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International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Cr localization and speciation in roots of chromate fed Helianthus annuus L. seedlings using synchrotron techniques a
b
c
Guadalupe de la Rosa , Hiram Castillo-Michel , Gustavo Cruz-Jiménez , Jesús Bernala
a
d
Alvarado , Teodoro Córdova-Fraga , Laura López-Moreno & Marine Cotte
b
a
División de Ciencias e Ingenierías , Universidad de Guanajuato , Loma del Bosque 103, Col. Lomas del Campestre, C.P. 37150, León , Gto. , México b
European Synchrotron Radiation Facility , B.P. 220—38043 Grenoble Cedex, France
c
División de Ciencias Naturales y Exactas , Universidad de Guanajuato , Col. N. Alta s/n C.P. 36050, Guanajuato , Gto. , México d
University of Puerto Rico at Mayagüez , P.O. Box 9019, Mayagüez , P.R. , 00681–9019 Accepted author version posted online: 09 Jul 2013.Published online: 09 Jul 2013.
To cite this article: International Journal of Phytoremediation (2013): Cr localization and speciation in roots of chromate fed Helianthus annuus L. seedlings using synchrotron techniques, International Journal of Phytoremediation, DOI: 10.1080/15226514.2013.810584 To link to this article: http://dx.doi.org/10.1080/15226514.2013.810584
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
ACCEPTED MANUSCRIPT Cr localization and speciation in roots of chromate fed Helianthus annuus L. seedlings using synchrotron techniques Guadalupe de la Rosaa*, Hiram Castillo-Michelb, Gustavo Cruz-Jiménezc, Jesús BernalAlvaradoa, Teodoro Córdova-Fragaa, Laura López-Morenod, Marine Cotteb
Downloaded by [Dalhousie University] at 04:36 13 November 2013
a
División de Ciencias e Ingenierías, Universidad de Guanajuato, Loma del Bosque 103, Col.
Lomas del Campestre, C.P. 37150, León, Gto., México b
European Synchrotron Radiation Facility, B.P. 220 – 38043 Grenoble Cedex, France
c
División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Col. N. Alta s/n C.P.
36050, Guanajuato, Gto., México d
University of Puerto Rico at Mayagüez, P.O. Box 9019, Mayagüez, P.R. 00681-9019
RUNNIG HEAD: Cr localisation and speciation in H. annuus L. roots
* Corresponding author Tel and fax: 52(477)7885100; E-mail: [email protected] and [email protected]
Abstract
In order to gain knowledge on the potential use of Helianthus annnuus L. for the remediation of Cr(VI) polluted waters, hydroponics experiments were set up to determine Cr uptake and tolerance in different Cr(VI)-sulfate conditions, and Cr biotransformations. Results indicated that Cr(VI) promoted seed germination, and plant tolerance was higher at younger
1
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT plant stages. Cr uptake was dependent on sulfate concentrations. The highest Cr levels in roots and shoots (13,700 and 2,500 mg kg -1 dry weight (DW), respectively) were obtained in 1 mM sulfate. The lowest Cr uptake in roots (10,600 mg kg-1DW) was observed in seedlings treated with no sulfate. In shoots, this number was of 1,500 mg kg -1DW for the 1 mM sulfate treatment, indicating a different level of interaction between chromate and sulfate in both tissues. For the
Downloaded by [Dalhousie University] at 04:36 13 November 2013
first time, using micro X-ray florescence (µXRF), we demonstrated Cr reaches the root stele and is located in the walls of xylem vessels. Bulk and micro X-ray Absorption Near-Edge Structure (µXANES) results showed that Cr in the roots is mostly in the form of Cr(III) phosphate (80%), with the remainder complexed to organic acids. Our results suggest this plant species may serve for Cr(VI) rhizofiltration purposes.
Keywords: Cr(VI) biotransformation, µXAS, µXRF, H. annuus L.
