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ISSN: 1600-5775

Minimizing experimental artefacts in synchrotron-based X-ray analyses of Fe speciation in tissues of rice plants

aCollege of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China, bSchool of Agriculture and Food Sciences, The University of Queensland, St Lucia, Queensland 4072, Australia, and cAustralian Synchrotron ANSTO, Clayton, Victoria 3168, Australia
*Correspondence e-mail: p.wang3@njau.edu.cn

Edited by R. W. Strange, University of Essex, UK (Received 13 December 2018; accepted 30 March 2019; online 6 June 2019)

Iron (Fe) plays an important role within environmental systems. Synchrotron-based X-ray approaches, including X-ray absorption spectroscopy (XAS), provide powerful tools for in situ analyses of Fe speciation, but beam damage during analysis may alter Fe speciation during its measurement. XAS was used to examine whether experimental conditions affect the analysis of Fe speciation in plant tissues. Even when analyzed in a cryostat at 12 K, it was found that FeIII can rapidly (within 0.5–1 min) photoreduce to FeII, although the magnitude of photoreduction varied depending upon the hydration of the sample, the coordination chemistry of the Fe, as well as other properties. For example, photoreduction of FeIII was considerably higher for aqueous standard compounds than for hydrated plant-root tissues. The use of freeze-dried samples in the cryostat (12 K) markedly reduced the magnitude of this FeIII photoreduction, and there was no evidence that the freeze-drying process itself resulted in experimental artefacts under the current experimental conditions, such as through the oxidation of FeII, although some comparatively small differences were observed when comparing spectra of hydrated and freeze-dried FeII compounds. The results of this study have demonstrated that FeIII photoreduction can occur during X-ray analysis, and provides suitable conditions to preserve Fe speciation to minimize the extent of beam damage when analyzing environmental samples. All studies utilizing XAS are encouraged to include a preliminary experiment to determine if beam damage is occurring, and, where appropriate, to take the necessary steps (such as freeze drying) to overcome these issues.

1. Introduction

Synchrotron-based X-ray approaches, including X-ray absorption spectroscopy (XAS), are valuable in examining elemental speciation within a wide variety of materials, including plant tissues and environmental samples. For example, XAS offers a range of advantages compared with other approaches for examining elemental speciation, perhaps the most important of which is the ability to examine samples in situ. Accordingly, XAS has been used to examine a wide range of environmental samples, including plant tissues (Castillo-Michel et al., 2017[Castillo-Michel, H. A., Larue, C., Pradas del Real, A. E., Cotte, M. & Sarret, G. (2017). Plant Physiol. Biochem. 110, 13-32.]; Wang et al., 2017[Wang, P., Lombi, E., Sun, S., Scheckel, K. G., Malysheva, A., McKenna, B. A., Menzies, N. W., Zhao, F. J. & Kopittke, P. M. (2017). Environ. Sci. Nano, 4, 448-460.]), soils and sediments (Burton et al., 2009[Burton, E. D., Bush, R. T., Sullivan, L. A., Hocking, R. K., Mitchell, D. R. G., Johnston, S. G., Fitzpatrick, R. W., Raven, M., McClure, S. & Jang, L. Y. (2009). Environ. Sci. Technol. 43, 3128-3134.]; Wang et al., 2018[Wang, P., Menzies, N. W., Chen, H., Yang, X., McGrath, S. P., Zhao, F. J. & Kopittke, P. M. (2018). Environ. Sci. Technol. 52, 4901-4909.]; Wu et al., 2018[Wu, Y., Kukkadapu, R. K., Livi, K. J. T., Xu, W., Li, W. & Sparks, D. L. (2018). ACS Earth Space Chem. 2, 256-268.]), and in aqua­tic samples (Bai et al., 2016[Bai, Y., Yang, T., Liang, J. & Qu, J. (2016). Water Res. 98, 119-127.]; von der Heyden et al., 2014[Heyden, B. P. von der, Hauser, E. J., Mishra, B., Martinez, G. A., Bowie, A. R., Tyliszczak, T., Mtshali, T. N., Roychoudhury, A. N. & Myneni, S. C. B. (2014). Environ. Sci. Technol. Lett. 1, 387-392.]).

Of particular interest in the present study is Fe, which is the fourth most abundant element in the earth's crust and soil (Sposito, 2008[Sposito, G. (2008). The Chemistry of Soils, 2nd ed., p. 329. New York: Oxford University Press.]), is an essential nutrient for plants and animals, and has important roles in aqua­tic ecosystems. Iron has a range of oxidation states (of which both FeII and FeIII are commonly found in environmental systems), and hence understanding the speciation of Fe within environmental systems is critical in understanding its behaviour and function. In this regard, synchrotron-based XAS analyses have been used to investigate a wide range of Fe-containing processes, including the oxidation of Fe in acid sulfate sediments (Morgan et al., 2009[Morgan, K. E., Burton, E. D., Cook, P., Raven, M. D., Fitzpatrick, R. W., Bush, R., Sullivan, L. A. & Hocking, R. K. (2009). J. Phys. Conf. Ser. 190, 012144.]), the speciation of Fe within soils (Prietzel et al., 2007[Prietzel, J., Thieme, J., Eusterhues, K. & Eichert, D. (2007). Eur. J. Soil Sci. 58, 1027-1041.]) and minerals (Shorttle et al., 2015[Shorttle, O., Moussallam, Y., Hartley, M. E., Maclennan, J., Edmonds, M. & Murton, B. J. (2015). Earth Planet. Sci. Lett. 427, 272-285.]), its speciation in waste materials including mine tailings (Essilfie-Dughan et al., 2017[Essilfie-Dughan, J., Hendry, M. J., Dynes, J. J., Hu, Y., Biswas, A., Lee Barbour, S. & Day, S. (2017). Sci. Total Environ. 586, 753-769.]; Hayes et al., 2014[Hayes, S. M., Root, R. A., Perdrial, N., Maier, R. M. & Chorover, J. (2014). Geochim. Cosmochim. Acta, 141, 240-257.]), its speciation in aqua­tic ecosystems (Bai et al., 2016[Bai, Y., Yang, T., Liang, J. & Qu, J. (2016). Water Res. 98, 119-127.]; von der Heyden et al., 2014[Heyden, B. P. von der, Hauser, E. J., Mishra, B., Martinez, G. A., Bowie, A. R., Tyliszczak, T., Mtshali, T. N., Roychoudhury, A. N. & Myneni, S. C. B. (2014). Environ. Sci. Technol. Lett. 1, 387-392.]), as well as the form of Fe within plant tissues and the associated human food products (De Brier et al., 2016[De Brier, N., Gomand, S. V., Donner, E., Paterson, D., Smolders, E., Delcour, J. A. & Lombi, E. (2016). Plant Cell Environ. 39, 1835-1847.]; Terzano et al., 2013[Terzano, R., Mimmo, T., Vekemans, B., Vincze, L., Falkenberg, G., Tomasi, N., Schnell Ramos, M., Pinton, R. & Cesco, S. (2013). Anal. Bioanal. Chem. 405, 5411-5419.]; Yoshimura et al., 2000[Yoshimura, E., Sakaguchi, T., Nakanishi, H., Nishizawa, N. K., Nakai, I. & Mori, S. (2000). Phytochem. Anal. 11, 160-162.]). The speciation of Fe within plant tissues is of particular importance, for example, given that Strategy I and Strategy II plants differ in their approach for the acquisition of Fe, either as FeII or as FeIII (Marschner & Römheld, 1994[Marschner, H. & Römheld, V. (1994). Plant Soil, 165, 261-274.]).

