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Soil Sci. Soc. Am. J. 74:1607–1612 Published online 12 Aug.2010 doi:10.2136/sssaj2009.0295 Received 11 Aug. 2009. *Corresponding author (cheesman@ufl.edu). © Soil Science Society of America, 5585 Guilford Rd., Madison WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Interaction of Phosphorus Compounds with Anion-Exchange Membranes:

Implications for Soil Analysis

Soil Chemistry

Anion exchange membranes are commonly used to study P dynamics in soils and sediments (Saunders, 1964; Skogley and Dobermann, 1996; Myers et al.,

2005). In particular, they are used to measure “readily exchangeable” phosphate in soils (Sibbesen, 1978) and, in conjunction with hexanol fumigation, to determine P contained within the soil microbial biomass (Kouno et al., 1995; Myers et al., 1999). Anion exchange membranes offer potential benefits over conventional soil P tests given their action as passive ion sinks analogous to biological uptake (Qian and Schoenau, 2002). Their use in the field can provide an integrated measure of P avail-ability (Cooperband and Logan, 1994; Drohan et al., 2005; Meason and Idol, 2008) and has led to the development of commercial products designed to determine nutri-ent supply rates (e.g., PRS probes, Western Ag Innovations, Saskatoon, SK, Canada).

Despite numerous assessments of the practical influence of experimental design on both batch and field-deployed membranes (Qian et al., 1992; Qian and Schoenau, 1997, 2002; Sato and Comerford, 2006; Mason et al., 2008), few studies have examined their interaction with organic and condensed inorganic P compounds. Yet there is a clear potential for sorption and recovery of organic P to both resin beads and membranes (Rubaek and Sibbesen, 1993; Cooperband et al., 1999; McDowell et al., 2008). Of particular note is the ~100% recovery of

Alexander W. Cheesman*Soil and Water Science Dep.106 Newell HallUniv. of FloridaGainesville, FL 32611

Benjamin L. TurnerSmithsonian Tropical Research Inst.Apartado 0843-03092Balboa, Ancon, Republic of Panama

K. Ramesh ReddySoil and Water Science Dep.106 Newell HallUniv. of FloridaGainesville, FL 32611

Anion exchange membranes are commonly used to measure readily exchangeable and microbial P in soil, yet there is little information on their interactions with organic and condensed inorganic P compounds, which can interfere with interpretation of the results. We addressed this by quantifying the sorption of a range of P compounds to a commonly used anion exchange membrane (551642S, BDH-Prolabo, VWR International, Lutterworth, UK). Sorption and recovery of orthophosphate by the membranes was complete up to 1.17 g P m−2. The membranes also completely recovered sodium pyrophosphate, glucose 6-phosphate, and adenosine 5´-monophosphate, as well as significant levels (20–60%) of 2-aminoethylphosphonic acid, sodium hexametaphosphate, and sodium phytate. Only sodium pyrophosphate, sodium hexametaphosphate, and d-glucose 6-phosphate were detected subsequently as molybdate-reactive P after elution with 0.25 mol L−1 H2SO4, however, indicating their hydrolysis in the acid eluant. Solution 31P nuclear magnetic resonance spectroscopy was used to confirm the stability of the tested compounds when exposed to the membrane and the absence of significant concentrations of orthophosphate as trace contaminants in the compound preparations. Finally, for a series of tropical wetland soils from the Republic of Panama, we found negligible difference in eluted P concentrations determined by molybdate colorimetry and inductively coupled plasma optical emission spectrometry for both unfumigated and hexanol-fumigated samples. We therefore conclude that although organic and condensed inorganic P compounds can be recovered by anion exchange membranes, this is likely to have limited impact on the analysis of soil samples.

Abbreviations: HDPE, high-density polyethylene; ICP-OES, inductively coupled plasma optical emission spectrometry; MDP, methylenediphosphonic acid; MRP, molybdate reactive P; NMR, nuclear magnetic resonance.

