use!of!thedonnanmembranetechnique!(dmt)!forfree ... · 6!...

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Use of the Donnan Membrane Technique (DMT) for free Zn measurements in nutrient solution compared to

speciation models and the Permeation Liquid Membrane Technique (PLM)

Report for Short-‐Term Scinetific Mission (STSM) in the framework of COST FA 0905 to Wageningen University, The Netherlands

Carried out by Anja Gramlich, ETH Zurich, Switzerland

From May, 29th until June, 24th 2011

Supervision Wageningen University: Dr. E. Hoffland, Dr. E. Temminghoff Supervision ETH Zürich: Prof. R. Schulin

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Objectives The permeation liquid membrane technique (PLM) is a speciation tool that has been used for free metal measurements of Cu, Pb and Cd Parthasarathy et al., 1997; Slaveykova et al., 2004; Bayen et al., 2006. In this PhD study, the PLM technique has for the first time been used for free Zn measurement. The ligands EDTA, citrate, histidine and cysteine have been used to alter the free Zn concentration while the total Zn concentration remained the same. The first results show for all tested ligands except EDTA, between 2-‐7 times higher Zn2+ concentrations than predicted by the models. We are now testing whether these higher concentrations are due to a contribution of labile complexes to the measurements. However, a further validation of the method by comparison with an independent measurement technique already used for Zn speciation, in our case the Donnan Membrane Technique (DMT), would be very valuable. The goal of this STSM would be to measure the samples analyzed by PLM with DMT and to compare both techniques with the modeled results. Background Naturally occurring organic ligands in soils are known to increase the mobility of heavy metals in soil solution (Meers et al., 2005; Khademi et al., 2009). It is not yet clear to what extent the metal-‐ligand complexes contribute to the bioavailability and bio-‐uptake of the metals. According to the FIAM (Free Ion Activity Model) and the BLM (Biotic Ligand Model) only ions that are occurring in the free form are available to organisms. The models are based on the assumption that metal uptake through biological membranes is slower than the metal diffusion towards the membrane, thus making only the free metal ion activity important for bio-‐uptake. These assumptions however have never been verified and recently, more emphasis has been put on the investigation of the contribution of ligands to bio-‐uptake of metals (Wang et al., 2009). One hypothesis is that there exist specific uptake systems for metal ligand complexes playing a role in plant nutrition. It is also possible that the complexes dissociate within the diffusion layer at the root surface and enhance the bioavailability of metals indirectly (Panfili et al., 2009; Wang et al., 2009). In some cases it has already been shown, that metal-‐organic complexes can in addition to the free metal ions, contribute to bio-‐uptake. Zn-‐phytosiderophore complexes for example are partly taken up as entire complexes (Vonwiren et al., 1996; White and Broadley, 2009). It is hypothesized also for two other organic ligands, citrate and histidine that specific uptake pathways exist, since enhanced uptake of labeled citrate and histidine by swiss chard was found compared to uptake of labeled EDTA. However, no correlation was made between ligand and metal uptake (Bell et al., 2003). On the other hand, many studies support the hypothesis that synthetic ligands such as EDTA are only taken up to a small extent, probably only due to leaks in the Casparian strip or by disrupting the membranes themselves (Haussling et al., 1988; Vassil et al., 1998; Nowack et al., 2008). To address questions about bioavailability of metal-‐ligand complexes and to compare the effects of different ligands on plant uptake, the free ion activity in nutrient solution is a very important parameter. There exist several measurement techniques suitable for measuring free metal ion concentrations in solution such as electrochemical speciation methods, ion selective electrodes, the donnan membrane technique (DMT) or the permeation liquid membrane technique (PLM) (Pesavento et al., 2009). In my PhD work the PLM and the DMT methods will be compared for Zn speciation in nutrient solutions. This work only focuses on the results using DMT.

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Methods The details of the methodological setup of the DMT cells are well described by Temminghoff et al., 2000. This method has already been used for free Zn measurements and good agreements with the models were found. Sample solutions The same sample types as used for PLM analyses will be analyzed by the DMT (Table 1). From each ligand treatment at least 3 samples have been analyzed (12 samples in total). Control 1 and 2 will not be analyzed in this context. Table 1: Treatments for PLM and DMT experiments. All solutions contain a background concentarion of 500 µM KNO3 and 400 µM Ca(NO3)2, buffered using MOPS at pH 7.2. Zn is added as ZnSO4.

Detection limit of the ICP-‐MS analysis In a first step the detection limit for Zn analysis of the ICP-‐MS needed to be determined in order to decide whether Zn concentrations of ca. 3µg/l (50nM Zn) can be detected. è The detection limit of the ICP-‐MS was found to be at 0.02µg/l. The contamination due to other chemicals used in the acceptor solution was 0.57µg/l.

