Evaluation of the factors affecting direct polarization solid state 31P-NMR spectroscopy of bulk soils

³¹P‐NMR spectroscopy on bulk soils is a powerful tool for the identification of the different phosphorus forms in soils and for the evaluation of the dynamics of soil P. Up to now the majority of the papers dealt with liquid state ³¹P‐NMR spectroscopy on soluble soil organic substances. Only few papers were addressed to the study of the different phosphorus forms directly in bulk soils. In the present paper, some organic and inorganic phosphates of known structures, which are likely to be present in soil systems, were studied by direct polarization (DP) magic angle spinning (MAS) ³¹P‐NMR spectroscopy in order to understand the electronic factors responsible for chemical shifts of the phosphorus (P) nucleus and to serve as guidelines to assign P resonances in soil spectra. Number of hydrating water molecules, type of counter‐cation, degree of covalence, and spatial conformation of P in phosphate structures were found to affect signal positions in ³¹P‐NMR spectra. Both hydrating water and increase in degree of covalence of the X‐O‐P bonds (X=H, Na) enhanced the electronic density (E_D) around P, thereby producing up‐field shifts in ³¹P‐NMR spectra. The exchange of the Na⁺ counter‐cation with NH₄⁺ resulted in an increase of the cation potential (P_C) that is a measure of the cation polarizing power, and induced a down‐field shift of P signals, due to a corresponding reduction in E_D around the P nucleus. Both NMR down‐ and up‐field shifts were observed in organic phosphates, and were dependent on the spatial orientation of the phosphate groups that may have been fixed anisotropically in the solid state. Based on the factors that influence P chemical shifts for standard phosphates, attempts to assign ³¹P‐NMR signals in the spectra of five different unperturbed bulk soils were made.     

Loss of phosphorus from soil in semi-arid northern Tanzania as a result of cropping: evidence from sequential extraction and 31P-NMR spectroscopy

In semi‐arid northern Tanzania, the native woodland is being rapidly cleared and replaced by low input agriculture. This has resulted in pronounced environmental degradation, and in particular loss of phosphorus (P) from the soil. We have used sequential extraction and ³¹P‐NMR to investigate the effects of land use changes, i.e. native woodland, degraded woodland, cultivation for 3 and 15 years and homestead fields where manure was applied, on the amount and structural composition of P in this soil. Clearing and continuous cultivation reduced both organic and inorganic P in the soil. The difference in the amount of organic P from the bulk soil of the fields cultivated for 3 and 15 years was not statistically significant (P < 0.05), suggesting that most of the depletion in organic P occurred during the first 3 years of cultivation. By contrast, in the homesteads, there was much organic and inorganic P in the soil. The ³¹P‐NMR revealed that cultivation resulted in a 53% depletion of orthophosphate diester P, whereas only a 30% and 39% reduction of orthophosphate monoester P was found in the bulk soil after 3 and 15 years of cultivation, respectively. These results concur with the suggestion that diester P constitutes more easily mineralizable forms of organic P in soil than does monoester P. Our ³¹P‐NMR also showed that 70% of the inorganic orthophosphate P was depleted from the coarse and fine sand separates as a result of cultivation. The influence of clearing and subsequent cropping on the amount and forms of P was more pronounced in the coarse and fine sand than in the silt and clay, stressing the importance of particle size and chemical properties such as organic matter and oxides in the availability of P in this soil. Our results show that the current low input agricultural practice is not sustainable, and that practices must be developed to combat the ongoing degradation of the soil. A combined use of available organic materials such as animal manure with the judicious use of inorganic fertilizers can replenish the soil's fertility.     

Land-use effects on the composition of organic matter in particle-size separates of soils: II. CPMAS and solution 13C NMR analysis

