Assessing the bioavailability of dissolved organic phosphorus in pasture and cultivated soils treated with different rates of nitrogen fertiliser

Soil Biology & Biochemistry 38 (2006) 61–70 www.elsevier.com/locate/soilbio

Assessing the bioavailability of dissolved organic phosphorus in pasture and cultivated soils treated with different rates of nitrogen fertiliser R.W. McDowella,*, G.F. Koopmansb,1 b

a AgResearch, Invermay Agricultural Centre, Private Bag 50034, Mosgiel, New Zealand Alterra, Wageningen University and Research Centre (WUR), P.O. Box 47, 6700 AA, Wageningen, The Netherlands

Received 24 November 2004; received in revised form 17 March 2005; accepted 21 March 2005

Abstract A proportion of dissolved organic phosphorus (DOP) in soil leachates is readily available for uptake by aquatic organisms and, therefore, can represent a hazard to surface water quality. A study was conducted to characterise DOP in water extracts and soil P fractions of lysimeter soils (pasture before and after, and cultivated soil after leaching to simulate a wet winter–autumn) from a field trial. Data on DOP in drainage waters from the field trial were also generated. In water extracts, used as a surrogate for soil solution and drainage water, 70–90% of the total dissolved P (TDP) concentration was made up of DOP, of which 40% was hydrolysable by phosphatase enzymes. Proportions of hydrolysable DOP to TDP in drainage waters of the field trial were less than in water extracts due to enhanced DRP loss via dung inputs, but still large at 35% of DOP. Analysis of lysimeter soils by sequential fractionation indicated that several organic P fractions changed with land use and due to leaching. Further investigation using NaOH–EDTA extracts and 31P nuclear magnetic resonance spectroscopy indicated that the greatest changes were a decrease in the concentrations of orthophosphate diester P and an increase in orthophosphate monoester P. This was attributed to mineralization by cultivation and plant roots and also to the leaching of mobile diester P. This study suggests that in such soils with a dynamic soil organic P pool, the concentration of readily bioavailable P in soil solution and drainage waters and the potential to impair surface water quality cannot be determined from the DRP concentration alone. q 2005 Elsevier Ltd. All rights reserved. Keywords: Dissolved organic phosphorus; Alkaline phosphatase; Phosphodiesterase; Phytase; 31P nuclear magnetic resonance spectroscopy

1. Introduction The loss of phosphorus (P) from soil to water represents a potential hazard to surface water quality leading to eutrophication, because P often limits primary production in freshwater ecosystems (Correll, 1998; McDowell et al., 2004). The impacts of eutrophication include excessive production of autotrophs, especially eukaryotic algae and cyanobacteria, causing green, turbid water with limited transparency. Eutrophic surface waters have reduced ecological value, because they are dominated by only a few species. Hence, recreation becomes less attractive and * Corresponding author. Tel.: C64 3 489 9262; fax: C64 3 489 3739. E-mail address: [email protected] (R.W. McDowell). 1 Present address: Department of Soil Quality, Wageningen University, WUR, P.O. Box 8005, 6700 EC, Wageningen, The Netherlands.

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.03.026

the use of eutrophic surface waters for fisheries, industry, and drinking is restricted (Sharpley et al., 1994). The loss of P in subsurface drainage waters is well established, as is the dominance of dissolved P forms (measured after filtration through a 0.45 mm filter), unless drainage occurs soon after drain installation when particulate P (PP) dominates (Djodjic et al., 2000). Dissolved reactive P (DRP; largely orthophosphate) often represents a major fraction of total dissolved P (TDP) in subsurface drainage waters and is considered as being readily available for uptake by algae in surface waters. However, DRP is not necessarily or indeed the major P fraction, because high organic P concentrations have been found in soil solution (Shand et al., 1994; Chapman et al., 1997) and leachate (Chardon et al., 1997), and organic P can, therefore, represent a large fraction of the total P load of surface waters. The hydrolysis of dissolved organic P (DOP) can act as a mechanism for aquatic flora and fauna to acquire P when orthophosphate P limits growth (Whitton et al., 1991). Phosphatase enzymes have become increasingly used to