Despite its usefulness, it is known that X-ray beams can also cause damage to the sample, resulting in experimental artefacts (George et al., 2012[George, G. N., Pickering, I. J., Pushie, M. J., Nienaber, K., Hackett, M. J., Ascone, I., Hedman, B., Hodgson, K. O., Aitken, J. B., Levina, A., Glover, C. & Lay, P. A. (2012). J. Synchrotron Rad. 19, 875-886.]). This is especially important when examining the speciation of Fe which is known to be particularly redox sensitive. For example, Terzano et al. (2013[Terzano, R., Mimmo, T., Vekemans, B., Vincze, L., Falkenberg, G., Tomasi, N., Schnell Ramos, M., Pinton, R. & Cesco, S. (2013). Anal. Bioanal. Chem. 405, 5411-5419.]) conducted a study to determine the speciation of Fe in the sap of tomato (Solanum lycopersicum L.), and found that FeIII in the samples can be photoreduced from FeIII to FeII during analysis. In a similar manner, Sigfridsson et al. (2013[Sigfridsson, K. G. V., Chernev, P., Leidel, N., Popović-Bijelić, A., Gräslund, A. & Haumann, M. (2013). J. Biol. Chem. 288, 9648-9661.]) demonstrated the photoreduction of Fe in enzymes during X-ray analyses. In contrast, other authors have reported that no photoreduction of Fe was observable under their experimental conditions (Grillet et al., 2014[Grillet, L., Ouerdane, L., Flis, P., Hoang, M. T. T., Isaure, M.-P., Lobinski, R., Curie, C. & Mari, S. (2014). J. Biol. Chem. 289, 2515-2525.]; James et al., 2016[James, S. A., Hare, D. J., Jenkins, N. L., de Jonge, M. D., Bush, A. I. & McColl, G. (2016). Sci. Rep. 6, 20350.]). However, perhaps most importantly, many studies examining Fe using X-ray based approaches in environmental investigation do not appear to explicitly consider the issue of beam damage (including photoreduction), with their findings potentially confounded by experimental artefacts.

The aim of the present study was to examine the experimental conditions that are suitable for the examination of Fe speciation using synchrotron-based X-ray analyses. We aimed to (i) highlight the types of problems associated with beam damage in X-ray studies of Fe, (ii) examine the factors which influence the severity of this beam damage and (iii) broadly provide information on the types of approaches which can be used to overcome these problems. For this study, we utilized XAS analyses, including both X-ray absorption near-edge structure (XANES) spectroscopy and extended X-ray absorption fine structure (EXAFS). However, the concepts developed in the present study also apply to other X-ray approaches, including µ-XANES analyses at X-ray fluorescence microscopy (XFM) beamlines. We have focused on the examination of root tissues of rice plants as examples of `environmental' samples, with these roots being highly hydrated (>95% water) and hence possibly more susceptible to beam damage than other types of samples. It is important to note that Fe is particularly redox sensitive, and studies investigating other metal(loid)s, such as Zn and Cd, are less likely to encounter issues with beam damage during XAS analyses as examined in the present study.

2. Materials and methods

2.1. Plant growth

Seeds of rice (Oryza sativa L.) were surface sterilized with 8% NaClO and rinsed using sterile water before being germinated in rolled paper towel suspended in tap water. After 4 days, the seedlings were transferred to containers with 11 l of nutrient solution (µM): 680 NO3–N, 120 NH4+–N, 100 Ca, 100 Mg, 310 K, 350 SO42−–S, 10 P, 25 Fe (supplied as FeIIIEDTA; EDTA: ethylenediaminetetraacetic acid), 3.0 B, 1.0 Mn, 0.05 Cu, 1.0 Zn and 0.02 Mo. The pH of the continuously aerated nutrient solutions was not adjusted (ca pH 5.6). Plants were grown at 25°C with 14 h of light at a photon flux density of 400 µM m−2 s−1. All nutrient solutions were renewed every 2 days. After 7 days, the roots were washed with flowing deionized water before the plants were separated into roots and shoots. Root tissues were snap frozen in liquid nitro­gen for later analysis using XAS, with approximately half of these frozen root tissues maintained in liquid nitro­gen whilst the other half were freeze dried. In addition, a further sub-sample of the root tissues was dried (65°C) and digested in a 1:5 mixture of nitric acid and perchloric acid and analyzed using inductively coupled plasma mass spectroscopy to determine the bulk Fe concentration of the root tissue. The average bulk Fe concentration of the root tissues was determined to be 20.3 mg kg−1 on a fresh-mass basis and 304 mg kg−1 on a dry-mass basis (93% water content).

2.2. XAS analyses

All analyses were undertaken at the XAS beamline at the Australian Synchrotron (Melbourne, Australia). The use of this beamline to examine environmental samples has been described previously (Kopittke et al., 2011[Kopittke, P. M., Menzies, N. W., de Jonge, M. D., McKenna, B. A., Donner, E., Webb, R. I., Paterson, D. J., Howard, D. L., Ryan, C. G., Glover, C. J., Scheckel, K. G. & Lombi, E. (2011). Plant Physiol. 156, 663-673.]). The XAS beamline utilizes a 1.6 T wiggler, Si collimating mirror cryo-cooled Si(111) monochromator and Rh-coated torodial refocusing mirror. The X-ray beam spot at the sample was set at ca 2 mm × 0.3 mm (H × V), which was defocused. The flux was determined to be 1.5 × 1012 photons s−1, resulting in a flux density of ca 3 × 1012 photons s−1 mm−2. Under the current experimental conditions, this was calculated as corresponding to a dose of 5.4 × 103 Gray s−1. The energy scale was calibrated by the simultaneous measurement of a metallic Fe reference foil in transmission. Under the experimental conditions of this experiment, the energy scale was typically stable to 0.05 eV or better. Fluorescence spectra were measured with a 100-element solid-state detector at 90° to the incident beam. The samples were analyzed in situ using the Fe K edge (7112 eV) using both XANES and EXAFS. All samples, whether hydrated or freeze dried, were analyzed in a He exchange gas cryostat at a temperature of ca 12 K.