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both phytic acid (myo-inositol hexakisphosphate, IP6) and glu-cose 6-phosphate by 204-U-386 membranes (Ionics, Watertown, MA) when exposed at 0.16 g P m−2 and loaded with a Cl− coun-terion (Cooperband et al., 1999)

Consideration of potential interactions between anion ex-change membranes and organic and condensed inorganic P com-pounds is important given the number of assumptions made dur-ing routine application of the exchange media to study P dynam-ics. First, if significant levels of organic and condensed inorganic P are recovered by anion exchange membranes and transferred to the eluant solution, their inclusion in subsequent measurements will lead to an overestimation of readily exchangeable orthophosphate.

Second, when anion exchange membranes are used as an ion sink during the measurement of microbial P (Kouno et al., 1995; Myers et al., 1999) the analysis of total P in the eluant is typically omitted because the difference between orthophosphate and total P is assumed to be negligible (Brookes et al., 1984), as in fumigation–extraction procedures (Brookes et al., 1982; Hedley et al., 1982). If organic or condensed inorganic P compounds re-leased from lysed microbial cells are adsorbed directly to anion exchange membranes without conversion to orthophosphate by enzymatic or matrix induced hydrolysis, however, this may lead to an underestimation of fumigation-released (microbial) P.

Finally, if organic and condensed inorganic P are recovered by anion exchange membranes, both the nature of the eluant used for P recovery and the method of P detection may influence the P concentration determined as recovered from a sample. The use of nonselective elemental analysis, such as inductively coupled plas-ma optical emission spectrometry (ICP-OES), or the hydrolysis of P-containing compounds during elution by mineral acids may erroneously attribute complex forms of P as bioavailable.

The aims of this study were to establish (i) the potential in-teractions between a commonly used anion exchange membrane and a range of model organic and condensed inorganic P com-pounds, and (ii) the implications of any interaction during the routine application of anion exchange membrane procedures to wetland soils expected to contain high levels of soluble organic and condensed inorganic P.

MATERIALS AND METHODSAnion Exchange Membranes

The anion exchange membrane characterized in this study (551642S, BDH-Prolabo, VWR International, Lutterworth, UK) is used routinely in field and laboratory procedures (McLaughlin et al., 1994; Myers et al., 2005; Roboredo and Coutinho, 2006; Turner and Romero, 2009). The membranes (previously sold under the BDH brand name) use a polystyrene–divinylbenzene copolymer base doped with quaternary ammonium as the ionogenic group and are supplied in 12.5- by 12.5-cm sheets preloaded with Cl− counterions.

The anion exchange membranes were cut into 1.5- by 6.25-cm strips, yielding a reactive area of 18.75 cm2 per strip. The strips were charged with HCO3

− counterions by shaking 25 strips in a 250-mL high-density polyethylene (HDPE) bottle with three sequential chang-es of 200 mL of 0.5 mol L−1 NaHCO3 during a 24-h period. The strips

were then rinsed three times in deionized water to remove adhering solu-tion. Bicarbonate was used as the counterion because it mimics biologi-cal uptake from the rhizosphere (Sibbesen, 1978; Qian and Schoenau, 2002) and is considered preferable when studying P dynamics due to its lower affinity for exchange sites compared with other commonly used counterions (e.g., Cl−) (Skogley and Dobermann, 1996).

After exposure to a test solution, the membranes were rinsed in deionized water, shaken dry of excess water, and immersed in a conical tube containing 50 mL of 0.25 mol L−1 H2SO4 and shaken for 3 h, after which a subsample of the eluant was decanted into a 20-mL scintilla-tion vial. The membranes were cleaned using a secondary acidic wash (0.25 mol L−1 H2SO4 for 1 h) and rinsed with multiple changes of de-ionized water before regeneration with NaHCO3 as described above. Phosphorus recovery was unaffected by repeated use of the membrane strips (data not shown) despite some discoloration following use with soil samples.

