant growth inhibition (Table 1). Mendonca et al. (2003) indicated that the Al exposure led to reduce in K, Mg, Ca, and P contents and uptake in rice plants, which toxic mechanism was similar with In toxicity of this study. Fig. 5 shows the dose-response curves obtained gave an EC10 value of 0.17 mg In L 1. The results of Kopittke et al. (2009) reported that the average elongation rate of cowpea roots was reduced by 50% when grown in a culture solution of 0.72 μM In concentration (0.083 mg In L 1). These results suggest that both paddy rice and cowpea are In-sensitive plants. The concentrations of Ga and In measured in the rice seedling roots were about 10-fold higher than in the shoots, and the content (total uptake) and distribution of Ga and In in the roots were also higher than in the shoots, suggesting that the roots were the dominant sink of Ga and In. Yu et al. (2015) and Yu and Zhang (2015) also reported that the concentration of Ga accumulated in roots of rice seedlings was much higher than in shoots. Wheeler and Power (1995) indicated that the average ratio of the Ga concentration in roots to those in tops of wheat was 12, a ratio close to the one for rice observed in the present study. Besides accumulating in roots, trivalent cations may be strongly bound by the cell walls of roots, which contain ligands such as aldehyde, carboxyl, and hydroxyl in the lignin and cellulose, preventing those cations from reaching beyond the roots to other parts of the plant (Reid et al., 1996; Ren et al., 2014). The distribution of In in different parts of plants has rarely been discussed in previous studies. We also found that the translocation factor from roots to shoots of Ga (0.69 7 0.18) was about 2-fold higher than that of In (0.33 7 0.10), indicating that translocation of Ga in rice plants is easier than translocation of In, which might be why Ga was less phytotoxic to rice seedlings than In was. The differences in the uptake and translocation in rice plants between Ga and In were further supported by the results of the accumulation kinetics of Ga and In in roots and shoots under Ga and In treatments (Fig. 3). A linear increase in Ga accumulation in roots with increased Ga concentration
C.-H. Syu et al. / Ecotoxicology and Environmental Safety 135 (2017) 32–39
in the culture solution suggests that the mechanism of uptake of Ga was not adversely affected by Ga treatment (Gao15 mg L 1, Fig. 3a), and the content of Ga that accumulated in shoots did so at a constant rate which may be determined by the rice plant's capability to translocate Ga (Fig. 3d). However, the content of In that accumulated in roots and shoots reached a plateau, which may have been a result of In phytotoxicity (Fig. 3b, c, e). The decrease of nutrient uptake by rice seedlings may also have resulted from In phytotoxicity to the roots, which further reduced translocation of those nutrients from root to shoot (Table S2, Supporting information). Moreover, under low-In treatments, a linear increase in In content in the shoots with In concentration in the culture solution was observed (Fig. 3f), which indicates that any In phytotoxicity that might have occurred was insufficient to noticeably affect the translocation of In from roots to shoots. Fig. 2c indicated that In content in the shoots was below the detection limit under 0.04– 0.15 mg In L 1 treatment, which may have resulted because most of the In were adsorbed by the root cell walls, reducing translocation of the element in the rice seedlings (Reid et al., 1996). In addition, we also found that there was negatively correlation between plant growth (shoot height and total biomass of rice seedlings) and In contents in rice seedlings, and the PT10 values (threshold value for 10% reduction in plant growth) based on shoot height and total biomass for In were 15.4 and 10.6 μg plant 1, respectively (Fig. 4).
5. Conclusion In summary, the results of this study indicated that qualitative differences exist between Ga and In with regard to their phytotoxicity, uptake, and translocation in rice seedlings. Treatment with Ga exerted a beneficial effect on growth in the rice seedlings, perhaps by increasing nutrient uptake (N, K, Mg, Ca, Mn) in plant tissue. In contrast, In treatment led to growth inhibition of rice seedlings, which may be attributed to a reduction in nutrient uptake (P, Mg, Ca, Fe, Zn, Mn) by plant tissue resulting from In phytotoxicity to roots. The EC10 value we observed for In treatment was 0.17 mg L 1, based on the dose-response curve. We found that the roots were the dominant sink of Ga and In in rice seedlings, and the concentrations of both elements in were about 10-fold higher in the roots of rice seedlings than in their shoots. Moreover, we found that the capability of translocation of Ga from roots to shoots were higher than In. From this study, we understand the effects of Ga and In on the growth of paddy rice grown in culture solutions. However, in paddy soils, the toxicity, uptake, and accumulation of Ga and In in rice plants may also be affected by the soil characteristics, thus requiring further investigation in a subsequent study.
Acknowledgements This work was supported by the Ministry of Science and Technology, Executive Yuan, Taiwan (grant no. MOST 104-2313-B002-015-MY3) is sincerely appreciated.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2016.09.016.