A range of samples and standard compounds were prepared for analyses using XAS. Where dehydrated (freeze-dried) plant-tissue samples were used, these were ground at room temperature using an agate ball mill grinder. The fine powder was then prepared into a pellet using a manual pellet press before being placed in a Perspex sample holder sealed with Kapton tape. For plant-tissue samples that were hydrated (frozen), the plant tissues were placed into an agate mortar and pestle and ground under liquid nitro­gen before being placed in a sample holder, sealed with Kapton tape, and transferred directly to the cryostat (12 K). Thus, for the frozen plant tissues, the samples were never permitted to thaw prior to analysis. The plant-tissue samples, both hydrated (frozen) and dehydrated (freeze dried), were then analyzed as outlined below.

A total of five different Fe-containing standard compounds were prepared for XAS analysis. These compounds consisted of four FeIII standards plus four FeII standards. The four FeIII and FeII standards had an Fe concentration of 3.6 mM (200 mg l−1), being prepared with either Fe2(SO4)3 or with FeSO4. For both FeIII and FeII, the final standard mixtures consisted of (i) 3.6 mM Fe, and (ii–iv) 3.6 mM Fe mixed with 36 mM of one of three ligands (citric acid or phytic acid, and cysteine). The ligand was provided at a concentration ten times higher than the metal to maximize the binding of the Fe to the ligand. For all eight of these FeIII and FeII standards, the solutions were either prepared in ambient conditions or inside an O2-free glovebox (LC Technology Solutions, USA) as described later. For the solutions where the Fe was mixed with other ligands (citric acid or phytic acid, and cysteine), solution pH was adjusted to ca 5.5 using 0.1 M NaOH. The pH of the FeIII sulfate and FeII sulfate solutions was not adjusted. A pH of 5.5 was selected as this is similar to values encountered in relevant plant cellular compartments, such as the vacuole. After preparation, these five standards were either directly utilized (i.e. hydrated, albeit frozen once placed in the cryostat) or they were first freeze dried. Where samples were analyzed hydrated, they also contained 30% glycerol to reduce ice-crystal formation during cooling (initial concentrations of Fe were adjusted so that the final Fe concentrations remained 3.6 mM). For the standards that were analyzed as solutions, the time from preparation to analysis was less than 30 min. Where the freeze-dried standard compounds were examined, they were first diluted by mixing with cellulose to achieve a final concentration of ca 200 mg kg−1.

Experiment 1 aimed to determine whether the presence of O2 during sample preparation resulted in the oxidation of FeII. For this experiment, XANES and EXAFS spectra were obtained for FeII sulfate solutions that had been prepared either in ambient conditions or in an O2-free glove box. Two replicate scans were performed on both of the standards, with the total exposure time being 600 s per scan. To minimize the risk of beam damage, the two replicate scans were performed at different locations on the sample (i.e. the sample position was moved between the two scans).

Experiment 2 examined whether the freeze-drying process resulted in a change in Fe speciation. First, two standard compounds were examined, being an FeII sulfate solution that was examined hydrated (frozen) plus the corresponding solution that had been freeze dried. In addition, two plant-root samples were examined, again being a hydrated (frozen) sample plus the corresponding freeze-dried sample. Samples were scanned as outlined above.

Experiment 3 aimed to determine the extent to which FeIII is photoreduced during analysis. Three samples were examined, being the FeIII sulfate solution, a freeze-dried FeIII sulfate standard, and the FeII sulfate solution. First, a normal XANES and EXAFS scan was performed for all three compounds as described earlier. Next, to provide a detailed analysis of the rate at which FeIII photoreduction occurs, a different type of scan was performed in which the energy of the incident X-rays was maintained constant. In this regard, by carefully selecting the energy, it was possible to determine the speed (and extent) of the photoreduction of FeIII. Specifically, we selected two individual energy values (7126.1 and 7131.0 eV) for study. The first of these energy values (7126.1 eV) corresponds to the white line peak where the spectrum for FeII compounds have the highest fluorescence intensity and thus the photoreduction of FeIII to FeII would be associated with a concomitant increase in fluorescence intensity at 7126.1 eV (see spectra in Results[link] for further illustration). The second value (7131.0 eV) corresponds to the white line peak for FeIII compounds [where FeIII compounds have a higher fluorescence intensity than FeII compounds] and thus the photoreduction of FeIII to FeII would be associated with a concomitant decrease in fluorescence intensity. At these two energy values, fluorescence intensity was measured every 0.4 s across a total exposure time of 300 s.

Experiment 4 examined whether the rate of FeIII photoreduction was influenced by the ligand to which the FeIII was bound. A total of nine samples were examined. For FeIII, the citrate, phytate and cysteine standards were examined, both as hydrated and as freeze-dried samples (yielding a total of six FeIII standards). For FeII, we also examined the citrate, phytate and cysteine standards, but only as hydrated (not freeze dried). For these samples, we conducted both a normal scan, as well as examining changes in fluorescence intensity while holding energy constant at the two values as described for Experiment 3. For the scans at constant energy, we examined only the three hydrated (frozen) FeIII standards.

Experiment 5 examined the potential photoreduction in roots of rice. For this experiment, we examined both hydrated (frozen) and freeze-dried sample of the roots. As described in Experiment 3, we examined changes in fluorescence intensity at two constant values for energy (7126.1 and 7131.0 eV).

The spectra were energy normalized using the reference energy of the foil, with replicate spectra merged using Athena 0.8.056 (Ravel & Newville, 2005[Ravel, B. & Newville, M. (2005). J. Synchrotron Rad. 12, 537-541.]).

3. Results

3.1. Oxidation of FeII during sample preparation

Our first aim (Experiment 1) was to determine if FeII compounds were likely to oxidize to FeIII during their initial preparation. For this, we chose an FeII sulfate solution which we considered to be the most susceptible to oxidation. However, it was evident from both the XANES and EXAFS analyses that the method of preparation did not influence speciation (Fig. 1[link]). Regardless of whether samples were prepared in an O2-free environment or in ambient conditions (i.e. atmospheric O2 concentrations), XANES and EXAFS analyses indicated that the Fe was present as FeII. It must be noted that there is a possibility that perhaps some of the FeII did indeed oxidize to FeIII during the sample preparation, but that during the subsequent X-ray scanning this FeIII was rapidly photoreduced back to FeII (see later). However, we contend that if this occurred, it could have only been a comparatively small proportion of the FeII that initially oxidized to FeIII, as not all FeIII is photoreduced to FeII during XANES and EXAFS analyses (see later).