1. Introduction The detrimental effects of hexavalent chromium [Cr(VI)] in living organisms are well documented (Gruber and Jenette, 1978; Shanker et al. 2005; Ali et al. 2010; D´ors et al. 2010). Due to its high toxicity, Cr(VI) should be promptly removed from contaminated media when detected. In leather tanning, one of the main anthropogenic sources of Cr contamination in wastewaters, Cr(III) salts are used to cross link the collagen protein chains (Abreu and Toffoli 2009). Even though Cr(III) is less toxic than Cr(VI), the former could eventually be oxidized to Cr(VI), posing a threat to environmental and human health (Congzheng et al. 2007). In order to remove this pollutant, rhizofiltration –the use of plant roots to reduce the presence of
2
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT contaminants in waters- represents an economically feasible option (Dushenkov et al. 1995). In plant species intended as rhizofiltrators, the ability to accumulate and tolerate elevated concentrations of contaminants is highly desirable (Dushenkov and Kapulnik, 2000). To measure tolerance, the effects of pollutants on seed germination, plant growth, chlorophyll production, and physiological distress are used as indicators (Sundaramoorthy et al. 2010; Redondo-Gómez
Downloaded by [Dalhousie University] at 04:36 13 November 2013
et al. 2011). In addition, potential biotransformations of the contaminants, and the effect of plant age, nutrient concentration, and other elements on metal absorption and tolerance are important (Peralta et al. 2004; De la Rosa et al. 2008). Unfortunately, this information is scarce. Regarding the mechanisms of metal uptake, chromate transport through sulfate carriers has been reported in some plant species (Kleiman and Cogliatti 1997; Schiavon et al. 2007, 2008). Thus, sulfate concentration in the growth media is an important parameter to consider. As per biotransformation -the chemical conversion of a substance by means of a living organism or its enzymes (Stephenson et al., 2006)- the use of synchrotron techniques has allowed to determine the reduction of Cr(VI) to Cr(III) in several plant species(Aldrich et al. 2003; Zhao et al. 2009; Mongkhonsin et al. 2011). Salsola kali L, Prosopis spp, and Prosopis laevigata (Humb. & Bonpl. ex Willd) are plant species that have proven Cr tolerance and/or potential hyperaccumulation (BuendíaGonzález et al. 2010; Aldrich et al. 2003; Gardea-Torresdey et al. 2005). However, they are desert plants and should not be introduced in other ecosystems. Helianthus annuus L. (sunflower), a crop cultivated in the state of Guanajuato in Mexico, may be suitable for Cr phytoremediation (Sudha et al. 2009). We are especially interested in exploring options for the rhizofiltration of tannery wastewaters containing Cr. Thus, in an effort to identify new
3
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT alternatives for Cr remediation in central Mexico, as well as to gain information on mechanistic aspects including biotransformation, the aim of this research was to study the effects of growth stage and sulfate concentration on Cr(VI) uptake and tolerance by H. annuus; Cr biotransformation and microdistribution in roots was studied using X-ray absorption spectroscopy (XAS) and X-ray Fluorescence microscopy (microXRF). These studies were set in
Downloaded by [Dalhousie University] at 04:36 13 November 2013
hydroponics to explore the potential use of H. annuus for the rhizofiltration of Cr(VI).
2. Material and Methods Experiments were performed using Helianthus annuus L. seeds, Sunbright supreme var., purchased at Sakata Seed AmericaTM (Morgan Hill, CA, USA)
2.1 Preparation of the hydroponics solutions A Hoagland modified nutrient solution was prepared as by Peralta et al. (2001) with minimum modifications. Growth medium contained: Ca(NO3)2·4H2O (0.35 mM); CaCl2·2H2O (2.1 mM); Mg(NO3)2·6H2O (0.91 mM); KH2PO4 (0.97 mM); KNO3 (0.255 mM) H3BO3 (23.13 µM); MnCl2·4H2O (3.9 µM); MoO3 (23.13µM); CuNO3 (0.44 µM); Fe(NO3)3·9H2O (10 µM); and Zn(NO3)2·6H2O (0.37 µM). When appropriate, MgSO4 was used as sulfate source. A 19.2 mM (1000 mg L-1) Cr(VI) solution was prepared using K2CrO4. Proper dilutions were performed using the nutrient solution to obtain different Cr(VI) concentrations, and pH was adjusted to 4.8. Reagents were purchased from Sigma-Aldrich (St. Louis, MO).