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indicate potential bioavailability of DOP in soil solution and drainage waters to aquatic flora and fauna (Whitton et al., 1991; Turner et al., 2002). Toor et al. (2003) found that 16–99% of DOP in leachate from lysimeters of a soil in pasture was susceptible to hydrolysis by phosphatase enzymes. Hence, knowledge on the proportion of readily available P in DOP in soil solution and subsurface drainage waters after enzymatic hydrolysis is important, because it helps to improve our understanding of the potential of the loss of DOP to impair the quality of surface waters. In a recent study of McDowell and Monaghan (2002), lysimeters were taken from a field trial reported by Monaghan et al. (2000, 2002). This study examined the P losses in response to increasing applications of nitrogen (N) fertiliser at rates of 200 or 400 kg N haK1 commensurate with the current intensification with dairying in New Zealand. A control, with no fertiliser N applied was also included. Up to 67% of TDP lost in leachate of a pasture soil over a period of 7 months was in the DOP form. When the pasture soil was cultivated, the proportion of DOP increased to 80% of TDP. This increase in DOP was attributed to the retention of DRP by the cultivated pasture soil via matrix flow while DOP was lost from the uncultivated pasture soil due to poor retention in macropores and loss via preferential flow. Because of the large amounts of DOP lost in leachate reported by McDowell and Monaghan (2002), we concluded it was necessary to assess the bioavailability of DOP released into solution with phosphatase enzymes. Consequently, our first objective was to examine the proportion of enzymatic hydrolysable DOP in water extracts of soils from lysimeters collected by McDowell and Monaghan (2002). Water extracts can be used as a surrogate for soil solution or drainage water (Sonneveld et al., 1990). However, examining the release of P to water extracts used to determine soil solution P in small-scale lysimeter soils does not necessarily translate to field drainage losses (Rodda and Cooper, 1996). Hence, our second objective was to compare the data obtained from the water extracts of soils from the lysimeters with DOP concentrations and fractions in drainage waters from an accompanying field trial (Monaghan et al., 2000, 2002). The lysimeters were taken from this field trial after its completion in 2000. The application of fertiliser N and changing land use can lead to changes in the soil organic P concentration and the composition of organic P and, consequently, to changes in concentrations and fractions of water soluble or leached organic P. Therefore, our third objective was to determine the chemical speciation of P in soils from lysimeters collected by McDowell and Monaghan (2002) from a pasture soil with three different N fertiliser application rates, using the detailed chemical fractionation scheme of Golterman (1996), Golterman et al. (1998), and 31P nuclear magnetic resonance (NMR) spectroscopy. The chemical P speciation in pasture soil was determined before and after leaching, equivalent to a wet-autumn–winter period.

Furthermore, an additional treatment was created by leaching pasture soil that had been broken-up to simulate cultivation prior to re-sowing in pasture.

2. Materials and methods 2.1. Field site The field site was located in eastern Southland, New Zealand, 4 km west of Edendale township. Average annual rainfall for this area is approximately 1000 mm, with an average annual surplus rainfall (drainage) of approximately 350 mm. The site had slopes ranging from 0 to 48. The soil was a Fleming silt loam (New Zealand classification mottled Fragic Pallic soil USDA Taxonomy; Typic Hapludalf) that had been in pasture for O15 years and had total C and N concentrations in the 0–7.5 cm topsoil of 53 and 4.8 g kgK1, respectively. The drainage trial was initiated in the spring of 1995, with clay drainage pipes (tiles) laid in each of nine plots at a depth of 75 cm, overlain with 50 cm of pea gravel, and trench spoil to the soil surface. The following summer, mole channels were pulled perpendicular to, and intercepting each tile drain, at a depth of 45 cm and spacing of 1.8 m. Experimental treatments were replicated three times in a randomised block design and began in spring 1996 with three rates of fertiliser N input: 0, 200 and 400 kg N haK1 yrK1 (as urea), hereafter referred to as 0, 200 and 400 N treatments, respectively. Urea applications were split into 50 kg N haK1 application and applied in spring and subsequent grazing rounds. In December each year, 450 kg of 15% potassium superphosphate per hectare was applied to maintain soil Olsen P at 20–25 mg P kgK1. Due to the extra pasture grown in the N-fertilised treatments, stocking rate varied between treatments to ensure all extra pasture grown was eaten. Flow-weighted drainage samples were collected from the outlet of each plot. Data for DRP and total P (TP) in drainage waters sampled in 1998 and 1999 from the field trial described above were taken from Monaghan et al. (2000, 2002). No data for DOP (as the difference of TDP and DRP) or PP (as the difference of TP and TDP) existed from this period and were generated in this study by re-analyzing storedfrozen samples (25 ml) for TDP. This involved filtering samples (!0.45 mm) immediately once thawed, and analyzing for TDP after persulphate digestion (0.15 g K2S2O8 in 1 ml, 0.5 M H2SO4, 150 8C for 2 h) within 3 h. Monaghan et al. (2002) found that reanalysing these samples for DRP within 3 h of thawing was identical to the results if analysed fresh. In addition to the normal sampling regime, larger samples (2 L) of drainage water were taken during five events from the 12th of June and 17th of November, 1998. These samples were frozen at the time of sampling and later thawed for analysis of DRP, TDP, TP and additional enzyme analyses (see below).