Based on these results, it was decided to try the measurements without additional adding of ligands in the acceptor solution. (Ligands with well known stability constants with Zn (e.g. purified humic acids) can be used in the acceptor solution to increase the detection limit in samples with very low free Zn concentrations (Kalis et al., 2006).) DTM setup (adapted from the DMT Manual from Erwin Kalis) Cells The cells need to be cleaned in 0.1 M HNO3 over night (2*). Then they need to be washed with ultrapure water over night (2*). Then they are dried on filter paper over night. Tubes Tubes are attached to the peristaltic pumps (4 tubes of 0.5m per cell) and they need to be cleaned at speed 5ml min-‐1 with 0.01M HNO3 over night (2*). In the following they are cleaned with ultrapure water at speed 2.5ml min-‐1 over night (2*). Then they need to be cleaned over night with the background solution at speed 7.5 ml min-‐1. Repeat this step twice (constitution of the Background solution see in section “sample solutions”). DMT Membranes The membranes should be shaken in 0.1 M HNO3 over night (2*). Then they should be cleaned with ultrapure water twice. Then a solution containing 500* the Ca and K concentration of the background solution is added. The pH needs to be adjusted to the one of the experimental solutions (pH 7.2 in this case) and solution must be exchanged 3 times.

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Then the membranes are again washed with ultrapure water and the background solution is added, exchanged 3* and is then left over night. Sampling: Time 0 / Time 1d / Time 2d / Time 3d:

• Acceptor solution: 5ml for ICP-‐MS analysis (Zn) 1ml for ICP-‐OES analysis (Ca, K) 1ml to measure pH

7ml in total; needs to be refilled after sampling

• Donor solution: 1ml for ICP-‐OES analysis: Zn, Ca, K 1ml to measure pH

2ml in total Sample analysis: Zn concentrations in the Acceptor solutions are determined by ICP-‐MS measurements. Zn, K and Ca concentrations in the Donor and K and Ca in the acceptor solutions are determined by ICP-‐OES. Results DMT analysis of Citrate solutions Composition of the solutions: Test 1 (Large differences in ionic strength between Donor and Acceptor solution): Donor solution: -‐1450µM K3Citrate -‐20µM ZnSO4 -‐400µM Ca(NO3)2 -‐500µM KNO3 -‐2.5mM MOPS -‐1.2mM NaOH pH: 7.2

Acceptor solution: -‐400µM Ca(NO3)2 -‐500µM KNO3 -‐2.5mM MOPS -‐1.3mM NaOH pH: 7.2

Test 2 (Due to differences in the ionic strength between acceptor and donor solutions in test 1, it had been decided to run another measurement with an adjusted acceptor solution): Donor solution: -‐1450µM K3Citrate -‐20µM ZnSO4 -‐400µM Ca(NO3)2 -‐500µM KNO3 -‐2.5mM MOPS -‐1.2mM NaOH pH: 7.2

Acceptor solution: -‐400µM Ca(NO3)2 -‐4.3mM KNO3 -‐500µM KNO3 -‐2.5mM MOPS -‐1.3mM NaOH pH: 7.2

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Both tests were carried out in duplicates. pH: pH’s in the Donor as well as in the Acceptor solution were constant over the course of both experiments (pH 7.15±0.04).

(a) (b)

(c) (d) Figure 1: (a) and (c) show the Zn concentration measured in the acceptor solution of the citrate treatment during the course of the experiment, while (a) represents Test 1 and (c) Test 2. The measured Zn values are adjusted using the correction factor derived from the squared K ratio between the donor and the acceptor solution. K concentrations in both acceptor and donor solutions are shown in (c) for Test 1 and in (d) for Test 2. Correction of the free Zn concentration at high differences in ionic strength The K concentrations of the Donor and acceptor solution need to be used for corrections of the free Zn in the donor solution especially in Test 1 using the following equation:

cfree Zn donor

= cZn acceptor

cK donor

cK acceptor

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K can be used for corrections since according to the model 99.5% of the total K in the donor solution are available in the free form. Using this equation, we get after 72 hours in test 1 a free Zn concentration in the donor solution of: 71±4.5nM. In test 2 we get a free Zn concentration of 43±0.32nM (Figure 1). The free Zn concentration predicted by the model was: 50 nM. Considering the large differences between total and free Zn in this solution, the difference of a factor of 1.4 in test 1 and a factor of 0.86 in test 2 between measurements and models lay in an acceptable range. DMT analysis of Histidine solutions Composition of the solutions: Donor solution: -‐1300µM L-‐Histidine -‐20µM ZnSO4 -‐400µM Ca(NO3)2 -‐500µM KNO3 -‐2.5mM MOPS -‐1.2mM NaOH pH: 7.2


Five replicates were analyzed. pH: pH’s in the Donor as well as in the Acceptor solution were constant over the course of both experiments between pH 7.05±0.08. K and Ca concentration were constant at a similar level in the donor and the acceptor solutions. Corrections of the free Zn concentration due to differences in ionic strength are therefore not necessary. No equilibrium in the Zn concentration in the acceptor solution was reached in the DMT system containing the histidine treatment after the experimental period of three days (Figure 2). Additionally, the measured Zn concentration in the acceptor solution leads to values that are about 30 times higher than one would expect according to the model calculations. However, between replicates the precision was found to be good. Possible explanations for the differences between the modeled and the measured values could be:

-‐ Extreme errors in the equilibrium models -‐> very inaccurate stability constants of histidine with Zn??