Soils from A horizons of Eutrochrepts under spruce forest (Sf), mixed deciduous forest (Df), permanent grassland (Gp), and arable rotation (Ar) were fractionated into clay- (<2 μm), silt-(2–20 μm) and sand- (20–2000 μm) sized separates. ¹³C NMR spectroscopy was used to compare SOM composition across size separates and between land-use regimes. CPMAS ¹³C NMR spectroscopy showed that the intensity of signals assigned to carbohydrates (representing most O-alkyl C) and lignin (phenolic and methoxyl C) declined with decreasing particle size. Concurrently, alkyl C and C-substitution of aromatic C increased in the order sand, silt, clay. The amount of alkyl C correlated well with microbial resynthesis of carbohydrates. Solution ¹³C NMR spectra suggested that humic acids (HA) extracted from the size separates were richer in carboxyl C and aromatic C than the bulk size separates. Also HA reflected increasing percentage of alkyl C with decreasing particle size. O-alkyl C were lower in silt HA than in clay HA whereas aromatic C tended to peak in silt HA. These results suggested that sand-sized separates were enriched in plant residues (primary resources) whereas clay-sized separates were dominated by products of microbial resynthesis (secondary resources). Silt was rich in selectively preserved and microbially transformed primary resources. ¹³C NMR spectroscopy showed only small differences in SOM composition between land-use regimes, except that silt and silt HA from Ar were richer in aromatic C than those from the other plots. But enrichment factors (E= content in fraction/content in whole soil) revealed differences in the distribution of C species across the size separates. Relatively high E_aromatic (0.9) and E_o-alkyl (1.0) for sand from Gp indicated high amounts of plant residues, probably due to intense rhizodeposition and to occlusion of plant debris within aggregates. Low E_aromatic (0.3) and E_o-alkyl (0.3) for sand from Ar suggested depletion of primary resources, which could be attributed to disintegration of soil aggregates upon cultivation. A pronounced enrichment of alkyl C in Ar clay-sized separates (E_alkyl= 3.1) suggested large amounts of microbial carbon. Microbial products attached to clay surfaces by a variety of physico-chemical bondings appeared more stable against mineralization induced by cultivation than plant residues sequestered in aggregates.     

Phosphorus speciation in temperate basaltic grassland soils by solution 31P NMR spectroscopy

Phosphorus (P) speciation in 21 basaltic and four non-basaltic Irish grassland soils was determined by NaOH–EDTA extraction and ³¹P NMR spectroscopy. Organic P in basaltic soils ranged between 30 and 697 mg P kg⁻¹ and consisted of phosphate monoesters (84–100%), DNA (0–16%) and phosphonates (0–5%). Inorganic P was mainly phosphate (83–100%) with small concentrations of pyrophosphate (0–17%). Phosphate monoesters were more important as a proportion of extracted P in basaltic soils, probably because of their greater oxalate-extractable Fe and Al contents. Phosphate monoesters appeared to be strongly associated with non-crystalline Al and increased with total soil P concentration, indicating that they do accumulate in grassland soils. In non-basaltic soils myo -inositol hexakisphosphate constituted between 20 and 52% of organic P, while scyllo -inositol hexakisphosphate constituted between 12 and 17%. These compounds were not quantified separately in basaltic soils because of poor NMR resolution in the phosphate monoester region, but appeared to represent a considerable proportion of the organic P in most samples. DNA concentrations were greater in basaltic soils compared with non-basaltic soils and were associated with acidic pH and large total C contents. The inability of the Olsen P test to assess effectively the P status of basaltic soils may result from strong phosphate sorption to Fe and Al oxides, inducing plant utilization of soil organic P. Phosphorus nutrient management should account for this to avoid over-application of P and associated financial and environmental costs.     

Forms and rates of release of 137Cs in two peat soils

Cation exchange resin saturated with H⁺ and Ca²⁺ was used to extract ¹³⁷Cs from peat soil at two sites in Britain affected by ^l37Cs deposition following the Chernobyl accident. The technique identified three classes of ¹³⁷Cs, similar to those observed for K⁺ in soils: ‘Fast’, ‘Intermediate’ and ‘Slow’. These classes are probably related to the selectivity for ¹³⁷Cs of the cation exchange sites on the organic matter and the clay minerals, and to the structure of the soil. With one exception, most ¹³⁷Cs was in the ‘Slow’ form and was only very slowly released to the resins, if at all. However, there was enough ^l37Cs in the ‘Fast’ and ‘Intermediate’ forms to contaminate pasture and thus grazing animals for some years. Based on the resin technique, it is estimated that contamination will persist for several decades in uplands contaminated at these activity concentrations.     