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2.2. Lysimeter sampling and additional treatment

2.4. P fractionation

After the cessation of the field trial in October 2000, in February 2001, nine lysimeters (internal diameter 18 cm and depth 24 cm) were taken from each treatment. The soil from three lysimeters of each treatment was broken up by hand to simulate cultivation prior to re-sowing in pasture. Over the next 7 months, nine cultivated and nine pasture lysimeters were subjected to a leaching regime equivalent to a wet-autumn–winter period yielding 480 mm of drainage. Soils from these leached lysimeters along with soils from nine unleached lysimeters in pasture were air-dried, ground, and sieved to !1 mm. Data for the concentrations and loads of DRP and DOP leached from these lysimeter were presented by McDowell and Monaghan (2002). Later in 2003, it was decided to re-examine the DOP in soil solution from these lysimeters. To determine P soluble in water as a surrogate for P in drainage waters, samples of soil (10 g) from each lysimeter (nZ27) were moistened with 5 ml of deionised water and allowed to equilibrate in the dark at 20 8C for 10 days (Brookes et al., 1984; Turner et al., 2002). Following equilibration, soils were shaken with deionized water for 30 min at a 1:5 (w/v) soil to solution ratio (McDowell and Sharpley, 2001) before centrifuging (4000!g) and filtering the supernatant to !0.45 mm. Dissolved reactive P and DOP were determined in each extract and additional enzyme analyses run to determine DOP bioavailability.

To elucidate the chemical speciation of P in the lysimeter soils, we used the detailed chemical fractionation scheme of Golterman (1996) and Golterman et al. (1998) (Fig. 1). Unlike many fractionation schemes used for soil P analysis, fractions within the scheme of Golterman (1996) and Golterman et al. (1998), along with those of the SEDEX method (Ruttenberg, 1992), have been quantified against known P compounds. Briefly, for each lysimeter soil, 1 g is sequentially extracted via shaking with 30 ml of deionised water (2 h), 0.05 M Ca–EDTA (C1% Na-dithionite, pH 7.8), 0.1 M Na–EDTA (pH 4.5), 0.5 M H2SO4, cold 0.5 M trichloroacetic acid (TCA; 0 8C, 4 h), hot 0.5 M TCA (95 8C, 30 min), and finally 2 M NaOH (90 8C, 1 h) before the remaining P is released by persulphate digestion. These fractions represent, in sequential order, water soluble or soil solution P (H2O–P), Fe associated P, Ca associated P, acid soluble organic P (ASOP), sugar bound P after digestion by K2S2O8 (cold TCA), nucleic P and polyphosphate after digestion by K2S2O8 (hot TCA; Golterman, 1960), humic bound P and phytate (NaOH Pi and NaOH Po), and residual P. Following extraction, each soil-suspension was centrifuged (4000!g) for 10 min, decanted, and an aliquot was taken for P determination. For Ca- and Na-EDTA extracts, a maximum of 2 ml could be used before EDTA interferes with the Mo–P colorimetric reaction. For H2O–P, Fe–P, Ca–P, and NaOH fractions, the organic P fraction was defined as the difference between P detectable before and after digestion by K2S2O8.

2.3. Enzyme analyses

2.5.

For the determination of organic P fractions in drainage waters and the water extracts, enzyme assays using alkaline phosphomonoesterase (EC 3.1.3.2.), phosphodiesterase (EC 3.1.4.1.) and phytase (EC 3.1.3.8) were ran using a modified method based on De Groot and Golterman (1993) and Turner et al. (2002). Briefly, 0.1 M sodium azide (to prevent microbial growth and P uptake) and the appropriate buffered enzyme mixture (Turner et al., 2002) were added to a sample of water extract in the ratio of 1:1:9, and incubated overnight at 37 8C in inert plastic centrifuge tubes. A blank sample containing no enzyme, just buffer, was also incubated. The next morning, DRP was determined in both samples and the difference in DRP concentration denoted as enzyme liberated P. Calibration curves for P also contained each enzyme to account for any P release by the enzyme itself or protein interference. For the determination of phosphodiesters, alkaline phosphomonoesterase was also added to fully convert esters to orthophosphate detectable by colorimetry. The concentration of diesters was then calculated as P released from the combined alkaline phosphomonoesterase and diesterase mix minus P released from alkaline phosphomonoesterase alone.