Phosphorus DeterminationMolybdate-reactive P, an operationally defined parameter, was

determined in the extracts by automated colorimetry with detec-tion at 880 nm using a flow injection analyzer (Lachat Quickchem 8500, Hach Ltd., Loveland, CO). Total P was determined in the ex-tracts by ICP-OES (Optima 2100, PerkinElmer, Waltham, MA). Orthophosphate standards were prepared in the same matrix as the samples (0.25 mol L−1 H2SO4) for both analyses.

Anion Exchange Membrane Exchange CapacityThe response of the exchange membranes to increased orthophos-

phate concentrations was tested to determine the exchange capacity of the membranes and the range across which the dynamic anion exchange mim-ics that of an infinite sink. Replicate (n = 3) preloaded and rinsed mem-branes were placed in 250-mL HDPE bottles with 75 mL of orthophos-phate standards between 0 and 2.5 mg P and shaken for 24 h. The adsorbed P was eluted and determined by molybdate colorimetry as described above.

Phosphorus Recovery by Anion Exchange Membrane Strips

A series of organic and condensed inorganic P compounds were prepared at an approximate concentration of 200 mg P L−1 in deion-ized water, with total P concentrations in the solutions determined subsequently by ICP-OES (Table 1). Duplicate preloaded and rinsed anion exchange membrane strips were loaded individually into 250-mL HDPE centrifuge bottles with 70 mL of deionized water and 5 mL of standard P-containing solutions. After exposure and elution from the membranes (see above), the eluants were stored in 20-mL HDPE scin-tillation vials at 4°C until analysis by molybdate colorimetry within 72 h and total P within 1 mo.

Purity and Stability of Organic and Condensed Inorganic Phosphates in Deionized Water

Standard solutions were analyzed by solution 31P nuclear mag-netic resonance (NMR) spectroscopy during 24 h to determine the stability of the P compounds during exposure to anion exchange mem-branes. Solutions (~200 mg P L−1) were mixed 1:1 with an internal

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standard (methylenediphosphonic acid; MDP) (~50 mg P L−1) made up in deionized water. Of the resulting mixture, 0.9 mL was added to 0.1 mL of deuterium oxide, the solution was vortexed, and then loaded into a 5-mm-diameter NMR tube. Solution 31P spectra were acquired after 15 min and 24 h using a Bruker Avance 500 Console, Magnex 11.75 T/54 mm magnet fitted with a 5-mm BBO probe. Acquisitions were run at a stabilized 25°C with a 2.47-µs (~30°) pulse length and a 2-s recycle delay. An average 2000 scans (run length, 1 h 21 min) were acquired and the resulting spectra referenced against internal MDP set as 17.46 ppm, determined with reference to an externally held 85% H3PO4 standard (chemical shift 0 ppm).

Extraction of Phosphorus Compounds from Wetland Soils by Anion Exchange Membranes

A comparison of total and molybdate-reactive P in the membrane eluants from wetland soil extractions was performed to determine the potential for organic and condensed inorganic P recovery when anion exchange membranes are used to measure readily exchangeable and mi-crobial P. A batch process using anion exchange membranes prepared as described above was applied to 27 soils collected from a nutrient gra-dient in the freshwater San San Pond Sak wetland, a domed peatland in Bocas del Toro province, western Panama (Phillips et al., 1997). The soils were all high in organic matter (total C 41–50%) but contained a range of total P concentrations (388–1028 mg P kg−1).

Two fresh samples of each soil (3.5 g dry weight equivalent) were weighed into 250-mL HDPE centrifuge bottles and sample-specific volumes of deionized water were added to bring the total water con-tent to 75 mL. Both samples received deionized water and a single anion exchange membrane strip (1.5 by 6.25 cm), with one subsample also receiving 1 mL of hexanol (95%, Sigma Aldrich Chemical Co., St Louis, MO). After 24 h of shaking, the anion exchange membrane strips were removed, rinsed, and eluted and the resulting solution ana-lyzed for total and molybdate-reactive P as described above. A signifi-cant difference between total and molybdate-reactive P would indi-cate the retention of organic or condensed inorganic P compounds that were not hydrolyzed to molybdate-reactive P during elution in 0.25 mol L−1 H2SO4.

