Characterizing Dissolved and Particulate Phosphorus in Water with

Nov 2, 2006 - (DP) and particulate (PP) P from river waters by solution. 31P nuclear magnetic ... for routine collection, and puts PP and DP into the ...
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Environ. Sci. Technol. 2006, 40, 7874-7880

Characterizing Dissolved and Particulate Phosphorus in Water with 31P Nuclear Magnetic Resonance Spectroscopy B A R B A R A J . C A D E - M E N U N , * ,† JOHN A. NAVARATNAM,‡ AND M A R K R . W A L B R I D G E ‡,§ Department of Geological and Environmental Sciences, Stanford University, Stanford, California 94305-2115, and Department of Biology, West Virginia University, Morgantown, West Virginia 26506-6057

Management of aquatic ecosystems is hampered because current methodology limits characterization of phosphorus (P) forms. We developed a procedure to characterize dissolved (DP) and particulate (PP) P from river waters by solution 31P nuclear magnetic resonance (NMR) spectroscopy, using 4-L samples, and tested this procedure with a spiking trial. Most P was orthophosphate. Organic P forms included phosphonates, myo-inositol hexakisphosphate, and orthophosphate diesters. This research represents an important technical advance to characterize DP and PP in natural waters. It is simple, uses samples small enough for routine collection, and puts PP and DP into the same chemical environment for direct comparison. The technique is sensitive, detecting changes in spectra from P additions as small as 2% of total P, and identifying differences from two points along the flow path of a single river. However, lyophilizing samples in NaOH-ethylenediaminetetraacetic acid (EDTA) may alter some P forms, which requires further investigation.

Introduction Phosphorus (P) is essential for all organisms. In aquatic ecosystems, too little P in bioavailable form limits growth, while too much causes eutrophication (1). For analytical purposes, P in water is divided into dissolved (DP) and particulate (PP) fractions, with DP passing through a 0.45-µm filter (2, 3). These fractions are further partitioned into organic (Po) and inorganic P (Pi) by indirect techniques. First, total P (TP) is determined by digestion and colorimetric techniques; total DP may also be determined without digestion by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Inorganic P is determined colorimetrically (4), and the difference between TP and Pi is called Po. However, colorimetric methods do not measure all Pi but instead measure only molybdate-reactive P (MRP), also called soluble reactive P (SRP), which is orthophosphate only; complex Pi forms such as pyrophosphate and polyphosphate are not molybdate-reactive and thus will be designated as Po (2, 5, 6). * Corresponding author phone: (650)725-0927; fax: (650)725-2199; e-mail: [email protected]. † Stanford University. ‡ West Virginia University. § Current address: National Program Staff, USDA-Agricultural Research Service, Beltsville, MD 20705-5140. 7874

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In addition to being arbitrary, these techniques to determine Po and Pi provide no information on P speciation. Because P forms vary in their bioavailability (7), ecosystem management to prevent eutrophication requires knowledge of P forms and their concentrations. However, P characterization has been limited by methodology. No single technique is suitable for both DP and PP, and current methods are often indirect (2), may require complex chromatographic procedures (3, 8), or may be specific for only one class of compound (8, 9). An alternative is nuclear magnetic resonance (NMR) spectroscopy. Although solution 31P NMR, in which samples are extracted to concentrate P and remove interfering paramagnetic ions, has been used extensively for more than 20 years to identify P forms in soil samples (10), there have been few applications of this technique to aquatic ecosystems. Solution 31P NMR has been used for extracts of sediments from lakes (11-14), estuaries (15), and oceans (16), and for ocean PP (17, 18). Dissolved P has been concentrated by ultrafiltration and reverse osmosis, to characterize lake and river DP by solution 31P NMR (19) and ocean DP by solid-state 31P NMR (20). However, large volumes of water were concentrated in these studies: 250-500 L of lake and river water (19), and 10 000 L of ocean water (20). Recently, a 31P NMR spectrum was obtained for leachate from a manure-treated soil, by lyophilizing a 6-L sample and then extracting the dried solid by soil extraction methods (21). Our objective was to test this procedure to characterize both DP and PP in water. Initially, river water samples at natural P concentrations were tested, followed by spiking and degradation experiments to test detection limits and possible alterations of P forms.

