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a Alterra, Wageningen University and Research Centre (WUR), P.O. Box 47, 6700 AA, Wageningen, the Netherlands
b ATO, WUR, P.O. Box 17, 6700 AA, Wageningen, the Netherlands
c Department of Soil Quality, Wageningen University, WUR, P.O. Box 8005, 6700 EC, Wageningen, the Netherlands
* Corresponding author (g.f.koopmans{at}alterra.wag-ur.nl)
Received for publication March 1, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: DPS, degree of phosphorus saturation • EDTA, ethylenediaminetetraacetic acid • NMR, nuclear magnetic resonance • Pw, water-extractable phosphorus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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In animal manure, P is composed of both inorganic and organic P fractions, the latter varying from 5 to 25% of total P (Gerritse, 1981; Dou et al., 2000; Sharpley and Moyer, 2000). Dissolved organic P compounds in liquid pig slurry (a mixture of feces, urine, and cleaning water), representing 1% of total P, were found to be of high molecular weight and related to deoxyribonucleic acids (DNA), that is, orthophosphate diesters (Gerritse and Eksteen, 1978). In sandy soils amended with large amounts of pig and cattle slurry, the P leaching through the soil profile was found to be mainly organic P (Gerritse, 1981; Chardon et al., 1997). In the study of Gerritse (1981), where pig slurry was used as an amendment, the organic P leached had molecular weight characteristics similar to dissolved organic P compounds in the liquid fraction of pig slurry found by Gerritse and Eksteen (1978). Therefore, some organic P compounds in animal manure may be more mobile in soil than inorganic P (Gerritse, 1981). In soils treated with large amounts of animal manure, increased inorganic and organic P contents can be expected. Since the contribution of these P compounds to P leaching may differ, it is necessary to determine the P speciation in these soils.
Various sequential extraction methods have been used to characterize P pools of different availability in relation to plant uptake of P (e.g., Tiessen and Moir, 1993). However, extraction methods do not give direct information on the structural composition of the various compounds of P in soil (Guggenberger et al., 1996). Liquid-state 31P nuclear magnetic resonance (NMR) spectroscopy, a relatively simple and direct technique, has been used to characterize P in soil extracts (e.g., Tate and Newman, 1982; Hawkes et al., 1984; Dai et al., 1996). Characterization of P in soil extracts with 31P NMR has indicated the presence of inorganic P compounds such as orthophosphate, pyrophosphate, and polyphosphate, and organic P compounds such as phosphonate, orthophosphate monoesters (e.g., inositol phosphates), and orthophosphate diesters (e.g., phospholipids and DNA) (Cade-Menun and Preston, 1996).
The method used for extracting P from soil may affect the results obtained by 31P NMR (Cade-Menun and Preston, 1996). In most 31P NMR studies, P has been extracted by alkaline extracts, such as 0.5 M NaOH (e.g., Tate and Newman, 1982; Hawkes et al., 1984; Bedrock et al., 1994). However, 0.5 M NaOH usually extracts <50% of the total P content from soil, whereas an ideal method should extract all P from soil without altering the P compounds in soil (Cade-Menun and Preston, 1996). The use of 0.25 M NaOH–0.05 M Na2EDTA extracts more P from soil with minimal alkaline hydrolysis of organic P to inorganic P (Bowman and Moir, 1993; Cade-Menun and Preston, 1996; Dai et al., 1996). Alkaline hydrolysis of orthophosphate diesters may occur, especially at higher NaOH concentrations (Leinweber et al., 1997).
