Chemosphere 136 (2015) 56–62
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Fluoride bioaccumulation by hydroponic cultures of camellia (Camellia japonica spp.) and sugar cane (Saccharum officinarum spp.) Nancy Camarena-Rangel a,1, Angel Natanael Rojas Velázquez b,2, María del Socorro Santos-Díaz a,⇑ a
Facultad de Ciencias Químicas de la Universidad Autónoma de San Luis Potosí, San Luis Potosí, Manuel Nava 6, 78210 San Luis Potosí, Mexico Facultad de Agronomía de la Universidad Autónoma de San Luis Potosí, San Luis Potosí, Km. 14.5 Carretera San Luis Potosí-Matehuala, Ejido Palma de la Cruz, Soledad de Graciano Sánchez, San Luis Potosí CP. 78321, Mexico b
h i g h l i g h t s We establish hydroponic cultures of camellia and sugar cane species. The capacity of these cultures to remove fluoride from water was measured. The sugarcane roots accumulated F with 86% of it absorbed and 14% adsorbed. In camellia plants the highest fluoride concentration was found in the leaves. Camelia plants bio-accumulated 74–221-fold while sugarcane 100–500-fold.
a r t i c l e
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Article history: Received 25 September 2014 Received in revised form 6 February 2015 Accepted 11 March 2015
Keywords: Fluoride Hydroponic cultures Camellia Sugar cane
a b s t r a c t The ability of hydroponic cultures of camellia and sugar cane adult plants to remove fluoride was investigated. Plants were grown in a 50% Steiner nutrient solution. After an adaptation period to hydroponic conditions, plants were exposed to different fluoride concentrations (0, 2.5, 5 and 10 mg L1). Fluoride concentration in the culture medium and in tissues was measured. In sugar cane, fluoride was mainly located in roots, with 86% of it absorbed and 14% adsorbed. Sugar cane plants removed 1000–1200 mg fluoride kg1 dry weight. In camellia plants the highest fluoride concentration was found in leaf. Roots accumulated fluoride mainly through absorption, which was 2–5 times higher than adsorption. At the end of the experiment, fluoride accumulation in camellia plants was 1000–1400 mg kg1 dry weight. Estimated concentration factors revealed that fluoride bioaccumulation is 74–221-fold in camellia plants and 100–500-fold in sugar cane plants. Thus, the latter appear as a suitable candidate for removing fluoride from water due to their bioaccumulation capacity and vigorous growth rate; therefore, sugar cane might be used for phytoremediation. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Fluoride (F) is a common environmental pollutant in groundwater. High fluoride concentrations have been reported in India (38.5 mg L1), China (>8 mg L1), Australia (9 mg L1), Mexico (11.89 mg L1), North America (3.3 mg L1), and South America (0.9–18.2 mg L1) (Fuhong and Shuqin, 1988; Paoloni et al., 2003; WHO, 2006). Additionally, human activities often lead to increased local fluoride levels. HF and particulate fluoride derive primarily from mining operations, fertilizers, chemicals, and metal ⇑ Corresponding author. Tel.: +52 (444) 8262300x6568. E-mail addresses: [email protected] (N. Camarena-Rangel), [email protected] (A.N. Rojas Velázquez), [email protected] (M.S. Santos-Díaz). 1 Tel.: +52 (444) 826 2440x6568. 2 Tel.: +52 (444) 852 40 56. http://dx.doi.org/10.1016/j.chemosphere.2015.03.071 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
processing. In particular, the use of phosphate fertilizers can increase fluoride levels in soil to as much as 5300 mg kg1 dry weight (DW) (Azbar and Turkman, 2000). Drinking water is deemed safe for human consumption if fluoride concentrations do not exceed 1 mg L1. Unfortunately, over 200 million people worldwide drink water with fluoride contents above the maximum allowable level. Excessive fluoride consumption causes dental fluorosis, and chronic intake leads to bone stiffness, rheumatism, and permanent crippling known as skeletal fluorosis (WHO, 2006; Ayoob et al., 2008). Considerable efforts have been made to lower fluoride content in drinking water. Physicochemical methods are commonly used, and can be broadly classified into membrane and adsorption techniques. Membrane techniques include reverse osmosis, nanofiltration, dialysis and electro-dialysis. Fluoride adsorption has been carried out using various adsorbents, including clay, alumina, rare-earth oxides,
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silica, carbonaceous materials, solid industrial waste, zeolites and, more recently, cellulose-supported double-layered hydroxides (Tripathy et al., 2006; Mohapatra et al., 2009). However, few adsorbents are able to lower fluoride levels below 1–1.5 mg L1, affect the taste of the treated water, and require large spaces for drying of sludge; additionally effective technologies have also drawbacks; for example, ion exchange and reverse osmosis are relatively expensive and require trained personnel for implementation (Mandal and Mayadevi, 2008). An alternative to achieve fluoride removal from water is phytoremediation, which is defined as the use of plants to reduce in situ levels of hazardous organic and inorganic pollutants from soil, sediments, water, and air. Biochemical processes carried out by plants can lead to the reduction, mineralization, degradation, volatilization, sequestration, and/or stabilization of various pollutants (Suresh and Ravishankar, 2004). A number of plants species including grasses, legumes, aquatic plants and the so-called hyperaccumulators have been used for these purposes. Field tests using plants have been conducted to remove petroleum hydrocarbons, pesticides and food waste, among others (Schnoor et al., 1997; Dushenkov et al., 1997). It has been reported that some plants are also able to take up fluoride from soil (Asada et al., 2006; Kang et al., 2008; Telesin´ski et al., 2010; Baunthiyal and Sharma, 2012; Saini et al., 2012). In addition, previous studies in our laboratory showed that hydroponic cultures of Camellia japonica, Pittosporum tobira, and Saccharum officinarum (Santos-Díaz and Zamora-Pedraza, 2010), and tree species from semiarid regions (Baunthiyal and Sharma, 2012) are able to remove fluoride from water. For the phytoextraction to be effective, vigorously growing plants are required. Thus, the purpose of this study was to explore the fluoride-uptake ability of hydroponic cultures of C. japonica spp. and S. officinarum spp. adult plants. The effects of fluoride on chlorophyll content, as well as fluoride concentration in leaves, stem and roots were determined to examine the accumulation pattern, and identify the primary tissues involved in fluoride uptake. Bioaccumulation factors were also calculated. 2. Materials and methods 2.1. Plant material Camellia (C. japonica) plants of the same age and height (60 cm) were purchased from a commercial nursery. Sugarcane (S. officinarum, SP-70-1284) plants about 1.5 m height were obtained from the ‘‘Plan de Ayala SA de CV’’ sugar mill located in Ciudad Valles, San Luis Potosí, Mexico. All plants were kept in a greenhouse (18–30 °C, 68.8% RH, light 127 lmol m2 s1) for at least 2 months; plants were irrigated twice per week during the winter, and three times per week in the summer. 2.2. Establishment of camellia and sugar cane hydroponic cultures Camellia and sugar cane plants were collected from the greenhouse; the soil was carefully removed and roots were thoroughly washed with running tap water followed by deionized water. Hydroponic cultures were established in opaque plastic containers (40 L) previously washed with 10% Dextran, rinsed with deionized water, treated with 10% HNO3 for 24 h, and rinsed three times with deionized water. A plastic mesh was fitted to each container’s mouth to provide physical support to plants, as well as to ensure that only roots were in contact with the culture medium. The nutrient solution described by Steiner (1961), prepared with deionized water, was used as growth medium; the nutrient solution (pH = 6) was continuously aerated employing a commercial
