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a AgResearch, Invermay Agricultural Centre, Private Bag 50034, Mosgiel, Otago, New Zealand
b Department of Chemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand
* Corresponding author (richard.mcdowell{at}agresearch.co.nz)
Received for publication July 21, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: NMR, nuclear magnetic resonance
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The ability of soil to assimilate P concentrated in dung patches at rates of up to 280 kg P ha–1 will depend on the form of P in dung (Williams and Haynes, 1995). It is well known that some organic P forms such as phytate are more strongly sorbed to soil than orthophosphate, while many others (e.g., orthophosphate diesters) are poorly sorbed to soil (Leytem et al., 2002). Depending on the concentration and form of P supplied this could lead to localized areas of soil P saturation and P loss. If P is in ready supply then pasture will also tend to utilize inorganic P before organic P forms (Magid et al., 1996). Consequently, it is important to know the relative quantities and forms of P in dung of grazing animals.
To date, the majority of literature has focused on P forms in animal manures, largely stored dung that is spread on soils. For example, using the sequential extraction procedure of Hedley et al. (1982), Leinweber et al. (1997) found that most P in freeze-dried hog and poultry manure was in the recalcitrant residual P pool (39–41%). Conversely, Ajiboye et al. (2004) found that the majority of P in fresh dairy, beef, and hog manure was in labile P pools (NaHCO3–NaOH extractable). These authors also found that oven-drying samples caused organic P in the labile pool to shift into inorganic P, extractable in water. While oven-drying is extreme, the work of Ajiboye et al. (2004) shows that labile organic P could transfer to inorganic P as moisture decreases. Our hypothesis was to test this in dung samples where, unlike stored manure, moisture content will vary from fresh to air-dry condition depending on soil and climatic conditions.
While techniques such as sequential extraction (fractionation) yield information on the relative proportions of inorganic and organic P, they cannot determine specific P forms. Some forms of organic P are more bioavailable than others (e.g., orthophosphate diesters compared with orthophosphate monoesters; Makarov et al., 2002) and hence information on specific P, especially organic, forms is necessary to know what transformations are occurring as dung dries. Using 31P NMR spectroscopy some researchers have looked at P forms in alkaline extracts. For example, Turner (2004) identified phytic acid in extracts of broiler litter and swine manure, whereas Leinweber et al. (1997) used 31P NMR to identify broad classes of P compounds (orthophosphate monoesters and diesters, pyrophosphates, orthophosphate, and techioc acid) in swine and poultry manure. Using solid state 31P NMR in conjunction with isotopic exchange kinetics, Frossard et al. (2002) showed that slowly or non-exchangeable inorganic P in composted solid organic waste could be in the form of apatites or octacalcium phosphates.
The objective of this work was to identify the P forms in the dung of pastoral grazing animals (dairy cattle, deer, and sheep) by sequential extraction and 31P NMR. A second objective was to compare how P forms change when fresh dung naturally air-dries, as would occur in the field following deposition.
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MATERIALS AND METHODS |
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Dung pellets of deer and sheep were gently pulled apart before mixing within each dung type. Cattle dung was sufficiently moist to allow samples to be stirred. Half of each dung type was left to air-dry at 25°C in a temperature-controlled glasshouse for 20 d until air-dried dung had moisture contents of <2% of fresh dung (determined gravimetrically by drying overnight at 105°C). Although not having diurnal variation and hence moisture from dew, this temperature was chosen to represent typical summer daily maximums. Fresh and air-dried dung was digested with aqua regia (0.15 g in 5 mL of 4:1 concentrated nitric to hydrochloric acid mix, heated at 150°C for 2 h and filtered through Whatman [Maidstone, UK] GF/A) for later chemical analysis of total Ca, Al, and Fe by inductively coupled plasma–magic angle spinning (ICP–MAS). Organic C was determined by a LECO (St. Joseph, MI) C/N analyzer and pH was determined in water (1:5 mix).
Sequential Extraction
The sequential extraction procedure of Hedley et al. (1982) as modified by Dou et al. (2000) was used to examine P fractions in fresh and air-dry dung samples. Briefly, 0.3 g of dung (oven-dry basis) was shaken with 30 mL of extractant for 1 h, the suspension centrifuged at 8000 x g for 20 min, and the supernatant removed and filtered (<0.45 µm) for later P analysis. The 0.45-µm membrane filter was added back to dung and the extraction process repeated twice before the next extracting reagent was used. The order of extractants used was distilled water, 0.5 M NaHCO3, 0.1 M NaOH, and finally 1 M HCl. Any P not extracted by these reagents was removed by sulfuric acid–persulfate digestion (0.3 g K2S2O8 in 2 mL 0.5 M H2SO4 added to sample and heated at 150°C for 2 h), and termed residual P. All P determinations were made via colorimetry at a wavelength of 712 nm (Watanabe and Olsen, 1965). Organic P in water, bicarbonate, and NaOH fractions was determined by difference of orthophosphate detected by colorimetry before and after an aliquot was digested by sulfuric acid–persulfate. Total organic P was organic P from these three fractions.
