Scientific registration n o : 471 Symposium n o : 6 Presentation : poster

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1 Scientific registration n o : 471 Symposium n o : 6 Presentation : poster Sequential extraction and 31 P NMR spectroscopy of whole soil and soil extracts Extraction séquentielle et spectroscopie RMN du 31 P des sols et des extraits de sol M c DOWELL Richard (1), MAHEIU Nathalie (2), CONDRON Leo (3), TRUDGILL Stephen (1) (1) Department of Geography, University of Cambridge, Downing Place, Cambridge CB2 3EN, UK (2) Department of Chemistry, Queen Mary and Westfield College, University of London, London E1 4NS, UK (3) Department of Soil Science, P.O. Box 84, Lincoln University, New Zealand Introduction The use of sequential extraction techniques (fractionation) to determine the plant availability and environmental significance of soil phosphorus (P) pools has been extensively developed and applied in a wide range of studies. However, the precise chemical nature of inorganic and organic P extracted from soil by different reagents is largely unknown. Nuclear magnetic resonance (NMR) spectroscopy has been shown to be a useful technique for the quantitative determination of P species in soil (solid-state) and soil extracts (liquid-state). There is considerable scope for the development of NMR to soil P studies to further our understanding of soil P cycling in relation to bioavailability and mobility in the soil environment. Materials and methods Soils All soils were taken from the Park Grass Continuous Hay Experiment, Hertfordshire, England (Warren and Johnston, 1964). They are silty clay loams of the Batcombe Series (paleo-argillic and argillic brown earths). The soils are from samples taken in 1991 from plots which receive annual applications of lime (since 1965) calculated to give a ph of 7. Plot treatments and soil characteristics are given in Table 1. Sequential extraction of soil P and additional soil analyses Soil samples (2 mm) were sequentially extracted using the scheme outline in Figure 1. The organic P (P o ) concentration of each soil extract was found as the difference between inorganic P (P i ) (Murphy & Riley, 1962) and total P (Brookes & Powlson, 1981). Organic carbon and total nitrogen were measured with a LECO C/N analyzer and soil ph determined in water (1:5 soil to solution ratio). 1

2 Table 1. Plot treatments and soil characteristics Plot Treatments ph Olsen P (mg P kg -1 ) % Total nitrogen % Organic carbon 4/2 N2 P P Na Mg /2 N2 P K Na Mg /1 N3 P K Na Mg FYM / Fishmeal /1 (N2 + ) P K Na Mg P K Na Mg N1 + P K Na Mg N2 K Na Mg Nitrogen (applied in spring) N1 N2 N3 sulphate of ammonia giving 48, 96, 144 kg N ha -1 N1 + nitrate of soda giving 48 kg N ha -1 (N2 + ) nitrate of soda giving 96 kg N ha -1 last applied in 1989 Minerals (applied in winter) P 35 kg P ha -1 as granular superphosphate (19% P 2 O 5 ) K 225 kg K ha -1 as sulphate of potash (50% K 2 O) Na 15 kg Na ha -1 as sulphate of soda (14% Na) Mg 10 kg Mg ha -1 as sulphate of magnesia (10% Mg) Organics (applied every fourth year) FYM 35 t ha -1 farmyard manure (bullocks) Fish meal about 6% N to give 63 kg N ha -1 All soils were sampled to 10 cm, air-dried and ground (< 210 µm) except those samples used for solid-state NMR (< 20 µm separated by sedimentation). Each replicate: triplicate 1 g samples of air-dried, sieved (< 210 µm) soil in 50 ml plastic centrifuge tubes. Add 30 ml 1M NH 4 Cl, shake 30 minutes, centrifuge and determine P i concentration of supernatant 25ml 0.1M NH 4 F, shake 4 hours, centrifuge (P i ; Chang & Jackson, 1959) 30ml 0.1M NaOH (I), shake 17 h, centrifuge (P i and P o ) 30ml deionised water, shake 30 minutes, centrifuge and discard supernatant 30ml 0.5M H 2 SO 4, shake 17 h, centrifuge (P i and P o ) 30ml deionised water, shake 30 minutes, centrifuge and discard supernatant 30ml 0.1M NaOH (II), shake 17 h, centrifuge (Pi and Po) 30ml deionised water, shake 30 minutes, centrifuge and discard supernatant residue (P i ; Brookes & Powlson, 1981) Total soil P (Brookes & Powlson, 1981). Figure 1. Scheme for the sequential extraction of soil P Solid-state 31 P NMR Solid-state 31 P HP/Dec (High Power Decoupling) NMR spectra were obtained using a Bruker MSL 300 operating at MHz. Soil samples were packed into 4 mm zirconia 2