In normal application of the method, the difference in molybdate-reactive P between the fumigated and unfumigated samples would be attributed to “fumigation-released P.” This provides an estimate of mi-

crobial P when adjusted for extraction efficiency, and microbial biomass not killed by fumigation (k factor) (Kouno et al., 1995). Given problems with both correction factors, however, some researchers have preferred simply to use uncorrected fumigation-released values (e.g., Bunemann et al., 2008). For this study, uncorrected total and molybdate-reactive P in the eluants of both unfumigated and fumigated samples were compared (mg P kg−1 dry weight).

RESULTSAnion Exchange Membrane Capacity

Orthophosphate recovered from a noncompetitive envi-ronment was within a 5% error for the majority of the exposure concentrations tested (Fig. 1). Only at >1.17 g P m−2 was re-covery unacceptable, defined here as <95%. Taking a conserva-tive working maximum of 1.00 g P m−2 of membrane and the utilized membrane area/soil ratio of 5.6, this would equate to an orthophosphate sorption capacity of 560 mg P kg−1—well

Table 1. Phosphorus compounds tested on anion exchange membranes. Stock solution concentrations determined by inductively coupled plasma optical emission spectrometry analysis.

Compound Chemical formula Standard conc.

mg P L−1

Potassium dihydrogen phosphate KH2PO4 100.0

Sodium hexametaphosphate (NaPO3)n ∙ Na2O 171.1

Sodium pyrophosphate, decahydrate Na4P2O7 ∙ 10H2O 207.6

d-Glucose 6-phosphate, disodium salt hydrate C6H11O9PNa2 ∙ xH2O 169.0

Adenosine 5´-monophosphate, monohydrate C10H14N5O7P ∙ H2O 157.5

2-Aminoethylphosphonic acid C2H8NPO3 195.6

Phytic acid†, dodecasodium salt C6H6O24P6Na12 183.6

RNA, Type VI from torula yeast (8.5% P) NA‡ 177.5DNA, from salmon testes (A260 = 17.5) NA 250.7† myo-Inositol hexakisphosphate, dodecasodium salt (sodium phytate).‡ NA, not appropriate.

Fig. 1. Exchange capacity of anion exchange membrane (AEM) strips. Values are plotted alongside limits of reasonable recovery (95–105%) of calculated exposure. A typical exposure level (dashed line A) of up to 0.25 g m−2 equates to 469 mg P kg−1 of exchangeable phosphate when using one strip (6.25 × 1.5 cm) with 1 g of soil. The limit of reasonable recovery rate (dashed line B) equates to 1.17 g P m−2 in a noncompetitive environment.

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above the maximum labile P likely to be encoun-tered in the natural environment.

Organic and Condensed Phosphorus Recovery by Anion Exchange Membrane Strips

The total P concentrations in the eluants demonstrated that most of the P compounds interacted with the anion exchange membranes (Fig. 2). Three compounds were recovered com-pletely (sodium pyrophosphate, glucose 6-phos-phate, and adenosine 5´-monophosphate), while three others showed recoveries between 20 and 60% (sodium hexametaphosphate, 2-aminoeth-ylphosphonic acid, and phytate). The macromol-ecules RNA and DNA showed very limited re-covery (~10 and 0.2%, respectively), although it was unclear if this represented true ion exchange at the ionogenic sites or physical adherence of the macromolecule to the polymeric membrane. Under the concentrated conditions of the experi-ment, a “sheen” was observed on the membrane strips exposed to DNA and RNA. We therefore considered that while DNA recovery was neg-ligible, the limited RNA recovery observed was inconclusive in the context of this experiment.