[Figure 1]
Figure 1
Fe K-edge XANES and k3-weighted EXAFS spectra of FeII sulfate solution (Experiment 1). The standard was prepared either in ambient conditions or in an O2-free glove box. The vertical dashed lines in (a) correspond to the white line peaks of FeII sulfate (7126.1 eV) and FeIII sulfate (7131.0 eV).

The observation that FeII sulfate solution was not rapidly oxidized to FeIII when prepared in ambient conditions was perhaps not unexpected given that free hydrated FeII often has an oxidation half life of days to weeks, although trace quantities of Fe(OH)20 can reduce the overall oxidation half life to a few seconds [see King (1998[King, D. W. (1998). Environ. Sci. Technol. 32, 2997-3003.]) and references therein]. Regardless, although the oxidation of FeII to FeIII was not observed during sample preparation in the present study when prepared in ambient conditions, care should still always be taken when preparing redox-sensitive samples for analysis to avoid changes in speciation during sample preparation in order to avoid potential experimental artefacts.

3.2. Freeze drying and potential oxidation of FeII

Next, we considered whether the freeze-drying process would result in the oxidation of FeII to FeIII (Experiment 2). In particular, it was of interest whether FeII would be oxidized during the preparation and drying process. We examined both a FeII sulfate solution and hydrated rice roots, and no oxi­dation to FeIII was observed for either sample as a result of the freeze-drying process (Fig. 2[link]). Furthermore, there was only comparatively small differences in the spectra between the hydrated and freeze-dried compounds [Fig. 2[link](a)]. Thus, when using X-ray approaches to examine Fe speciation, the data suggest that freeze drying would be suitable for sample preparation where it was not either possible or desirable to utilize hydrated samples (for example, see later).

[Figure 2]
Figure 2
Fe K-edge XANES spectra of (a) FeII sulfate, either as a solution (frozen) or as a solid after freeze drying, and (b) rice-root tissues grown in solutions containing 25 µM FeIII EDTA, either hydrated (frozen) or following freeze drying (Experiment 2). The vertical dashed lines in (a) correspond to the white line peaks of FeII sulfate (7126.1 eV) and FeIII sulfate (7131.0 eV).

3.3. Photoreduction of FeIII and the influence of sample hydration and coordination chemistry

Given that oxidation of FeII to FeIII did not appear to be of concern under the current experimental conditions (Figs. 1[link] and 2[link]), we next examined whether the X-ray beam caused photoreduction of FeIII (Experiment 3). First, we performed XANES and EXAFS, comparing the FeIII sulfate solution, FeIII sulfate solid and FeII sulfate solution [Fig. 3[link](a)]. It was found that the white line peak of the FeII sulfate solution was at 7126.1 eV, corresponding to the approximate value expected for FeII (for example, see Wilke et al., 2001[Wilke, M., Farges, F., Petit, P.-E., Brown, G. E. Jr & Martin, F. (2001). Am. Mineral. 86, 714-730.]). In contrast, for the FeIII sulfate solution, although there was a small shoulder at 7131.0 eV (corresponding to the FeIII white line peak; for example, see Wilke et al., 2001[Wilke, M., Farges, F., Petit, P.-E., Brown, G. E. Jr & Martin, F. (2001). Am. Mineral. 86, 714-730.]), the white line peak was actually at ca 7126 eV and was thus similar to FeII rather than FeIII [Fig. 3[link](a)]. This observation was unexpected and is attributed to the comparatively rapid photoreduction of FeIII to FeII in this hydrated (frozen) standard. It was apparent that much of the FeIII had already been reduced to FeII by the time that the XANES scan had reached 7126 eV, with this corresponding to an exposure of ca 230 s to this location within the XANES scan. This photoreduction of FeIII to FeII was also evident by examining the EXAFS spectra [Fig. 3[link](b)] and by examining the derivative of the XANES spectra [Fig. 3[link](c)]. Importantly, however, it was also observed that the FeIII sulfate solid (freeze-dried) standard was substantially less sensitive to photoreduction, with the white line peak for this FeIII compound corresponding to the expected value of 7131.0 eV and with no noticeable shoulder at 7126.1 eV [Fig. 3[link](a)].

[Figure 3]
Figure 3
X-ray induced photoreduction of FeIII, comparing FeIII sulfate (either as hydrated or freeze-dried forms) with FeII sulfate (hydrated) (Experiment 3). (a) Fe K-edge XANES, (b) k3-weighted EXAFS, and (c) derivative of normalized XANES. In (d), a hydrated (frozen) FeIII sulfate sample was scanned at constant energy corresponding to the white line peaks of FeII and FeIII sulfates (7126.1 or 7131.0 eV) for 300 s. At 7126.1 eV, a progressive increase in fluorescence intensity would correspond to the conversion of FeIII to FeII (and vice versa), while at 7131.0 eV, a progressive increase in fluorescence intensity would correspond to the conversion of FeII to FeIII (and vice versa).

To examine the kinetics of the photoreduction of FeIII to FeII more closely, we measured changes in fluorescence intensity over time whilst scanning at a constant energy of either 7126.1 or 7131.0 eV (these values corresponding to the white line peaks of FeII and FeIII, respectively). Thus, at these constant energy values, a change in fluorescence intensity would correspond to a change in Fe speciation. Specifically, at 7126.1 eV the photoreduction of FeIII would cause an increase in fluorescence intensity, whilst at 7131.0 eV the photo­reduction of FeIII would cause a decrease in fluorescence intensity [compare the XANES spectra in Fig. 3[link](a)]. From these analyses, it was apparent that the extent of FeIII photoreduction in the hydrated FeIII standard was both substantial and rapid, with the majority of photoreduction occurring within 0.5–1 min of exposure [Fig. 3[link](d)].

Next, we examined whether the rate of photoreduction was also influenced by the ligand to which the FeIII was bound (i.e. coordination chemistry). Specifically, we found that the rate of photoreduction was greatest for free FeIII ions (i.e. the FeIII sulfate solution), followed by FeIII complexed with phytate and citrate (cysteine is considered later) (Figs. 3[link] and 4[link]). Thus, when conducting preliminary studies to determine whether photoreduction (beam damage) is of concern within a particular experimental system, care should be taken to ensure that the most sensitive sample is examined. It was interesting to note that the photoreduction of FeIII within the highly hydrated plant-root tissues was negligible (Fig. 5[link]) despite the Fe being present as FeIII [Fig. 2[link](b)], suggesting that the sample properties influence the magnitude of the beam damage.