Materials and Methods Sample Collection and Preparation: (1) Natural Waters. Waters were collected from the river inlet (RI) and floodplain (FP) of the Pee Dee River, South Carolina, in July 2003. Samples were filtered within 24 h to separate DP from PP (retained on filter) with 0.45-µm membrane filters (Whatman Inc., Clifton, NJ), using 500 mL of water per filter; filtrates and filters were then refrigerated. After an aliquot of each filtrate was removed for chemical analysis, portions (4 L) of each filtrate were frozen in 150-mL plastic cups and subsequently lyophilized by placing frozen samples (without cups) directly into freeze-dryer flasks. (2) Spiking Trial. Water (∼60 L) was collected from the RI of the Pee Dee River in February 2005 and divided into 13 4.1-L subsamples. The first sample was left unspiked and was filtered as above. The next six subsamples were each spiked with one of the following compounds prior to filtration: (1) (2-aminoethyl)phosphonic acid (AEP), Sigma A6037, 0.14 mg added to 4.1 L (3.47 µg of P); (2) deoxyribonucleic acid (DNA), sodium salt, from salmon testes, Sigma D1626, 0.03 mg (0.34 µg of P); (3) glucose 6-phosphate (g6P), disodium salt hydrate, Sigma G7250, 0.165 mg (16.81 µg of P); (4) myo-inositol hexakis(dihydrogen) phosphate (IHP), dipotassium salt, Sigma P5681, 0.106 mg (26.76 µg of P); (5) dicalcium pyrophosphate (pyroP), Sigma P5681, 0.02 mg (4.876 µg of P); and (6) pentasodium tripolyphosphate (polyP), Sigma T5883, 0.02 mg (5.052 µg of P). Masses of spiking compounds were determined by using twice the concentration of these compounds determined in natural river water samples (part 1), to allow a measure of the sensitivity of the technique and to avoid swamping peaks from natural P forms with those from spiking compounds. The exception was DNA, which was added at a much lower concentration than other compounds due to a miscalculation of the DNA TP con10.1021/es061843e CCC: $33.50

 2006 American Chemical Society Published on Web 11/02/2006

TABLE 1. Distribution of Dissolved and Particulate P Species in River Inlet and Floodplain Samples from the Pee Dee River dissolved P

particulate P

river inlet (%)

floodplain (%)

river inlet (%)

floodplain (%)

orthophosphate pyrophosphate phosphonates orthophosphate monoesters orthophosphate diesters