Of the compounds showing significant interaction with the membranes, some were re-covered as molybdate-unreactive P (adenosine 5´-monophosphate, 2-aminoethlphosphonic acid, and phytate), while large proportions of others were recovered as molybdate-reactive P (Fig. 2). For example, of the ~100% of pyrophos-phate and glucose 6-phosphate recovered by the anion exchange membrane, 36 and 69%, respec-tively, were detected as molybdate-reactive P.

Purity and Stability of Phosphorus Compounds in Deionized Water

Comparison of the 31P NMR spectra from test compounds with a known orthophosphate standard (Fig. 3) showed neither contamination of the original compounds with orthophosphate nor orthophosphate released by hydrolysis in de-ionized water during a period of 24 h. The pres-ence of molybdate-reactive P in the eluant solu-tions was therefore considered to be due entirely to acidic hydrolysis during elution of P from the membranes and subsequent storage before colo-rimetric analysis. Hydrolysis during colorimetric analysis itself (<1-min contact time) was likely to be negligible given the relatively long period of elution and storage in 0.25 mol L−1 H2SO4.

Fig. 3. Solution 31P nuclear magnetic resonance spectra of P compounds measured after 24 h in deionized water. Spectra were acquired using a 2.47-μs (~30°) pulse length and a 2-s recycle delay, with approximately 2000 scans required to achieve suitable signal to noise. Spectra are presented using 8-Hz line broadening and referenced to an internal methylenediphosphonic acid standard (17.46 ppm).

Fig. 2. Recovery of P compounds by anion exchange membrane (AEM) strips. Average values (n = 2) are shown, with all repeats within 2.3%. Recovered P plotted as molybdate-reactive P (MRP) or as non-molybdate-reactive P (difference between total P and MRP).

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Application to Wetland Soils for Exchangeable and Fumigation-released Phosphorus

For the total and molybdate-reactive P recovered by the an-ion exchange membranes in a standard extraction from wetland soils, paired t-tests showed significant (P < 0.05) differences be-tween molybdate-reactive P and total P determined in both un-fumigated and fumigated samples; however, the average differ-ences between molybdate-reactive and total P were only 3.4 and 1.3% within unfumigated and fumigated samples, respectively. Such small relative differences were below the margin of error due to calibration. We conclude, therefore, that there was no evidence that acid-stable P forms (i.e., adenosine 5´-monophos-phate, 2-aminoethylphosphonic acid, and phytate) were recov-ered in appreciable amounts by the anion exchange membranes.

DISCUSSIONThe orthophosphate exchange capacity for the studied anion

exchange membranes agrees well with the published information on the performance of similar membranes, including the linear re-sponse observed to 0.221 g P m−2 (Schoenau and Huang, 2001) and the total exchange capacity of commercially available anion ex-change membrane products. For example, The P exchange capacity of PRS probes is reported to be 3.014 g P m−2 based on the charge capacity (www.westernag.ca/innov/technical_1.php; verified 14 July 2010). Our window of acceptable recovery appears more robust than that demonstrated by Mason et al. (2008), who, assum-ing that a 6.26- by 2.5-cm strip has a reactive surface of 31.3 cm2, found a linear response to only 0.046 g P m−2. While this may be due to their use of a counterion (Cl−) with a higher affinity for the membrane exchange sites, it is also likely to have been influenced by their conservative interpretation of acceptable recovery (S. Mason, Univ. of Adelaide, personal communication, 2008).

Several organic and condensed inorganic P compounds in-teracted with, and in some cases were fully recovered by, a routine-

ly used anion exchange membrane method. Of these compounds, glucose 6-phosphate and the condensed inorganic P compounds pyrophosphate and hexametaphosphate were recovered partially as molybdate-reactive P. Given that orthophosphate contamina-tion in the original compounds was negligible and that the com-pounds were stable in deionized water during a 24-h period, we conclude that these compounds were hydrolyzed to orthophos-phate during elution from the membranes in 0.25 mol L−1 H2SO4 and storage before colorimetric analysis. The potential for acidic hydrolysis of organic and condensed inorganic P is a recognized source of error in the determination of orthophosphate by mo-lybdate colorimetry (Dick and Tabatabai, 1977; Worsfold et al., 2005), although hydrolysis during the acidic molybdate reaction (<1-min contact time) in the flow injection analysis procedure used here was considered to be negligible given the relatively long period of elution and storage in 0.25 mol L−1 H2SO4.