-‐ According to the model, about 25% of the total Zn occur in the form of positively charged ZnHis+ complexes. It has been stated, that it is possible that such complexes can pass across the negatively charged DMT membrane. Also membrane transport of

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neutral complexes can occur (Temminghoff et al., 2000). This might be of particular importance in this experiment since the remaining 75% of the total Zn form neutral Zn(His)2 complexes.

Figure 2: Zn concentrations measured in the acceptor solution of the histidine treatment during the course of the experiment (error bars represent Standard deviances between replicates). DMT analysis of EDTA solutions Composition of the solutions: Donor solution: -‐20.2µM H4EDTA -‐20µM ZnSO4 -‐400µM Ca(NO3)2 -‐500µM KNO3 -‐2.5mM MOPS -‐1.3mM NaOH pH: 7.2


Five replicates were analyzed. pH: pH’s in the Donor as well as in the Acceptor solution were constant over the course of both experiments between pH 7.08±0.04. K and Ca concentration were constant at a similar level in the donor and the acceptor solutions. Corrections of the free Zn concentration due to differences in ionic strength are therefore not needed. According to the model, the measured Zn values in the acceptor solution are about 10 times too high. In the case of EDTA however, it seems that the slightly too high total Zn concentration added to the donor solution had caused this problem. In average, the Zn concentration was 20.7µM instead of 20µM.

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(Accuracy in the experimental adding of EDTA was assured by preparing two different stock solutions (10mM and 1mM -‐> agreement between these two experiments was very well) Applying 20.7µM Zn to the speciation model, results in a free Zn concentration of: 510nM. This value fits quite well to the measured concentration of 488±41 (Figure 3). In order to achieve more accurate values, the experiment might need to be repeated using also a higher diluted stock solution for Zn.

Figure 3: Zn concentrations measured in the acceptor solution of the EDTA treatment during the course of the experiment (error bars represent Standard deviances between replicates). DMT analysis of Cysteine solutions Composition of the solutions: Donor solution: -‐355µM L-‐Cysteine -‐20µM ZnSO4 -‐400µM Ca(NO3)2 -‐500µM KNO3 -‐2.5mM MOPS -‐1.3mM NaOH pH: 7.2


Three replicates were analyzed. pH: pH’s in the Donor as well as in the Acceptor solution were constant over the course of both experiments between pH 7.07±0.01. Accuracy and precision of the Zn concentrations measured in the cysteine treatments were very bad (Figure 4). So far, I do not have an explanation for the decrease of the measured

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free Zn concentration in the acceptor solution after the first measurement after one day in two of the replicates. It also seems that no equilibrium is reached in the cysteine treatment. Speculations about the reasons that may be responsible for the problems in the measurements: It is probable that the 10% neutral complexes occurring in the solution according to the speciation model influenced the results, by being able to cross the DMT membrane. However, it is not possible that these neutral complexes are responsible alone for these unexpected results. A second very important point is the fact that cysteine is oxidized to cysteine very quickly when it’s in contact with O2. Cystine has a different stability constant with Zn and therefore, the speciation can be altered.

Figure 4: Zn concentrations measured in the acceptor solution of the cysteine treatment during the course of the experiment (error bars represent standard deviances between replicates).

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Conclusion and outlook Only for the citrate treatment a good agreement between the measured and the modeled free Zn values could be found. In the case of EDTA, the differences in the free Zn values found between the DMT measurements and the model predictions seem to be caused by an experimental error, as ZnEDTA speciation is extremely sensitive to small changes in the total Zn concentration. A better result could probably be achieved by using a more diluted stock solution for Zn addition. In the histidine treatment, it is likely that positive or neutral complexes are transported across the membrane. Therefore, the measured Zn values in the acceptor solution are much higher than expected by the models and they probably give no approximation of the free Zn concentration. In the case of cysteine, the results that are much higher than expected seem to be caused by a combination of different reasons. With the data collected so far, it is difficult to say, what the exact reasons are. For that purpose, more measurements would be needed. Presentations of the work I gave a presentation of my previous work carried out in Zurich in the Soil Chemistry Group at Wageningen University (NL). The presentation included plant experiments with relation to Zn speciation in the nutrient solution as well as the data collected using PLM. Back in Zurich I will present my DMT results achieved in Wageningen within the Soil Protection Group-‐Seminar. And many thanks to…

… COST FA 0905 for the financial support of my STSM … Ellis Hoffland for giving me the opportunity to carry out the STSM in Wageningen … Erwin Temminghoff for supervising my DMT work … Andreas Duffner for practical help with the DMT, for interesting discussions, for administrative support and for lending me a bike!