2.2 Effect of Cr(VI) on seed germination
4
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT For this study, H. annuus seeds were disinfected for 30 min in 2% w/v sodium hypochlorite followed by rinsing 3 times with sterilized deionized (DI) water. After disinfection, seeds were placed on Petri dishes with paper soaked in nutrient solution containing different Cr(VI) concentrations (0, 0.09, 0.19, 0.38, 0.77, and 1.5 mM). Dishes were covered with aluminum paper to avoid light exposure and germination was performed at 25 °C. For statistical
Downloaded by [Dalhousie University] at 04:36 13 November 2013
purposes, experiment was conducted in triplicate with 30 seeds per replicate per treatment. After four days, germination was measured as the percent of seedlings having a radicle of at least 2 mm. 2.3 Effect of the growth stage on plant tolerance to Cr(VI) In this experiment, H. annuus was exposed to Cr(VI) at different stages. For this purpose, seeds were germinated in rolled paper and placed on Mason Jars containing nutrient solution (Carrillo-Castañeda et al. 2002). After germination, seedlings at different ages (4, 8, 16, and 20 days) were exposed to 0.19 mM Cr(VI). These ages were selected according to De la Rosa et al. (2008) for comparison purposes. A separate set of seeds was germinated in rolled paper imbibed in nutrient solution containing 0.19 mM Cr(VI). Seedlings were maintained in the treatments with a light photoperiod of 12/12 h, at 25 °C and 39.5 µmol m-2 s-1 photon irradiance until visible signs of toxicity appeared (loss of turgidity, chlorosis). Experiment was conducted in triplicate with 10 seedlings per replicate per treatment.
2.4 Effect of sulfate concentration on Cr(VI) uptake and tolerance Seeds of H. annuus were germinated in paper as previously described. The nutrient solution used for paper imbibition contained 0.19 mM Cr(VI) added with either 0, 0.1, 1, or 10
5
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT mM sulfate (SO42-). Sulfate solutions were prepared from MgSO4 according to Kaszycki et al. (2005). After four days, seedlings were transferred to a hydroponics system (water culture) containing the same Cr(VI)-sulfate concentrations used for germination with a light photoperiod of 12/12, at 25 °C and a photon irradiance of 39.5 µmol m-2 s-1. Root and shoot sizes of ten plants per triplicate per treatment were measured after 15 days of exposure in order to determine the
Downloaded by [Dalhousie University] at 04:36 13 November 2013
effect of treatments on plant growth. Roots were measured from the main root tip to the crown. Stems were measured from the crown to the main apex.In addition, seedlings were sampled every 3 days for 15 days in order to determine Cr absorption as a function of time and sulfate concentration in the media. After harvesting, plant roots were rinsed with 0.01M HNO3 and deionized water. Roots and shoots were separated, oven-dried at 65°C for 72 h, and prepared for acid digestion and metal quantification.
2.5 Quantification of Cr in plant tissues Plant samples were manually ground using a mortar and pestle and passed through an 18mesh screen. Subsequently, a sample of 100 mg was acid digested using trace pure HNO3 and H2O2 as per Kalra (1998). The determination of Cr concentrations in tissues was performed using a flame atomic absorption spectrometer (Perkin Elmer Aanalyst 100, New Jersey, USA). The equipment was setup as follows: λ=228 nm, slit 0.7 and lamp current of 15mA. The Reference Standard Material used for QC/QA analysis was tomato leaves (SRM-1573), chromium recovery was 92%. Detection limit for Cr was of 0.1 mg L-1.