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air pump (Elite 800). Once the plant-container assembly was prepared, containers were individually wrapped with a black plastic bag to reduce water evaporation and prevent the incidence of light on the hydroponic medium. Plants were maintained in the hydroponic solution (35 L) for 30 days for adaptation. 2.3. Fluoride removal by camellia and sugar cane hydroponic cultures Camellia and sugar cane hydroponic cultures were exposed to a NaF solution (2.5, 5 or 10 mg fluoride L1) for 28 days. Control plants were grown under the same conditions, but with no NaF supplementation. Four plants of each species were used for each treatment. A 15 ml sample of the nutrient solution was collected at 3 d intervals for 28 d, to make triplicate fluoride concentration measurements using an ion selective electrode (960 900, Thermo Orion). To calculate anion concentration, a standard curve was elaborated (n = 10) using NaF; in addition, a certificated standard of fluoride was included (High Purity Standard, 100 ppm, Charleston, SC, USA) to determine repeatability and reproducibility. The curve presented a linear correlation coefficient of 0.99, repeatability, expressed as a variation coefficient was 2.51%, and the exactitude was 98% (Standard Methods, 1998). The presence of oxidation or chlorosis spots, wilting, formation of new shoots and roots was recorded on individual plants. Total chlorophyll contents were also measured according to Bruinsma (1961), as indicators of cellular integrity. 2.4. Fluoride accumulation in camellia and sugar cane tissues Six leaves from the basal, medial and apical aerial portions of camellia plants were harvested each week for four weeks. At the same time, a single leaf from each sugar cane plant was collected and excised to obtain the basal, media and apical sections. The roots of camellia and sugar cane plants, and the stems of sugar cane plants were fully harvested on day 28. The stems were segmented into basal, media, and apical sections. Plant materials were dried at 105 °C for 24 h, pulverized, sieved through a 0.425 mm mesh sieve and kept frozen until analysis. Both adsorbed and absorbed fluoride were determined in roots. Two desorption kinetics were performed. For the first protocol, 5 mg of dried roots were placed on high-density polyethylene (HDPE) flasks and treated with 3 ml of 1 mM HNO3. Flasks were kept under continuous stirring (135 rpm) at 25 °C, and the supernatant was collected every 10 min for 300 min (Santos-Díaz and Barrón-Cruz, 2011). For the second procedure, 100 mg of dried roots were treated with 50 ml of 0.1 M HCl and incubated at 25 °C for 1 h under continuous stirring at 135 rpm (Murugan and Subramanian, 2006). The supernatant was collected every 10 min for 60 min and fluoride content in it was determined using a potenciometric method. The procedure described by Menzies et al. (1993) was used for quantifying the amount of fluoride absorbed by camellia and sugar cane leaves and roots. Roots were first washed with 1 mm HNO3 for 300 min to desorb fluoride from the surface. Concentration factors (CF) were estimated following EspinozaQuinones et al. (2005), as follows:
CF ¼ fluoride concentration in plant tissue ðmg g1 DWÞ=fluoride concentration in mediumðmg L1 Þ: 2.5. Statistical analyses Results are shown as mean ± SD (standard deviation) and were analyzed with ANOVA, using a 95% significance level, by means of the SAS software.
In previous studies, we showed that camellia and sugar cane hydroponic cultures are able to remove fluoride. However, toxic effects, including wilting and oxidation, were observed in seedlings exposed to 10 mg L1 fluoride (Santos-Díaz and Zamora-Pedraza, 2010). To improve fluoride tolerance, vigorously growing adult camellia and sugar cane plants were used in this investigation. Hydroponic cultures showed evidence of oxidation and signs of salt toxicity after 7 d in Steiner solution. Therefore, plants were transferred to a medium with reduced (50%) salt concentration. Camellia and sugar cane adult plants successfully adapted to these conditions, with no evidence of oxidation, wilting or chlorosis after 28 d (data not shown). Once the plants were fully adapted to hydroponic conditions, these were exposed to fluoride. No negative effects on morphological parameters were observed in any species, and new shoots and roots developed in sugar cane plants grown in 5 and 10 mg L1 fluoride (data not shown). Chlorophyll content was determined in order to examine the integrity of plants, as photosynthesis has been reported as being highly sensitive to various types of stress (metals, drought, nutrient depletion). In camellia leaves, the contents of total chlorophyll was not significantly different in basal, media and apical sections. However, chlorophyll concentration after 28 d decreased significantly (p < 0.5) in the presence of fluoride at all concentration levels tested (Fig. 1A), thus suggesting that prolonged exposure to this anion affects the photosynthetic process. In the sugar cane leaves, the lowest concentrations of total chlorophyll occurred in the basal section, while the highest accumulation was observed in the apical region, hence suggesting the presence of a concentration gradient. This pattern was independent of fluoride concentration. On the other hand, chlorophyll content was significantly higher (p < 0.05) in plants exposed to 2.5 and 5 mg L1 fluoride relative to control plants. The chlorophyll content of plants grown at a fluoride concentration of 10 mg L1 was similar to that of control cultures (Fig. 1B). Thus, exposure to fluoride did not affect the integrity of sugar cane tissues. 3.2. Fluoride removal by camellia and sugar cane hydroponic cultures Fig. 2A depicts fluoride removal by camellia plants. At 2.5 mg L1, fluoride concentration decreased steadily until day 18. In the presence of 5 or 10 mg L1, fluoride in the medium decreased rapidly by day 3 and then remained virtually unchanged thereafter. The amount of fluoride removed by camellia plants from the nutrient solution containing fluoride levels of 2.5, 5 or 10 mg L1 was 1.3 mg L1, 1.25 mg L1 and 3.4 mg L1, respectively. In sugar cane hydroponic cultures, fluoride was rapidly taken up during the first 3 days in media containing levels of 5 or 10 mg L1 fluoride, and after 3 days in medium with 2.5 mg L1 fluoride (Fig. 2B). Uptake continued steadily until day 9, without noticeable changes thereafter. The amount of fluoride taken up by sugar cane plants from media containing fluoride levels of 2.5, 5 and 10 mg L1 was 2.3 mg L1, 1.94 mg L1 and 4.57 mg L1, respectively. 3.3. Fluoride desorption from the root surface In order to identify which tissues were involved in fluoride accumulation, we determined fluoride concentration in leaves, stems and roots. Any fluoride adsorbed to the root surface was released by administering an acid treatment. Nitric acid has been
250
A
200 150 100 50 0 Control
2.50
5.00
10.00
Fluoride concentration (mg L-1) Chlorophyll concentration (mg 100g -1)
3.1. Adaptation of camellia and sugar cane plants to hydroponic conditions, and quantification of chlorophyll
180
B
160 140 120 100 80 60 40 20 0 Control
2.50
5.00
10.00
Fluoride concentration (mg L-1) Fig. 1. Content of total chlorophyll in (A) camellia, and (B) sugar cane leaves exposed to 2.5, 5 and 10 mg L1 fluoride during 28 days. Basal (j), medium (h) and apical ( ).
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Fluoride in medium (mg l-1)
3. Results
Chlorophyll concentration (mg 100g -1)
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A
10 8 6 4 2 0 0
3
6
9
12
15
18
21
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Time (days)
Fluoride in medium (mg l -1)
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12
B
10 8 6 4 2 0 0
3
6
9
12
15
18
21
24
27
Time (days) Fig. 2. Fluoride removed from the medium by hydroponic cultures of (A) camellia and (B) sugar cane. Control (), 2.5 mg L1 (j), 5 mg L1 (N) and 10 mg L1 (d) fluoride.
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Fluoride desorbed (mg kg-1)
1200 1000 800 600 400 200 0 0
10
20
30
40
50
60
70
Time (minutes) Fig. 3. Desorption of fluoride from sugar cane roots using HCl 0.1 M. Control (), 2.5 mg L1 (j), 5 mg L1 (N) and 10 mg L1 (d) fluoride.