[Figure 4]
Figure 4
X-ray induced photoreduction of FeIII as influenced by chelation, comparing FeIII and FeII chelated with phytate (a, b), citrate (c, d) or cysteine (e, f) (Experiment 4). Both hydrated and freeze-dried forms were compared. (a), (c), (e) Fe K-edge XANES spectra of the FeIII complex (hydrated and freeze dried) compared with the FeII complex (hydrated). In (b, d, f), hydrated (frozen) FeIII samples complexed with (b) phytate, (d) citrate and (f) cysteine, were scanned at constant energy corresponding to the white line peaks of FeII and FeIII sulfates (7126.1 or 7131.0 eV) for 300 s. At 7126.1 eV, a progressive increase in fluorescence intensity would correspond to the conversion of FeIII to FeII (and vice versa), while at 7131.0 eV, a progressive increase in fluorescence intensity would correspond to the conversion of FeII to FeIII (and vice versa).
[Figure 5]
Figure 5
Changes in Fe speciation in roots of rice, either (a) hydrated or (b) freeze-dried roots, following growth in 25 µM FeIII EDTA for 7 days (Experiment 5). Samples were scanned at constant energy corresponding to the white line peaks of FeII and FeIII sulfates (7126.1 or 7131.0 eV) for 300 s. At 7126.1 eV, a progressive increase in fluorescence intensity would correspond to the conversion of FeIII to FeII (and vice versa), while at 7131.0 eV, a progressive increase in fluorescence intensity would correspond to the conversion of FeII to FeIII (and vice versa).

3.4. Reduction of FeIII by cysteine

For FeIII cysteine, it was noted that all three XANES spectra had white line peaks that corresponded to FeII [Fig. 4[link](e)] and that there was no change in fluorescence intensity over time when measured at constant energy [Fig. 4[link](f)]. Thus, the data suggest that the Fe in all three samples was already present as FeII at the commencement of XAS analyses. We suggest that this is probably because of the reduction of FeIII by cysteine itself (which is a reducing agent) upon sample preparation. For example, cysteine has been reported to cause the reductive dissolution of FeIII (Doong & Schink, 2002[Doong, R. & Schink, B. (2002). Environ. Sci. Technol. 36, 2939-2945.]). Thus, these data demonstrate that care must be taken when analyzing and interpreting cysteine standards prepared using FeIII, as the FeIII would potentially have been reduced to FeII.

3.5. Implications for study of plant tissues

The results of the present study have implications for reducing the formation of experimental artefacts during X-ray analyses of Fe speciation in environmental samples. Most importantly, we have shown that in comparatively high-flux beamlines (such as the XAS beamline at the Australian Synchrotron), hydrated samples are susceptible to rapid photoreduction of FeIII to FeII, with much of this occurring within only 0.5–1 min of exposure [Fig. 3[link](d)]. By using freeze-dried samples rather than hydrated samples, it was found that the rate of the photoreduction of FeIII to FeII decreased markedly [Figs. 3[link](a), 4[link](a) and 4[link](c)]. Interestingly, however, the photoreduction of FeIII within hydrated plant-root tissues was negligible (Fig. 5[link]) despite the Fe being present as FeIII [Fig. 2[link](b)], confirming that the sample properties influence the likelihood of beam damage in any given experiment.

Not only did the hydration of samples influence the rate of the photoreduction of FeIII, but the coordination chemistry of the FeIII was also important. For example, it was found that the photoreduction of FeIII decreased in the order: free Fe3+ ions (FeIII sulfate solution) > FeIII phytate > FeIII citrate (Fig. 4[link]). We have also confirmed that in the present experimental conditions the process of freeze-drying hydrated standards and plant tissues did not result in oxidation of FeII to FeIII during preparation, either for standard compounds or for highly hydrated plant tissues (Fig. 2[link]).

4. Discussion

Our current study has demonstrated the photoreduction of FeIII during X-ray analysis of standards and (to a much lesser extent) plant tissues during an experiment investigating the speciation of Fe in tissues of rice. These findings in regard to the photoreduction of FeIII are in agreement with the data reported previously. For example, Terzano et al. (2013[Terzano, R., Mimmo, T., Vekemans, B., Vincze, L., Falkenberg, G., Tomasi, N., Schnell Ramos, M., Pinton, R. & Cesco, S. (2013). Anal. Bioanal. Chem. 405, 5411-5419.]) found photoreduction of FeIII when examining Fe in hydrated plant sap at the Fe K edge, with these authors reporting a photon flux of ca 1 × 1012 (similar to the present study) by comparing XANES spectra following exposure for 1, 5 and 10 XANES scans, corresponding to 600, 3000 and 6000 s exposure in their experimental system. Similarly, using soft X-rays at the Fe L edge, George et al. (2008[George, S. J., Fu, J., Guo, Y., Drury, O. B., Friedrich, S., Rauchfuss, T., Volkers, P. I., Peters, J. C., Scott, V., Brown, S. D., Thomas, C. M. & Cramer, S. P. (2008). Inorg. Chim. Acta, 361, 1157-1165.]) examined metalloproteins, finding rapid photoreduction of FeIII to FeII. Furthermore, Gonçalves Ferreira et al. (2013[Gonçalves Ferreira, P., de Ligny, D., Lazzari, O., Jean, A., Cintora Gonzalez, O. & Neuville, D. R. (2013). Chem. Geol. 346, 106-112.]) examined an inorganic substrate (soda-lime silicate glass), finding photoreduction of FeIII caused by the X-ray beam at the Fe K edge during analysis at room temperature.

Importantly, our study has provided conditions that are suitable for preserving Fe speciation and minimizing the extent of beam damage during analysis. Specifically, we demonstrate that although the use of highly hydrated samples for the analysis of Fe speciation can result in large changes in Fe speciation caused by beam damage, the freeze-drying process largely prevented this photoreduction and caused only comparatively small changes in the XANES spectra. This is important, given the crucial roles of Fe within soil–plant systems. Freeze drying also has the advantage of increasing the concentrations of absorbers, which is important in experiments using dilute samples (such as plant tissues) (George et al., 2012[George, G. N., Pickering, I. J., Pushie, M. J., Nienaber, K., Hackett, M. J., Ascone, I., Hedman, B., Hodgson, K. O., Aitken, J. B., Levina, A., Glover, C. & Lay, P. A. (2012). J. Synchrotron Rad. 19, 875-886.]).