Land-use related organic matter dynamics in North Cameroon soils assessed by 13C analysis of soil organic matter fractions

Topsoil samples from cultivated and adjacent non‐cultivated fields on three major agricultural soils in North Cameroon were fractionated into particle‐size fractions that were analysed subsequently for their C and ¹³C contents. The aim was to obtain further insight into the dynamics of soil organic matter (SOM) in relation to land use in Cameroon. Since organic carbon contents of the fractions were often very small, samples and analyses were extensively replicated to obtain robust statistical estimates of observed differences. For each soil type, differences in δ¹³C values between fields could be related to changes in the input and decomposition of organic matter arising from soil type, land management and, for example, the nature and abundance of weeds. Turnover of organic matter appeared to be fastest in the sand fraction, which is in line with results from earlier studies. In the finer fractions, clear differences in reaction to changes in input and decomposition were observed, that seem to be linked to differences in clay mineralogy. The results illustrate that SOM in the various fractions is much less stable and more strongly affected by changes in land use than might be assumed on the basis of changes in total SOM contents alone. At the same time, they demonstrate the relevance of ¹³C isotope analyses of SOM for studies on the impact of land use on these savannah soils with little SOM that are highly susceptible to degradation.     

Phosphate speciation in excessively fertilized soil: a 31P and 27Al MAS NMR spectroscopy study

Both P and Al MAS NMR spectra of samples of excessively fertilized sandy soil provided information about the P and Al speciation. Peak deconvolution was used to interpret reliably and quantitatively the ³¹P NMR spectra recorded. Most of the P was found to be associated with Al. Part of the P exhibited a chemical shift that could be attributed to octocalcium phosphate, amorphous calcium phosphate or apatite. Apatite has, however, never been reported to occur in sandy soils of temperate climates. A dithionite extraction used to remove interfering Fe from the samples also removed most of the octahedral Al-P phase. After oxalate extraction more than 99% of the original P signal disappeared. About 7.5 to 11 % of the total oxalate extractable P of the excessively fertilized soil was present as a Ca-P phase, even though these soils are slightly acid to acid. The estimated size of the Ca-P phase roughly corresponds to the size of the labile P pool of these soils, as assessed in long-term batch desorption experiments. It still remains unclear whether the labile P pool should be attributed solely to such a Ca-P phase.     

13C and 15N natural abundances of urban soils and herbaceous vegetation in Karlsruhe, Germany

We undertook what we believe to be a unique survey of the natural abundances of ¹³C and ¹⁵N in urban soils and plants in Karlsruhe (Germany), a European city of average size. We found broad patterns of these abundances in both soils and plants, which reflected geology and land use. In contrast with studies on smaller areas (showing the direct effect of human activities), our study first determined the extent to which the abundances correlated with land use or underlying geology and then assessed how we could further test such relationships. The spatial pattern of δ¹³C in surface soil correlated with that of the underlying parent material; construction activities superimposed a secondary signal. Maize cultivation was a source of less negative soil δ¹³C, whereas the C₃ vegetation is a source of more negative soil δ¹³C. There was a footprint of less negative plant δ¹³C in the industrial and port areas; plant δ¹³C downwind of the city was less negative than upwind, which might relate to atmospheric pollution from the port area or to differences in soil properties. There was no significant effect of wind direction or geology on soil or plant δ¹⁵N, which was correlated mainly with land use. The largest soil δ¹⁵N was under agriculture and the smallest under woodland. The abundance of ¹⁵N in inner-urban soil and plants was intermediate between those of agriculture and forests. This study represents a major advance in the use of stable isotope geochemistry in understanding urban environments.     

Nitrogen in particle size fractions of soils incubated for five years with 15N-ammonium and 14C-hemicellulose

Four soils with a range of clay and silt contents were incubated for 5 a with ¹⁵N-labelled (NH₄)SO₄ and ¹⁴C-labelled hemicellulose and then fractionated according to particle size by ultrasonic dispersion and sedimentation. The distribution of labelled and native N between clay, silt and sand fractions was determined and elated to previous results on the C distributions. Between 29% and 48% of the added N was found in organic form. The ¹⁵N atom percentage excess decreased in the order: clay > whole soil > silt > sand. For both clay and silt, the enrichment factor for labelled and native N decreased with increasing fraction weight. Clay enrichment was higher for labelled than for native N, the converse being true for silt. The distribution of whole soil labelled organic N was: clay 77–91%, silt 4–11%, and sand <0.5%. Corresponding values for native N were 69–74%, 16–22%, and 1–2%, respectively. All soils had higher proportions of labelled than of native N in the clay, the converse was true for the silt. The C/N ratio of the native silt organic matter was higher and that of clay organic matter lower than whole soil C/N ratios. Differences between the C/N ratio distributions of native and labelled organic matter were small. The relative distribution of labelled N and C was very similar confirming that the turnover of C and N in soil organic matter is closely interrelated.     