3 rotors, with Kel-F caps and spun at magic angle spinning rates of 12 khz. Depending upon the sample, between and scans were accumulated at a repetition rate of 500 ms (9 42 hours). Spectra were convoluted with a Lorentzian lineshape of width 100 Hz. Chemical shifts were determined relative to 85% orthophosphoric acid (H 3 PO 4 ) and are expressed as δ values in ppm. Spectra were deconvoluted using the Bruker Winfit programme. Liquid-state 31 P NMR The liquid state 31 P NMR spectra were obtained using a Bruker AMX 600 operating at 243 MHz. Samples were spun at 20 Hz, and a total acquisition time of 2 s was used. Depending on the concentration of the samples, between 3000 and scans were added ( 2 22 hours). After convolution of the spectra with a Lorentzian of width 5 to 20 Hz, the areas were measured electronically using Bruker software. The peak positions are relative to 85% H 3 PO 4. Results and discussion The concentration of total P ranged from 601 to 2048 µg g -1 of soil. The higher concentrations were found in those plots receiving regular applications of P fertilizer (Table 2). Within this group (1575 to 2148 µg g -1 ) total P is dependent upon how much P the crop has removed. Where FYM was applied larger proportions of NaOH I P o and II P o were present compared to soils with a higher total P (e.g. plots 9/2 and 15). Higher levels of NaOH I extractable (labile) P o and especially NaOH II extractable (recalcitrant) P o were found in soils which have received N as ammonium sulphate (9/2 and 11/1) than those plots (of equivalent total P) which have received N as sodium nitrate (14/1 and 16). This may be the legacy of decreased organic matter mineralization and contamination of the soil from the litter mat formed (evident from the higher organic C content of these soils [Table 1]) when those plots were very acid (< ph 4 in 1923), before a new liming regime was started in Table 2. Concentrations of P fractions in whole soil samples Plot NH 4 Cl-P NH 4 F-P NaOH P i NaOH P o H 2 SO 4 -P NaOH II P i NaOH II P o (µg g -1 ) (µg g -1 ) (µg g -1 ) (µg g -1 ) (µg g -1 ) (µg g -1 ) (µg g -1 ) Residual P (µg g -1 ) Total P (µg g -1 ) 4/ / / For solid state (SS) NMR peak integration data ranged from to 658 ppm. These correspond to the total P of samples by sequential extraction. This relationship is confirmed by a significant regression function (y = 0.31x r = *** ). Discrepancies between total P by SS NMR and wet chemical methods can be accounted for by different particle sizes (< 20 µm for SS NMR and < 2 mm for sequential extraction and liquid-state NMR), paramagnetics and manual error. Peak integration 3