Although we recommend in the present study that samples should be freeze dried prior to XAS analyses for Fe, we are not recommending that this be standard practice in all bio­logical experiments. For example, using the present experimental setup at the XAS beamline of the Australian Synchrotron, we have previously found that the use of hydrated (frozen) plant tissues to examine a range of metal(loid)s did not result in beam damage when examining Zn (Kopittke et al., 2011[Kopittke, P. M., Menzies, N. W., de Jonge, M. D., McKenna, B. A., Donner, E., Webb, R. I., Paterson, D. J., Howard, D. L., Ryan, C. G., Glover, C. J., Scheckel, K. G. & Lombi, E. (2011). Plant Physiol. 156, 663-673.]), Mn (Kopittke et al., 2013[Kopittke, P. M., Lombi, E., McKenna, B. A., Wang, P., Donner, E., Webb, R. I., Blamey, F. P. C., de Jonge, M. D., Paterson, D., Howard, D. L. & Menzies, N. W. (2013). Physiol. Plant. 147, 453-464.]), Ag (Wang et al., 2015[Wang, P., Menzies, N. W., Lombi, E., Sekine, R., Blamey, F. P. C., Hernandez-Soriano, M. C., Cheng, M., Kappen, P., Peijnenburg, W. J. G. M., Tang, C. & Kopittke, P. M. (2015). Nanotoxicology 9, 1041-1049.]) and Cd (Cheng et al., 2016[Cheng, M., Wang, P., Kopittke, P. M., Wang, A., Sale, P. W. G. & Tang, C. (2016). J. Exp. Bot. 67, 5041-5050.]). Nevertheless, Fe is not the only element for which photoreduction has been observed. For example, in plant tissues, photoreduction has also been reported for As (Lombi et al., 2011[Lombi, E., Scheckel, K. G. & Kempson, I. M. (2011). Environ. Exp. Bot. 72, 3-17.]), Se (Wang et al., 2013[Wang, P., Menzies, N. W., Lombi, E., McKenna, B. A., de Jonge, M. D., Paterson, D. J., Howard, D. L., Glover, C. J., James, S., Kappen, P., Johannessen, B. & Kopittke, P. M. (2013). Plant Physiol. 163, 407-418.]) and Cu (Yang et al., 2011[Yang, J., Regier, T., Dynes, J. J., Wang, J., Shi, J., Peak, D., Zhao, Y., Hu, T., Chen, Y. & Tse, J. S. (2011). Anal. Chem. 83, 7856-7862.]). Thus, we contend that it is imperative that studies examining biological samples at XAS beamlines conduct preliminary studies to investigate whether beam damage is occurring. Where no beam damage is evident, the use of hydrated (frozen) samples is generally the preferred option. Instead, freeze drying of samples should only be considered where beam damage is evident, such as in the current study where we examined Fe, which is particularly redox sensitive.

It is also important to note that, in some instances, freeze drying may not be the preferred method of sample prep­aration. For example, if the primary interest of a study is to determine the ligand to which Fe is bound rather than determining the oxidation state of the Fe, then freeze drying may not necessarily be the most suitable approach. This is because it is possible that strong ligands may remain bound to Fe even if the oxidation state of Fe changes. However, it is possible that sample-preparation techniques (such as freeze drying) may alter the ligand to which Fe is bound despite maintaining its oxidation state, thereby causing experimental artefacts. Thus, careful consideration must always be given to the experimental system and the nature of the information required.

In the present study, we have focused on changes in the sample environment (freeze drying) as a method for limiting beam damage. It is also possible sometimes to take other precautions in order to reduce beam damage, including by altering properties of the beamline itself. For example, George et al. (2012[George, G. N., Pickering, I. J., Pushie, M. J., Nienaber, K., Hackett, M. J., Ascone, I., Hedman, B., Hodgson, K. O., Aitken, J. B., Levina, A., Glover, C. & Lay, P. A. (2012). J. Synchrotron Rad. 19, 875-886.]) identifies that damage can be reduced by decreasing the X-ray beam flux density, the use of cryogenic temperatures, sample translation, use of a photon shutter to reduce X-ray exposure and the use of electrochemical XAS cells. Such approaches should also be considered and implemented where possible. However, in the present study, despite defocusing the beam to the maximum possible as well as using a cryostat (ca 12 K) for all samples, rapid photoreduction was still observed. Thus, a multi-faceted approach is likely to be required in such experiments.

We also observed that the ligand alters the rate of photoreduction of FeIII, with this being in agreement with previous reports. For example, Abrahamson et al. (1994[Abrahamson, H. B., Rezvani, A. B. & Brushmiller, J. G. (1994). Inorg. Chim. Acta, 226, 117-127.]) examined photosensitivity of a range of Fe-containing samples complexed with carb­oxy­lic acids, finding that photoreduction of FeIII to FeII decreased in the order: oxalate > tartate > citrate > malate > isocitrate. In a similar manner, Terzano et al. (2013[Terzano, R., Mimmo, T., Vekemans, B., Vincze, L., Falkenberg, G., Tomasi, N., Schnell Ramos, M., Pinton, R. & Cesco, S. (2013). Anal. Bioanal. Chem. 405, 5411-5419.]) found that whilst FeIII was photoreduced when complexed with succinate, nicotianamine and α-ketoglutarate, it was not photoreduced when complexed with citrate, acetate, malate, shikimate or fumarate – results which differ somewhat from the present study.

In the present study, we have examined speciation of Fe in bulk plant tissues at an XAS beamline. However, speciation can also be examined using µ-XANES at XFM beamlines. In such analyses, XANES spectra are often collected from samples at room temperature, with the samples also often hydrated. Thus, for such analyses, considerable care is required to avoid the photoreduction of Fe. It is noteworthy in this regard that fast fluorescence detector systems at XFM beamlines are now allowing `fluorescence XANES imaging' (Kopittke et al., 2014[Kopittke, P. M., de Jonge, M. D., Wang, P., McKenna, B. A., Lombi, E., Paterson, D. J., Howard, D. L., James, S. A., Spiers, K. M., Ryan, C. G., Johnson, A. A. T. & Menzies, N. W. (2014). New Phytol. 201, 1251-1262.]). In this approach, XANES spectra can potentially be extracted from every pixel in the map. Importantly, given that the total dwell can be in the range of <1 s per pixel (Blamey et al., 2015[Blamey, F. P. C., Hernandez-Soriano, M. C., Cheng, M., Tang, C., Paterson, D. J., Lombi, E., Wang, W. H., Scheckel, K. G. & Kopittke, P. M. (2015). Plant Physiol. 169, 2006-2020.]; Kopittke et al., 2014[Kopittke, P. M., de Jonge, M. D., Wang, P., McKenna, B. A., Lombi, E., Paterson, D. J., Howard, D. L., James, S. A., Spiers, K. M., Ryan, C. G., Johnson, A. A. T. & Menzies, N. W. (2014). New Phytol. 201, 1251-1262.]), this approach is potentially of value for overcoming issues of photoreduction of redox-sensitive metal(loid)s.