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Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
The growth and uptake of Ga and In of rice (Oryza sative L.) seedlings as affected by Ga and In concentrations in hydroponic cultures Chien-Hui Syu a,1, Po-Hsuan Chien b,1, Chia-Chen Huang b, Pei-Yu Jiang b, Kai-Wei Juang c, Dar-Yuan Lee b,n a
Division of Agricultural Chemistry, Taiwan Agricultural Research Institute, No.189, Zhongzheng Rd., Wufeng Dist., Taichung 41362, Taiwan Department of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan c Department of Agronomy, National Chiayi University, No.300 Syuefu Rd., Chiayi 60004, Taiwan b
art ic l e i nf o
a b s t r a c t
Article history: Received 11 July 2016 Received in revised form 9 September 2016 Accepted 19 September 2016
Limited information is available on the effects of gallium (Ga) and indium (In) on the growth of paddy rice. The Ga and In are emerging contaminants and widely used in high-tech industries nowadays. Understanding the toxicity and accumulation of Ga and In by rice plants is important for reducing the effect on rice production and exposure risk to human by rice consumption. Therefore, this study investigates the effect of Ga and In on the growth of rice seedlings and examines the accumulation and distribution of those elements in plant tissues. Hydroponic cultures were conducted in phytotron glasshouse with controlled temperature and relative humidity conditions, and the rice seedlings were treated with different levels of Ga and In in the nutrient solutions. The growth index and the concentrations of Ga and In in roots and shoots of rice seedlings were measured after harvesting. A significant increase in growth index with increasing Ga concentrations in culture solutions (o10 mg Ga L 1) was observed. In addition, the uptake of N, K, Mg, Ca, Mn by rice plants was also enhanced by Ga. However, the growth inhibition were observed while the In concentrations higher than 0.08 mg L 1, and the nutrients accumulated in rice plants were also significant decreased after In treatments. Based on the dose-response curve, we observed that the EC10 (effective concentration resulting in 10% growth inhibition) value for In treatment was 0.17 mg L 1. The results of plant analysis indicated that the roots were the dominant sink of Ga and In in rice seedlings, and it was also found that the capability of translocation of Ga from roots to shoots were higher than In. In addition, it was also found that the PT10 (threshold concentration of phytotoxicity resulting in 10% growth retardation) values based on shoot height and total biomass for In were 15.4 and 10.6 μg plant 1, respectively. The beneficial effects on the plant growth of rice seedlings were found by the addition of Ga in culture solutions. In contrast, the In treatments led to growth inhibition of rice seedlings. There were differences in the phytotoxicity, uptake, and translocation of the two emerging contaminants in rice seedlings. & 2016 Elsevier Inc. All rights reserved.
Keywords: Gallium Indium Emerging contaminants Paddy rice Hydroponic culture
1. Introduction Gallium (Ga) and indium (In) are regarded as toxic substances to humans based on previous reports (Fowler et al.. 1993; Ivanoff et al.. 2012; Kabata-Pendias and Mukherjee, 2007; Tanaka 2004). In general, Ga and In are produced as byproducts in the production of Al (bauxite), Pb (galena) and Zn (sphalerite), and these two elements are widely used in semiconductor manufacturing and the electro-optical and medical industries (Alfantazi and Moskalyk, 2003; Kabata-Pendias and Mukherjee, 2007; Kabata-Pendias 2011; Yu and Liao, 2011).
n
Corresponding author. E-mail address: [email protected] (D.-Y. Lee). 1 Dr. Chien-Hui Syu and Mr. Po-Hsuan Chien are equal contributors to this paper. http://dx.doi.org/10.1016/j.ecoenv.2016.09.016 0147-6513/& 2016 Elsevier Inc. All rights reserved.
Nowadays, due to progressively increasing usage of Ga and In, wastewater containing them may be discharged into farmland soils by the irrigation system, further raising the risk of human exposure to Ga and In through the food chain. Chen (2006) reported the high concentrations of Ga (up to 41 μg L 1) and In (up to 20 μg L 1) in groundwater contaminated by wastewater from semiconductor manufacturing area of Taiwan. To date, the information about the contents of Ga and In in soils contaminated by semiconductor manufacturing is limited. However, there were some studies indicated that the contents of Ga and In in soils near area of automobile and ZnPb smelting industries were 10.89–22.46 mg kg 1 and 0.11– 1.92 mg kg 1, respectively (Asami et al., 1990; Yu et al., 2015). Nevertheless, studies about the effects of Ga and In on the growth of edible crops are still scarce. Therefore, understanding the interaction between Ga and In and edible crops is necessary.