Further analysis of soil P forms was conducted using 31P nuclear magnetic resonance spectroscopy (31P NMR). Soils from each replicate were bulked within treatments (pasture before, and pasture and cultivated soil after leaching) and 5 g sub-sampled for analysis. This sub-sample was extracted twice with 100 ml of 0.25 M NaOHC0.05 M EDTA (Na form), shaken overnight, centrifuged (4000!g), decanted, and the combined extract analyzed for P after digestion by K2S2O8. Each combined extract was frozen and freeze-dried. Approximately, 0.5 g of the freeze-dried material was then re-dissolved in 2 ml of 1 M NaOH and 0.2 ml of D2O (for signal lock) and transferred into the NMR tubes. The pH of the suspension was O13. Solution 31 P NMR spectra were obtained using a Bruker (Rheinstetten, Germany) DPX 300 spectrophotometer operating at 121.49 MHz at 20 8C. For each sample, 1024 scans were accumulated using a pulse angle of 908, a pulse delay of 1 s and an acquisition time of 0.82 s. Chemical shifts (d, ppm) were recorded relative to an external 0.98 mM methylenediphosphonic acid standard (MDP; 98%, trisodium salt tetrahydrate) measured simultaneously in a capillary tube. Spectra were deconvoluted using a Lorentzian line shape of 10 Hz and measured using Mestre-C software

31

P NMR

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Fig. 1. Flow chart of the sequential P fractionation scheme (after Golterman (1996) and Golterman et al. (1998)). Note that Pi is orthophosphate and Po organic phosphate, determined as the difference in orthophosphate before and after persulphate digestion.

(Go´mez and Lo´pez, 2004). Organic P compounds within spectra were determined quantitatively using the peak assignments of Cade-Menun and Preston (1996) and Turner et al. (2003), the percentage spectral area occupied by each compound, and the P concentration of the corresponding NaOH–EDTA extract.

to water seepage across two plots, a balanced statistical model for drainage waters was not possible. Data for one 400 N plot was omitted. All statistical analyses were performed with SPSS v10.0 (SPSS, Inc., 1999).

3. Results 2.6. Statistical analyses Analysis of DOP enzyme fractions and P fractionation data used a design with three land uses (pasture soils before drainage, pasture soils after drainage, and cultivated soils after drainage) by three N application rates (0, 200 and 400 N) as a factorial, randomised block design with three replicates. The F-statistic is presented to compare means and indicate significant differences. Due to the expense of 31 P NMR, analysis of only one sample was possible for each land use and N application rate. In the field experiment due

3.1. DOP bioavailability in water extracts of lysimeter soils and field drainage In Table 1, data are presented on water extractable DRP, DOP and hydrolysable DOP fractions in water extracts of lysimeter soils. Of the TDP extractable by water from lysimeter soils, on average 89% was DOP, ranging from 80% in the 0 N pasture soil before leaching to 95% in the 400 N pasture soil after leaching. The majority of DOP hydrolysable was classified as either alkaline

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Table 1 Mean and G95% confidence intervals in parentheses, of water extractable DRP, DOP and hydrolysable DOP fractions (all mg LK1) before and after leaching of pasture lysimeter soils and after leaching of cultivated soils at each N rate Land use and N application rate (kg N haK1)

DRP

DOP

Pasture soil before leaching 0 96 (46) 200 61 (37) 400 51 (45) Pasture soil after leaching 0 20 (20) 200 25 (22) 400 15 (7) Cultivated soil after leaching 0 51 (57) 200 41 (57) 400 30 (38) Land use leaching !0.001 N application rate ns Land use!N applins cation rate Field drainage (1998, nZ5) 0 109 (51) 200 98 (42) 400 74 (24)

Alkaline phosphomonoesterase

Phosphodiesterase

Phytase

388 (324) 360 (62) 352 (86)

48 (59) 55 (17) 57 (20)

33 (72) 32 (48) 30 (28)