It must be noted that in the present study we have only examined FeII and FeIII (with these being the most environmentally relevant forms of Fe), but other studies have previously found that Fe0 and FeI can potentially photooxidize in soft X-ray beams (George et al., 2008[George, S. J., Fu, J., Guo, Y., Drury, O. B., Friedrich, S., Rauchfuss, T., Volkers, P. I., Peters, J. C., Scott, V., Brown, S. D., Thomas, C. M. & Cramer, S. P. (2008). Inorg. Chim. Acta, 361, 1157-1165.]). Finally, the issue of radiation damage and experimental artefacts is also not limited to XAS analyses. For example, using X-ray crystallography to examine proteins, Weik et al. (2000[Weik, M., Ravelli, R. B. G., Kryger, G., McSweeney, S., Raves, M. L., Harel, M., Gros, P., Silman, I., Kroon, J. & Sussman, J. L. (2000). Proc. Natl Acad. Sci. USA, 97, 623-628.]) also demonstrated problems with radiation damage. Specifically, these authors demonstrated cleavage of di­sulfide bridges and loss of definition of carboxyl groups of acidic residues, with these authors also using a third-generation synchrotron with a photon flux of 5 × 1012 photons s−1 through a collimator 0.15 mm in diameter.

In conclusion, our data demonstrate how to minimize the likelihood of beam damage during X-ray analyses of Fe speciation in plant tissues, with only comparatively small differences between the use of freeze-dried samples and highly hydrated samples in regard to Fe speciation analysis. However, substantial differences were observed in the rate of photoreduction, depending upon the coordination chemistry of the Fe as well as the properties of the samples themselves (for example, hydrated standards compared with hydrated plant tissues). Thus, whilst we have shown that studies should always give consideration to the potential formation of beam damage, care must also be taken to ensure that the most sensitive samples are examined when assessing the impact of beam damage. Where beam damage cannot be reduced through altering sample conditions, consideration should also be given to altering the conditions of the beamline. For example, it may be possible to reduce flux density, either by increasing beam size or by decreasing total flux. Also, consideration should be given to conducting multiple shorter scans in different parts of the sample which can then be averaged to yield a single spectrum. It is important to note that the configuration of each individual X-ray beamline is important and will influence the magnitude of the beam damage observed. Finally, for studies examining metal(loid)s which are less redox sensitive than Fe, it is likely that the freeze drying of samples is not required, with this being preferable as the freeze-drying process itself could potentially cause experimental artefacts in some situations. Indeed, it is our hope that our work will encourage others to explicitly examine issues of beam damage within their experimental systems and thereby take steps to avoid these potential problems. In the present study, we show that even if beam damage occurs within seconds or minutes of exposure, this can be assessed by examining fluorescence intensity at a constant energy over time.

Acknowledgements

This research was undertaken on the XAS beamline at the Australian Synchrotron, part of ANSTO (Australian Nuclear Science and Technology Organisation).

Funding information

This work was supported by the Natural Science Foundation of China (grant No. 21661132001 and 41671309), the Innovative Research Team Development Plan of the Ministry of Education of China (grant No. IRT_17R56) and the fundamental research funds for the central universities of China (grant No. KYT201802 and No. KYT201624).