Fluoride desorbed (mg kg -1)
700
A
600 500 400 300 200 100 0 0
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300
Time (minutes)
Fluoride desorbed (mg kg -1)
300
B
250 200 150 100 50 0 0
50
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Time (minutes) Fig. 4. Desorption of fluoride from (A) sugar cane and (B) camellia roots using HNO3 1 mM. Control (), 2.5 mg L1 (j), 5 mg L1 (N) and 10 mg L1 (d) fluoride.
reported to release heavy metals from roots (Santos-Díaz and Barrón-Cruz, 2011); likewise, HCl is effective in releasing fluoride from plant materials packed in columns (Murugan and Subramanian, 2006). In this work we tested both HCl and HNO3, and the respective release kinetics were examined to determine the time needed to achieve fluoride desorption from root surfaces. HCl-treated sugar cane roots displayed a continuous fluoride release at all concentrations tested, and did not reach a stationary phase after 60 min (Fig. 3). This treatment also caused tissue discoloration and fragmentation, thus indicating that the procedure was too aggressive. Fluoride release was also observed in the roots of control plants, thus suggesting that those plants had been previously in contact with fluoride, probably through irrigation water. On the other hand, the amount of fluoride desorbed with HNO3 from sugar cane roots was proportional to fluoride concentration in the medium (at 5 and 10 mg L1), increased gradually for 150 min, and stabilized by minute 300 (Fig. 4A). Fluoride desorption at 2.5 mg L1 became virtually constant after 60 min. In camellia roots exposed to 5 or 10 mg L1 fluoride, a rapid desorption with HNO3 was observed for the first 30 min (Fig. 4B). Then, a steady release occurred until minute 120, with no further relevant variations thereafter. Fluoride release from roots of plants grown in 2.5 mg L1 fluoride was slower and peaked by minute 120, with no further noticeable changes thereafter. The use of HNO3 caused no apparent effect on the roots of sugar cane or camellia plants and, therefore, this procedure was selected for further experiments. To note, the roots of camellia and sugar cane control plants released 50 and 200 lg fluoride g1 of tissue, respectively, hence indicating their previous contact with fluoride. 3.4. Fluoride accumulation in camellia and sugar cane tissues Table 1 shows the amount of fluoride accumulated in different tissues of camellia plants after 28 days. The largest accumulation occurred in leaves, but the final concentration was similar in both control and fluoride-exposed plants (p > 0.05). However, marked differences were observed in roots. Fluoride concentrations were about 3–4 higher (p < 0.05) in fluoride-exposed than in control plants. The amount of fluoride absorbed within roots was not statistically different in plants grown in 2.5 or 5 mg L1 fluoride and in control plants, but fluoride concentration almost doubled in plants exposed to 10 mg L1. Camellia roots accumulated fluoride mainly through absorption, which was 2–5 times the amount of fluoride accumulated through adsorption. At the end of the experiment, camellia plants had accumulated some 1000–1400 mg fluoride kg1 DW. In the sugar cane plants, no statistically significant differences (p < 0.05) were found in fluoride content between basal, medial, and apical sections of stems (Fig. 5A); total fluoride content in stems was similar in non-exposed and exposed plants (Table 1). An accumulation gradient was observed in leaves (Fig. 5B), as
Table 1 Accumulated fluoride in tissues of hydroponic cultures at 28 days. Species
Fluoride (mg L)
Stem (mg kg1)
Leaf (mg kg1)
Root Absorbed (mg kg
1
)
1
Adsorbed (mg kg
Total (mg kg1)
CF1
)
Sugar cane
0 2.5 5 10
52 ± 2.3a 46 ± 0.4a 48 ± 0.79a 47 ± 0.56a
67 ± 0.98a 55 ± 1.0a 86 ± 15.5b 120 ± 25b
443 ± 5.3d 985 ± 20.5a 634 ± 66.3b 521 ± 25.3c
124 ± 87.3d 159 ± 92.1c 261 ± 169.2b 325 ± 152.8a
686 ± 96.0b 1245 ± 114a 1030 ± 216 a 1012 ± 204.1a
1 497.94 205.97 101.99
Camellia
0 2.5 5 10
– – – –
741 ± 25a 768 ± 9.5a 760 ± 20a 697 ± 8.3a
273 ± 1.8b 363 ± 4.3b 275 ± 9.3b 553 ± 4.6a
45 ± 12c 190 ± 6.4a 114 ± 47b 192 ± 71a
1059 ± 38.8a 1321 ± 20.2b 1149 ± 76.3a 1442 ± 83.9c
1 221.2 77.8 74.5
Different letters in the same column are statistically different (Tukey test, p < 0.05) 1 CF: concentration factor.
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Fluoride absorbed (mg kg-1)
60
A
b
a
20
b
b
b
b
b
b
b
15
b
b b
10 5 0 Control
2.5
5
Fluoride absorbed (mg kg-1)
10
35 30 25 20 15 10 5 0
B
a b
a a
ba
ba
a
ba
ba
b b b
Control
2.5
5
10
Fig. 5. Accumulation of fluoride in stems (A) and leaf (B) from sugar cane. Basal basal (h), medium ( ) and apical (j) sections.
higher fluoride levels (p < 0.05) were recorded in basal vs. apical sections. Leaves of plants grown in media containing 5 and 10 mg L1 fluoride had total fluoride levels higher than those in control plants (Table 1). Most of the fluoride removed by sugar cane hydroponic cultures was located in roots, with 86% being absorbed and 14% adsorbed. Sugar cane plants removed between 1000 and 1200 mg fluoride kg1 DW. 4. Discussion Phytoremediation is not the only alternative available for removing pollutants, but it offers substantial economic advantages over other systems. Conventional remediation methods cost between $10 and $1000 US dollars (USD) per cubic meter, while the estimated cost of phytoextraction may be as low as $0.05 USD per cubic meter (Ghosh and Singh, 2005), making of this a very appealing option. For a practical phytoremediation treatment, a vigorously-growing, easily harvested plant that accumulates large amounts of the pollutant is desired (Suresh and Ravishankar, 2004). Therefore, in this study, actively-growing adult camellia and sugar cane plants were used instead of seedlings. Camellia plants grown in fluoride-containing media did not show evidence of toxic effects. Nevertheless, a small—but significant— reduction in chlorophyll content was observed after 28 days at all the fluoride levels tested. Thus, chlorophyll content was a better indicator of cellular integrity than other parameters such as oxidation or wilting. Camellia species have been previously reported to display a high fluoride tolerance (Ruan et al., 2003, 2004); however, those studies were performed using Camellia sinensis grown in soil where fluoride availability is affected by the chemical complexity of the substrate. Under hydroponic conditions, no restrictions for fluoride uptake occur. Toxic effects of fluoride have also been described in almond (Elloumi et al., 2005), sorghum (Schender and Maclean, 1970) and barley (Gautam and Bhardwaj, 2010). Sugar cane plants were not affected by fluoride; moreover, plants grown in medium with 5 or 10 mg L1 fluoride showed a
higher chlorophyll concentration than control plants and developed new shoots and roots, thus confirming the tolerance of sugar cane to fluoride. Camellia plants took up fluoride faster than sugar cane plants, removing up to 40–50% of the anion available in the medium by the 3th day, while sugar cane required about 6 days to achieve similar results. In order to identify which tissue was mainly involved in fluoride accumulation, the fluoride content in leaves, stem and roots was measured. Camellia and sugar cane roots internalized about 65–85% of all the fluoride taken. However, the amount of fluoride accumulated in sugar cane roots grown in medium containing 2.5 and 5 mg L1 fluoride was 2–2.3 times higher than the amount accumulated in camellia roots, and 1.2 times higher when grown in 10 mg L1 fluoride (Table 1). It has been suggested that fluoride uptake by roots is a passive process involving an extracellular pathway traversing the endodermal barrier at either the root tips or the endodermis where lateral roots are formed, following the transpiration stream (Stevens et al., 1998). However, participation of other mechanisms, for example anion channels, cannot be ruled out. Anion channels are integral proteins located in the cell membrane, tonoplast, endoplasmic reticulum, mitochondria, and chloroplasts. It has been reported that the slow-activation (S-type) anion channels show differential selectivity according to the following order: NO 3 > Br > F > Cl > I > malate (Schmidt and Schroeder, 1994; Lurin et al., 2000; White and Broadley, 2001; Clausen et al., 2004). Thus, S-type anion channels are able to transport fluoride with moderate selectivity and might be involved in fluoride uptake. Further research is necessary to determine whether these channels are present in sugar cane and camellia roots and whether they participate in fluoride transport. On the other hand, the amount of fluoride adsorbed onto the sugar cane root surface increased proportionally to its concentration in the medium. In camellia roots, the amount of fluoride adsorbed was 2–3 times higher than in control plants, but no clear relationship with concentration could be observed. Fluoride adsorbed onto roots might be attached to cell-wall components, such as calcium, or to ionizable compounds. Previous studies show that several species from semiarid regions accumulated fluoride in cytosolic and cell-wall fractions (Baunthiyal and Sharma, 2012). It is well known that the ionic and electric properties of the cell wall are mainly due to the dissociation of weak acids. In monocotyledon roots, galactouronic acids, carboxylic acids, and phenolics are particularly abundant. As galactouronic acids are more readily ionizable than carboxylic acids and phenolics, they are likely involved in fluoride adsorption (Meychik, 2001; Meychik et al., 2010). Another possibility is the binding of fluoride to OH-rich arabinoxylanes. Burke et al. (1974) showed that arabinoxylanes are the major components of cell walls obtained from a sugar cane cell suspension. Thus, fluoride ions might replace the –OH group in arabinoxylanes, in a similar way as they replace the –OH group from hydroxyapatite in bone. Further experiments are required to identify which cell-wall components are involved in fluoride uptake. In camellia plants, the highest fluoride concentration was found in leaves; this, however, did not seem to be the result of translocation from the hydroponic medium, since fluoride levels were similar in both control plants and plants grown in fluoridesupplemented media. Camellia plants were likely in contact with fluoride in irrigation water and accumulated it throughout their development. Fluoride may also have deposited on the leaf surface from air pollution. Gaseous fluoride is an important phytotoxic air pollutant, which is absorbed through leaf stomata and moves in the transpiration stream to the leaf tip and margins. High fluoride accumulation in leaves has been attributed to air pollution in several species (Horntvedt, 1997; Domingos et al., 2003). To rule out this possibility, leaves must be washed prior to fluoride quantification. This is a subject that deserves further investigation.