Very little information exists on changes of inorganic and organic P fractions and the distribution of P among the various P compounds within these fractions in soils exposed to long-term application with animal manure. We therefore selected soil samples from the top 5 cm of grassland exposed to different fertilization regimes in a long-term field experiment, started in 1970 and ending in 1980 (van der Veen, 1985). These soil samples are part of an extensive archive for technical information and soil samples (TAGA) containing the results of about 20 000 field experiments, performed in the period between 1879 and 1998 in the Netherlands, and about 250 000 soil samples from selected experiments (de Willigen et al., 2001). Our first objective was to determine the P speciation in 0.25 M NaOH–0.05 M Na2EDTA extracts of soils exposed to no fertilizer (control), or applications of N (no P application), N–P–K, or different animal manures. Phosphorus in the NaOH–Na2EDTA extracts was characterized with wet chemical analysis and 31P NMR. Our second objective was to characterize P in water extracts at a soil to solution ratio of 1:5 (w/v), representing the fraction of P in soil relevant for potential mobilization of P to surface waters (e.g., McDowell and Sharpley, 2001), again with wet chemical analysis and 31P NMR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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NaOH–Na2EDTA Extracts
A slight modification of the single-step extraction method (0.25 M NaOH–0.05 M Na2EDTA) of Bowman and Moir (1993) was used to extract P from soil. It is a simple method needing minimal analytical prowess and extracts a large part of total P from soil with minimal alkaline hydrolysis of organic P (Bowman and Moir, 1993; Cade-Menun and Preston, 1996). The NaOH solubilizes the organic material and EDTA increases the effectiveness of organic P extraction by breaking P containing Al and Fe complexes from soil. Three grams of soil were suspended in 60 mL of 0.25 M NaOH–0.05 M Na2EDTA in a centrifuge tube and shaken reciprocatively for 16 h at 85 strokes min-1 and 20°C. The suspension was centrifuged at 2100 x g for 10 min and filtered through a Schleicher & Schuell (Dassel, Germany) 589/5 filter (pore size 2 to 4 µm). An aliquot of 30 mL of filtrate was freeze-dried and stored at -18°C. Before 31P NMR analysis, the freeze-dried extract was dissolved in D2O, vortexed for 2 min, and kept at room temperature for 2 h. The remaining filtrate was used to determine inorganic P and total P, Fe, and Mn, and the pH. Inorganic P measurements were made colorimetrically (Murphy and Riley, 1962) after removal of a large fraction of the solubilized organic matter through acidification and centrifugation (Tiessen and Moir, 1993). Total P, Fe, and Mn concentrations were measured by ICP. Organic P was calculated as the difference between total and inorganic P. The pH was measured by pH indicator strips (pH of approximately 13). The 31P NMR spectra were obtained on a Bruker (Rheinstetten, Germany) DPX 300 spectrometer operating at 121.49 MHz and room temperature. Conditions used for 31P analysis were a pulse angle of 90°, a pulse delay of 2 s, and an acquisition time of 0.67 s. For each sample, 512 scans were used to obtain an acceptable signal to background noise ratio. Chemical shifts (
) of the peaks were measured according to an external 0.98 mM methylenediphosphonic acid trisodium salt tetrahydrate (MDP; 98%) standard, contained in a capillary tube measured simultaneously with each sample. In most other 31P NMR studies, orthophosphoric acid is used as a standard. For comparison of our results with these studies, MDP was set at = 18.1 ppm. Interpretation of the 31P NMR spectra was based on literature assignments (Bedrock et al., 1994; Cade-Menun and Preston, 1996; Amelung et al., 2001). Peak areas were calculated by instrumental integration. The 31P NMR spectra were evaluated semiquantitatively, that is, the fractions of the different P compounds were calculated by relating the respective peak areas to the total area.
Water Extracts
In addition to the Pw results of van der Veen (1985), we measured water-extractable P at a soil to solution ratio of 1:5 (w/v). Twelve grams of soil were suspended in 60 mL of water and shaken reciprocatively for 16 h at 85 strokes min-1 and 20°C. The suspension was centrifuged at 2100 x g for 10 min and filtered through a Schleicher & Schuell 589/5 filter. An aliquot of 40 mL of filtrate was freeze-dried and stored at -18°C. Before 31P NMR analysis, the freeze-dried extract was dissolved in D2O, vortexed for 2 min, and kept at room temperature for 2 h. The 31P NMR spectra of the water extracts were obtained with the same 31P NMR parameters as for the NaOH–Na2EDTA extracts. Interpretation of the spectra was based on our experience with 31P NMR analysis of solutions exhibiting a slightly acidic pH. The remaining filtrate was used to determine inorganic and organic P and total P, Fe, and Mn.