2.6 Bulk and microXANES studies
6
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Bulk-XAS was performed on beam line 7-3 at Stanford Synchrotron Radiation Lightsource (SSRL, Palo Alto, CA). Roots of H. annuus exposed to 0.19 mM Cr(VI) and 1 mM sulfate for 15 days were frozen in liquid nitrogen and lyophilized using a Freezone 4.5 freeze dryer at -45ºC and 70 × 10-3 mbar (Labconco, Kansas City, MO). Samples were ground, loaded in aluminum sample holders, and sealed with ®Kapton tape. Spectra of sample and Cr model
Downloaded by [Dalhousie University] at 04:36 13 November 2013
compounds were collected at the Cr-K edge (E0 5.989 keV). SSRL beamline was operated with a Si (111) double crystal monochromator, and a 1 × 10 mm slit. The output was detuned by 30% to reduce higher order harmonics. Sample spectrum was collected in fluorescence mode using a 30 element Canberra Ge detector. A chromium foil [Cr(0)] was used as a calibration standard to determine the correct sample edge energy. Experiments at the European Synchrotron Radiation Facility were done in beamline ID21. Roots of H. annuus exposed to 0.19 mM Cr(VI) and 1 mM sulfate were rinsed with 0.01M HNO3 and deionized water, immersed into Tissue Tek resin, and rapidly frozen in liquid nitrogen. After embedded in resin, the samples were axially sectioned at 25 µm thick using a cryomicrotome, freeze dried and mounted in between Ultralene window film. MicroXRF mapping of Cr K-edge was performed with an incident beam at 6.1KeV during the continuous operation mode. The beam was focused to a size of 0.300 x 0.700 µm² (VxH) using a Fresnel zone plate. The fluorescence signal was detected by a large Si drift detector (Bruker). Two photodiodes were used to measure the incident and transmitted beam intensities. Dwell time and distance of the detector were optimized for each XRF map maintaining the dead time below 15%. The XRF data was processed using the PyMCA software (Solé et al. 2007). The Cr, S and P images were obtained by fitting each pixel in the XRF maps. Thus, the net counts in the images
7
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT are those of the elements of interest. The S to Cr and P to Cr ratios were calculated using the fitted images. For microXANES data acquisition, the energy was selected using a Si111 monochromator and scanned from 5980 to 6090 eV. The zone plate was translated in the beam axis in order to maintain the beam focus. XANES data analysis was carried out using the Athena software (Ravel and Newville 2005). XANES spectra from samples were fitted using the linear
Downloaded by [Dalhousie University] at 04:36 13 November 2013
combination procedure provided in the Athena software. Reference materials were chemical grade reagents analyzed as fine powder pellets in transmission and fluorescence mode. 2.7 Statistical analyses For this investigation, completely randomized design experiments were used. For statistical purposes, three replicates per treatment were set up. Data was analyzed using the statistical package SPSS 11.0 (Chicago, IL, USA). One way ANOVA and Tukey tests were performed to determine statistically significant differences between treatment means.
3. Results and Discussion 3.1 Effect of Cr(VI) on seed germination Germination rate in seeds treated with Cr(VI) at 0.09, 0.19, and 0.38 mM, significantly increased (P
International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Cr localization and speciation in roots of chromate fed Helianthus annuus L. seedlings using synchrotron techniques a
b
c
Guadalupe de la Rosa , Hiram Castillo-Michel , Gustavo Cruz-Jiménez , Jesús Bernala
a
d
Alvarado , Teodoro Córdova-Fraga , Laura López-Moreno & Marine Cotte
b
a
División de Ciencias e Ingenierías , Universidad de Guanajuato , Loma del Bosque 103, Col. Lomas del Campestre, C.P. 37150, León , Gto. , México b
European Synchrotron Radiation Facility , B.P. 220—38043 Grenoble Cedex, France
c
División de Ciencias Naturales y Exactas , Universidad de Guanajuato , Col. N. Alta s/n C.P. 36050, Guanajuato , Gto. , México d
University of Puerto Rico at Mayagüez , P.O. Box 9019, Mayagüez , P.R. , 00681–9019 Accepted author version posted online: 09 Jul 2013.Published online: 09 Jul 2013.
To cite this article: International Journal of Phytoremediation (2013): Cr localization and speciation in roots of chromate fed Helianthus annuus L. seedlings using synchrotron techniques, International Journal of Phytoremediation, DOI: 10.1080/15226514.2013.810584 To link to this article: http://dx.doi.org/10.1080/15226514.2013.810584
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
ACCEPTED MANUSCRIPT Cr localization and speciation in roots of chromate fed Helianthus annuus L. seedlings using synchrotron techniques Guadalupe de la Rosaa*, Hiram Castillo-Michelb, Gustavo Cruz-Jiménezc, Jesús BernalAlvaradoa, Teodoro Córdova-Fragaa, Laura López-Morenod, Marine Cotteb
Downloaded by [Dalhousie University] at 04:36 13 November 2013
a
División de Ciencias e Ingenierías, Universidad de Guanajuato, Loma del Bosque 103, Col.
Lomas del Campestre, C.P. 37150, León, Gto., México b
European Synchrotron Radiation Facility, B.P. 220 – 38043 Grenoble Cedex, France
c
División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Col. N. Alta s/n C.P.
