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

article info abstract

Article history: Limited information is available on the effects of gallium (Ga) and indium (In) on the growth of paddy rice. The Ga and In are
Received 11 July 2016 emerging contaminants and widely used in high-tech industries nowadays. Under-standing the toxicity and accumulation of
Received in revised form Ga and In by rice plants is important for reducing the effect on rice production and exposure risk to human by rice
9 September 2016 consumption. Therefore, this study investigates the effect of Ga and In on the growth of rice seedlings and examines the
Accepted 19 September 2016 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
Keywords:
Gallium
were measured after harvesting. A significant increase in growth index with increasing Ga concentrations in culture solutions
1
Indium (o10 mg Ga L ) was observed. In addition, the uptake of N, K, Mg, Ca, Mn by rice plants was also enhanced by Ga.
Emerging contaminants However, the growth inhibition were observed while the In concentrations higher than 0.08 mg L 1, and the nutrients
Paddy rice accumulated in rice plants were also significant decreased after In treatments. Based on the dose-response curve, we observed
Hydroponic culture
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 PT 10
(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.

1. Introduction Nowadays, due to progressively increasing usage of Ga and In, was-tewater

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-Pen-dias and Mukherjee, 2007; Kabata-Pendias
2011; Yu and Liao, 2011).
n
Corresponding author.
E-mail address: dylee@ntu.edu.tw (D.-Y. Lee).
1 necessary.
Dr. Chien-Hui Syu and Mr. Po-Hsuan Chien are equal contributors to this paper.
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 con-centrations 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 man-
ufacturing is limited. However, there were some studies indicated that the
contents of Ga and In in soils near area of automobile and Zn-Pb 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

http://dx.doi.org/10.1016/j.ecoenv.2016.09.016
0147-6513/& 2016 Elsevier Inc. All rights reserved.
 C.-H. Syu et al. / Ecotoxicology and Environmental Safety 135 (2017) 32–39
Gallium and indium are grouped among the IIIA group of ele-ments 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 re-spectively (Kabata-Pendias, 2011). In general,
the oxidation states of Ga and In are þ3, but oxidation states of þ1 and þ2
may also occur under anaerobic conditions. At 25 °C, the soluble species of
Ga and In present in acidic conditions are Ga 3 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, transpira-tion
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; Ko-pittke 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 ele-vation 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) re-ported 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 ele-ments 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 dis-tilled 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 (pre-liminary 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). De-spite 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 concentra-tions 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 sig-nificant 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 solu-tion was used
in this study, which contains the following compo-sitions: 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 ele-
ments 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 mea-sured. 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-citrate-bicarbonate) solution
(Liu et al., 2004) before performing plant di-gestion. 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-Al-drich), and then
the DCB extracts were discarded. The roots were then washed three times
with deionized water, removing the re-sidues 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 sepa-rately with
concentrated HNO3 (69–70%, J.T. Baker)/H2O2 (30%(w/w) in H2O, Sigma-
Aldrich) in heating blocks (Meharg and Rahman,
34 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 dif-ferences between the
treatments, we used the least significant dif-ference (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, ex-periments with lower concentrations of
In (low-In treatments) were performed to further understand the toxicity and
accumu-lation 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. Sig-nificant 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 de-creased 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 (Po0.001), shoot height (Po0.01), biomass of
root (Po0.001) and shoot (Po0.001), and chlorophyll SPAD value (Po0.01) of
rice seedlings among the Ga treatments, and these growth indices all
increased as Ga con-centration 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 treat-ment
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 con-centration of Ga in the roots and shoots of rice seedlings under
dif-ferent Ga treatments were N.D. 774 and N.D. 74 mg kg 1, re-spectively.
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.

Root length Shoot height Root biomass Shoot biomass Chlorophyll

mg L 1 cm cm mg plant 1 mg plant 1 SPAD value
Ga treatments
0 17.770.1 cn 61.971.3 d 51.572.2c 282712.0c 31.271.1c
1 17.270.9 c 63.473.9 dc 49.573.5c 284712.7c 32.271.0 bc
3 17.671.0c 67.170.6 abc 52.775.3 bc 313727.4b c 33.971.2 ab
5 19.57 0.8 b 69.170.7 a 60.573.7 ab 3517 5.6 a 33.371.1 ab
10 20.970.3 a 67.971.6 ab 69.174.5 a 366 721.3 a 34.271.1 a
15 21.3 7 0.4 a 64.372.7 bcd 68.578.2 a 336724.5 ab 35.270.7 a