Phosphate sorption by synthetic amorphous aluminium hydroxides: a 27Al and 31P solid-state MAS NMR spectroscopy study

The sorption of phosphate on amorphous aluminium hydroxides was investigated using ²⁷Al and ⁷¹P solid-state magic-angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy, following the effect of different exposures to soluble phosphate. The spectra obtained were compared with the spectrum of amorphous aluminium phosphate. Aluminium in the unreacted hydroxide had a 100% octahedral co-ordination. When dried at 200°C and exposed to soluble phosphate, very little (maximum 0.1%) amorphous aluminium hydroxide transformed to a tetrahedral co-ordination (A1 bound by oxygen bridges to four P atoms), even after 120d. The tetrahedral co-ordination exists in aluminium phosphate gel, although most of its A1 atoms exhibit an octahedral co-ordination. For the aluminium hydroxide dried at 200°C, no formation of aluminium phosphate in which aluminium is in octahedral co-ordination could be detected, not even when the aluminium hydroxide was exposed to a phosphate solution for 120 d. We concluded that the formation of aluminium phosphate is restricted to the surface of the hydroxide. Most of the phosphate which is bound to the aluminium oxide however may not have formed a ‘bulk solid’ aluminium phosphate, but is adsorbed on the internal and external surface of the oxide. The same amorphous aluminium hydroxide, dried at 70°C instead of 200°C, is converted much more rapidly to aluminium phosphate when exposed to soluble phosphate. We propose a P-induced weathering mechanism to describe P sorption on amorphous aluminium hydroxides at high P concentrations. In addition to NMR, phosphate adsorption experiments conducted on aluminium hydroxides dried at different temperatures produced evidence that the porosity of the aluminium hydroxide aggregated particles can also be a factor controlling the rate of phosphate uptake from solution, if the aggregate is stable (is not resuspended) in solution.     

Organic phosphorus speciation and pedogenesis: analysis by solution 31P nuclear magnetic resonance spectroscopy

Changes in phosphorus (P) during soil development are central to the understanding of labile P for plant productivity and soil P management. We used NaOH‐EDTA extraction with ³¹P nuclear magnetic resonance spectroscopy (³¹P NMR), sequential P fractionation, and general soil chemical characterization to better our understanding of P dynamics within two chronosequences (Manawatu and Reefton) and one Basalt maturity sequence under original native vegetation. With time, orthophosphate and orthophosphate monoesters tended to increase with organic C to a maximum of about two‐thirds of NaOH‐EDTA‐extractable P in young soils (16 000 years in the Reefton chronosequence), but gradually declined thereafter to about one‐third of NaOH‐EDTA‐extractable P in the oldest soils (130 000 years old). This coincided with a depletion of P from primary minerals (e.g. apatite) and readily available P for plant production. This depletion of inorganic P resulted in a greater reliance on organic P cycling via mineralization, hence the depletion of the normally recalcitrant monoester‐P pool. Concomitantly, the build‐up of labile P species (diesters and pyrophosphate) and scyllo‐ over myo‐inositol hexakisphosphate occurred as soils developed, and might be attributed to microbial activity, including scavenging for P. This work highlights the importance of organic P cycling during pedogenesis.     

Fertilization effects on organic matter in physically fractionated soils as studied by 13C NMR: Results from two long-term field experiments