Phosphorus-31 Nuclear Magnetic Resonance
Analysis of dung by 31P NMR was made on resuspended NaOH + EDTA extracts. Briefly, samples (5 g, dry weight equivalent) were shaken with 100 mL of 0.25 M NaOH + 0.05 M EDTA (Na form) for 16 h, centrifuged (4000 x g, 20 min), and the supernatant filtered (Whatman GF/A). The extract was analyzed via colorimetry for orthophosphate before and after digestion by K2S2O8 and 0.5 M H2SO4 (yielding total P). Each extract was then frozen and freeze-dried.
Solution 31P NMR spectra were obtained using a 500-MHz Inova NMR spectrometer (Varian, Palo Alto, CA) with a 51-mm standard superconducting magnet (Oxford Instruments, Oxford, UK), FTS temperature controller (Varian), dual fullband channels, one low band decoupler channel, and a 28 shim set. A 5-mm Varian z-axis PFG direct detection probe was used for all the samples. The Sun Microsystems (Santa Clara, CA) Ultra 10 workstation uses Solaris 8 OS and Varian VNMR 6.1C NMR software. Each sample was prepared to a pH > 13 by taking 0.1 g of the dried extract and adding 600 µL of D2O and 100 µL of 10 M NaOH. Samples were ultrasonicated (Model HT 710-3; Crest Ultrasonics, Trenton, NJ) for 3 min, equilibrated for 20 min, and then centrifuged (Qualitron micro centrifuge; Chiron Scientific, Sylvania, OH) for 5 min. The supernatant was transferred to a 5-mm NMR tube and 31P NMR spectra obtained at 202.298 MHz at 20°C. Accumulation of data for each sample was halted when a sufficient signal to noise ratio was obtained (3600–20000 scans). Scans were accumulated using a pulse angle of 45°, a pulse delay of 8 s, and an acquisition time of 1.99 s with 64K data points. Chemical shifts were recorded relative to an external phosphoric acid standard (
= 0 ppm) in a capillary tube.
Spectra were deconvoluted using a Lorentzian line shape of 5 Hz and measured using Mestre-C software (Gómez and López, 2004). Peak assignments for classes of P compounds were taken from the literature (Newman and Tate, 1980; Condron et al., 1990; Cade-Menun and Preston, 1996; Makarov et al., 2002; Turner et al., 2003) and classified as orthophosphate, orthophosphate monoesters, and orthophosphate diesters (DNA and phospholipids-techoic acid-P), polyphosphates, pyrophosphates, and phosphonates. Classes of P compounds were quantified by combining the percentage spectral area occupied by each class of compound with total P concentration in the corresponding NaOH-EDTA extract.
Statistical Analyses
All statistical procedures (analysis of variance, means, and standard error of the difference between treatment means) were conducted using Genstat (Genstat Committee, 2002).
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RESULTS |
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Phosphorus Forms in NaOH-EDTA Dung Extracts and Phosphorus-31 Nuclear Magnetic Resonance Analysis
Orthophosphate and total P in NaOH-EDTA dung extracts are shown in Table 3. Between 517 and 1191 mg kg–1 of organic P was extracted, which in some cases was more than the sum of water, bicarbonate, and NaOH organic P fractions. For fresh dung the values were 111, 129, and 139% of the sum of these organic P fractions for cattle, deer, and sheep dung, respectively, while for dry dung the values were 61, 89, and 91%. Recent work has argued for the reclassification of the residual P fraction as organic P in manures from inorganic P in soils, as manures contain no mineral material (Ajiboye et al., 2004). If residual P is classified as organic P in our fractionation scheme, then organic P extracted by NaOH-EDTA varied from 27 to 77% of total organic P. Furthermore, on average, more P was extracted from fresh dung (68%) than from dry dung (33%).
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DISCUSSION |
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Detailed sequential fractionation studies, using the same extractants as used in this study, highlight the importance of water- and bicarbonate-extractable P. For instance, Sharpley and Moyer (2000) found that for a range of animal manures that had been stored wet for 4 wk, water-extractable P as a percentage of total P varied from 15 to 47% and bicarbonate P from 22 to 30%. Dou et al. (2000) found 71% of dairy manure P was extractable by water, whereas Ajiboye et al. (2004) report around 20% of total P as water extractable, and 50% of total P as bicarbonate extractable in fresh dairy manure. Our data indicate that water-extractable P as a percentage of total P was at the low end of this range, whereas bicarbonate-extractable P was at the high end.