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Fig. 6. Fluoride accumulation in sugarcane plants. Fluoride adsorbed (d) and absorbed ( ). Plants exposed to 2.5 mg l1 accumulated fluoride mainly inside the roots and a lower content remained adsorbed to root surface. At 5 and 10 mg l1 the content of fluoride adsorbed increase 1.5–2.1 folds and initiate the translocation of fluoride to aerial parts.
Large amounts of fluoride being translocated to mature camellia leaves have been reported. (Yumada and Hattori, 1977; Xie et al., 2001; Ruan et al., 2003). However, those studies were conducted with a different species (C. sinensis) an under different conditions, i.e. plants were grown in soil and in the presence of aluminum, a cation that improves fluoride translocation to shoots. On the other hand, fluoride concentration in sugar cane leaves was 1.3 and 1.8 times higher in plants grown in 5 and 10 mg L1, respectively, relative to control plants, suggesting that fluoride was translocated to aerial parts. Moreover, a concentration gradient was observed, with apical sections having a higher fluoride content than basal sections. Increased fluoride levels in leaves were coupled with a reduction in the amount of fluoride absorbed in roots, thus confirming that fluoride was translocated. Fig. 6 presents a putative diagram of fluoride accumulation in sugar cane plants. The amount of fluoride taken up by sugar cane and camellia adult plants at the end of the experiment was 6–10 times higher than the amount removed by seedlings in previous studies (Santos-Díaz and Zamora-Pedraza, 2010). Both species have a moderate capacity to accumulate fluoride (1200–1400 g kg1) in comparison to other species. For example, spinach, amaranth, cabbage, okra and tomato plants irrigated with water containing 10 mg L1 fluoride accumulated 1.7, 20.9, 0.17 and 0.2 g kg1 DW, respectively (Khandare and Rao, 2006); barley plants exposed to 20 mg L1 accumulated 13 g kg1 (Gautam and Bhardwaj, 2010); Acacia tortilis, Cassia fistula and Prosopis juliflora took up about 1200 g kg1 (Baunthiyal and Sharma, 2012); hybrid willow, black willow and basket willow accumulated over 5000 g kg1 (Kang et al., 2008; Telesin´ski et al., 2011). While willow species seem suitable for phytoremediation studies, their growth rate is lower than that of sugar cane, which can grow up to 2–3 m per year. Finally, CFs were calculated for both species. As fluoride content in camellia leaves was similar in control and fluoride-exposed plants, these data were not included in the CF calculation. Our data showed that camellia plants bioaccumulate fluoride 74–221 times, while sugar cane plants do it 100–500 times (Table 1). In both species, CF decreased proportionally as fluoride concentration in the medium increased, suggesting that the efficiency of cellular mechanisms to remove fluoride decreases when anion concentration increased. Sugar cane plants exposed to 2.5 mg L1 accumulated fluoride mainly inside the roots and a lower content remained adsorbed to root surface. At 5 and 10 mg L1 the content of fluoride adsorbed
increase 1.5–2.1 folds and initiate the translocation of fluoride to aerial parts. Since, the sugar cane hydroponic cultures exposed to 2.5 mg L1 removed 93% of fluoride, this species could be useful to reduce F from water containing 62.5 mg L1 F. It is well known that among 1.5–3 mg L1 fluoride in drinking water causes dental fluorosis. According to WHO (2006), around 70 million persons, distributed in China, India, Africa and eastern Mediterranean regions, suffer dental fluorosis. Then, the use of sugar cane hydroponic cultures could reduce the incidence of this affection. As sugar cane is cultivated in great extensions, the disposal of plant material would not be a problem for their installation in small containers, or for studies on artificial wetlands. These options must be explored in more detail in further studies. In addition, the use of sugar cane plants could be combined with others methodologies, as osmosis reverse, improving the half life of filters and reducing costs. 5. Conclusions The highest concentration of fluoride in camellia plants was found in leaves but this accumulation was not the result of a translocation process since similar levels of fluoride were detected in non-exposed and exposed plants. This specie bio-concentrated fluoride between 74 and 221 folds. On the contrary, the sugar cane plants bio-accumulate the anion mainly in roots where 86% is internalized and 14% is adsorbed. This species translocated fluoride to leaves at 5 and 10 mg L1 and concentrate the anion between 100 and 500 folds. Taking into account its bio-accumulation capacity and fast growth rate the sugar cane plants can be used for phytoremediation purposes. Acknowledgements The authors thank Dra. María Degracias Ortiz Pérez for her valuable comments to the work; and to CONACYT for a scholarship granted to NCR (365990). References Asada, M., Parkpian, P., Horiuchi, S., 2006. Remediation technology for boron and fluoride contaminated sediments using green plants. In: Fukue, M., Kita, K., Ohtsubo, M., Chaney, R. (Eds.), Contaminated Sediments: Evaluation and Remediation Techniques. ASTM, West Conshohocken PA, USA.