36050, Guanajuato, Gto., México d
University of Puerto Rico at Mayagüez, P.O. Box 9019, Mayagüez, P.R. 00681-9019
RUNNIG HEAD: Cr localisation and speciation in H. annuus L. roots
* Corresponding author Tel and fax: 52(477)7885100; E-mail: [email protected] and [email protected]
Abstract
In order to gain knowledge on the potential use of Helianthus annnuus L. for the remediation of Cr(VI) polluted waters, hydroponics experiments were set up to determine Cr uptake and tolerance in different Cr(VI)-sulfate conditions, and Cr biotransformations. Results indicated that Cr(VI) promoted seed germination, and plant tolerance was higher at younger
1
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT plant stages. Cr uptake was dependent on sulfate concentrations. The highest Cr levels in roots and shoots (13,700 and 2,500 mg kg -1 dry weight (DW), respectively) were obtained in 1 mM sulfate. The lowest Cr uptake in roots (10,600 mg kg-1DW) was observed in seedlings treated with no sulfate. In shoots, this number was of 1,500 mg kg -1DW for the 1 mM sulfate treatment, indicating a different level of interaction between chromate and sulfate in both tissues. For the
Downloaded by [Dalhousie University] at 04:36 13 November 2013
first time, using micro X-ray florescence (µXRF), we demonstrated Cr reaches the root stele and is located in the walls of xylem vessels. Bulk and micro X-ray Absorption Near-Edge Structure (µXANES) results showed that Cr in the roots is mostly in the form of Cr(III) phosphate (80%), with the remainder complexed to organic acids. Our results suggest this plant species may serve for Cr(VI) rhizofiltration purposes.
Keywords: Cr(VI) biotransformation, µXAS, µXRF, H. annuus L.
1. Introduction The detrimental effects of hexavalent chromium [Cr(VI)] in living organisms are well documented (Gruber and Jenette, 1978; Shanker et al. 2005; Ali et al. 2010; D´ors et al. 2010). Due to its high toxicity, Cr(VI) should be promptly removed from contaminated media when detected. In leather tanning, one of the main anthropogenic sources of Cr contamination in wastewaters, Cr(III) salts are used to cross link the collagen protein chains (Abreu and Toffoli 2009). Even though Cr(III) is less toxic than Cr(VI), the former could eventually be oxidized to Cr(VI), posing a threat to environmental and human health (Congzheng et al. 2007). In order to remove this pollutant, rhizofiltration –the use of plant roots to reduce the presence of
2
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT contaminants in waters- represents an economically feasible option (Dushenkov et al. 1995). In plant species intended as rhizofiltrators, the ability to accumulate and tolerate elevated concentrations of contaminants is highly desirable (Dushenkov and Kapulnik, 2000). To measure tolerance, the effects of pollutants on seed germination, plant growth, chlorophyll production, and physiological distress are used as indicators (Sundaramoorthy et al. 2010; Redondo-Gómez
Downloaded by [Dalhousie University] at 04:36 13 November 2013
et al. 2011). In addition, potential biotransformations of the contaminants, and the effect of plant age, nutrient concentration, and other elements on metal absorption and tolerance are important (Peralta et al. 2004; De la Rosa et al. 2008). Unfortunately, this information is scarce. Regarding the mechanisms of metal uptake, chromate transport through sulfate carriers has been reported in some plant species (Kleiman and Cogliatti 1997; Schiavon et al. 2007, 2008). Thus, sulfate concentration in the growth media is an important parameter to consider. As per biotransformation -the chemical conversion of a substance by means of a living organism or its enzymes (Stephenson et al., 2006)- the use of synchrotron techniques has allowed to determine the reduction of Cr(VI) to Cr(III) in several plant species(Aldrich et al. 2003; Zhao et al. 2009; Mongkhonsin et al. 2011). Salsola kali L, Prosopis spp, and Prosopis laevigata (Humb. & Bonpl. ex Willd) are plant species that have proven Cr tolerance and/or potential hyperaccumulation (BuendíaGonzález et al. 2010; Aldrich et al. 2003; Gardea-Torresdey et al. 2005). However, they are desert plants and should not be introduced in other ecosystems. Helianthus annuus L. (sunflower), a crop cultivated in the state of Guanajuato in Mexico, may be suitable for Cr phytoremediation (Sudha et al. 2009). We are especially interested in exploring options for the rhizofiltration of tannery wastewaters containing Cr. Thus, in an effort to identify new
3
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT alternatives for Cr remediation in central Mexico, as well as to gain information on mechanistic aspects including biotransformation, the aim of this research was to study the effects of growth stage and sulfate concentration on Cr(VI) uptake and tolerance by H. annuus; Cr biotransformation and microdistribution in roots was studied using X-ray absorption spectroscopy (XAS) and X-ray Fluorescence microscopy (microXRF). These studies were set in
Downloaded by [Dalhousie University] at 04:36 13 November 2013
hydroponics to explore the potential use of H. annuus for the rhizofiltration of Cr(VI).