28 (52) 41 (32) 59 (26)

278 (22) 287 (87) 283 (33)

85 (63) 73 (35) 113 (112)

15 (49) 18 (10) 18 (20)

71 (88) 46 (77) 120 (123)

343 (14) 312 (12) 300 (14) 0.020 ns ns

69 (51) 61 (41) 91 (46)

49 (43) 52 (60) 41 (32) ns ns 0.016

16 (22) 13 (5) 18 (5) ns ns ns

16 (10) 14 (12) 21 (14)

20 (10) 19 (10) 21 (11)

60 (71) 57 (46) 43 (22) ns ns 0.013

8 (7) 15 (8) 17 (6)

ProbabilityOF statistic from ANOVA for land use-leaching, N application rate, and the interaction between L and use-leaching and N application rate on P fractions, ns: not significant. Additional data is given for five events from the field trial sampled in 1998.

phosphomonoesterase or phytase hydrolysable, while the total sum of hydrolysable DOP was about 45% ranging from 28 to 89% of TDP. A significant difference was noted for alkaline phosphomonoesterase and phytase hydrolysable DOP for the interaction of N application rate with land use-leaching largely due to their greater concentrations in the pasture soil after leaching. However, no significant effects on these DOP fractions were noted for land use-leaching or N application rates individually, despite there being a significant effect of land use-leaching on total DOP concentration. Variation in non-hydrolysable DOP was probably to blame. In Table 2, data are presented on mean flow weighted concentrations and loads for P fractions in drainage waters during 1998 and 1999 for each land use and N application rate. Concentrations of DRP in the water extracts of the pasture lysimeter soils before leaching were similar to

concentrations of DRP in drainage waters from the field trial (Tables 2 and 3). Many other studies have shown similar results for both manured pasture and cropped soils (e.g. McDowell and Sharpley, 2001). However, in both water extracts and field drainage waters, the DRP concentrations exceed the recommended guideline of 10 mg DRP LK1 for lowland freshwater quality (ANZECC, 2000). The limit for TP at 33 mg LK1 is also exceeded in field drainage waters, whereas no data is available for TP in the water extracts from the lysimeter soils. For DOP fractions in field drainage waters collected during five events in 1998, apart from the 0 N treatment, more DOP was hydrolysable by either alkaline phosphomonoesterase or phosphodiesterase than by phytase (Table 1). This contrasts to water extracts of lysimeter soils where phosphodiesterase hydrolysable DOP was commonly poor.

Table 2 Mean flow weighted concentrations and loads and the standard error of the mean of all treatments for P fractions in drainage waters during 1998 and 1999 for each N application rate N application rate (kg N haK1 yrK1) 1998 0 200 400 1999 0 200 400

DRP (mg LK1)

DOP (mg LK1)

PP (mg LK1)

TP (mg LK1)

DRP (g haK1)

DOP (g haK1)

PP (g haK1)

TP (g haK1)

82 53 71

63 75 55

80 95 67

225 223 193

0.164 0.165 0.121

0.126 0.232 0.094

0.160 0.293 0.115

0.450 0.690 0.330

42 29 52

24 33 21

81 59 88

147 121 161

0.097 0.103 0.083

0.056 0.116 0.033

0.188 0.211 0.143

0.341 0.430 0.259

ProbabilityOF statistic from analysis of variance for land use-leaching, N application rate, and interaction between land use-leaching and N application rate on P fractions, ns: not significant.

19 16 19 ns ns ns 285 298 311 !0.01 ns 0.03 19 18 12 0.03 !0.01 !0.01 3.2 3.2 2.8 0.04 ns ns

17 12 16 ns ns ns

106 90 76 ns 0.04 ns

29 29 26 !0.001 ns 0.04

166 181 145 ns ns ns

55 54 44 0.04 ns ns

17 18 13 0.04 ns ns

151 166 148 0.03 ns ns

928 845 850 21 19 17 235 274 291 26 22 19 5.1 4.2 4.2

18 22 18

148 92 91

22 34 29

217 162 161

58 51 49

18 14 14

160 148 154

999 947 829 16 31 16 29 22 20

Pasture soil before leaching 0 2.5 200 2.0 400 1.5 Pasture soil after leaching 0 2.5 200 1.9 400 1.5 Cultivated soil after leaching 0 1.8 200 1.6 400 1.2 Land use-leaching ns N application rate !0.01 Land use!N rate !0.01