86.2 3.4 1.7 5.2 3.5

80.0 3.2 1.6 13.6 1.6

62.6 10.3 2.4 22.3 2.4

54.2 13.5 1.8 27.1 3.4

inorganic P organic P organic P from chemical determination

89.6 10.4 12.5

83.2 16.8 18.2

73.9 27.1 nd

67.7 32.3 nd

centration prior to spiking (Sigma does not certify DNA P concentration). The final six subsamples were each filtered as above, and then each filtrate was spiked with one of the six compounds. Next, 4.1 L of deionized (DI) water was spiked with all six compounds, at the above concentrations. For all spiked filtrates, 100 mL of each was removed for chemical analysis, and 4 L was frozen in acid-washed plastic ice cube trays. The ice cubes were subsequently placed directly into freeze-dryer flasks for lyophilization. This increased the surface area of the frozen samples, decreasing the lyophilization time. Finally, the above compounds were individually dissolved for NMR, to determine expected chemical shifts for each compound and also to determine what, if any, other P forms were present in each compound. For this test, AEP (0.022 g), DNA (0.047 g) g6P (0.629 g), IHP (0.098 g), pyroP (0.069 g) and polyP (0.091 g) were each dissolved into separate aliquots containing 0.75 mL of D2O, 1 mL of NaOHethylenediaminetetraacetic acid (EDTA) extractant, and 0.4 mL of 10 M NaOH, as described below for NMR analysis. Chemical Analysis. Filtrates were analyzed for TP by ICPAES (Thermo-Jarrell Ash; IRIS Advantage) and for SRP colorimetrically (4). Two filters from each water source were oven-dried, digested (22), and analyzed colorimetrically (4) to determine PP TP. NMR Extraction and Analysis. Lyophilized water samples were extracted in 20 mL of 0.5 M NaOH and 0.1 M Na2EDTA (1:1) overnight and centrifuged (23). Filters were similarly extracted: eight filters (not dried) from each water source were used for the natural waters, with four filters per 20 mL of extractant, and six filters (three per 20 mL) for the spike trial; the 20-mL extracts were subsequently combined to a single 40-mL sample for each PP sample. A 1-mL aliquot (later diluted to 10 mL) was removed from all extracts to determine total extracted P by ICP; the remainder was lyophilized. For NMR spectroscopy, samples were dissolved in 1 mL of DI water, 1.6 mL of D2O, and 0.4 mL of 10 M NaOH. Spectra were acquired within 12 h at 202.45 MHz with a Varian Unity INOVA 500 MHz spectrometer equipped with a 10-mm broadband probe. Parameters were 20 °C temperature; 90° pulse; 0.68 s acquisition time; 4.32 s relaxation delay; 15 Hz spinning; and an external H3PO4 standard. The number of scans ranged from 4808 (FP particulates) to 9815 (RI water). Spectra were processed with 7 Hz of line-broadening; peak identification was based on the literature (10, 24). Degradation Test. Several permutations were tested to see if NaOH-EDTA extraction caused hydrolysis of polyphosphate, glucose 6-phosphate and pyrophosphate, with 0.1 g of each compound: (1) 25-mL aliquots of the NaOHEDTA extractant were frozen and lyophilized, and then polyP, g6P, or pyroP (as described above) was added during preparation for NMR; (2) polyP, g6P, and pyroP were dissolved in separate samples with 25 mL of DI water, frozen, lyophilized, and prepared for NMR; (3) polyP, g6P, and pyroP were dissolved in separate samples with 25 mL of NaOHEDTA extractant, frozen, lyophilized, and prepared for NMR;

(4) polyP, g6P, and pyroP were dissolved in separate samples with 25 mL of DI water, frozen, and lyophilized and then dissolved in 25 mL of NaOH-EDTA extractant, frozen, lyophilized, and prepared for NMR; (5) polyP and g6P were separately dissolved in separate samples with 25 mL of NaOH-EDTA extractant, neutralized to pH 6.6-7.0, frozen, lyophilized, and prepared for NMR; and (6) polyP and g6P were dissolved in separate samples with 25 mL of DI water, frozen, and lyophilized and then dissolved in 25 mL of NaOHEDTA extractant, neutralized to pH 6.6-7.0, frozen, lyophilized, and prepared for NMR analysis. For NMR analysis, lyophilized samples were dissolved in 1 mL of D2O and 1 mL of NaOH-EDTA extracting solution, plus 0.4 mL of 10 M NaOH. The NMR parameters were as above, but only 40-50 scans per sample were collected.