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Gallium and indium are grouped among the IIIA group of elements in the periodic table, and the content of Ga and In in worldwide soils ranges from 3 to 70 and 0.11–0.25 mg kg 1 respectively (Kabata-Pendias, 2011). In general, the oxidation states of Ga and In are þ3, but oxidation states of þ 1 and þ2 may also occur under anaerobic conditions. At 25 °C, the soluble species of Ga and In present in acidic conditions are Ga3 þ , Ga(OH)2 þ , Ga(OH)2 þ , and In3 þ , In(OH)2 þ , In(OH)2 þ ; the soluble species present in alkaline conditions are Ga(OH)4 and In(OH)4 , and the insoluble species Ga (OH)3 and In(OH)3 are present in conditions of pH 4–6 and pH 5–9 (Kabata-Pendias, 2011; Wood and Samson 2006). These solubility characteristics are similar to those of Al, which suggests that Ga and In are amphoteric elements. Orians and Bruland (1988) showed that the geochemistry and aquatic chemistry properties of Ga are similar to Al, but that the reactivity of Ga is less than that of Al. Rice is a dietary food for about half of the world's population, and for over 90% of the population in Asia (Meharg et al.. 2009). Because paddy fields may suffer from Ga- and In-associated wastewater contamination, study of the accumulation of Ga and In in rice plants and the effect of these two emerging contaminants on rice plant growth is merited. The chemical properties of Ga and In are similar to those of Al, and several studies have investigated Al toxicity and tolerance in rice plants (Roy and Bhadra, 2014; Silva 2012; Tanaka and Navasero, 1966). However, little information about the effects of Ga and In on the growth of rice plants exists to date. Yu et al.. (2015) reported an associated reduction in relative growth rate, transpiration rate and water use efficiency of rice seedlings grown for 2 days in solution culture of increasing Ga concentration. Yu and Zhang (2015) found that over-accumulation of Ga in plant tissue resulted in cell death and growth inhibition in rice seedlings. Some studies have also investigated the toxic effect and accumulation of Ga and In in other plant species (Berg and Steinnes, 1997; Fergusson 1990; Kopittke et al., 2009; Shacklette et al., 1978). Shacklette et al. (1978) reported that the concentration of Ga in a variety of native species ranged from 3 to 30 mg kg 1 in the United States. Berg and Steinnes (1997) indicated that atmospheric deposition may result in the elevation of Ga content by up to 16 mg kg 1 in moss growing wild in Norway. Fergusson (1990) found concentrations of In ranging from 80 to 300 μg/kg FW in beets, 0.64–1.8 μg/kg FW in leaves of fruit trees, and 30–710 μg/kg FW in vegetables. Kopittke et al. (2009) reported that soluble Ga and In reduced cowpea root growth and caused cell rupture in hydroponic experiments. To the best of our knowledge, the uptake and accumulation of Ga and In in rice plants and their effect on rice growth is still unclear. Therefore, our objective in this study was to investigate the effect of Ga and In on rice plant growth and how uptake of these two elements affects rice seedlings grown in solution cultures treated with different concentrations of Ga and In. In particular, Ga and In may impact the uptake of nutrients through competitive uptake by roots or through phytotoxicity; thus we also investigated the effect of Ga and In treatments on the uptake of nutrients by rice plants.
2. Material and methods 2.1. Hydroponic cultures The hydroponic cultures were performed in a phytotron with a controlled temperature (25/20 °C, day/night) and relative humidity (70–95%) under sunlight. The cultivar of paddy rice (Oryza sative L., cv Taikeng 9) was used in this study because it is commonly planted in Taiwan and considered to be of high quality. Rice seeds were sterilized in a solution containing 1% sodium hydrochloride solution and one drop of Tween 20 for 30 min, and then washed with distilled water for 30 min. The seeds were then germinated in Petri dishes containing tissue paper under moist conditions at 37 °C for
33
2 days. After germination, thirty seedlings were then transferred to an iron mesh set on the surface of the culture solution, contained in a 0.6-L beaker. Seedlings were then raised in half-strength modified Kimura B nutrient solution (pH was adjusted to 4.8–5.0 and the solution was renewed every three days) for 14 days until they reached the three-leaf age. Afterward, the solution was replaced with full-strength nutrient solution and treated with the indicated amount of Ga or In stock solution. The stock solutions of Ga and In were prepared using GaCl3 (99.999%, ultra dry, Alfa Aesar) and InCl3 (99.999%, anhydrous, Alfa Aesar) respectively. The Ga treatment concentrations were 0, 1, 3, 5, 10, 15 mg L 1, and the exposure time was 40 days (growth period: Nov.–Dec., 2014) The In treatments were separated into two parts: (a) For the high-In treatment (preliminary experiment), the In treatment concentrations were 0, 0.1, 1, 3, 5, 10 mg L 1, and the exposure time was 25 days (growth period: Jul.–Aug., 2014); (b) for the low-In treatment, the In treatment concentrations were 0, 0.04, 0.08, 0.1, 0.15, 1, 2 mg L 1, and the exposure time was 40 days (growth period: Nov.