3.6 3.5 3.2

16 17 18

135 116 100

98 102 72

191 181 151

62 61 61

24 17 24

160 160 154

313 304 265

Total H2O, Pi

H2O, Po

Ca–EDTA, Pi

Ca–EDTA, Po

Na–EDTA, Pi

Na–EDTA, Po

ASOP, Po

Cold TCA, Po

Hot TCA, Pi/o

NaOH, Pi

NaOH, Po

Residual

857 855 805 !0.01 !0.01 ns

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Land use and N application rate (kg N haK1)

Table 3 Mean inorganic (Pi) and organic (Po) P forms (mg kgK1) in pasture soils before, and pasture and cultivated soils after leaching at each N treatment rate (0, 200 or 400 kg N haK1 yrK1)

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Concentrations of DOP in the water extracts of the soils from the lysimeters are similar to those reported by McDowell and Monaghan (2002) in lysimeter leachate. However, DOP in field drainage waters for both years (1998 and 1999) and all treatments is on average only 44% of TDP, half the proportion detected in the water extracts of the lysimeter soils or that reported by McDowell and Monaghan (2002) (Tables 2 and 3). 3.2. Lysimeter soil P fractions and forms In Table 3, data are presented of the sequential P fractionation of replicated lysimeter soils for each land useleaching and N application rate. The largest fraction was NaOH extractable P, representing P associated with humic material and phytate (De Groot and Golterman, 1993). The next largest fraction was either ASOP or Ca–P (Na–EDTA–P). Up to 47% of the Ca–P extractable was in organic form. On an average, organic P in the fractionation scheme represented 64% of total P (range 60–68%). Among land use-leaching treatments, significant differences were noted for water soluble or soil solution Po, Fe–Pi (Ca–EDTA), Ca–Po, sugar bound P (cold TCA), nucleic and polyphosphate P (hot TCA), humic associated Pi (NaOH–Pi), and Po (NaOH–Po) (Table 3). In contrast, only water soluble Pi and Fe–Pi were significantly different among N application rates, while the interaction of land useleaching with N application rate yielded significant differences in the soil solution, Fe–Pi, Ca–Po, and humic associated-Po fractions. The later interaction emphasized the decrease in organic P in both leached pasture and cultivated soils compared to the pasture soil before leaching. Further investigation of organic P by 31P NMR spectroscopy of NaOH–EDTA extracts indicated the presence of six classes of P compounds. In Table 4, data are presented on the concentrations of these P compounds in each soil (Table 4, Fig. 2). On the basis of spectral area measured by 31P NMR and the total P concentration measured in the NaOH–EDTA extracts by wet chemical analysis (colorimetry), a calculated 349 and 481 mg kgK1 of orthophosphate P was detected by 31 P NMR, which accounted for 48–59% of the total P concentration in the NaOH–EDTA extracts. For other inorganic P species, polyphosphate (K20 ppm) was only detected in very small concentrations and only in the 400 N pasture soil after leaching. In contrast, pyrophosphate was detected in all spectra, but still only accounted for a small proportion of total P (1–3%). For organic P species, the concentration of monoesters, assigned to peaks between 6 and 3 ppm, appeared to be less in pasture than in cultivated soils after leaching. However, the overall concentration of diesters, assigned to peaks between 2 and K1 ppm (phospholipids plus remaining diesters such as DNA and RNA), decreased in the pasture soils after leaching and was completely absent from the 0 and 200 N cultivated soils after leaching. Generally, phospholipids were assigned to the region of 1.83–1.97 ppm,

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Table 4 Concentration (mg kgK1), and percentage in parentheses, of P forms in each soil as detected by 31P liquid state NMR spectroscopy N rate (kg haK1)

Ortho-phosphate {w6.2}

Pasture soil before leaching 0 481 (59) 200 463 (59) 400 424 (55) Pasture soil after leaching 0 491 (57) 200 527 (64) 400 430 (53) Cultivated soil after leaching 0 415 (53) 200 381 (51) 400 349 (48)

Monoesters {6 to 3}

Diesters {2 to K1}

Pyro-phosphate {K3 to K4}

Poly-phosphate {K20}

Phosphonates {20}

295 (36) 265 (34) 277 (36)

9 (1) 10 (1) 45 (8)

17 (2) 7 (1) 21 (1)

– – –

12 (2) 7 (1) –

347 (41) 284 (35) 378 (45)

5 (1) 7 (1) 10 (1)

14 (2) 2 (1) 5 (1)