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Mohapatra, M., Anand, S., Mishra, B.K., Giles, D.E., Singh, P., 2009. Review of fluoride removal from drinking water. J. Environ. Manage. 91, 67–77. Murugan, M., Subramanian, E., 2006. Studies on defluoridation of water by tamarind seed, an unconventional biosorbent. Water Health 4, 453–461. Paoloni, J.D., Florentino, C.E., Sequeira, M.E., 2003. Fluoride contamination of aquifers in the southeast subhumid Pampa, Argentina. Environ. Toxicol. 18, 317–321. Ruan, J., Ma, L., Shi, Y., Han, W., 2003. Uptake of fluoride by tea plant (Camellia sinenesis L) and the impact of aluminium. J. Sci. Food Agric. 83, 1342–1348. Ruan, J., Ma, L., Shi, Y., Han, W., 2004. The impact of pH and calcium on the uptake of fluoride by tea plants (Camellia sinensis L). Ann. Bot. 93, 97–105. Saini, P., Khan, S., Baunthiyal, M., Sharma, V., 2012. Organ-wise accumulation of fluoride in Prosopis juliflora and its potential for phytoremediation of fluoride contaminated soil. Chemosphere 89, 633–635. Santos-Díaz, M.S., Barrón-Cruz, M.C., 2011. Lead, chromium and manganese removal by in vitro root cultures of two aquatic macrophytes species: Typha latifolia L. and Scirpus americanus Pers. Int. J. Phytorem. 13, 538–551. Santos-Díaz, M.S., Zamora-Pedraza, C., 2010. Fluoride removal from water by plant species that are tolerant and highly tolerant to hydrogen fluoride. Fluoride 43, 150–156. Schender, R.E., Maclean, D.C., 1970. Relative susceptibility of seven grain sorghum hybrids to hydrogen fluoride. Contrib. Boyce Thompson Inst. 24, 216–219. Schmidt, C., Schroeder, J.I., 1994. Anion selectivity of slow anion channels in the plasma-membrane of guard-cells: large nitrate permeability. Plant Physiol. 106, 383–391. Schnoor, J.L., Aitchison, E.W., Kelley, S.L., Alvarez, P.J., Wakefield, S., Burken, J.G., Just, C.L., 1997. Phytoremediation of 1,4-Dioxane by Hybrid Poplars. ACS. 213, Symposium Paper. Environ. Chem. 195. Standard Methods, 1998. Standard Methods for the Examination of Water and Wastewater, 20 ed. American Public Health Association, Washington, EUA. 1325 p.. Steiner, A.A., 1961. A universal method for preparing nutrient solutions of a certain desired composition. Plant Soil 15, 134–154. Stevens, D.P., McLaughlin, M.J., Alston, A.M., 1998. Phytotoxicity of fluoride ion and its uptake from solution culture by Lycopersicon esculentum and Avena sativa. Plant Soil 200, 119–129. Suresh, B., Ravishankar, G.A., 2004. Phytoremediation-a novel and promising approach for environmental clean-up. Crit. Rev. Biotechnol. 24, 97–124. Telesin´ski, A., Smolik, B., Grabczyn´ska, E., 2010. Formation of adenylate energy charge (aec) versus the fluorine content in forest soil in the area affected by emission from police chemical plant. J. Elementol. 15, 355–362. Telesin´ski, A., S´nioszek, M., Smolik, B., Malinowska, K., Mikiciuk, M., Cichocka, J., Zakrzewskaa, H., 2011. Fluoride uptake in hydroponic culture by different clones of basket willow, Salix viminalis L. Fluoride 44, 255–259. Tripathy, S.S., Bersillon, J.L., Gopal, K., 2006. Removal of fluoride from drinking water by adsorption onto alum-impregnated activated alumina. Sep. Purif. Technol. 50, 310–317. White, P.J., Broadley, M.R., 2001. Chloride in soils and its uptake and movement within the plant: a review. Ann. Bot. 88, 967–988. WHO, 2006. Fluoride in Drinking Water. Background Document for Development of WHO Guidelines for Drinking-Water Quality. Xie, Z., Ye, Z.H., Wong, M.H., 2001. Distribution characteristics of fluoride and aluminium in soil profiles of an abandoned tea plantation and their uptake by six woody species. Environ. Int. 26, 341–346. Yumada, H., Hattori, T., 1977. Relationship between A1 and F in plants. I. About tea leaves. J. Soil Sci. Manure Jpn. 48, 253–261.
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Fluoride bioaccumulation by hydroponic cultures of camellia (Camellia japonica spp.) and sugar cane (Saccharum officinarum spp.) Nancy Camarena-Rangel a,1, Angel Natanael Rojas Velázquez b,2, María del Socorro Santos-Díaz a,⇑ a
Facultad de Ciencias Químicas de la Universidad Autónoma de San Luis Potosí, San Luis Potosí, Manuel Nava 6, 78210 San Luis Potosí, Mexico Facultad de Agronomía de la Universidad Autónoma de San Luis Potosí, San Luis Potosí, Km. 14.5 Carretera San Luis Potosí-Matehuala, Ejido Palma de la Cruz, Soledad de Graciano Sánchez, San Luis Potosí CP. 78321, Mexico b
h i g h l i g h t s We establish hydroponic cultures of camellia and sugar cane species. The capacity of these cultures to remove fluoride from water was measured. The sugarcane roots accumulated F with 86% of it absorbed and 14% adsorbed. In camellia plants the highest fluoride concentration was found in the leaves. Camelia plants bio-accumulated 74–221-fold while sugarcane 100–500-fold.
a r t i c l e
i n f o
Article history: Received 25 September 2014 Received in revised form 6 February 2015 Accepted 11 March 2015
Keywords: Fluoride Hydroponic cultures Camellia Sugar cane
a b s t r a c t The ability of hydroponic cultures of camellia and sugar cane adult plants to remove fluoride was investigated. Plants were grown in a 50% Steiner nutrient solution. After an adaptation period to hydroponic conditions, plants were exposed to different fluoride concentrations (0, 2.5, 5 and 10 mg L1). Fluoride concentration in the culture medium and in tissues was measured. In sugar cane, fluoride was mainly located in roots, with 86% of it absorbed and 14% adsorbed. Sugar cane plants removed 1000–1200 mg fluoride kg1 dry weight. In camellia plants the highest fluoride concentration was found in leaf. Roots accumulated fluoride mainly through absorption, which was 2–5 times higher than adsorption. At the end of the experiment, fluoride accumulation in camellia plants was 1000–1400 mg kg1 dry weight. Estimated concentration factors revealed that fluoride bioaccumulation is 74–221-fold in camellia plants and 100–500-fold in sugar cane plants. Thus, the latter appear as a suitable candidate for removing fluoride from water due to their bioaccumulation capacity and vigorous growth rate; therefore, sugar cane might be used for phytoremediation. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Fluoride (F) is a common environmental pollutant in groundwater. High fluoride concentrations have been reported in India (38.5 mg L1), China (>8 mg L1), Australia (9 mg L1), Mexico (11.89 mg L1), North America (3.3 mg L1), and South America (0.9–18.2 mg L1) (Fuhong and Shuqin, 1988; Paoloni et al., 2003; WHO, 2006). Additionally, human activities often lead to increased local fluoride levels. HF and particulate fluoride derive primarily from mining operations, fertilizers, chemicals, and metal ⇑ Corresponding author. Tel.: +52 (444) 8262300x6568. E-mail addresses: [email protected] (N. Camarena-Rangel), [email protected] (A.N. Rojas Velázquez), [email protected] (M.S. Santos-Díaz). 1 Tel.: +52 (444) 826 2440x6568. 2 Tel.: +52 (444) 852 40 56. http://dx.doi.org/10.1016/j.chemosphere.2015.03.071 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
processing. In particular, the use of phosphate fertilizers can increase fluoride levels in soil to as much as 5300 mg kg1 dry weight (DW) (Azbar and Turkman, 2000). Drinking water is deemed safe for human consumption if fluoride concentrations do not exceed 1 mg L1. Unfortunately, over 200 million people worldwide drink water with fluoride contents above the maximum allowable level. Excessive fluoride consumption causes dental fluorosis, and chronic intake leads to bone stiffness, rheumatism, and permanent crippling known as skeletal fluorosis (WHO, 2006; Ayoob et al., 2008). Considerable efforts have been made to lower fluoride content in drinking water. Physicochemical methods are commonly used, and can be broadly classified into membrane and adsorption techniques. Membrane techniques include reverse osmosis, nanofiltration, dialysis and electro-dialysis. Fluoride adsorption has been carried out using various adsorbents, including clay, alumina, rare-earth oxides,
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silica, carbonaceous materials, solid industrial waste, zeolites and, more recently, cellulose-supported double-layered hydroxides (Tripathy et al., 2006; Mohapatra et al., 2009). However, few adsorbents are able to lower fluoride levels below 1–1.5 mg L1, affect the taste of the treated water, and require large spaces for drying of sludge; additionally effective technologies have also drawbacks; for example, ion exchange and reverse osmosis are relatively expensive and require trained personnel for implementation (Mandal and Mayadevi, 2008). An alternative to achieve fluoride removal from water is phytoremediation, which is defined as the use of plants to reduce in situ levels of hazardous organic and inorganic pollutants from soil, sediments, water, and air. Biochemical processes carried out by plants can lead to the reduction, mineralization, degradation, volatilization, sequestration, and/or stabilization of various pollutants (Suresh and Ravishankar, 2004). A number of plants species including grasses, legumes, aquatic plants and the so-called hyperaccumulators have been used for these purposes. Field tests using plants have been conducted to remove petroleum hydrocarbons, pesticides and food waste, among others (Schnoor et al., 1997; Dushenkov et al., 1997). It has been reported that some plants are also able to take up fluoride from soil (Asada et al., 2006; Kang et al., 2008; Telesin´ski et al., 2010; Baunthiyal and Sharma, 2012; Saini et al., 2012). In addition, previous studies in our laboratory showed that hydroponic cultures of Camellia japonica, Pittosporum tobira, and Saccharum officinarum (Santos-Díaz and Zamora-Pedraza, 2010), and tree species from semiarid regions (Baunthiyal and Sharma, 2012) are able to remove fluoride from water. For the phytoextraction to be effective, vigorously growing plants are required. Thus, the purpose of this study was to explore the fluoride-uptake ability of hydroponic cultures of C. japonica spp. and S. officinarum spp. adult plants. The effects of fluoride on chlorophyll content, as well as fluoride concentration in leaves, stem and roots were determined to examine the accumulation pattern, and identify the primary tissues involved in fluoride uptake. Bioaccumulation factors were also calculated. 