Dou et al. (2000) stated that while an exact parallel between the fractionation schemes used for manure and that of Hedley et al. (1982) for soils cannot be drawn, it is likely that elements such as Al, Fe, and Ca are, in part, selectively extracted into each fraction. Consequently, water-, bicarbonate-, and NaOH-extractable P were defined by Dou et al. (2000) as labile pools of P, while HCl and residual P were more recalcitrant pools of P. Bicarbonate- and NaOH-extractable P were thought associated with Al and Fe, while HCl-extractable P was assigned as P associated with Ca. Studies by Ajiboye et al. (2004), Sharpley and Moyer (2000), and Dou et al. (2000) showed that the concentration of Ca, Al, and Fe control P solubility in manure. Furthermore, Dou et al. (2000) hypothesized that since Ca is commonly the dominant cation in manure, Ca-P forms would control P solubility. Our data partly support this hypothesis in that sheep and deer dung have a high pH and contain more Ca and HCl-P than cattle dung. However, Ajiboye et al. (2004) also attributed large Al and Fe concentrations in biosolids to poor water extractability of P. Compared with the study of Ajiboye et al. (2004), Ca concentrations in our dung samples were much less suggesting Al- and Fe-P compounds played a greater role.
In each type of fresh or dry dung, organic P extracted in water, bicarbonate, and NaOH fractions was a small part of total P and did not vary among types (organic P was a maximum of 15% of total P; Table 2). This contrasts with 59, 35, and 25% of total P as organic P for some studies of dairy manure (Zhang et al., 1994; Ajiboye et al., 2004; and Sharpley and Moyer, 2000, respectively), and just under 20% as determined by Dou et al. (2000). However, Sharpley and Moyer (2000) note that differences are likely to be caused by mineral supplementation of the animals' diet. In our case, dairy cattle, deer, and sheep were grazed year-round on the same dominant pasture species (ryegrass, Lolium perenne L.). Furthermore, phytate, an orthophosphate monoester, is rich in the diet of grains fed to swine and broilers, but poor in pastures (Peperzak et al., 1959). Barnett (1994b) noted that poultry litter commonly contains much organic P as phytic acid (4.5–5% of total P), while Kemme et al. (1999) noted a similar concentration (4%) in swine manure.
When dung was allowed to air-dry, significant changes occurred in the sequentially extracted P fractions (Table 2). In a similar study, but of hog and dairy manures that had been oven-dried at 105°C, Ajiboye et al. (2004) found that inorganic P extractable in water increased approximately 15 to 24% at the expense of organic P extractable by water or, as in the case of dairy manure, bicarbonate-extractable inorganic and organic P. This was attributed to a combination of hydrolysis and increased P desorption. However, our data show that inorganic P, especially in water- and bicarbonate-extractable P pools, decreased in air-dry dung, which led to a significant increase in HCl and residual P pools (Table 2). Of the labile organic P pools, only organic P extractable by NaOH decreased, while water- and bicarbonate-extractable organic P generally increased. Clearly, the mechanisms of P transformation involved with air-drying dung are not simple. Some clues may be gleaned from the sequential fractionation of peat soils, which like dung contain much plant matter and a lesser mineral proportion than most soil types. Schlichting and Leinweber (2002) and Schlichting et al. (2002) sequentially extracted P from 29 peat soils using the Hedley et al. (1982) fractionation. They also examined the effect of air-drying peat soil at 40°C compared with fresh samples and found that drying caused large increases in residual P thought to be mainly organic P. This was attributed to the binding and inclusion of P in poor pedogenic oxides, or more likely, the structural rearrangement of humic macromolecules and increased hydrophobicity. This leads to the greater exposure of hydrophobic surfaces to extractants in dried than fresh samples, causing poor extractability.
Phosphorus-31 Nuclear Magnetic Resonance Examination of Phosphorus Forms in Dung
To elucidate P forms in fresh and dry dung, samples were extracted with NaOH-EDTA, which commonly removes 70 to 90% of organic P from soils (Bowman and Moir, 1993). Comparing data in Tables 2 and 3 shows that the proportion of organic P varies, and would change depending if residual P is classified as either inorganic or organic P. Ajiboye et al. (2004) classify residual P as organic P, as unlike soil, manures contain little clay capable of occluding P minerals and preventing extraction. However, dairy cattle and sheep do intake a proportion of soil with pasture [2% for dairy cattle (Healy, 1968) and 5% for sheep (Field and Purves, 1964)]. While only a minor proportion, some residual P in dung will be occluded. Consequently, data for the extraction of organic P from dung contain some uncertainty. Despite this uncertainty, extraction with NaOH-EDTA will remove P that is more than likely to be labile and interact to some extent with soil or runoff waters. The remaining P as shown by sequential fractionation is held within more recalcitrant pools.