– – 2 (1)

– – –

339 (44) 344 (46) 360 (50)

– – 5 (1)

17 (2) 21 (3) 6 (1)

– – –

– – –

The chemical shift (d, ppm) of each peak assignment is given in brackets.

similar to that assigned to phosphatidyl ethanolamine (1.8 ppm) by Turner et al. (2003). Signals between K0.35 and 0.64 ppm, assigned to other diesters such as DNA and RNA (K0.3 ppm by Turner, 2004), were much broader (Fig. 2). Although RNA has been found in alkaline soil extracts at 0 and 1 ppm, degradation of this P compound is rapid, leading to a 82% decrease in concentration over 30 min and an increase in monoesters (Makarov et al., 2002; Turner et al., 2003). While only a small percentage of the total P concentration in NaOH–EDTA extracts was present as diester P in pasture soils before leaching (1–8%), degradation and transformation of diester P may help to

explain the increase in monoester P in the pasture and cultivated soils after leaching (Table 3).

4. Discussion 4.1. Bioavailability of DOP in water extracts of lysimeter soils and field drainage waters The application of N and changing land use can lead to changes in the soil organic P concentration and the composition of organic P and, consequently, to changes in

Fig. 2. Example 31P NMR spectrum of a NaOH–EDTA extract of the unleached pasture soil receiving 400 kg N haK1 yrK1, showing the assignment of different P species. Note that MDP is the reference compound.

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concentrations and fractions of water soluble or leached organic P. For water extracts of the lysimeter soils, slightly less DOP was found in leached pasture soils than in either the leached cultivated soils or the pasture soils before leaching (Table 1). Differences were noted in the concentration of alkaline phosphomonoesterase and phytase hydrolysable DOP when land use-leaching interacted with N application rate. More specifically, the concentrations of alkaline phosphomonoesterase and phytase hydrolysable DOP were greater in the pasture soils after leaching, especially in the 400 N treatment. The proportion of DOP hydrolysed by alkaline phosphomonoesterase compared well to those in water extracts and leachate from lysimeter soils in other studies (Pant et al., 1994; Toor et al., 2003). Similarly, the proportion of phosphodiesterase and phytase hydrolysable DOP in extracts was similar to the range detected in leachate by Toor et al. (2003) (2–37%). In general, the bioavailability of DOP species to terrestrial and aquatic plants is greater for orthophosphate monoesters (detected by alkaline phosphomonoesterase) and orthophosphate diesters (detected by phosphodiesterase) than for DOP hydrolysable by phytase (mainly inositol phosphate but also some diesters and monoesters) (Turner et al., 2002). Turner et al. (2003) noted that the presence of labile esters in water extracts was important for surface water quality. Our results confirm that both monoesters and diesters were important components of our lysimeter soil water extracts and drainage waters as a potential source of orthophosphate (Table 1). Typically, past studies have found !60% of DOP is hydrolysable by phosphatase enzymes (e.g. Fox and Comerford, 1992; Pant et al., 1994). Similarly, in our study, on average only 45% of DOP was hydrolysable. The remaining material is probably made up of DOP strongly sorbed to small (!0.45 mm) colloids or sorbed within pockets of small colloids that are not accessible for enzymes (Kretzschmar et al., 1999), high molecular weight compounds (Pant et al., 1994), cell fragments such as phospholipids, and live bacteria (Salema et al., 1982). The bioavailability of many of these compounds is poor, emphasizing phosphatase hydrolysable DOP fractions as the main potential source of orthophosphate. Data suggested that these non-hydrolysable compounds play a role in DOP loss as a result of land use change and leaching. However, the exact role is unclear and warrants further work. McDowell and Monaghan (2002) showed that P uptake by pasture increased with N application rate causing a decrease in the load of P leached from lysimeters. Increased pasture growth also boosts labile monoester input into the soil since plant root and microbe turnover are their major source (Webley and Jones, 1971). Consequently, in the pasture lysimeters inorganic P will have been sequestered by pasture growth leaving behind less available DOP fractions. As a comparison, absence of plant growth and reduced root turnover in the cultivated soil has resulted in