2. Materials and methods 2.1. Plant material Camellia (C. japonica) plants of the same age and height (60 cm) were purchased from a commercial nursery. Sugarcane (S. officinarum, SP-70-1284) plants about 1.5 m height were obtained from the ‘‘Plan de Ayala SA de CV’’ sugar mill located in Ciudad Valles, San Luis Potosí, Mexico. All plants were kept in a greenhouse (18–30 °C, 68.8% RH, light 127 lmol m2 s1) for at least 2 months; plants were irrigated twice per week during the winter, and three times per week in the summer. 2.2. Establishment of camellia and sugar cane hydroponic cultures Camellia and sugar cane plants were collected from the greenhouse; the soil was carefully removed and roots were thoroughly washed with running tap water followed by deionized water. Hydroponic cultures were established in opaque plastic containers (40 L) previously washed with 10% Dextran, rinsed with deionized water, treated with 10% HNO3 for 24 h, and rinsed three times with deionized water. A plastic mesh was fitted to each container’s mouth to provide physical support to plants, as well as to ensure that only roots were in contact with the culture medium. The nutrient solution described by Steiner (1961), prepared with deionized water, was used as growth medium; the nutrient solution (pH = 6) was continuously aerated employing a commercial
57
air pump (Elite 800). Once the plant-container assembly was prepared, containers were individually wrapped with a black plastic bag to reduce water evaporation and prevent the incidence of light on the hydroponic medium. Plants were maintained in the hydroponic solution (35 L) for 30 days for adaptation. 2.3. Fluoride removal by camellia and sugar cane hydroponic cultures Camellia and sugar cane hydroponic cultures were exposed to a NaF solution (2.5, 5 or 10 mg fluoride L1) for 28 days. Control plants were grown under the same conditions, but with no NaF supplementation. Four plants of each species were used for each treatment. A 15 ml sample of the nutrient solution was collected at 3 d intervals for 28 d, to make triplicate fluoride concentration measurements using an ion selective electrode (960 900, Thermo Orion). To calculate anion concentration, a standard curve was elaborated (n = 10) using NaF; in addition, a certificated standard of fluoride was included (High Purity Standard, 100 ppm, Charleston, SC, USA) to determine repeatability and reproducibility. The curve presented a linear correlation coefficient of 0.99, repeatability, expressed as a variation coefficient was 2.51%, and the exactitude was 98% (Standard Methods, 1998). The presence of oxidation or chlorosis spots, wilting, formation of new shoots and roots was recorded on individual plants. Total chlorophyll contents were also measured according to Bruinsma (1961), as indicators of cellular integrity. 2.4. Fluoride accumulation in camellia and sugar cane tissues Six leaves from the basal, medial and apical aerial portions of camellia plants were harvested each week for four weeks. At the same time, a single leaf from each sugar cane plant was collected and excised to obtain the basal, media and apical sections. The roots of camellia and sugar cane plants, and the stems of sugar cane plants were fully harvested on day 28. The stems were segmented into basal, media, and apical sections. Plant materials were dried at 105 °C for 24 h, pulverized, sieved through a 0.425 mm mesh sieve and kept frozen until analysis. Both adsorbed and absorbed fluoride were determined in roots. Two desorption kinetics were performed. For the first protocol, 5 mg of dried roots were placed on high-density polyethylene (HDPE) flasks and treated with 3 ml of 1 mM HNO3. Flasks were kept under continuous stirring (135 rpm) at 25 °C, and the supernatant was collected every 10 min for 300 min (Santos-Díaz and Barrón-Cruz, 2011). For the second procedure, 100 mg of dried roots were treated with 50 ml of 0.1 M HCl and incubated at 25 °C for 1 h under continuous stirring at 135 rpm (Murugan and Subramanian, 2006). The supernatant was collected every 10 min for 60 min and fluoride content in it was determined using a potenciometric method. The procedure described by Menzies et al. (1993) was used for quantifying the amount of fluoride absorbed by camellia and sugar cane leaves and roots. Roots were first washed with 1 mm HNO3 for 300 min to desorb fluoride from the surface. Concentration factors (CF) were estimated following EspinozaQuinones et al. (2005), as follows:
CF ¼ fluoride concentration in plant tissue ðmg g1 DWÞ=fluoride concentration in mediumðmg L1 Þ: 2.5. Statistical analyses Results are shown as mean ± SD (standard deviation) and were analyzed with ANOVA, using a 95% significance level, by means of the SAS software.
In previous studies, we showed that camellia and sugar cane hydroponic cultures are able to remove fluoride. However, toxic effects, including wilting and oxidation, were observed in seedlings exposed to 10 mg L1 fluoride (Santos-Díaz and Zamora-Pedraza, 2010). To improve fluoride tolerance, vigorously growing adult camellia and sugar cane plants were used in this investigation. Hydroponic cultures showed evidence of oxidation and signs of salt toxicity after 7 d in Steiner solution. Therefore, plants were transferred to a medium with reduced (50%) salt concentration. Camellia and sugar cane adult plants successfully adapted to these conditions, with no evidence of oxidation, wilting or chlorosis after 28 d (data not shown). Once the plants were fully adapted to hydroponic conditions, these were exposed to fluoride. No negative effects on morphological parameters were observed in any species, and new shoots and roots developed in sugar cane plants grown in 5 and 10 mg L1 fluoride (data not shown). Chlorophyll content was determined in order to examine the integrity of plants, as photosynthesis has been reported as being highly sensitive to various types of stress (metals, drought, nutrient depletion). In camellia leaves, the contents of total chlorophyll was not significantly different in basal, media and apical sections. However, chlorophyll concentration after 28 d decreased significantly (p < 0.5) in the presence of fluoride at all concentration levels tested (Fig. 1A), thus suggesting that prolonged exposure to this anion affects the photosynthetic process. In the sugar cane leaves, the lowest concentrations of total chlorophyll occurred in the basal section, while the highest accumulation was observed in the apical region, hence suggesting the presence of a concentration gradient. This pattern was independent of fluoride concentration. On the other hand, chlorophyll content was significantly higher (p < 0.05) in plants exposed to 2.5 and 5 mg L1 fluoride relative to control plants. The chlorophyll content of plants grown at a fluoride concentration of 10 mg L1 was similar to that of control cultures (Fig. 1B). Thus, exposure to fluoride did not affect the integrity of sugar cane tissues. 3.2. Fluoride removal by camellia and sugar cane hydroponic cultures Fig. 2A depicts fluoride removal by camellia plants. At 2.5 mg L1, fluoride concentration decreased steadily until day 18. In the presence of 5 or 10 mg L1, fluoride in the medium decreased rapidly by day 3 and then remained virtually unchanged thereafter. The amount of fluoride removed by camellia plants from the nutrient solution containing fluoride levels of 2.5, 5 or 10 mg L1 was 1.3 mg L1, 1.25 mg L1 and 3.4 mg L1, respectively. In sugar cane hydroponic cultures, fluoride was rapidly taken up during the first 3 days in media containing levels of 5 or 10 mg L1 fluoride, and after 3 days in medium with 2.5 mg L1 fluoride (Fig. 2B). Uptake continued steadily until day 9, without noticeable changes thereafter. The amount of fluoride taken up by sugar cane plants from media containing fluoride levels of 2.5, 5 and 10 mg L1 was 2.3 mg L1, 1.94 mg L1 and 4.57 mg L1, respectively. 3.3. Fluoride desorption from the root surface In order to identify which tissues were involved in fluoride accumulation, we determined fluoride concentration in leaves, stems and roots. Any fluoride adsorbed to the root surface was released by administering an acid treatment. Nitric acid has been
250
A
200 150 100 50 0 Control
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Fluoride concentration (mg L-1) Chlorophyll concentration (mg 100g -1)
3.1. Adaptation of camellia and sugar cane plants to hydroponic conditions, and quantification of chlorophyll
180
B
160 140 120 100 80 60 40 20 0 Control
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10.00
Fluoride concentration (mg L-1) Fig. 1. Content of total chlorophyll in (A) camellia, and (B) sugar cane leaves exposed to 2.5, 5 and 10 mg L1 fluoride during 28 days. Basal (j), medium (h) and apical ( ).