High-In treatments
0 18.370.3 a 43.870.9 a 63.478.7 a 1827 15.1 a 30.370.7 a
0.1 14.77 1.8 b 39.970.7 b 39.9717.4 b 1427 25.5 b 26.475.1 ab
1 13.77 1.1 b 35.971.2c 33.075.9 b 123715.9 bc 22.474.6 b
3 13.87 0.8 b 36.171.9c 36.776.1 b 131720.9 bc 23.272.4 b
5 14.170.2 b 33.972.1 cd 29.472.2 b 1057 14.3c 22.474.5 b
10 13.97 0.8 b 32.770.6 d 28.471.7 b 11075.3c 21.671.2 b
Low-In treatments
0 17.770.1 a 61.971.3 a 51.572.2 a 282712.0 a 31.271.1 d
0.04 16.67 0.8 b 58.270.5 b 51.471.8 a 2717 14.8 ab 32.972.0 dc
0.08 16.67 0.4 b 56.970.8 bc 46.171.6 b 25475.9 bc 34.570.8 bc
0.10 16.37 0.9 b 56.171.5c 44.673.2 b 25478.9 bc 34.770.9 bc
0.15 15.77 0.1 b 57.070.3 bc 43.573.5 b 249720.8 bc 34.671.0 bc
1 16.07 0.2 b 55.671.6 cd 43.371.2 b 244 710.8 c 36.270.4 ab
2 15.87 0.2 b 54.171.2 d 48.375.5 ab 2477 17.0 c 36.770.1 a

n
Different letters indicated the differences in the value among the Ga/In treatments based on the LSD test (Po0.05).
 C.-H. Syu et al. / Ecotoxicology and Environmental Safety 135 (2017) 32–39 35

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 means7SD (n¼3). Different letters
above the bars indicate significant difference in the value among the Ga or In treatments based on the LSD test (Po0.05). 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 dif-ference 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
36 C.-H. Syu et al. / Ecotoxicology and Environmental Safety 135 (2017) 32–39
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 (Po0.05). 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/con-tent in roots) decreased as the concentration
increased in the culture solution. The average translocation factor of Ga
(0.6970.18) was higher than that of In (0.3370.10).
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 con-centration in culture solution was
best described by a linear model (R2 ¼0.9953, Po0.001), but the Ga content
in shoots was best de-scribed by a logarithmic model (R2 ¼0.9649, 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 ¼0.9497, Po0.01) and In
concentration in culture solution were best described by logarith-mic models,
except for the shoot content under low-In treatment (linear model, R2
¼0.9980, Po0.001). These results indicate that the accumulation kinetics of In
by the roots and by the shoots under high-In 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
Table S1 (Supporting information) shows the content (total up-take) 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 ob-served. The content of Mg in roots
significantly increased with in-creased 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 so-lution 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 de-crease 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%, re-spectively. 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 cul-ture
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 il-lustrating the phytotoxicity of In. The threshold
of In phytotoxicity is
 C.-H. Syu et al. / Ecotoxicology and Environmental Safety 135 (2017) 32–39 37

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 de-scribed by a logarithmic model
(R2¼0.8354, 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 re-gard 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, tran-spiration 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.
38 C.-H. Syu et al. / Ecotoxicology and Environmental Safety 135 (2017) 32–39
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-accu-mulation of
Ga in plants tissue caused the formation of DNA-protein cross-links (DPCs)
in roots, which led to cell death and growth in-hibition 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 seed-lings 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 con-centration.
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 un-clear. 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 con-tent of P, Mg, Ca, Fe, Zn, Mn measured in shoots significantly
de-creased under high-In treatment (Table S2, Supporting informa-tion),
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 low-In 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,
Sup-porting 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 con-
centration 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 hy-droxyl 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), in-dicating 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 sup-
ported by the results of the accumulation kinetics of Ga and In in roots and
shoots under Ga and In treatments (Fig. 3). A linear in-crease in Ga
accumulation in roots with increased Ga concentration
 C.-H. Syu et al. / Ecotoxicology and Environmental Safety 135 (2017) 32–39 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 cap-ability 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 nu-trient 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 informa-tion). 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 phyto-toxicity, uptake, and
translocation in rice seedlings. Treatment with Ga exerted a beneficial effect
on growth in the rice seedlings, per-haps by increasing nutrient uptake (N, K,
Mg, Ca, Mn) in plant tis-sue. In contrast, In treatment led to growth inhibition
of rice seedlings, which may be attributed to a reduction in nutrient up-take
(P, Mg, Ca, Fe, Zn, Mn) by plant tissue resulting from In phy-totoxicity 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.