2. Material and Methods Experiments were performed using Helianthus annuus L. seeds, Sunbright supreme var., purchased at Sakata Seed AmericaTM (Morgan Hill, CA, USA)
2.1 Preparation of the hydroponics solutions A Hoagland modified nutrient solution was prepared as by Peralta et al. (2001) with minimum modifications. Growth medium contained: Ca(NO3)2·4H2O (0.35 mM); CaCl2·2H2O (2.1 mM); Mg(NO3)2·6H2O (0.91 mM); KH2PO4 (0.97 mM); KNO3 (0.255 mM) H3BO3 (23.13 µM); MnCl2·4H2O (3.9 µM); MoO3 (23.13µM); CuNO3 (0.44 µM); Fe(NO3)3·9H2O (10 µM); and Zn(NO3)2·6H2O (0.37 µM). When appropriate, MgSO4 was used as sulfate source. A 19.2 mM (1000 mg L-1) Cr(VI) solution was prepared using K2CrO4. Proper dilutions were performed using the nutrient solution to obtain different Cr(VI) concentrations, and pH was adjusted to 4.8. Reagents were purchased from Sigma-Aldrich (St. Louis, MO).
2.2 Effect of Cr(VI) on seed germination
4
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT For this study, H. annuus seeds were disinfected for 30 min in 2% w/v sodium hypochlorite followed by rinsing 3 times with sterilized deionized (DI) water. After disinfection, seeds were placed on Petri dishes with paper soaked in nutrient solution containing different Cr(VI) concentrations (0, 0.09, 0.19, 0.38, 0.77, and 1.5 mM). Dishes were covered with aluminum paper to avoid light exposure and germination was performed at 25 °C. For statistical
Downloaded by [Dalhousie University] at 04:36 13 November 2013
purposes, experiment was conducted in triplicate with 30 seeds per replicate per treatment. After four days, germination was measured as the percent of seedlings having a radicle of at least 2 mm. 2.3 Effect of the growth stage on plant tolerance to Cr(VI) In this experiment, H. annuus was exposed to Cr(VI) at different stages. For this purpose, seeds were germinated in rolled paper and placed on Mason Jars containing nutrient solution (Carrillo-Castañeda et al. 2002). After germination, seedlings at different ages (4, 8, 16, and 20 days) were exposed to 0.19 mM Cr(VI). These ages were selected according to De la Rosa et al. (2008) for comparison purposes. A separate set of seeds was germinated in rolled paper imbibed in nutrient solution containing 0.19 mM Cr(VI). Seedlings were maintained in the treatments with a light photoperiod of 12/12 h, at 25 °C and 39.5 µmol m-2 s-1 photon irradiance until visible signs of toxicity appeared (loss of turgidity, chlorosis). Experiment was conducted in triplicate with 10 seedlings per replicate per treatment.
2.4 Effect of sulfate concentration on Cr(VI) uptake and tolerance Seeds of H. annuus were germinated in paper as previously described. The nutrient solution used for paper imbibition contained 0.19 mM Cr(VI) added with either 0, 0.1, 1, or 10
5
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT mM sulfate (SO42-). Sulfate solutions were prepared from MgSO4 according to Kaszycki et al. (2005). After four days, seedlings were transferred to a hydroponics system (water culture) containing the same Cr(VI)-sulfate concentrations used for germination with a light photoperiod of 12/12, at 25 °C and a photon irradiance of 39.5 µmol m-2 s-1. Root and shoot sizes of ten plants per triplicate per treatment were measured after 15 days of exposure in order to determine the
Downloaded by [Dalhousie University] at 04:36 13 November 2013
effect of treatments on plant growth. Roots were measured from the main root tip to the crown. Stems were measured from the crown to the main apex.In addition, seedlings were sampled every 3 days for 15 days in order to determine Cr absorption as a function of time and sulfate concentration in the media. After harvesting, plant roots were rinsed with 0.01M HNO3 and deionized water. Roots and shoots were separated, oven-dried at 65°C for 72 h, and prepared for acid digestion and metal quantification.