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Fluoride in medium (mg l-1)
3. Results
Chlorophyll concentration (mg 100g -1)
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A
10 8 6 4 2 0 0
3
6
9
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Fluoride in medium (mg l -1)
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B
10 8 6 4 2 0 0
3
6
9
12
15
18
21
24
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Time (days) Fig. 2. Fluoride removed from the medium by hydroponic cultures of (A) camellia and (B) sugar cane. Control (), 2.5 mg L1 (j), 5 mg L1 (N) and 10 mg L1 (d) fluoride.
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Fluoride desorbed (mg kg-1)
1200 1000 800 600 400 200 0 0
10
20
30
40
50
60
70
Time (minutes) Fig. 3. Desorption of fluoride from sugar cane roots using HCl 0.1 M. Control (), 2.5 mg L1 (j), 5 mg L1 (N) and 10 mg L1 (d) fluoride.
Fluoride desorbed (mg kg -1)
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250 200 150 100 50 0 0
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Time (minutes) Fig. 4. Desorption of fluoride from (A) sugar cane and (B) camellia roots using HNO3 1 mM. Control (), 2.5 mg L1 (j), 5 mg L1 (N) and 10 mg L1 (d) fluoride.
reported to release heavy metals from roots (Santos-Díaz and Barrón-Cruz, 2011); likewise, HCl is effective in releasing fluoride from plant materials packed in columns (Murugan and Subramanian, 2006). In this work we tested both HCl and HNO3, and the respective release kinetics were examined to determine the time needed to achieve fluoride desorption from root surfaces. HCl-treated sugar cane roots displayed a continuous fluoride release at all concentrations tested, and did not reach a stationary phase after 60 min (Fig. 3). This treatment also caused tissue discoloration and fragmentation, thus indicating that the procedure was too aggressive. Fluoride release was also observed in the roots of control plants, thus suggesting that those plants had been previously in contact with fluoride, probably through irrigation water. On the other hand, the amount of fluoride desorbed with HNO3 from sugar cane roots was proportional to fluoride concentration in the medium (at 5 and 10 mg L1), increased gradually for 150 min, and stabilized by minute 300 (Fig. 4A). Fluoride desorption at 2.5 mg L1 became virtually constant after 60 min. In camellia roots exposed to 5 or 10 mg L1 fluoride, a rapid desorption with HNO3 was observed for the first 30 min (Fig. 4B). Then, a steady release occurred until minute 120, with no further relevant variations thereafter. Fluoride release from roots of plants grown in 2.5 mg L1 fluoride was slower and peaked by minute 120, with no further noticeable changes thereafter. The use of HNO3 caused no apparent effect on the roots of sugar cane or camellia plants and, therefore, this procedure was selected for further experiments. To note, the roots of camellia and sugar cane control plants released 50 and 200 lg fluoride g1 of tissue, respectively, hence indicating their previous contact with fluoride. 3.4. Fluoride accumulation in camellia and sugar cane tissues Table 1 shows the amount of fluoride accumulated in different tissues of camellia plants after 28 days. The largest accumulation occurred in leaves, but the final concentration was similar in both control and fluoride-exposed plants (p > 0.05). However, marked differences were observed in roots. Fluoride concentrations were about 3–4 higher (p < 0.05) in fluoride-exposed than in control plants. The amount of fluoride absorbed within roots was not statistically different in plants grown in 2.5 or 5 mg L1 fluoride and in control plants, but fluoride concentration almost doubled in plants exposed to 10 mg L1. Camellia roots accumulated fluoride mainly through absorption, which was 2–5 times the amount of fluoride accumulated through adsorption. At the end of the experiment, camellia plants had accumulated some 1000–1400 mg fluoride kg1 DW. In the sugar cane plants, no statistically significant differences (p < 0.05) were found in fluoride content between basal, medial, and apical sections of stems (Fig. 5A); total fluoride content in stems was similar in non-exposed and exposed plants (Table 1). An accumulation gradient was observed in leaves (Fig. 5B), as
Table 1 Accumulated fluoride in tissues of hydroponic cultures at 28 days. Species
Fluoride (mg L)
Stem (mg kg1)
Leaf (mg kg1)
Root Absorbed (mg kg
1
)
1
Adsorbed (mg kg
Total (mg kg1)
CF1
)
Sugar cane
0 2.5 5 10
52 ± 2.3a 46 ± 0.4a 48 ± 0.79a 47 ± 0.56a
67 ± 0.98a 55 ± 1.0a 86 ± 15.5b 120 ± 25b
443 ± 5.3d 985 ± 20.5a 634 ± 66.3b 521 ± 25.3c
124 ± 87.3d 159 ± 92.1c 261 ± 169.2b 325 ± 152.8a
686 ± 96.0b 1245 ± 114a 1030 ± 216 a 1012 ± 204.1a
1 497.94 205.97 101.99
Camellia
0 2.5 5 10
– – – –
741 ± 25a 768 ± 9.5a 760 ± 20a 697 ± 8.3a
273 ± 1.8b 363 ± 4.3b 275 ± 9.3b 553 ± 4.6a
45 ± 12c 190 ± 6.4a 114 ± 47b 192 ± 71a
1059 ± 38.8a 1321 ± 20.2b 1149 ± 76.3a 1442 ± 83.9c
1 221.2 77.8 74.5
Different letters in the same column are statistically different (Tukey test, p < 0.05) 1 CF: concentration factor.
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Fluoride absorbed (mg kg-1)
60
A
b
a
20
b
b
b
b
b
b
b
15
b
b b
10 5 0 Control
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5
Fluoride absorbed (mg kg-1)
10
35 30 25 20 15 10 5 0
B
a b
a a
ba
ba
a
ba
ba
b b b
Control
2.5
5
10
Fig. 5. Accumulation of fluoride in stems (A) and leaf (B) from sugar cane. Basal basal (h), medium ( ) and apical (j) sections.