4 data (table 3) suggests that 57% and 33% of P detected in sample 14/1 is Ca-P is Al-P respectively. After treatment with citrate-dithionite the proportion of Ca-P in the sample by peak integration increased, suggesting citrate-dithionite selectively removes some Al-P. Subsequent studies suggest 0.5M NH 4 F (ph 8.5) extracts more Al-P without removing significant amounts of Ca-P. Due to overlapping peaks (see Figure 2), peak integration overestimates the size of the Ca-P fraction in non-citrate dithionite treated samples. Calibration of spectra usinbg proportions of total P, Al and Fe by oxalate extraction (Lookman et al., 1996) gives a more reliable estimate of different P pool sizes. Proportions of Ca-P by peak integration were linearly associated with Ca-P measured by sequential extraction with 0.5M H 2 SO 4 (y = 0.755x r = 0.734). Using calibrated data this relationship was significantly improved (y = 0.097x r = *** ). A similar relationship (y = x r = *** ) is obtained for the sum of Al-P and Fe-P by calibrated SS NMR to the sum of extracted Al-P and Fe-P (0.5M NH 4 F, 0.1M NaOH [I and II] P i and P o ). All samples (except 18, no P fertilizer) contained a high percentage of P ox as Ca-P (by calibrated SS NMR). This reflects fertilizer-manure history and soil ph (Table 1). Lookman et al. (1996) found most P was associated with Al and a much smaller fraction with Ca. They suggested Ca-P contributes a considerable proportion of P ox desorbable into a labile soil solution pool. Using this rationale P concentrations in dilute CaCl 2 extracts (used to simulate soil solution and mobile P) used in liquid NMR could be a reflection of Ca-P as determined by calibrated SS NMR. However, while high P concentrations of CaCl 2 extractable P were present in high fertility soils (those with an Olsen-P greater than that of plot 13), this is not related directly to the quantity of Ca-P by calibrated SS NMR. This is supported by the work by Holford and Mattingly (1976) on the same soil type which suggests Al-P and Fe-P play a more dominant role. The physical movement of soil particles results in the redistribution of nutrients. If the soils used in this study were disaggregated the lighter clay and small silt size fractions would be selectively transported to aquatic systems over long distances and time periods (allowing labile P to move and dissolve). The high levels of labile P found in the < 20 µm particle size used for SS NMR would therefore be a source of P contamination to aquatic systems. Figure P SS NMR spectra of sample 14/1 after treatment with citrate-dithionite showing possible peak assignments. 4

5 Table 3. Peak integration values (ppm) and quantitative estimation of the different P forms in the soil samples investigated based on deconvolution of 31 P SS NMR spectra Plot Sum Ca-P minerals (ppm) Sum Al-P minerals (ppm) Total P (ppm) P ox mmol kg -1 5 Fe ox mmol kg -1 Al ox Ca-P Al-P Fe-P a mmol kg / (100%) b (34%) (35%) (31%) (50%) (31%) (19%) 9/ (44%) (33%) (23%) 11/ (47%) (34%) 9.50 (17%) (51%) 9.55 (33%) (51%) 14/ (57%) (33%) (52%) (31%) 8.62 (17%) (44%) (36%) (20%) (49%) (32%) (19%) (43%) 2.77 (37%) 1.52 (20%) 14/1 cit-di c (62%) (28%) ND d ND ND a The amount of Fe-P cannot be determined from the NMR spectra, but were calculated from the Fe ox /Al ox ratio and total P ox of the samples, assuming that Fe and Al have the same affinity for P on a molar basis as per Lookman et al. (1996). b Percent of P ox in the NMR sample c Citrate-dithionite treated sample. d ND = not determined. For liquid state NMR between and and and µg P g -1 soil were measured in CaCl 2 and NaOH extracts respectively. Concentrations of each component P fraction individually or in any combination follow no direct sequentially extracted or mineral fraction (by calibrated SS NMR), but appear to be a function of Al- P, Fe-P and Ca-P by sequential extraction and calibrated SS NMR. To the authors knowledge this is the first reported NMR spectra of CaCl 2 extractants designed to simulate soil solution. It is interesting to note the larger variety of organic P fractions present in the CaCl 2 extracts compared to the NaOH extracts (although the latter extracts more organic P). Aromatic phosphate diesters appear in the spectra of all CaCl 2 extracts and do not appear in the spectra of NaOH extracts. This indicates either the different fractions extracted, or the decomposition of aromatic phosphate diesters during extraction. In NaOH extracts monoester P comprises most of the organic P fraction in those plots receiving long term P fertilizer. A much higher diester P fraction is present in plot 18 which has received no P fertilizer. The large amount of organic P and the presence of diester P in the NaOH extract of plot 13 may reflect organic P applied in FYM and associated microbial activity (Newman & Tate, 1982). Table 4. Concentrations (µg g -1 soil) and peak assignments (δ [ppm]) of P compounds and percentages of total P in re-suspended extract analyzed by liquid-state NMR.