^l3C–nuclear magnetic resonance (NMR) spectra taken using magic–angle spinning (MAS), cross polarization (CP) and with total suppression of side bands (TOSS) are reported for soils from two long–term field experiments. One set of soils was from the Broadbalk Experiment at Rothamsted, UK (monoculture of winter wheat since 1843) and the other was from the Lermarken site of the Askov Long–Term Experiment on Animal Manure and Mineral Fertilizers (arable rotation since 1894). At both sites soil samples were taken from three fertilizer treatments: nil, inorganic fertilizers, animal manure. Spectra were obtained from whole soil samples and from the size fractions clay (<2 μrn), silt (2–20 μm) and, in some cases, sand (20–2000 μm). Comparison of the total strengths of the ¹³C–NMR signal for each size separate in relation to its total organic C content shows that clay, particularly, contains large percentages of C not detected by NMR because of the large magnetic susceptibilities of the soil minerals. It is proposed that the observed signals come from the more labile pools of soil organic matter (SOM), on the presumption that these pools are less closely associated with soil minerals and iron oxides and are likely to be less protected from microbial or enzymic decomposition. For both Rothamsted and Askov, functional groups in the 45–110 ppm region (N– and O–alkyls) dominate in the spectra for whole soils, with aromatics (110–160 ppm) and alkyls (0–45 ppm) signals being the next prominent. In the Askov whole soil samples ¹³C–NMR revealed no differences between nil, inorganic fertilizer and animal manure treatments but in the Rothamsted whole soil there were some small differences. Clay and silt fractions from Askov contain more alkyls and less aromatics than those from Rothamsted. For both sites clay in enriched in alkyls and depleted in aromatics relative to silt. Clay from Askov, but not Rothamsted, contains more N–alkyls (45–65 ppm) and less acetals (90–110 ppm) than silt. O–alkyls (65–90 ppm) account for more than 20% of the total signal in clay and silt from both sites. Fertilization regimes have not significantly affected the chemical composition of SOM associated with clay– and silt–sized fractions in the soils at either site. We conclude that the chemical composition of SOM is determined primarily by the interaction between the organisms responsible for decomposition and the mineral soil matrix rather than the nature of substrate input.     

Availability of P from 32P-labelled endogenous soil P and 33P-labelled fertilizer in an alkaline soil producing cotton in Australia

Yield responses of irrigated, field‐grown cotton to phosphorus fertilizer application in Australia have been variable. In an attempt to understand better this variability, the distribution of fertilizer P within soil P fractions was identified using ³²P and ³³P radioisotopes. The soil chosen, an alkaline, grey, cracking clay (Vertosol), was representative of those used for growing cotton in Australia. Chang and Jackson fractionation of soil P from samples collected within 1 h of application indicated that 49, 7 and 13% of the P fertilizer was present as 0.5 m NH₄F, 0.1 m NaOH and 1 m H₂SO₄ extractable P, respectively. Over 89% of the P fertilizer was recovered as Colwell extractable P in these samples, suggesting that the majority of these reaction products was in a highly plant‐available form. Fertilizer‐P remained in an available form within the band 51 days after application, and 68% of the applied fertilizer‐P was recovered as Colwell‐P (1071 mg kg^?1). The Colwell‐P concentration in the band was 35 times that in the unfertilized soil. Thus, the variability in crop response to P fertilizer application in these soils is not a consequence of fertilizer‐P becoming unavailable to plants. These results confirm the suitability of the Colwell (1963) sodium bicarbonate extraction method for measuring available P in these soils.     

34S natural abundance variations in prairie and boreal forest soils

The systematic nature of the ³⁴S natural abundance variations (δ³⁴S) in a prairie and boreal forest ecosystem enabled construction of hypotheses regarding the origin and cycling of S in the two soil-plant systems. By considering the ³⁴S abundance variations in relation to soil S transformations, a better understanding of S isotope fractionation in soils was also achieved.
The δ³⁴S values suggest that atmospheric S becomes increasingly more important as a S input as pedogenesis proceeds in these soils. The origin and movement of sulphate salt in a saline seep was evaluated using δ³⁴S values, demonstrating the usefulness of the δ³⁴S technique in soil salinity studies. The ³⁴S-enrichments and depletions found in soil organic S fractions were consistent with postulated differences in lability, mobility, and turnover rate. Wheat plants growing on the saline, sulphate-saturated prairie soil were found to be enriched in ³⁴S relative to surrounding S sources, providing indirect evidence for release of ³⁴S-depleted H₂S by the plants as a S-stress relief mechanism.     

Nature and distribution of soil phosphorus as revealed by a sequential extraction method followed by 31P nuclear magnetic resonance analysis

Soil samples from long-term plots annually fertilized with superphosphate since 1952 and also unfertilized (control) plots of an irrigated and intensively grazed pasture in Canterbury, New Zealand, were subjected to a sequential extraction procedure followed by ³¹P nuclear magnetic resonance (NMR) analysis of the soil phosphorus (P) forms present. Overall, 80% of the total organic P (as determined by ignition) was removed from the soil by the sequential extraction procedure. Most of the organic P (90%) detected by NMR was in the monoester fraction. Small quantities of diester and pyrophosphate were also found. Choline phosphate constituted a significant proportion (18–25%) of the monoester P in the acetylacetone and 0.5 M NaOH extracts. Long-term superphosphate additions resulted in almost all of the P accumulated in the monoester P fraction (up to 99%).     