Results and Discussion (1) Natural River Waters. Total DP was 0.032 (RI) and 0.056 mg L-1 (FP), and total PP was 0.9 (RI) and 1.27 mg g-1 (FP), or 0.04 (RI) and 0.188 mg L-1 (FP). Thus, TP for RI was 0.073 mg L-1, of which 56% was PP. For FP, TP was 0.244 mg L-1, of which 77% was PP. These PP values fall within seasonal variations observed in other river systems (25, 26) and confirm that PP predominates in rivers (27). The higher TP, PP, and DP concentrations measured for FP compared to RI are consistent with nutrient cycling in these systems (26, 28). Lyophilization of 4 L of each filtrate produced 0.107 (RI) and 0.123 g (FP) of dried powder. Upon extraction with NaOH-EDTA, 89% of total DP (0.029 mg L-1) was recovered for RI and 97% (0.054 mg L-1) for FP. The range of recoveries of total DP from our samples was greater than that reported by Minear (29), who recovered 60-90% of TP from lyophilized lake water extracted with 0.1 M NaOH. As no material remained after extraction and the recovery of calcium was comparable to that of P (93% for RI; 97% for FP), any P loss probably occurred during transfer of lyophilized material from freeze-dryer flasks rather than by precipitation during lyophilization (19, 29). Dry weights of particulates from 4 L of water were 0.180 (RI) and 0.592 g (FP). After extraction with NaOH-EDTA, 54% (0.48 mg g-1) and 56% (0.71 mg g-1) of PP was recovered from RI and FP, respectively. These recovery rates are comparable to those of soil and marine particulates (17). Most extracted P was orthophosphate, HPO42- or H2PO4(Table 1; Figure 1). In filtered water, SRP concentrations were 0.028 mg L-1 (87.5% of total P) for RI and 0.048 mg L-1 (85.7%) for FP. If SRP is predominantly orthophosphate (2, 3), then NMR results are consistent with chemical analysis. Dissolved orthophosphate is the most readily bioavailable P form (2). Particulate orthophosphate may be adsorbed to the surface of clay and mineral particles, bound to Ca or oxides of Fe, Al, or Mn, or bound to organic matter by metal ligands (2, 6). Orthophosphate in DP and PP is thought to be in equilibrium in river waters (31), with seasonal variations in concentration (25, 26, 30). VOL. 40, NO. 24, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Solution 31P NMR spectra of dissolved phosphorus (top) and particulate phosphorus (bottom) from water samples collected from the river inlet and the floodplain of the Pee Dee River. Spectra are plotted with 7 Hz line broadening. Pyrophosphate was present in all samples, at -3.97 to -4.14 ppm. These do not appear to be polyphosphate end groups, due to their size and the lack of corresponding peaks in the polyphosphate region. Pyrophosphate is common in soil samples (10) and has been reported in marine particulates (17) and estuary sediments (15). Pyrophosphate function in terrestrial and aquatic P cycles is unclear, though it has been shown to be bioavailable even in the presence of high ambient SRP (15). This extraction technique will detect polyphosphate if present (13, 14, 17), but no polyphosphate peaks were observed in our initial water samples. Although TP was predominantly inorganic in both water samples, a range of Po forms was present. Phosphonates were detected at 22.4-8.2 ppm (Figure 1). Phosphonates have also been detected in marine (17, 20) and freshwater (19) samples. Phosphonates, which contain a C-P bond rather than the 7876

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C-O-P bond of other Po compounds, are thought to be chemically stable and more resistant to hydrolysis and biodegradation than other Po forms (20). A marine cyanobacterium was recently demonstrated to use phosphonates as a P source (31), but nothing is known about phosphonate bioavailability or cycling in fresh water ecosystems. In the orthophosphate monoester chemical shift range, peaks were detected at 5.8, 5.5, 4.8, 4.7, 4.5, 4.4, 4.3, 4.2, 4.0, and 3.7 ppm (Figure 1). At least one of the four peaks for IHP was observed at 5.5, 4.4, 4.0, or 3.7 ppm for all samples. With a high anionic charge, IHP will adsorb strongly to clays, mineral surfaces and organic matter, resisting hydrolysis and biodegradation (32). Inositol hexakisphosphate accumulates in soils and can move to aquatic environments by erosion, where it may be present in PP or DP (8, 33). Inositol hexakisphosphate is bioavailable to aquatic organisms, but

TABLE 2. Results from Testing Pure P Compounds P added chemical shift (ppm) separatelya chemical shift (ppm) in DI/NEb AEPc (µg of P) g6Pc (µg of P) IHPc (µg of P) DNAc (µg of P) pyroPc (µg of P) polyPc (µg of P) total addedc (µg of P)c % total P calcdd % total P obsde