Similar studies examining the hydrolysis of standard organic P during extraction in strong acids reported variable results. For example, Ivanoff et al. (1998) detected 38% hydrolysis of para-nitrophenyl phosphate during a 3-h extraction in 1 mol L−1 HCl, whereas Bowman (1989) reported “negligible” hydrolysis of a se-ries of organic P compounds (para-nitrophenyl phosphate, glyc-erophosphate, phytic acid, and bis-para-nitrophenyl phosphate) during a short extraction in 1.08 mol L−1 H2SO4, including an initial exposure to concentrated H2SO4. Further analysis using nondestructive direct monitoring by 31P NMR spectroscopy would be required to establish the impact of pH-dependant hy-drolysis on model compounds during eluant holding times.

For a series of wetland soils with contrasting P contents, no detectable molybdate-unreactive P was recovered by the anion ex-change membranes (Fig. 4). The organic nature of the samples sug-gested that they might contain relatively high organic P concentra-tions, while fumigated samples were expected to contain high con-centrations of both organic and condensed inorganic P from lysed

Fig. 4. Comparison of total and molybdate-reactive P detected in anion exchange membrane eluants from wetland soil extractions. Unfumigated (X) and hexanol fumigated (O) samples show significant differences (paired t-test P < 0.05) between P determined by inductively coupled plasma optical emission spectrometry and colorimetric analysis. Linear correlations are: unfumigated y = 0.98x + 0.36, R2 = 0.999; fumigated y = 0.96x − 4.08, R2 = 0.997.

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microbial cells, although it is probable that much of these would be hydrolyzed to orthophosphate during fumigation (Brookes et al., 1982). This study demonstrates that, given the use of an acidic eluant, there is no significant difference between microbial P re-covered by anion exchange membranes as determined from total P analysis (ICP-OES) and operationally defined molybdate colo-rimetric analysis. This does not rule out the inclusion of acid-labile organic and condensed inorganic P within the molybdate-reactive P, especially because both types of compounds have been reported in soil solutions (Espinosa et al., 1999). Yet the absence of any molybdate-unreactive P in these samples suggests that acid-labile compounds are likely to be negligible, although the use of anion exchange membranes with a more benign eluant (e.g., NaHCO3 or KCl) would be needed to confirm this assumption.

In studies that have reported the recovery of organic P by ex-change media, an assumption was made, due to their mode of action, that resin-extractable organic P provided an estimate of labile soil or-ganic P (Rubaek and Sibbesen, 1993; McDowell et al., 2008). A pre-vious study of the nature of organic P in pasture soils using phospha-tase hydrolysis, however, reported negligible concentrations of labile phosphomonoesters (including polyphosphoric compounds), which was presumed to indicate a rapid hydrolysis of these compounds fol-lowing release into the soil solution (Turner et al., 2002). Further in-vestigation is needed to determine whether labile organic P may be recovered from soils by the anion exchange membranes tested here.

In conclusion, although anion exchange membranes can interact with a diverse range of P species, this does not appear to limit their application to the determination of readily exchangeable phosphate or microbial P in soils, particularly for samples from nat-ural environments. Care must be taken, however, in assigning P re-covered by anion exchange membranes to “labile orthophosphate,” because acid-labile organic and condensed inorganic phosphates may also be included. This could be especially problematic if mem-branes are deployed after the application of condensed inorganic P fertilizer (Bertrand et al., 2006). Although not fully resolved, the adaptation of the anion exchange membrane procedure to include mild membrane eluants (e.g., NaHCO3 or KCl) and analysis of molybdate-reactive and total P may allow the parallel assessment of labile orthophosphate and organic and condensed inorganic P.

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