2.5 Quantification of Cr in plant tissues Plant samples were manually ground using a mortar and pestle and passed through an 18mesh screen. Subsequently, a sample of 100 mg was acid digested using trace pure HNO3 and H2O2 as per Kalra (1998). The determination of Cr concentrations in tissues was performed using a flame atomic absorption spectrometer (Perkin Elmer Aanalyst 100, New Jersey, USA). The equipment was setup as follows: λ=228 nm, slit 0.7 and lamp current of 15mA. The Reference Standard Material used for QC/QA analysis was tomato leaves (SRM-1573), chromium recovery was 92%. Detection limit for Cr was of 0.1 mg L-1.
2.6 Bulk and microXANES studies
6
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Bulk-XAS was performed on beam line 7-3 at Stanford Synchrotron Radiation Lightsource (SSRL, Palo Alto, CA). Roots of H. annuus exposed to 0.19 mM Cr(VI) and 1 mM sulfate for 15 days were frozen in liquid nitrogen and lyophilized using a Freezone 4.5 freeze dryer at -45ºC and 70 × 10-3 mbar (Labconco, Kansas City, MO). Samples were ground, loaded in aluminum sample holders, and sealed with ®Kapton tape. Spectra of sample and Cr model
Downloaded by [Dalhousie University] at 04:36 13 November 2013
compounds were collected at the Cr-K edge (E0 5.989 keV). SSRL beamline was operated with a Si (111) double crystal monochromator, and a 1 × 10 mm slit. The output was detuned by 30% to reduce higher order harmonics. Sample spectrum was collected in fluorescence mode using a 30 element Canberra Ge detector. A chromium foil [Cr(0)] was used as a calibration standard to determine the correct sample edge energy. Experiments at the European Synchrotron Radiation Facility were done in beamline ID21. Roots of H. annuus exposed to 0.19 mM Cr(VI) and 1 mM sulfate were rinsed with 0.01M HNO3 and deionized water, immersed into Tissue Tek resin, and rapidly frozen in liquid nitrogen. After embedded in resin, the samples were axially sectioned at 25 µm thick using a cryomicrotome, freeze dried and mounted in between Ultralene window film. MicroXRF mapping of Cr K-edge was performed with an incident beam at 6.1KeV during the continuous operation mode. The beam was focused to a size of 0.300 x 0.700 µm² (VxH) using a Fresnel zone plate. The fluorescence signal was detected by a large Si drift detector (Bruker). Two photodiodes were used to measure the incident and transmitted beam intensities. Dwell time and distance of the detector were optimized for each XRF map maintaining the dead time below 15%. The XRF data was processed using the PyMCA software (Solé et al. 2007). The Cr, S and P images were obtained by fitting each pixel in the XRF maps. Thus, the net counts in the images
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ACCEPTED MANUSCRIPT are those of the elements of interest. The S to Cr and P to Cr ratios were calculated using the fitted images. For microXANES data acquisition, the energy was selected using a Si111 monochromator and scanned from 5980 to 6090 eV. The zone plate was translated in the beam axis in order to maintain the beam focus. XANES data analysis was carried out using the Athena software (Ravel and Newville 2005). XANES spectra from samples were fitted using the linear
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combination procedure provided in the Athena software. Reference materials were chemical grade reagents analyzed as fine powder pellets in transmission and fluorescence mode. 2.7 Statistical analyses For this investigation, completely randomized design experiments were used. For statistical purposes, three replicates per treatment were set up. Data was analyzed using the statistical package SPSS 11.0 (Chicago, IL, USA). One way ANOVA and Tukey tests were performed to determine statistically significant differences between treatment means.
3. Results and Discussion 3.1 Effect of Cr(VI) on seed germination Germination rate in seeds treated with Cr(VI) at 0.09, 0.19, and 0.38 mM, significantly increased (P