13C NMR of humic substances: pH and solvent effects

Since the concentration of free radicals in humic subtances increases at high pH the use of basic solutions for ¹³C NMR spectroscopy may cause broadening and loss of aromatic signals, with distortion of intensity distributions. No such effects were found in ¹³C spectra of soil humic and fulvic acid, an aquatic fulvic acid, and two phenolic polymers run in aqueous solutions at different pH values, and in dimethylsulphoxide. With increasing pH, the peak in the carboxyl region shifted in a manner consistent with greater dissociation of carboxyl and phenolic groups, and also certain features in the aliphatic and carboxyl regions were enhanced under some solution conditions. Elevated solution temperature (70°C) caused only slight improvement in the resolution of some lines. Chemical shifts were determined for some known phenolic and benzenecarboxylic acid compounds in DMSO and NaOD. The range for phenolic carbons extended to 173 ppm in NaOD, while some aromatic carbons occurred around 105 ppm, in the same region as anomeric carbons. Thus, even under quantitative acquisition conditions, relative areas may be used only to estimate proportions of different types of carbons and functional groups.     

Soil organic phosphorus in tropical forests: an assessment of the NaOH–EDTA extraction procedure for quantitative analysis by solution 31P NMR spectroscopy

The extraction of soil organic phosphorus by the NaOH–EDTA procedure was assessed in detail for a tropical forest soil (clay‐loam, pH 4.3, total carbon 2.7%). Optimum conditions for the quantification of soil organic phosphorus and characterization of its composition by solution ³¹P NMR spectroscopy were extraction in a solution containing 0.25 m NaOH and 50 mm Na₂EDTA in a 1:20 solid to solution ratio for 4 hours at ambient laboratory temperature. Replicate analyses yielded a coefficient of variation of 3% for organic phosphorus as a proportion of the spectral area. There was no significant difference in total phosphorus extraction from fresh and air‐dried soil, although slightly more organic phosphorus and less paramagnetic ions were extracted from dried soil. The procedure was not improved by changing the concentration of NaOH or EDTA, extraction time, or solid to solution ratio. Pre‐extraction with HCl or Na₂EDTA did not increase subsequent organic phosphorus extraction in NaOH–EDTA or improve spectral resolution in solution ³¹P NMR spectroscopy. Post‐extraction treatment with Chelex resin did not improve spectral resolution, but removed small concentrations of phosphorus from the extracts. Increasing the pH of NaOH–EDTA extracts (up to 1.0 m NaOH) increased the concentration of phosphate monoesters, but decreased DNA to an undetectable level, indicating its hydrolysis in strong alkali. The standardized NaOH–EDTA extraction procedure is therefore recommended for the analysis of organic phosphorus in tropical forest soils.     

A 31P nuclear magnetic resonance study of the phosphorus species in alkali extracts of soils from long-term field experiments

The different forms of phosphorus (P) in 0.5 m sodium hydroxide extracts of soils from long-term field experiments at Rothamsted were characterized by ³¹P-nuclear magnetic resonance spectroscopy (NMR). The extract from an old grassland soil (pH 4.6) from a plot of the Park Grass Continuous Hay Experiment that had received no fertilizer or lime for at least 125 years contained the following forms of P: inorganic orthophosphate (22% of the extracted P), orthophosphate monoesters (49%), orthophosphate diesters (14%), phosphonates (3%), pyrophosphate (4%) and two unidentified forms of P (7%). The soil extract from a Park Grass plot given inorganic phosphate fertilizer (35 kg P ha^?1) annually for 121 years contained the same forms of P and, in addition, a small amount of polyphosphate. There was also evidence of an increase in the orthophosphate monoester fraction. Another old grassland soil, of pH 6.1, contained more total and organic P than Park Grass but the extract contained fewer forms of P: inorganic orthophosphate (14% of the extracted P), orthophosphate monoesters (39%), orthophosphate diesters (34%) and an unidentified form (13%). An area of this grassland that had been ploughed up 20 years previously, and kept bare since, contained less organic P. The extract contained less of the phosphate diesters but the more stable monoesters remained relatively unchanged.