–Dec., 2014). Despite the concentrations of Ga and In in wastewater higher than 10 mg L 1 were very rare, the concentration ranges of Ga and In used in this study were intended to clarify the toxicity concentrations to rice seedlings such as EC10 (effective concentration resulting in 10% growth inhibition) and PT10 (threshold concentration of phytotoxicity resulting in 10% growth retardation) values. In order to avoid Ga and In precipitation, the culture solutions were made just before use and renewed every day. We verified that no significant changes in Ga and In concentrations in the culture solutions had occurred at 24 h. Three replicates (pots) for each of the Ga and In treatments. The half-strength modified Kimura B nutrient solution was used in this study, which contains the following compositions: 0.18 mM (NH4)2SO4, 0.09 mM KNO3, 0.27 mM MgSO4‧7H2O, 0.09 mM KH2PO4, 30.6 μM Fe-citrate, 183 μM Ca(NO3)2‧4H20, 25.1 μM H3BO3, 2.01 μM MnSO4‧4H2O, 2.02 μM ZnSO4‧7H2O, 1.19 μM CuSO4‧5H2O and 0.49 μM MoO3. The concentrations of micro-elements (B, Mn, Zn, Cu and Mo) in full-strength modified nutrient solution were identical with the half-strength modified Kimura B nutrient solution, and the concentrations of other elements in full-strength modified nutrient solution were two folds higher than half-strength modified Kimura B nutrient solution. After harvesting, the rice seedlings were separated into root and shoot and rinsed first with tap water and then with deionized water. The biomass and lengths of each root and shoot were measured. In addition, their chlorophyll content was also measured with a chlorophyll meter (SPAD-502, Spectrum Technologies). Briefly, 10 plants per pot were selected and the chlorophyll meter was used to measure three points on the least expanded leaf of each plant (the interval between two points was 5 cm). Iron plaque on the roots was removed using modified cold DCB (dithionite-citratebicarbonate) solution (Liu et al., 2004) before performing plant digestion. The procedure of DCB extraction was the same as that described in our previous research (Lee et al., 2013). One gram of fresh roots was extracted for 1 h at ambient temperature (20–25 °C) in a 40 mL solution containing 0.03 M sodium citrate (Z99.0%, J.T. Baker) and 0.125 M sodium bicarbonate (Z99.7%, J.T. Baker), with the addition of 0.6 g sodium dithionite powder (Z82%, Sigma-Aldrich), and then the DCB extracts were discarded. The roots were then washed three times with deionized water, removing the residues of DCB extracts. Afterward, the roots without iron plaque and the shoots were oven dried at 70 °C for 72 h, and then ground to a fine powder and stored in a desiccator cabinet. 2.2. Plant digestion and analysis The dried root and shoot samples (0.1 g) were digested separately with concentrated HNO3 (69–70%, J.T. Baker)/H2O2 (30%(w/w) in H2O, Sigma-Aldrich) in heating blocks (Meharg and Rahman,
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C.-H. Syu et al. / Ecotoxicology and Environmental Safety 135 (2017) 32–39
2003). The volume of the digests was diluted by deionized water to 50 mL, filtered through a 0.45 μm filter and stored in plastic bottles for subsequent element analysis. The concentrations of Ca, In, Ca, Mg, K, Fe and P in the digests were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES, Optima 8000 DV, Perkin Elmer), and the concentrations of Mn and Zn in the digests were determined by inductively coupled plasma-mass spectrometry (ICP-MS 7700, Agilent Technologies). Total nitrogen (N) content in rice plants was determined by the Kjeldahl method (Bremner and Mulvaney, 1982). 2.3. Data analysis The statistical analysis was carried out using ANOVA (analysis of variance) to test the effect of Ga and In treatment on plant growth (biomass, root length and shoot height) and on the uptake of Ga, In, and essential nutrient elements in the rice seedlings. To test for differences between the treatments, we used the least significant difference (LSD) test at the level of P¼0.05. ANOVA and LSD tests were performed using the SAS 9.2 software package. Data presented in this study are means (n¼3) plus or minus standard deviations (SD).
In the preliminary experiment (high-In treatments), it was found that In treatment inhibited plant growth across the whole range of concentrations tested (0.1–10 mg L 1). Therefore, experiments with lower concentrations of In (low-In treatments) were performed to further understand the toxicity and accumulation of In in rice seedlings under low In conditions (0.04– 2 mg L 1). Table 1 shows the effect of In treatments on the growth of rice seedlings under both high-In and low-In treatments. Significant differences in the root length, shoot height, biomass of root and shoot, and chlorophyll SPAD value of rice seedlings among various In concentrations were found in the One-Way ANOVA analysis, and the values of these growth indices all decreased as In concentration increased, except for the chlorophyll SPAD value under the low-In treatments. The extent of decrease of these growth indices was 9–25%, 9–25%, 37–55%, 22–42% and 13– 29% for root length, shoot height, biomass of root and shoot, and chlorophyll SPAD value, respectively, under the high In treatments. Under the low-In treatments, the extent of decrease of these growth indices was 7–11%, 6–13%, 0.2–16%, and 4–13% for root length, shoot height, biomass of root and shoot, respectively. 3.2. Concentration of Ga and In in rice seedlings
3. Results 3.1. The effect of Ga and In treatment on rice seedling growth Table 1 shows the root length, shoot height, plant biomass, and chlorophyll SPAD value of rice seedlings grown in culture solutions with different Ga treatments. ANOVA analysis indicated that there were significant differences in the root length (P o0.001), shoot height (P o0.01), biomass of root (Po 0.001) and shoot (P o0.001), and chlorophyll SPAD value (Po 0.01) of rice seedlings among the Ga treatments, and these growth indices all increased as Ga concentration was increased in the culture solution. The extent of increase of these growth indices was 10–20%, 2–12%, 2–34%, 1–30% and 3–13% for root length, shoot height, biomass of root and shoot, and chlorophyll SPAD value of rice seedlings, respectively.