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References

Bayen, S., Worms, I., Parthasarathy, N., Wilkinson, K., Buffle, J., 2006. Cadmium bioavailability and speciation using the permeation liquid membrane. Analytica Chimica Acta 575(2): 267-‐273. Bell, P. F., McLaughlin, M. J., Cozens, G., Stevens, D. P., Owens, G., South, H., 2003. Plant uptake of C-‐14-‐EDTA, C-‐14-‐Citrate, and C-‐14-‐Histidine from chelator-‐buffered and conventional hydroponic solutions. Plant and Soil 253(2): 311-‐319. Haussling, M., Jorns, C. A., Lehmbecker, G., Hechtbuchholz, C., Marschner, H., 1988. ION AND WATER-‐UPTAKE IN RELATION TO ROOT DEVELOPMENT IN NORWAY SPRUCE (PICEA-‐ABIES (L) KARST). Journal of Plant Physiology 133(4): 486-‐491. Kalis, E. J. J., Weng, L. P., Dousma, F., Temminghoff, E. J. M., Van Riemsdijk, W. H., 2006. Measuring free metal ion concentrations in situ in natural waters using the Donnan Membrane Technique. Environmental Science & Technology 40(3): 955-‐961. Khademi, Z., Jones, D. L., Malakouti, M. J., Asadi, F., Ardebili, M., 2009. Organic acid mediated nutrient extraction efficiency in three calcareous soils. Australian Journal of Soil Research 47(2): 213-‐220. Meers, E., Lesage, E., Lamsal, S., Hopgood, M., Vervaeke, P., Tack, F. M. G., Verloo, M. G., 2005. Enhanced phytoextraction: I. Effect of EDTA and citric acid on heavy metal mobility in a calcareous soil. International Journal of Phytoremediation 7(2): 129-‐142. Nowack, B., Schwyzer, I., Schulin, R., 2008. Uptake of Zn and Fe by wheat (Triticum aestivum var. Greina) and transfer to the grains in the presence of chelating agents (ethylenediaminedisuccinic acid and ethylenediaminetetraacetic acid). Journal of Agricultural and Food Chemistry 56(12): 4643-‐4649. Panfili, F., Schneider, A., Vives, A., Perrot, F., Hubert, P., Pellerin, S., 2009. Cadmium uptake by durum wheat in presence of citrate. Plant and Soil 316(1-‐2): 299-‐309. Parthasarathy, N., Pelletier, M., Buffle, J., 1997. Hollow fiber based supported liquid membrane: a novel analytical system for trace metal analysis. Analytica Chimica Acta 350(1-‐2): 183-‐195. Pesavento, M., Alberti, G., Biesuz, R., 2009. Analytical methods for determination of free metal ion concentration, labile species fraction and metal complexation capacity of environmental waters: A review. Analytica Chimica Acta 631(2): 129-‐141. Slaveykova, V. I., Parthasarathy, N., Buffle, J., Wilkinson, K. J., 2004. Permeation liquid membrane as a tool for monitoring bioavailable Pb in natural waters. Science of the Total Environment 328(1-‐3): 55-‐68.

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Temminghoff, E. J. M., Plette, A. C. C., Van Eck, R., Van Riemsdijk, W. H., 2000. Determination of the chemical speciation of trace metals in aqueous systems by the Wageningen Donnan Membrane Technique. Analytica Chimica Acta 417(2): 149-‐157. Vassil, A. D., Kapulnik, Y., Raskin, I., Salt, D. E., 1998. The role of EDTA in lead transport and accumulation by Indian mustard. Plant Physiology 117(2): 447-‐453. vonWiren, N., Marschner, H., Romheld, V., 1996. Roots of iron-‐efficient maize also absorb phytosiderophore-‐chelated zinc. Plant Physiology 111(4): 1119-‐1125. Wang, P., Zhou, D. M., Luo, X. S., Li, L. Z., 2009. Effects of Zn-‐complexes on zinc uptake by wheat (Triticum aestivum) roots: a comprehensive consideration of physical, chemical and biological processes on biouptake. Plant and Soil 316(1-‐2): 177-‐192. White, P. J., Broadley, M. R., 2009. Biofortification of crops with seven mineral elements often lacking in human diets -‐ iron, zinc, copper, calcium, magnesium, selenium and iodine. New Phytologist 182(1): 49-‐84.

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