3.47 16.81 26.76 0.34 4.88 5.05 57.3

AEP

orthoP

20.46 20.2

6.00 6.0

5.60 5.04 4.75 4.62 4.25 4.13 -0.51 -4.10 -4.40 -18.26 5.5 5.1 4.7 4.6 4.2 4.1 -0.5 -3.7 -4.0

IHP

g6P

g6P

IHP

IHP

IHP

DNA

polyP

pyroP

polyP

3.43

0.04 0.17 0.54

4.28

15.3 1.35 8.83 9.10 4.01 0.34

3.43

1.22 0.05 2.01

6.0 4.4

3.5 16.1

4.28 15.3 1.35 8.83 9.10 4.01 0.34

2.73 2.73

3.66 0.86 4.52

1.42 1.42

7.5 7.3

4.8 4.4

7.9 8.7

2.5 0.0

26.7 2.3 16.1 2.9

15.4 15.9 7.0 16.1 17.5 5.8

0.6 0.7

a Chemical shift value when the individual compound was analyzed by 31P NMR spectroscopy. b Chemical shift value (ppm) when a mixture of all compounds was dissolved together in deionized water (DI), lyophilized, extracted with NaOH-EDTA (NE), relyophilized, and analyzed by 31 P NMR spectroscopy (procedure abbreviated DI/NE). c The amount of P indicated in the second column was added to 4 L of DI, and the expected distribution of this P across the DI/NE spectrum, on the basis of the individual tests, is shown in the following columns. d Relative percentage of P represented by each peak, calculated from the individual tests. e Relative percentage of P represented by each peak, observed in the DI/NE spectrum.

less so than orthophosphate and many other Po compounds (32). In our samples, IHP was 3% (RI) and 9% (FP) of total DP and 15% (RI) and 20% (FP) of PP. This higher IHP concentration in PP is consistent with increased sorption to particulates. Other orthophosphate monoesters that may be present, based on chemical shifts of peaks in the orthophosphate monoester region, include sugar phosphates and degradation products from phospholipids (10, 24). These compounds may be transported into water from terrestrial material or formed by aquatic organisms, and they vary in bioavailability (7). In the orthophosphate diester region, phospholipid peaks at 1.41 and 1.65 ppm were present in both DP samples. Phospholipids are thought to indicate bacterial and plankton biomass or the remains thereof (13). They vary seasonally in lakes (13) and are bioavailable (7). DNA was present at -0.3 to -0.81 ppm (10, 24). Both dissolved and particulate DNA and RNA have been identified in aquatic ecosystems and are readily degraded in the water column (9). (2) Spiking Trial: Individual Compounds, DI Water. Results from NMR analysis of individual P compounds and of DI spiked with all compounds are shown in Table 2 and Supporting Information Figure 1. The phosphonate compound (AEP) had one dominant peak (99%) at 20.48 ppm, and a smaller orthophosphate peak (1%) at 6.0 ppm. There was only one peak for DNA, at -0.51 ppm. Phytic acid had five peaks: an orthophosphate peak at 6.0 (2%) and peaks at 5.6 (16%), 4.62 (33%), 4.25 (34%), and 4.13 ppm (15%). The 4.13-ppm peak appears as a shoulder on the 4.25-ppm peak. Orthophosphate is 1% of g6P; other peaks are at 5.04 (91%) and 4.75 ppm (8%). Pyrophosphate has a peak at -4.44 ppm (75%) and an orthophosphate peak at 6 ppm (25%), while polyphosphate has peaks at 6.0 (orthophosphate, 1%), -4.10 (polyP end groups, 54%), -4.40 (pyroP, 17%) and -18.26 ppm (midchain polyP, 28%). Using these peak distributions, relative percentages, and the mass of each compound dissolved in deionized water, we estimated the relative percentage that each peak should comprise in the DI water spectrum and then compared this calculation to the experimental results obtained by NMR (Table 2). There were slight differences in chemical shifts for some peaks, due to salt and pH differences from extracting the lyophilized DI water with NaOH-EDTA. There are also slight differences in these chemical shifts compared to those in spectra from DP and PP samples, again due to differences in pH and salt and also to the presence of paramagnetic ions such as Fe and Mn in the natural samples. Most of the calculated percentages were very similar to those observed. However, the observed percentage for orthophosphate was markedly higher than