Fig. 1 shows the concentration of Ga and In measured in the roots and shoots of rice seedlings grown under different Ga and In treatment conditions. Ga and In concentration in the roots and shoots increased as the Ga and the In concentration in the culture solution was increased, and there were significant differences among the various Ga and In treatments according to the ANOVA and the LSD analysis. The concentration of Ga and In in the control treatments were below the detection limit (N.D.: o10 mg kg 1). The concentration of Ga in the roots and shoots of rice seedlings under different Ga treatments were N.D. 774 and N.D. 74 mg kg 1, respectively. The concentration of In in the roots and shoots of rice seedlings under high-In treatments (root: N.D. 1320 mg kg 1, shoot: N.D. 151 mg kg 1) was higher than that under low-In treatments (root: N.D. 566 mg kg 1, shoot: N.D. 33 mg kg 1). It was found that the Ga and In concentration in the roots was about
Table 1 The root length, shoot height, biomass of root and shoot (dry weight), and chlorophyll SPAD value of rice seedlings grown in culture solutions with different Ga and In treatments.
mg L 1
Root length cm
Shoot height cm
Root biomass mg plant 1
Shoot biomass mg plant 1
Chlorophyll SPAD value
Ga treatments 0 1 3 5 10 15
17.7 70.1 cn 17.2 70.9 c 17.6 71.0c 19.5 7 0.8 b 20.9 7 0.3 a 21.3 7 0.4 a
61.9 7 1.3 d 63.4 73.9 dc 67.17 0.6 abc 69.17 0.7 a 67.9 71.6 ab 64.37 2.7 bcd
51.5 72.2c 49.57 3.5c 52.7 75.3 bc 60.5 73.7 ab 69.17 4.5 a 68.57 8.2 a
282 7 12.0c 284 7 12.7c 3137 27.4b c 3517 5.6 a 366 721.3 a 3367 24.5 ab
31.2 7 1.1c 32.2 71.0 bc 33.9 7 1.2 ab 33.3 7 1.1 ab 34.2 71.1 a 35.2 7 0.7 a
High-In treatments 0 0.1 1 3 5 10
18.3 7 0.3 a 14.7 7 1.8 b 13.7 7 1.1 b 13.8 7 0.8 b 14.17 0.2 b 13.9 7 0.8 b
43.8 70.9 a 39.9 70.7 b 35.9 7 1.2c 36.1 7 1.9c 33.9 7 2.1 cd 32.7 70.6 d
63.4 78.7 a 39.9 717.4 b 33.0 7 5.9 b 36.7 76.1 b 29.4 72.2 b 28.4 71.7 b
1827 15.1 a 1427 25.5 b 1237 15.9 bc 1317 20.9 bc 1057 14.3c 1107 5.3c
30.3 70.7 a 26.4 75.1 ab 22.4 74.6 b 23.2 72.4 b 22.4 74.5 b 21.6 7 1.2 b
Low-In treatments 0 0.04 0.08 0.10 0.15 1 2
17.7 70.1 a 16.6 7 0.8 b 16.6 7 0.4 b 16.3 7 0.9 b 15.7 7 0.1 b 16.0 7 0.2 b 15.8 7 0.2 b
61.9 7 1.3 a 58.2 70.5 b 56.9 70.8 bc 56.17 1.5c 57.0 7 0.3 bc 55.6 71.6 cd 54.17 1.2 d
51.5 72.2 a 51.4 71.8 a 46.17 1.6 b 44.6 7 3.2 b 43.5 73.5 b 43.3 71.2 b 48.37 5.5 ab
282 7 12.0 a 2717 14.8 ab 254 7 5.9 bc 254 7 8.9 bc 249 7 20.8 bc 244 710.8 c 2477 17.0 c
31.2 7 1.1 d 32.9 72.0 dc 34.5 70.8 bc 34.7 70.9 bc 34.6 71.0 bc 36.2 70.4 ab 36.7 70.1 a
n
Different letters indicated the differences in the value among the Ga/In treatments based on the LSD test (P o0.05).