References

First citationAbrahamson, H. B., Rezvani, A. B. & Brushmiller, J. G. (1994). Inorg. Chim. Acta, 226, 117–127.  CrossRef CAS Web of Science Google Scholar
First citationBai, Y., Yang, T., Liang, J. & Qu, J. (2016). Water Res. 98, 119–127.  Web of Science CrossRef CAS PubMed Google Scholar
First citationBlamey, F. P. C., Hernandez-Soriano, M. C., Cheng, M., Tang, C., Paterson, D. J., Lombi, E., Wang, W. H., Scheckel, K. G. & Kopittke, P. M. (2015). Plant Physiol. 169, 2006–2020.  Web of Science PubMed Google Scholar
First citationBurton, E. D., Bush, R. T., Sullivan, L. A., Hocking, R. K., Mitchell, D. R. G., Johnston, S. G., Fitzpatrick, R. W., Raven, M., McClure, S. & Jang, L. Y. (2009). Environ. Sci. Technol. 43, 3128–3134.  Web of Science CrossRef PubMed CAS Google Scholar
First citationCastillo-Michel, H. A., Larue, C., Pradas del Real, A. E., Cotte, M. & Sarret, G. (2017). Plant Physiol. Biochem. 110, 13–32.  Web of Science CAS PubMed Google Scholar
First citationCheng, M., Wang, P., Kopittke, P. M., Wang, A., Sale, P. W. G. & Tang, C. (2016). J. Exp. Bot. 67, 5041–5050.  Web of Science CrossRef CAS PubMed Google Scholar
First citationDe Brier, N., Gomand, S. V., Donner, E., Paterson, D., Smolders, E., Delcour, J. A. & Lombi, E. (2016). Plant Cell Environ. 39, 1835–1847.  Web of Science CrossRef CAS PubMed Google Scholar
First citationDoong, R. & Schink, B. (2002). Environ. Sci. Technol. 36, 2939–2945.  Web of Science CrossRef PubMed CAS Google Scholar
First citationEssilfie-Dughan, J., Hendry, M. J., Dynes, J. J., Hu, Y., Biswas, A., Lee Barbour, S. & Day, S. (2017). Sci. Total Environ. 586, 753–769.  Web of Science CAS PubMed Google Scholar
First citationGeorge, G. N., Pickering, I. J., Pushie, M. J., Nienaber, K., Hackett, M. J., Ascone, I., Hedman, B., Hodgson, K. O., Aitken, J. B., Levina, A., Glover, C. & Lay, P. A. (2012). J. Synchrotron Rad. 19, 875–886.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationGeorge, S. J., Fu, J., Guo, Y., Drury, O. B., Friedrich, S., Rauchfuss, T., Volkers, P. I., Peters, J. C., Scott, V., Brown, S. D., Thomas, C. M. & Cramer, S. P. (2008). Inorg. Chim. Acta, 361, 1157–1165.  Web of Science CrossRef CAS Google Scholar
First citationGonçalves Ferreira, P., de Ligny, D., Lazzari, O., Jean, A., Cintora Gonzalez, O. & Neuville, D. R. (2013). Chem. Geol. 346, 106–112.  Google Scholar
First citationGrillet, L., Ouerdane, L., Flis, P., Hoang, M. T. T., Isaure, M.-P., Lobinski, R., Curie, C. & Mari, S. (2014). J. Biol. Chem. 289, 2515–2525.  Web of Science CrossRef CAS PubMed Google Scholar
First citationHayes, S. M., Root, R. A., Perdrial, N., Maier, R. M. & Chorover, J. (2014). Geochim. Cosmochim. Acta, 141, 240–257.  Web of Science CrossRef CAS PubMed Google Scholar
First citationHeyden, B. P. von der, Hauser, E. J., Mishra, B., Martinez, G. A., Bowie, A. R., Tyliszczak, T., Mtshali, T. N., Roychoudhury, A. N. & Myneni, S. C. B. (2014). Environ. Sci. Technol. Lett. 1, 387–392.  Google Scholar
First citationJames, S. A., Hare, D. J., Jenkins, N. L., de Jonge, M. D., Bush, A. I. & McColl, G. (2016). Sci. Rep. 6, 20350.  Web of Science CrossRef PubMed Google Scholar
First citationKing, D. W. (1998). Environ. Sci. Technol. 32, 2997–3003.  Web of Science CrossRef CAS Google Scholar
First citationKopittke, P. M., de Jonge, M. D., Wang, P., McKenna, B. A., Lombi, E., Paterson, D. J., Howard, D. L., James, S. A., Spiers, K. M., Ryan, C. G., Johnson, A. A. T. & Menzies, N. W. (2014). New Phytol. 201, 1251–1262.  Web of Science CrossRef CAS PubMed Google Scholar
First citationKopittke, P. M., Lombi, E., McKenna, B. A., Wang, P., Donner, E., Webb, R. I., Blamey, F. P. C., de Jonge, M. D., Paterson, D., Howard, D. L. & Menzies, N. W. (2013). Physiol. Plant. 147, 453–464.  Web of Science CrossRef CAS PubMed Google Scholar
First citationKopittke, P. M., Menzies, N. W., de Jonge, M. D., McKenna, B. A., Donner, E., Webb, R. I., Paterson, D. J., Howard, D. L., Ryan, C. G., Glover, C. J., Scheckel, K. G. & Lombi, E. (2011). Plant Physiol. 156, 663–673.  Web of Science CrossRef CAS PubMed Google Scholar
First citationLombi, E., Scheckel, K. G. & Kempson, I. M. (2011). Environ. Exp. Bot. 72, 3–17.  Web of Science CrossRef CAS Google Scholar
First citationMarschner, H. & Römheld, V. (1994). Plant Soil, 165, 261–274.  CrossRef CAS Web of Science Google Scholar
First citationMorgan, K. E., Burton, E. D., Cook, P., Raven, M. D., Fitzpatrick, R. W., Bush, R., Sullivan, L. A. & Hocking, R. K. (2009). J. Phys. Conf. Ser. 190, 012144.  CrossRef Google Scholar
First citationPrietzel, J., Thieme, J., Eusterhues, K. & Eichert, D. (2007). Eur. J. Soil Sci. 58, 1027–1041.  Web of Science CrossRef CAS Google Scholar
First citationRavel, B. & Newville, M. (2005). J. Synchrotron Rad. 12, 537–541.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationShorttle, O., Moussallam, Y., Hartley, M. E., Maclennan, J., Edmonds, M. & Murton, B. J. (2015). Earth Planet. Sci. Lett. 427, 272–285.  Web of Science CrossRef CAS Google Scholar
First citationSigfridsson, K. G. V., Chernev, P., Leidel, N., Popović-Bijelić, A., Gräslund, A. & Haumann, M. (2013). J. Biol. Chem. 288, 9648–9661.  Web of Science CrossRef CAS PubMed Google Scholar
First citationSposito, G. (2008). The Chemistry of Soils, 2nd ed., p. 329. New York: Oxford University Press.  Google Scholar
First citationTerzano, R., Mimmo, T., Vekemans, B., Vincze, L., Falkenberg, G., Tomasi, N., Schnell Ramos, M., Pinton, R. & Cesco, S. (2013). Anal. Bioanal. Chem. 405, 5411–5419.  Web of Science CrossRef CAS PubMed Google Scholar
First citationWang, P., Lombi, E., Sun, S., Scheckel, K. G., Malysheva, A., McKenna, B. A., Menzies, N. W., Zhao, F. J. & Kopittke, P. M. (2017). Environ. Sci. Nano, 4, 448–460.  Web of Science CrossRef CAS Google Scholar
First citationWang, P., Menzies, N. W., Chen, H., Yang, X., McGrath, S. P., Zhao, F. J. & Kopittke, P. M. (2018). Environ. Sci. Technol. 52, 4901–4909.  Web of Science CrossRef CAS PubMed Google Scholar
First citationWang, P., Menzies, N. W., Lombi, E., McKenna, B. A., de Jonge, M. D., Paterson, D. J., Howard, D. L., Glover, C. J., James, S., Kappen, P., Johannessen, B. & Kopittke, P. M. (2013). Plant Physiol. 163, 407–418.  Web of Science CrossRef CAS PubMed Google Scholar
First citationWang, P., Menzies, N. W., Lombi, E., Sekine, R., Blamey, F. P. C., Hernandez-Soriano, M. C., Cheng, M., Kappen, P., Peijnenburg, W. J. G. M., Tang, C. & Kopittke, P. M. (2015). Nanotoxicology 9, 1041–1049.  Web of Science CrossRef PubMed Google Scholar
First citationWeik, M., Ravelli, R. B. G., Kryger, G., McSweeney, S., Raves, M. L., Harel, M., Gros, P., Silman, I., Kroon, J. & Sussman, J. L. (2000). Proc. Natl Acad. Sci. USA, 97, 623–628.  Web of Science CrossRef PubMed CAS Google Scholar
First citationWilke, M., Farges, F., Petit, P.-E., Brown, G. E. Jr & Martin, F. (2001). Am. Mineral. 86, 714–730.  CrossRef CAS Google Scholar
First citationWu, Y., Kukkadapu, R. K., Livi, K. J. T., Xu, W., Li, W. & Sparks, D. L. (2018). ACS Earth Space Chem. 2, 256–268.  Web of Science CrossRef CAS Google Scholar
First citationYang, J., Regier, T., Dynes, J. J., Wang, J., Shi, J., Peak, D., Zhao, Y., Hu, T., Chen, Y. & Tse, J. S. (2011). Anal. Chem. 83, 7856–7862.  Web of Science CrossRef CAS PubMed Google Scholar
First citationYoshimura, E., Sakaguchi, T., Nakanishi, H., Nishizawa, N. K., Nakai, I. & Mori, S. (2000). Phytochem. Anal. 11, 160–162.  CrossRef CAS Google Scholar

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