Due largely to expense, there are few 31P NMR studies of P forms in manures, and no studies comparing fresh and dry dung. Frossard et al. (2002) used solid-state 31P magic angle spinning NMR to show the presence of Ca-P compounds (apatite or octacalcium phosphate) and organic P in solid organic wastes, but few samples contained animal manure or dung. Studies by Crouse et al. (2000), Leinweber et al. (1997), and Turner (2004) have examined P in NaOH extracts of poultry, swine, and dairy manure. All found that orthophosphate was the major component, and that monoesters due to the presence of phytic acid was the next major compound class. Both Leinweber et al. (1997) and Turner (2004) found that the distribution and extractability of organic P compounds was influenced by the concentration of NaOH. Leinweber et al. (1997) noted that diesters comprising teichoic acid (sugar phosphate structures from bacterial cell walls; Condron et al., 1990), and compounds at 0.4 ppm, later defined as RNA or DNA by Turner (2004), were easily hydrolyzed in 0.5 M NaOH. However, Turner (2004) found this hydrolysis was minimized in 0.25 M NaOH-EDTA extracts. All dung samples presented here were extracted with 0.25 M NaOH and 50 mM EDTA and therefore have similar conditions and potential for change. Similarly, the delay time between pulses (approximately 10 s) is sufficient for relaxation of P nuclei (T1) and yield quantitative spectra (T1 determined at 1–5 s for peaks in spectra of extracts of fresh sheep dung; McDowell, unpublished data, 2004). However, for some manures, insufficient concentration of paramagnetics or a wide pulse angle (e.g., 90° instead of 45° as used here) may mean T1 is not reached. Although this can be countered by adding paramagnetics, this also causes line-broadening, which can make peak resolving difficult. Coupled to this, peaks in the diester region are prone to line-broadening for a number of reasons such as anisotropic effects, subtle structural perturbations and degradation into fragments. Since DNA is a large molecule it tumbles slowly in the NMR whereas for small molecules, rapid tumbling averages out changes in chemical shift. Furthermore, local changes in the helical structure of DNA change the phosphate ester geometry and chemical shift. Finally, degradation due to hydrolysis separates DNA into smaller fragments resulting in more species and overlapping peaks (Gorenstein, 1994). Despite these problems, deconvolution software allowed for the determination of compound classes into phospholipids and DNA (Table 4).
Some differences were noted in chemical shift (ppm) of P compounds in Fig. 1 to 3 compared with those assigned in the literature by Turner et al. (2003), but probably result from subtle changes in pH caused by differences in Ca concentration (Fig. 1–3, Table 1; Crouse et al., 2000). As evident from Table 4, on drying, organic P concentration in NaOH-EDTA extracts was different to that in fresh samples. Drying also caused a significant effect on organic P extractable by NaOH during fractionation (Table 2). The main difference in spectra was the decrease and elimination of diesters in all samples except deer dung. These compounds are known to be more labile in soil extracts than either monoesters or inorganic P species such as pyrophosphate (Condron et al., 1990). For soils, Hinedi et al. (1988) found that diesters could be converted into monoesters, while monoesters remained stable. Our data clearly show that diesters in the region of 1 to –1 ppm (assigned to RNA and DNA) disappear completely in extracts of dry dung, while increases occur in orthophosphate, and in sheep dung monoesters. Although some of the increase in orthophosphate may be due to degradation of pyrophosphate, it is more likely that diesters either degrade into their constituent components, forming monoesters and orthophosphate, or are insoluble in NaOH-EDTA extracts when dried. Nevertheless, these findings have significant implications for soil fertility and environmental health.
Diesters are known to be bioavailable to aquatic algae and plants (Whitton et al., 1991). Consequently, a decrease in their concentration coupled with a decrease in orthophosphate solubility with drying indicates the potential for P loss to surface waters has decreased. Field studies have shown that P loss decreases with time since a field last had manure spread on it or was last grazed (Smith et al., 2001; Withers et al., 2003). Several mechanisms may cause this such as infiltration into and interaction with the soil. In addition, if dung behaves similarly to organic soil and sediment then repeated wetting and drying cycles also promote P movement into recalcitrant pools that are less available to runoff or streamflow (McDowell and Sharpley, 2001; Schlichting and Leinweber, 2002). Our work suggests that one wet–air-dry cycle of the dung from dairy cattle, sheep, and deer grazing pasture was sufficient to decrease P soluble in labile pools and hence may also influence the potential for P transport in runoff. Clearly there is a need to determine the relative influence of this effect on the potential for P transport in runoff, and when and where dairy cattle, deer, and sheep should be best placed on a farm to graze and minimize the potential for P transport. Additional work should look at the effect of wet–dry cycles on the P chemistry of manures from different animals and diets.
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CONCLUSIONS |
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ACKNOWLEDGMENTS |
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