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Fig. 1. The concentrations of Ga and In in (a–c) root and (d–f) shoot of rice seedlings grown in culture solutions with different Ga and In treatments. Data are means 7SD (n ¼3). Different letters above the bars indicate significant difference in the value among the Ga or In treatments based on the LSD test (P o 0.05). The treatment without bar shown on the diagram represents the concentrations of Ga/In in the treatment below the detection limit.
ten times higher than in the shoots. For the low-In treatments, the concentration of In in the shoots was below the detection limit when rice seedlings were grown in culture solutions with In concentration below 0.15 mg L 1 (Fig. 1f). 3.3. The content and distribution of Ga and In in rice seedlings Fig. 2 shows the content (total uptake) and distribution of Ga and In in rice seedlings grown in culture solutions of different Ga and In concentration. The content of Ga and In in plant tissues was calculated as the concentration of Ga and In in plant tissue the
biomass of plant tissue. The results indicate that the content of Ga and In in roots and shoots increased as the concentration in the culture solution increased, and the content in roots was higher than in shoots under all Ga treatment conditions (root: 68–100%, shoot: 0–32%) and all In treatment conditions (root: 70–100%, shoot: 0–31%). No significant difference in the Ga content in the shoots was found between the Ga 10 treatment and the Ga 15 treatment. For high-In treatments, there was no significant difference in In content in roots when the rice seedlings were grown in culture solutions with In concentrations higher than 3 mg L 1, and for low-In treatments, there was no significant difference in In
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Fig. 3 shows the relationship between Ga (In) content in rice plants and the Ga (In) concentration in culture solutions, respectively, which clearly indicates a significantly positive correlation in both cases. The relationship between Ga content in roots and Ga concentration in culture solution was best described by a linear model (R2 ¼0.9953, Po0.001), but the Ga content in shoots was best described by a logarithmic model (R2 ¼ 0.9649, Po0.01). These results indicate that the rice seedling shoot and root accumulation kinetics of Ga were different. For high-In and low-In treatments, the relationship between In content in roots (high-In: R2 ¼9463, Po0.01; low-In: R2 ¼0.9627, Po0.001) and shoots (high-In: R2 ¼ 0.9497, Po0.01) and In concentration in culture solution were best described by logarithmic models, except for the shoot content under low-In treatment (linear model, R2 ¼ 0.9980, Po0.001). These results indicate that the accumulation kinetics of In by the roots and by the shoots under highIn treatments and that by the roots under low-In treatments followed a saturation curve, but the accumulation behavior of In by the shoots under low-In treatments exhibited a linear tendency. 3.4. The effect of Ga and In treatment on accumulation of nutrients in rice seedlings
Fig. 2. The contents and distribution of (a) Ga and (b–c) In in rice seedlings grown in culture solutions with different Ga and In treatments. Different small and capital letters above the bars indicate significant difference in the Ga or In concentrations in root and shoot, respectively among the Ga or In treatments based on the LSD test (P o0.05). Content (μg Ga or In plant 1): Ga or In concentration (μg Ga or In g 1) biomass (g plant 1). The treatment without bar shown on the diagram represents the concentrations of Ga/In in the treatment below the detection limit, thus the contents of Ga/In are regarded as 0.
content both in the roots and in the shoots when the rice seedlings were grown in culture solutions with In concentrations ranging from 0.04 to 0.15 mg L 1. In addition, both for Ga and for In, the translocation factor from roots to shoots (content in shoots/content in roots) decreased as the concentration increased in the culture solution. The average translocation factor of Ga (0.69 70.18) was higher than that of In (0.33 70.10).
Table S1 (Supporting information) shows the content (total uptake) of nutrients in rice seedlings grown in culture solution under different Ga treatments. No significant effect on the accumulation of P, K, Zn, and Mn in roots under different Ga treatments was observed. The content of Mg in roots significantly increased with increased Ga concentration, whereas the content of Ca and Fe in roots significantly decreased. In shoots, the content of N, K, Mg, Ca, Mn significantly increased as Ga concentration increased, and the extent of increase of those nutrients was 5–25%, 7–21%, 2–25%, 3–27% and 12–51%, respectively. The content of P and Fe in shoots significantly decreased as Ga concentration in culture solution increased, but no significant difference in Zn content among different Ga treatments was found. The nutrient content in rice seedlings as effected by In treatment is presented in Table S2 (Supporting Information). Under high-In treatment, no significant difference in the content of Ca in roots and in the content of N and K in shoots was observed among different In treatments, whereas the content of the other nutrients tested significantly decreased as In concentration in the culture solution increased. The extent of decrease of P, Mg, Ca, Fe, Zn, Mn content in shoots was 13–17%, 36–64%, 43–62%, 14–29, 59–77% and 32–58%, respectively. Under low-In treatment, no significant decrease in the content of P, K and Mg was observed in the roots as In concentration increased, and in the shoots no significant decrease in the content of P and K were observed. Mg content in the shoots did decrease significantly with increased concentration of In as did the content of the other nutrients considered, both in the roots and the shoots. The extent of decrease of N, Mg, Ca, Fe, Zn, Mn content in the shoots was 5–13%, 8–36%, 18–39%, 16–49, 10–20% and 7–12%, respectively. In addition, it was found that the percentage of decrease of nutrient content in shoots under high-In treatment was higher than under low-In treatment for all nutrients tested except for Fe. 3.5. Phytotoxicity of In to rice seedlings grown in culture solutions No growth inhibition to rice seedlings grown in Ga-containing culture solution was observed; instead, a significant increase of plant growth was found as the concentration of Ga increased (Table 1). However, under In treatment, a significant decrease in plant growth was observed as the amount of In in culture solution increased (Table 1). Thus, the relationship between plant growth response and In uptake by plant could be used to assess the phytotoxicity of In. Fig. 4 shows the shoot height and total biomass of rice seedlings grown in In-containing cultural solutions as a function of plant (rootþshoot) In content for illustrating the phytotoxicity of In. The threshold of In phytotoxicity is
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Fig. 3. Relationship between the contents of Ga and In in (a–c) root and (d–f) shoot of rice seedlings and the concentrations of Ga and In in culture solutions.