higher fluoride levels (p < 0.05) were recorded in basal vs. apical sections. Leaves of plants grown in media containing 5 and 10 mg L1 fluoride had total fluoride levels higher than those in control plants (Table 1). Most of the fluoride removed by sugar cane hydroponic cultures was located in roots, with 86% being absorbed and 14% adsorbed. Sugar cane plants removed between 1000 and 1200 mg fluoride kg1 DW. 4. Discussion Phytoremediation is not the only alternative available for removing pollutants, but it offers substantial economic advantages over other systems. Conventional remediation methods cost between $10 and $1000 US dollars (USD) per cubic meter, while the estimated cost of phytoextraction may be as low as $0.05 USD per cubic meter (Ghosh and Singh, 2005), making of this a very appealing option. For a practical phytoremediation treatment, a vigorously-growing, easily harvested plant that accumulates large amounts of the pollutant is desired (Suresh and Ravishankar, 2004). Therefore, in this study, actively-growing adult camellia and sugar cane plants were used instead of seedlings. Camellia plants grown in fluoride-containing media did not show evidence of toxic effects. Nevertheless, a small—but significant— reduction in chlorophyll content was observed after 28 days at all the fluoride levels tested. Thus, chlorophyll content was a better indicator of cellular integrity than other parameters such as oxidation or wilting. Camellia species have been previously reported to display a high fluoride tolerance (Ruan et al., 2003, 2004); however, those studies were performed using Camellia sinensis grown in soil where fluoride availability is affected by the chemical complexity of the substrate. Under hydroponic conditions, no restrictions for fluoride uptake occur. Toxic effects of fluoride have also been described in almond (Elloumi et al., 2005), sorghum (Schender and Maclean, 1970) and barley (Gautam and Bhardwaj, 2010). Sugar cane plants were not affected by fluoride; moreover, plants grown in medium with 5 or 10 mg L1 fluoride showed a
higher chlorophyll concentration than control plants and developed new shoots and roots, thus confirming the tolerance of sugar cane to fluoride. Camellia plants took up fluoride faster than sugar cane plants, removing up to 40–50% of the anion available in the medium by the 3th day, while sugar cane required about 6 days to achieve similar results. In order to identify which tissue was mainly involved in fluoride accumulation, the fluoride content in leaves, stem and roots was measured. Camellia and sugar cane roots internalized about 65–85% of all the fluoride taken. However, the amount of fluoride accumulated in sugar cane roots grown in medium containing 2.5 and 5 mg L1 fluoride was 2–2.3 times higher than the amount accumulated in camellia roots, and 1.2 times higher when grown in 10 mg L1 fluoride (Table 1). It has been suggested that fluoride uptake by roots is a passive process involving an extracellular pathway traversing the endodermal barrier at either the root tips or the endodermis where lateral roots are formed, following the transpiration stream (Stevens et al., 1998). However, participation of other mechanisms, for example anion channels, cannot be ruled out. Anion channels are integral proteins located in the cell membrane, tonoplast, endoplasmic reticulum, mitochondria, and chloroplasts. It has been reported that the slow-activation (S-type) anion channels show differential selectivity according to the following order: NO 3 > Br > F > Cl > I > malate (Schmidt and Schroeder, 1994; Lurin et al., 2000; White and Broadley, 2001; Clausen et al., 2004). Thus, S-type anion channels are able to transport fluoride with moderate selectivity and might be involved in fluoride uptake. Further research is necessary to determine whether these channels are present in sugar cane and camellia roots and whether they participate in fluoride transport. On the other hand, the amount of fluoride adsorbed onto the sugar cane root surface increased proportionally to its concentration in the medium. In camellia roots, the amount of fluoride adsorbed was 2–3 times higher than in control plants, but no clear relationship with concentration could be observed. Fluoride adsorbed onto roots might be attached to cell-wall components, such as calcium, or to ionizable compounds. Previous studies show that several species from semiarid regions accumulated fluoride in cytosolic and cell-wall fractions (Baunthiyal and Sharma, 2012). It is well known that the ionic and electric properties of the cell wall are mainly due to the dissociation of weak acids. In monocotyledon roots, galactouronic acids, carboxylic acids, and phenolics are particularly abundant. As galactouronic acids are more readily ionizable than carboxylic acids and phenolics, they are likely involved in fluoride adsorption (Meychik, 2001; Meychik et al., 2010). Another possibility is the binding of fluoride to OH-rich arabinoxylanes. Burke et al. (1974) showed that arabinoxylanes are the major components of cell walls obtained from a sugar cane cell suspension. Thus, fluoride ions might replace the –OH group in arabinoxylanes, in a similar way as they replace the –OH group from hydroxyapatite in bone. Further experiments are required to identify which cell-wall components are involved in fluoride uptake. In camellia plants, the highest fluoride concentration was found in leaves; this, however, did not seem to be the result of translocation from the hydroponic medium, since fluoride levels were similar in both control plants and plants grown in fluoridesupplemented media. Camellia plants were likely in contact with fluoride in irrigation water and accumulated it throughout their development. Fluoride may also have deposited on the leaf surface from air pollution. Gaseous fluoride is an important phytotoxic air pollutant, which is absorbed through leaf stomata and moves in the transpiration stream to the leaf tip and margins. High fluoride accumulation in leaves has been attributed to air pollution in several species (Horntvedt, 1997; Domingos et al., 2003). To rule out this possibility, leaves must be washed prior to fluoride quantification. This is a subject that deserves further investigation.
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Fig. 6. Fluoride accumulation in sugarcane plants. Fluoride adsorbed (d) and absorbed ( ). Plants exposed to 2.5 mg l1 accumulated fluoride mainly inside the roots and a lower content remained adsorbed to root surface. At 5 and 10 mg l1 the content of fluoride adsorbed increase 1.5–2.1 folds and initiate the translocation of fluoride to aerial parts.
Large amounts of fluoride being translocated to mature camellia leaves have been reported. (Yumada and Hattori, 1977; Xie et al., 2001; Ruan et al., 2003). However, those studies were conducted with a different species (C. sinensis) an under different conditions, i.e. plants were grown in soil and in the presence of aluminum, a cation that improves fluoride translocation to shoots. On the other hand, fluoride concentration in sugar cane leaves was 1.3 and 1.8 times higher in plants grown in 5 and 10 mg L1, respectively, relative to control plants, suggesting that fluoride was translocated to aerial parts. Moreover, a concentration gradient was observed, with apical sections having a higher fluoride content than basal sections. Increased fluoride levels in leaves were coupled with a reduction in the amount of fluoride absorbed in roots, thus confirming that fluoride was translocated. Fig. 6 presents a putative diagram of fluoride accumulation in sugar cane plants. The amount of fluoride taken up by sugar cane and camellia adult plants at the end of the experiment was 6–10 times higher than the amount removed by seedlings in previous studies (Santos-Díaz and Zamora-Pedraza, 2010). Both species have a moderate capacity to accumulate fluoride (1200–1400 g kg1) in comparison to other species. For example, spinach, amaranth, cabbage, okra and tomato plants irrigated with water containing 10 mg L1 fluoride accumulated 1.7, 20.9, 0.17 and 0.2 g kg1 DW, respectively (Khandare and Rao, 2006); barley plants exposed to 20 mg L1 accumulated 13 g kg1 (Gautam and Bhardwaj, 2010); Acacia tortilis, Cassia fistula and Prosopis juliflora took up about 1200 g kg1 (Baunthiyal and Sharma, 2012); hybrid willow, black willow and basket willow accumulated over 5000 g kg1 (Kang et al., 2008; Telesin´ski et al., 2011). While willow species seem suitable for phytoremediation studies, their growth rate is lower than that of sugar cane, which can grow up to 2–3 m per year. Finally, CFs were calculated for both species. As fluoride content in camellia leaves was similar in control and fluoride-exposed plants, these data were not included in the CF calculation. Our data showed that camellia plants bioaccumulate fluoride 74–221 times, while sugar cane plants do it 100–500 times (Table 1). In both species, CF decreased proportionally as fluoride concentration in the medium increased, suggesting that the efficiency of cellular mechanisms to remove fluoride decreases when anion concentration increased. Sugar cane plants exposed to 2.5 mg L1 accumulated fluoride mainly inside the roots and a lower content remained adsorbed to root surface. At 5 and 10 mg L1 the content of fluoride adsorbed
increase 1.5–2.1 folds and initiate the translocation of fluoride to aerial parts. Since, the sugar cane hydroponic cultures exposed to 2.5 mg L1 removed 93% of fluoride, this species could be useful to reduce F from water containing 62.5 mg L1 F. It is well known that among 1.5–3 mg L1 fluoride in drinking water causes dental fluorosis. According to WHO (2006), around 70 million persons, distributed in China, India, Africa and eastern Mediterranean regions, suffer dental fluorosis. Then, the use of sugar cane hydroponic cultures could reduce the incidence of this affection. As sugar cane is cultivated in great extensions, the disposal of plant material would not be a problem for their installation in small containers, or for studies on artificial wetlands. These options must be explored in more detail in further studies. In addition, the use of sugar cane plants could be combined with others methodologies, as osmosis reverse, improving the half life of filters and reducing costs. 5. Conclusions The highest concentration of fluoride in camellia plants was found in leaves but this accumulation was not the result of a translocation process since similar levels of fluoride were detected in non-exposed and exposed plants. This specie bio-concentrated fluoride between 74 and 221 folds. On the contrary, the sugar cane plants bio-accumulate the anion mainly in roots where 86% is internalized and 14% is adsorbed. This species translocated fluoride to leaves at 5 and 10 mg L1 and concentrate the anion between 100 and 500 folds. Taking into account its bio-accumulation capacity and fast growth rate the sugar cane plants can be used for phytoremediation purposes. Acknowledgements The authors thank Dra. María Degracias Ortiz Pérez for her valuable comments to the work; and to CONACYT for a scholarship granted to NCR (365990). References Asada, M., Parkpian, P., Horiuchi, S., 2006. Remediation technology for boron and fluoride contaminated sediments using green plants. In: Fukue, M., Kita, K., Ohtsubo, M., Chaney, R. (Eds.), Contaminated Sediments: Evaluation and Remediation Techniques. ASTM, West Conshohocken PA, USA.
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