Test for Alkaline Hydrolysis of Organic Phosphorus
Alkaline hydrolysis of organic P to inorganic P was tested by measuring inorganic P before and after the addition of a volume of 0.25 M NaOH to a soil water extract. Five grams of soil were suspended in 25 mL of water and shaken reciprocatively for 16 h at 85 strokes min-1 and 20°C. The suspension was centrifuged at 2100 x g for 10 min and filtered through a Schleicher & Schuell 589/5 filter, and inorganic P and the pH were measured. On average, the pH was 5.34 ± 1.06. Three milliliters of 0.25 M NaOH were added to an aliquot of 10 mL of filtrate to increase the pH to approximately 13. The filtrate was shaken again for 16 h at 85 strikes min-1 and 20°C. Afterwards, inorganic P and the pH were measured (pH = 12.90 ± 0.03).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The Pw (plant-available P) and total P content of the soils amended with N–P–K or animal manures were 6.6 to 19.0 and 1.9 to 6.1 times higher than the corresponding values in the initial soil, respectively, due to the positive P balance. Interestingly, the relative increase of P available for plant uptake was much higher than the increase of total P. The DPS showed a large increase in the N–P–K- and animal manure–amended soils (Table 1). The sum of oxalate-extractable Al and Fe remained relatively constant. As soils become increasingly saturated with P, any additional soluble P applied through P fertilizer or animal manure remains as (readily) available P in soil, causing a large increase of Pw. In the soils treated with animal manures, the DPS is, however, (much) higher than expected on basis of the theoretical maximum (DPS = 100%) predicted by the sum of oxalate-extractable Al and Fe. This may indicate the buildup of a pool of Ca–P compounds in soil originating from the animal manures applied. In acidic to neutral soils, heavily enriched with P, Ca–P compounds may exist as metastable solid phases. De Haan and van Riemsdijk (1986) found indications for the existence of brushite (CaHPO4·2H2O) in heavily pig manured sandy soils. The Pw value and total P content of the soil treated with N fertilizer (no P application) slightly decreased, due to the negative P balance. In contrast, Pw and total P of the control soil slightly increased. In the soils treated with animal manures, organic P was 1.4 to 2.2 times higher than in the initial soil, due to the accumulation of organic P resulting from animal manure application. In the control and N- and N–P–K-amended soils, organic P was 1.1 to 1.2 times higher. This slight increase may be explained by accumulation of organic P from plant residues. Since the increase of the total P content was much higher than the increase of organic P in the soils treated with N–P–K or animal manures, P had mainly accumulated in soil as inorganic P (results not shown). Similar results have been found in soils treated with large amounts of cattle feedlot manure over several years; this was attributed to the high inorganic P fraction (78%) found in the cattle feedlot manure (Sharpley et al., 1984).
Wet Chemical Analysis of NaOH–Na2EDTA-Extractable Phosphorus
Figure 1
and Table 2 show inorganic and organic P measured in the NaOH–Na2EDTA extracts. Total P in the NaOH–Na2EDTA extracts represented 70 to 78% of the total P content in soil (Table 2), implying that it is a reasonably good extractant for total P in our sandy soil samples, although the method was originally developed for extracting organic soil P (Bowman and Moir, 1993). Our results correspond well with recoveries of total P found in other studies where 0.25 M NaOH–0.05 M Na2EDTA was used as an extractant: Cade-Menun and Preston (1996) and Dai et al. (1996) extracted 71 to 91% and 39 to 99% of total P from forest soils, respectively. The P remaining in soil after extraction with NaOH–Na2EDTA may represent some insoluble inorganic P compounds (Dai et al., 1996). Organic P in our NaOH–Na2EDTA extracts represented 27 to 70% of organic soil P (Table 2). These results agree with the 22 to 103% extracted by Dai et al. (1996). Interestingly, the recovery of extracted organic P relative to organic soil P decreased with increasing soil pH (Fig. 2)
. In the soils with pH values close to neutral, only 27 to 37% of organic soil P was extracted (Table 1 and 2; the solid poultry manure, solid poultry manure mixed with litter, and poultry and calf slurry treatments). Because of the higher soil pH of the latter treatments, the microbial mineralization rate may have been higher than in the soils of the other treatments; the remaining organic soil P may therefore have a lower extractability in NaOH–Na2EDTA. Another explanation includes a lower extractability of organic P originating from solid poultry manure, solid poultry manure mixed with litter, and poultry and calf slurry. According to the sequential extraction method of Tiessen and Moir (1993), 0.1 M NaOH represents less available inorganic and organic P compounds. Since we did not use any other extraction prior to the NaOH–Na2EDTA extraction, it thus contains both available and less available inorganic and organic P compounds, made available for characterization by 31P NMR. Total P measured in NaOH–Na2EDTA was 32 to 188 times higher than the corresponding Pw value (results not shown).