defined by the content of In in rice seedlings corresponding to 10% growth retardation (PT10). The PT10 values based on shoot height and total biomass for In were 15.4 and 10.6 μg plant 1, respectively. The dose-response curves for In treatment, shown in Fig. 5, indicates a significant reduction in shoot height as In concentration increased. The relationship between shoot height and In concentration was well described by a logarithmic model (R2 ¼0.8354, Po0.001). The EC10 value (effective concentration resulting in 10% growth inhibition) calculated by the dose-response curve of In treatment was 0.17 mg L 1. 4. Discussion In this study, we found differences between Ga and In with regard to their phytotoxicity, translocation, and accumulation in rice
plants, despite their being classified into the same group (IIIA) of the periodic table. Analysis of the various growth indices showed that the addition of Ga in culture solution enhanced the growth of rice seedlings grown in culture solution with Ga concentrations from 0 to 15 mg L 1 (Table 1). The significant increase in nutrient content (N, K, Mg, Ca, Mn) in shoots after Ga treatment (Table S1, Supporting information) might be one reason for the enhancement of plant growth associated with Ga treatment. These results differ from reports on the effect of Ga treatment in previous studies. Yu et al. (2015) reported a reduction of relative growth rate, transpiration rate and water use efficiency of rice seedlings while the Ga concentrations in the culture solution studied were higher 2.14, 12.83 and 4.28 mg L 1, respectively (exposure periods were 2 days), and found that the inhibition rate increased with Ga concentration.
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Fig. 4. Phytotoxicity thresholds (PT10) defined as the contents of In in rice seedling corresponding to 10% growth inhibition, respectively, for shoot height (a) and total biomass (b) of rice seedlings.
Fig. 5. Dose-response relation model as a function of the concentration of In for the relative shoot height of rice seedlings grown in culture solutions under In treatments.
The results of Yu and Zhang (2015) also indicated that over-accumulation of Ga in plants tissue caused the formation of DNA-protein cross-links (DPCs) in roots, which led to cell death and growth inhibition of rice seedlings (Ga treatments: 1.06–15.63 mg L 1, 4-d exposure period). This discrepant finding regarding the effect of Ga on rice plants may be attributed to differences in the Ga exposure time and rice cultivars considered. Given that we observed that the extent of increase of shoot height and biomass of root and shoot diminished when Ga concentration exceeded 10 mg L 1, further study is needed to investigate whether phytotoxicity to rice seedlings of the cultivar we used would occur at Ga concentrations higher than 15 mg L 1. Kopittke et al. (2009) reported that the average elongation rate of cowpea root was reduced by 90% when
grown in culture solution of 0.59 μM (0.041 mg Ga L 1) Ga concentration. This Ga concentration is much lower than those used in the present study, which indicates that cowpeas are more sensitive to Ga phytotoxicity than paddy rice. The physiological mechanisms resulting in the positive effect on the plant growth and nutrients accumulation of rice seedlings under Ga treatments were still unclear. It needs further investigation. The growth responses of rice seedlings under In treatments were different from those under Ga treatments, which resulted in a significant reduction in all the growth indices studied, namely root length, shoot height, and plant biomass (Table 1). Obvious toxicity symptoms of chlorosis appeared on the leaf of rice shoots grown under high-In treatment conditions. In addition, the nutrient content of P, Mg, Ca, Fe, Zn, Mn measured in shoots significantly decreased under high-In treatment (Table S2, Supporting information), which may have resulted in the observed growth inhibition of the rice seedlings. Whether this reduction in nutrient uptake by the rice seedlings resulted from In toxicity or from competitive uptake between In and nutrients merits further investigation. For the lowIn treatments, although the extent of decrease of the growth indices was lower than that of the high-In treatments, growth inhibition and decrease of nutrients in the rice seedlings still occurred at In concentrations higher than 0.08 mg L 1. Kopittke et al. (2008) suggested that the toxicity to plants of trace elements, including In, Ga, Al, Cu, Gd, Hg, La, Ru and Sc, is due to their strong binding to the cell walls of roots, which leads to increased rigidity of the cell walls located in the zone of elongation, thereby reducing root growth and forming cell ruptures. Therefore, in this study, it predicted that the rice root suffered from In toxicity under In treatments, and induced the nutritional imbalances and reduction of nutrients (P, Mg, Ca, Fe, Zn, Mn) uptake and accumulated in rice seedlings (Table S2, Supporting information), thus leading to the plant growth inhibition (Table 1). Mendonca et al. (2003) indicated that the Al exposure led to reduce in K, Mg, Ca, and P contents and uptake in rice plants, which toxic mechanism was