a similar pattern of DOP species extractable by water to that present in the pasture soil before leaching. On an average, the proportions of DOP to TDP in field drainage were about half those in water extracts of the lysimeter soils. The disparity in concentrations may reflect the increase in scale and depth. The lysimeters drain the top 24 cm depth of soil while drains are set at 45 cm depth. This could decrease P loss due to dilution or sorption by P-poor (sub)soil or water. However, if this was the only factor, then DRP concentrations should have been less in field drainage and not similar to those in the water extracts of the lysimeter soils: they were not. On the other hand, the disparity may be explained by different P inputs due to management. In the field, stock grazed plots depositing dung until the end of the experiment in 2000, while no dung was deposited on the lysimeters since they were collected in February 2001. Recent data show that dung can account for much P loss in drainage (Jensen et al., 2000). The major form of P in dung is inorganic (up to 90%), and hence DRP will likely dominate any loss of P in field drainage compared to lysimeters. 4.2. Soil P fractions and forms Data in Table 3 indicated that land use-leaching had a significant effect on the concentration of total P in the lysimeter soils. The lowest total P concentration was found in the cultivated soil after leaching. However, in a review of the effects of tillage and mineralization on organic P, Addiscott and Thomas (2000) concluded that mineralization was probably too slow to cause large losses in drainage as with nitrate, and furthermore much of the mineralised P will be immobilised in the soil. Consequently, an explanation for this decrease in total P is unclear. However, our data showed that the distribution of soil P among fractions had changed (Table 2). Significant changes were noted in organic P fractions associated with soil solution, sugars, nucleic acids and humic substances, while changes were also noted in the inorganic P fractions associated with soil solution and Fe–P. The changes in the soil solution fractions are especially important as the soil solution represents the medium through which P is either taken up by plant roots or lost in flow. However, although sequential fractionation aims to isolate dynamic pools of soil P, exact characterisation of specific forms is poor. To address this issue, soils were extracted with NaOH–EDTA and specific P forms in the extracts were analysed by 31P NMR spectroscopy, a direct and relatively simple technique which has frequently been used in chemical P speciation studies (Cade-Menun and Preston, 1996; Koopmans et al., 2003; Turner et al., 2003; Turner, 2004). The NaOH–EDTA extraction method was originally developed as a measure of total organic P (Bowman and Moir, 1993), but extractions using NaOH have also been used to estimate algal available P (Dorich et al., 1985;

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Williams et al., 1980). Data in Table 4 showed that after leaching the proportion of diester P in the pasture and cultivated soils had decreased. Much previous work has shown that the concentration of diesters decreases following long-term cultivation. For instance, Condron et al. (1990) showed that diester P had completely disappeared from cultivated soils that had been subject to rotational cerealfallow cropping for 70 years without manure or fertilizer input in southwestern Saskatchewan. While our data confirm that diester P appears to be labile and easily mineralised following cultivation, the decrease in diester P concentration in the pasture soils also indicated that mineralization induced by cultivation was not the only mechanism. Mineralisation of diester P from plant root exudates is likely (Fox and Comerford, 1992), and additional diester P may be lost in leachate since diester P is more mobile in soil than either orthophosphate, orthophosphate monoester or pyrophosphate P (Leytem et al., 2002). The exact proportion of each of these P compounds in drainage waters is unknown since the leachate was not kept. However, evidence exists for both mechanisms. For instance, DOP hydrolysable by phosphodiesterase was commonly greater than DOP hydrolysable by alkaline phosphomonesterase or phytase in field drainage waters collected during five events in 1998, but not in the water extracts obtained from lysimeter soils. In addition, the concentration of monoester P increased in both pasture and cultivated soils after leaching, possibly as by-products of mineralised diester P.

5. Conclusions Isolation of DOP by phosphatase enzymes indicated that on average about 45% of the large (89%) TDP fraction was hydrolysable. This in-effect increased the concentrations of readily bioavailable dissolved P by 2–3 times over DRP alone. Further investigation of P in the lysimeter soils indicated that a significant change occurred in many organic P fractions with changing land use and after leaching. The large DOP fraction present in water extracts of lysimeter soils and loss in field drainage waters was paralleled by the decrease in soil diester P due to mineralisation by cultivation and plant roots and loss in leachate. In such soils with a large and dynamic soil organic P pool, the concentration of readily bioavailable P in soil solution and drainage waters and the potential to impair surface water quality cannot be determined from DRP concentration alone.

Acknowledgements Funding for this work was provided by the New Zealand Foundation for Research, Science and Technology. Dr Ross Monaghan is thanked for the supply of frozen field trial

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drainage samples and flow volumes, and DRP and TP data. Dr Wim Chardon is thanked for his valuable comments to a previous version of this manuscript.

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