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Test for Alkaline Hydrolysis of Organic Phosphorus
With any extraction method, soil P compounds can be chemically altered during or after extraction (Cade-Menun and Preston, 1996). Hydrolysis of organic P in alkaline extracts has been reported in literature (e.g., Leinweber et al., 1997). Alkaline hydrolysis was tested by increasing the pH of the water extracts from 5.34 ± 1.06 to 12.90 ± 0.03. After shaking for 16 h, inorganic P in the water extracts increased to a small extent (on average 6.5 ± 5.4%), probably due to alkaline hydrolysis of chemically labile organic P compounds into inorganic P. Thus, the possibility of some transformation of organic P to inorganic P during our NaOH–Na2EDTA extraction cannot be excluded.
Phosphorus-31 Nuclear Magnetic Resonance Analysis of NaOH–Na2EDTA-Extractable Phosphorus
Figure 3
shows representative 31P NMR spectra of the NaOH–Na2EDTA extracts from the soils either amended with solid poultry manure mixed with litter or amended with calf slurry. Four distinct P compounds were detected: orthophosphate ( = 7.0–7.1 ppm), orthophosphate monoesters ( = 4.7–5.8 ppm), orthophosphate diesters ( = 0.6–0.8 ppm), and pyrophosphate ( = -3.6 to -3.7 ppm) (Cade-Menun and Preston, 1996). A peak appearing at = 7.6 to 7.9 ppm may reflect the presence of aromatic orthophosphate diesters (Bedrock et al., 1994; Amelung et al., 2001). The quality of our 31P NMR spectra was good, with a good separation of the orthophosphate and orthophosphate monoester peaks. This contrasts with the 31P NMR spectra obtained by Cade-Menun and Preston (1996), with a relatively poor separation of the orthophosphate and orthophosphate monoester peaks. This was attributed to the complexing of Fe and Mn by EDTA. Extraction of paramagnetic ions, such as Fe and Mn, causes line broadening and distortion of the 31P NMR spectra (Hawkes et al., 1984). In our study, however, Fe and Mn concentrations after extraction were on average only 45 and 3% of those of Cade-Menun and Preston (1996), respectively.
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Table 3 shows the P compounds as a fraction of total P (% of total area) measured by 31P NMR. In all treatments, inorganic P was mainly found as orthophosphate, although some very small fractions of pyrophosphate were found in the solid poultry manure–, solid poultry manure mixed with litter–, and poultry- and pig slurry–treated soils. Pyrophosphate can be a product of biological cycling of P in soil, but it can also be formed by hydrolization of organic esters during or after the extraction by NaOH–Na2EDTA (Condron et al., 1985). Pyrophosphate is a short-chain polyphosphate (n = 2), which is used as a microbial storage product appearing from microbial activity (Condron et al., 1985; Cade-Menun et al., 2000a,b). The presence of pyrophosphate in the solid poultry manure–, solid poultry manure mixed with litter–, and poultry slurry–amended soils, with a pH close to neutral, further supports the idea of a higher microbial mineralization rate in these soils. Organic P was mainly found as orthophosphate monoesters. This agrees with results reported in other studies (e.g., Tate and Newman, 1982; Hawkes et al., 1984; Condron et al., 1985, 1990; Bedrock et al., 1994). Orthophosphate diesters were detected in the solid poultry manure– and calf slurry–amended soils. Orthophosphate monoesters and diesters in soil can be derived from a combination of plant and microbial residues (Anderson, 1967; Bedrock et al., 1994; Guggenberger et al., 1996) and animal manure application. Using 31P NMR, Leinweber et al. (1997) detected these P compounds in poultry and pig manure. Orthophosphate monoesters consist mainly of inositol phosphates, amounting to >50% of total organic P in soil (Anderson, 1967). Due to the high charge density of inositol phosphates, these P compounds are strongly sorbed by (hydr)oxides of Al and Fe in soil (Turrión et al., 2001), protecting inositol phosphates from mineralization. Orthophosphate diesters comprise