similar with In toxicity of this study. Fig. 5 shows the dose-response curves obtained gave an EC10 value of 0.17 mg In L 1. The results of Kopittke et al. (2009) reported that the average elongation rate of cowpea roots was reduced by 50% when grown in a culture solution of 0.72 μM In concentration (0.083 mg In L 1). These results suggest that both paddy rice and cowpea are In-sensitive plants. The concentrations of Ga and In measured in the rice seedling roots were about 10-fold higher than in the shoots, and the content (total uptake) and distribution of Ga and In in the roots were also higher than in the shoots, suggesting that the roots were the dominant sink of Ga and In. Yu et al. (2015) and Yu and Zhang (2015) also reported that the concentration of Ga accumulated in roots of rice seedlings was much higher than in shoots. Wheeler and Power (1995) indicated that the average ratio of the Ga concentration in roots to those in tops of wheat was 12, a ratio close to the one for rice observed in the present study. Besides accumulating in roots, trivalent cations may be strongly bound by the cell walls of roots, which contain ligands such as aldehyde, carboxyl, and hydroxyl in the lignin and cellulose, preventing those cations from reaching beyond the roots to other parts of the plant (Reid et al., 1996; Ren et al., 2014). The distribution of In in different parts of plants has rarely been discussed in previous studies. We also found that the translocation factor from roots to shoots of Ga (0.69 7 0.18) was about 2-fold higher than that of In (0.33 7 0.10), indicating that translocation of Ga in rice plants is easier than translocation of In, which might be why Ga was less phytotoxic to rice seedlings than In was. The differences in the uptake and translocation in rice plants between Ga and In were further supported by the results of the accumulation kinetics of Ga and In in roots and shoots under Ga and In treatments (Fig. 3). A linear increase in Ga accumulation in roots with increased Ga concentration
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in the culture solution suggests that the mechanism of uptake of Ga was not adversely affected by Ga treatment (Gao15 mg L 1, Fig. 3a), and the content of Ga that accumulated in shoots did so at a constant rate which may be determined by the rice plant's capability to translocate Ga (Fig. 3d). However, the content of In that accumulated in roots and shoots reached a plateau, which may have been a result of In phytotoxicity (Fig. 3b, c, e). The decrease of nutrient uptake by rice seedlings may also have resulted from In phytotoxicity to the roots, which further reduced translocation of those nutrients from root to shoot (Table S2, Supporting information). Moreover, under low-In treatments, a linear increase in In content in the shoots with In concentration in the culture solution was observed (Fig. 3f), which indicates that any In phytotoxicity that might have occurred was insufficient to noticeably affect the translocation of In from roots to shoots. Fig. 2c indicated that In content in the shoots was below the detection limit under 0.04– 0.15 mg In L 1 treatment, which may have resulted because most of the In were adsorbed by the root cell walls, reducing translocation of the element in the rice seedlings (Reid et al., 1996). In addition, we also found that there was negatively correlation between plant growth (shoot height and total biomass of rice seedlings) and In contents in rice seedlings, and the PT10 values (threshold value for 10% reduction in plant growth) based on shoot height and total biomass for In were 15.4 and 10.6 μg plant 1, respectively (Fig. 4).
5. Conclusion In summary, the results of this study indicated that qualitative differences exist between Ga and In with regard to their phytotoxicity, uptake, and translocation in rice seedlings. Treatment with Ga exerted a beneficial effect on growth in the rice seedlings, perhaps by increasing nutrient uptake (N, K, Mg, Ca, Mn) in plant tissue. In contrast, In treatment led to growth inhibition of rice seedlings, which may be attributed to a reduction in nutrient uptake (P, Mg, Ca, Fe, Zn, Mn) by plant tissue resulting from In phytotoxicity to roots. The EC10 value we observed for In treatment was 0.17 mg L 1, based on the dose-response curve. We found that the roots were the dominant sink of Ga and In in rice seedlings, and the concentrations of both elements in were about 10-fold higher in the roots of rice seedlings than in their shoots. Moreover, we found that the capability of translocation of Ga from roots to shoots were higher than In. From this study, we understand the effects of Ga and In on the growth of paddy rice grown in culture solutions. However, in paddy soils, the toxicity, uptake, and accumulation of Ga and In in rice plants may also be affected by the soil characteristics, thus requiring further investigation in a subsequent study.
Acknowledgements This work was supported by the Ministry of Science and Technology, Executive Yuan, Taiwan (grant no. MOST 104-2313-B002-015-MY3) is sincerely appreciated.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2016.09.016.
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