6 Plot phosphonate ( ) orthophosphate ( ) CaCl 2 4/ (1.7%) (47.5%) (56.6%) 14/ (73.6%) (47.4%) NaOH 4/ (1.4%) (58.1%) (39.8%) 14/ (65.2%) (34.4%) Conclusions phosphate monoesters ( ) 9.52 (41.8%) 5.41 (40.0%) 6.09 (24.18%) 4.75 (37.1%) (23.7%) (42.4%) (27.3%) (48.0%) phosphate diesters ( ) aromatic phosphate diesters ( ) 1.53 (6.5%) 0.52 (2.3) (1.5%) (1.9%) (0.6%) (10.4%) (2.0%) 8.10 (6.3%) 3.2 (6.4%) 3.70 (4.6%) 6.40 (15.3%) pyrophosphate ( ) polyphosphate ( ) unknown ( ) (2.1%) (3.74%) 4.97 (3.9%) (7.5%) (3.0%) (2.2%) It was found that calibrated SS NMR spectra gave better resolution of individual Al-P, Fe-P and Ca-P phases in-situ while still following general trends of sequentially extracted P fractions. Solid-state NMR can therefore be used as a refined tool to measure P transformations in soil. Liquid-state NMR spectra of mild CaCl 2 extracts gave a wider range of labile organic P compounds, than spectra of NaOH extracts. This is the first time CaCl 2 extracts designed to simulate soil solution have been successfully measured by NMR, and therefore, provide a method to study P transformations from soil to soil solution and potential transfer to aquatic ecosystems. Acknowledgments This work was supported by Grant AO 6462 from the United Kingdom Biotechnology and Biological Sciences Research Council. The NMR instrumentation was provided by the University of London Intercollegiate Research scheme and is located at University College (Bruker MSL 300, Dr. A. E. Aliev) and Queen Mary and Westfield College (Bruker AMX 600, Dr. H. Toms). References Brookes, P.C. & Powlson, D.C Preventing phosphorus losses during perchloric acid digestion of sodium bicarbonate soil extracts. Journal of the Science of Food and Agriculture, 32, Chang, S.C. & Jackson, M.L Fractionation of soil phosphorus. Soil Science, 84, Holford, I.C.R. & Mattingly, G.E.G The high- and low-energy phosphate adsorbing surfaces in calcareous soils. Journal of Soil Science, 26,

7 Leinweber, P., Haumaier, L. & Zech, W Sequential extractions and 31 P-NMR spectroscopy of phosphorus forms in animal manures, whole soils and particle-size separates from a densely populated livestock area in northwest Germany. Biology and Fertility of Soils, 25, Lookman, R., Geerts, H., Grobet, P., Merckx, R. & Vlassak, K Phosphate speciation in excessively fertilized soil: a 31 P and 27 Al MAS NMR spectroscopic study. European Journal of Soil Science, 47, Murphy, J. & Riley, J.B A modified single solution method for the determination of phosphorus in natural waters. Analytica Chimica Acta, 27, Nanny, M.A. & Minear, R.A P FT-NMR of concentrated lake water samples. In NMR Spectroscopy in Environmental Chemistry (eds. Nanny, M.A., Minear, R.A. & Leehneer, J.A), pp Oxford University Press, New York. Tate, K.R. & Newman, R.H Phosphorus fractions of a climosequence of soils in New Zealand tussock grassland. Soil Biology and Biochemistry, 4, Keywords : phosphorus, fractionation, liquid state 31 P NMR, solid state 31 P NMR Mots clés : phosphore, fractionnement, 31 P RMN du liquide, 31 P RMN du solide 7