P compounds such as phospholipids, ribonucleic acids (RNA), and DNA. Because of their lower charge density, orthophosphate diesters are only slightly retained in soil, and are more mobile and more accessible for microbial degradation than monoesters (Gerritse, 1981; Turrión et al., 2001). Under favorable soil conditions, such as an optimal pH and moisture content for microbial activity, orthophosphate diesters can be transformed into monoesters (Hinedi et al., 1988). In intensively managed grassland soils with favorable conditions for mineralization, such as in our soils, orthophosphate diesters can be absent (Condron et al., 1990). In contrast, in nonagricultural soils where conditions favored a lower rate of mineralization (low pH and high moisture content), significant amounts of orthophosphate diesters have been found (Condron et al., 1990; Bedrock et al., 1994; Cade-Menun et al., 2000a). Moreover, alkaline hydrolysis of orthophosphate diesters into monoesters may have occurred to a small extent. Especially at higher NaOH concentrations (0.5 M NaOH), alkaline hydrolysis of orthophosphate diesters can occur (Leinweber et al., 1997). We used a lower NaOH concentration (0.25 M NaOH), possibly decreasing the potential of alkaline hydrolysis.
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Wet Chemical and Phosphorus-31 Nuclear Magnetic Resonance Analysis of Water-Extractable Phosphorus
Characterization of P in NaOH–Na2EDTA extracts gives limited information on the soil potential for P mobilization, since both available and less-available inorganic and organic P compounds are extracted. For a better understanding of the potential mobility of P in our soils, water- or 0.01 M CaCl2–extractable P needs to be characterized, since these extractants represent the P fraction in soil relevant for potential P loss to flowing water (e.g., McDowell and Sharpley, 2001). In addition to results of Pw, which has a low soil to solution ratio (1:60 [v/v]), taken from van der Veen (1985) (Table 1), we measured water-extractable P at a soil to solution ratio of 1:5 (w/v). Because of the higher soil to solution ratio of these water extracts, these results may be more indicative for the leaching of soil solution through the soil profile where a relatively large amount of soil comes into contact with a relatively limited volume of solution. Table 4 shows total, inorganic, and organic P measured in the water extracts by wet chemical analysis. Water extracted 1.5 to 3.7% of the total P content from soil (Table 4). The trend shown by inorganic P is similar to the one in the NaOH–Na2EDTA extracts. Inorganic P increased by 7.5 to 24.8 times in the N–P–K- and animal manure–amended soils. In contrast, organic P increased only by 1.5 to 3.2 times. These results are further illustrated by organic P as a fraction of total P. The organic P fraction decreased from 67% in the initial soil to 20 to 32% in the soils amended with N–P–K or animal manures, but remained relatively constant in the control and N-amended soils (Table 4).
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= 2.2–2.4 ppm) and orthophosphate monoesters ( = 2.8–3.0 ppm). The chemical shifts of orthophosphate and orthophosphate monoesters in the water extracts differed from those in the NaOH–Na2EDTA extracts. Since we did not adjust the pH of the solution containing the freeze-dried water extract prior to 31P NMR analysis, the pH must have been lower than the pH of the solution with the freeze-dried NaOH–Na2EDTA extract. As demonstrated by Crouse et al. (2000), the pH significantly affects the chemical shifts of the various P compounds in the 31P NMR spectra: changing the pH causes a change in the chemical environment surrounding the P nuclei and as pH changes so do the chemical shifts. Thus, the differences between the chemical shifts of orthophosphate and orthophosphate monoesters in our NaOH–Na2 EDTA and water extracts can be attributed to a change in pH.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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