calculated, while those for g6P (5.1 ppm) and polyP (-18.26 ppm) were much lower. Because no relaxation agents were added and no paramagnetics were present, the results from the DI test may not be fully quantitative, due to differences in relaxation rates among the compounds (33). However, these differences also suggest that these P compounds may be altered by the extraction or lyophilization procedure (see below). For the DI test, 71% of the added P was recovered after lyophilization and extraction with NaOH-EDTA, with loss most likely occurring during transfer from the freeze-dryer flasks. (3) Degradation Test. The degradation test results support those seen with spiked DI water (Supporting Information Figure 2), in that g6P and polyP were altered depending on sample preparation. No changes were observed for pyrophosphate. When polyP was added to lyophilized NaOHEDTA, there were peaks at 6.0 (orthophosphate, 1%), -4.0 (74%), and -17.9 ppm (25%). When polyP was lyophilized in water and prepared for NMR (without lyophilized NaOHEDTA), the peak at 6.0 ppm was 2%, the peak at -4.0 ppm was 49%, and peaks were present at -4.5 (24%), -18.4 (23%), and -18.9 ppm (2%). When polyP was lyophilized in NaOHEDTA, peaks were detected at 6.0 (44%), -4.0 (44%), and -17.9 ppm (12%); and when polyP was lyophilized in water, redissolved in NaOH-EDTA, and lyophilized again (to represent the treatment of the natural waters), there were peaks at 6.0 (35%), -4.05 (49%), and -17.9 ppm (16%). Neutralization reduced the degradation to orthophosphate: lyophilizing in neutralized NaOH-EDTA resulted in peaks at 6.0 (2%), -3.6 (15%), -3.8 (55%), -17.4 (26%), and -18.4 ppm (2%). For the polyP sample lyophilized in water and then in neutralized NaOH-EDTA, peaks in the same regions were observed, with the exception of -18.4 ppm, at 3%, 34%, 45%, and 18% respectively. The presence of an additional peak at -18.4 to -18.9 ppm suggests that a polyphosphate with a different chain length than the added tripolyphosphate may be formed under some conditions, although not consistently. In alkaline samples, there appears to be considerable degradation of polyphosphate when samples are lyophilized in NaOH-EDTA, based on the sharp increase in orthophosphate. Neutralizing before lyophilization appears to minimize this degradation. Similar results were observed for glucose 6-phosphate: g6P lyophilized in water and prepared for NMR without lyophilized NaOH-EDTA had peaks at 6.0 (1%), 5.0 (91%), and 4.7 ppm (8%), and when g6P was lyophilized in water and added to lyophilized NaOH-EDTA, peaks were present at 6.0 (5%), 4.5 (87%), and 4.4 ppm (8%). When g6P was VOL. 40, NO. 24, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 3. Relative Distribution of P in Dissolved P (DP) Forms in Water Samples Spiked with Various P Compounds before or after Filtrationa spike compdb none AEP G6P IHP DN1A pyroP polyP

before after before after before after before after before after before after mean (std dev)

orthoP (%)

pyroP (%)

polyP (%)

phon (%)

mono (%)

di (%)

NMR Pi (%)

NMR Po (%)

total P (mg L-1)

SRP (mg L-1)

SRP (% total P)

82.7 80.0 76.9 73.3 72.1 76.9 64.8 84.3 84.0 80.4 78.0 75.6 79.0

4.1 2.1 2.1 1.9 1.3 1.1 1.1 1.1 1.9 2.5 5.4 4.7 3.0

2.1 3.3 3.0 4.0 2.6 1.8 1.1 2.9 2.8 3.0 2.0 3.6 3.0

2.1 5.3 4.1 1.0 3.9 1.2 1.1 1.1 3.7 1.2 1.3 1.7 2.0

8.3 8.2 12.9 19.8 17.5 17.9 30.8 7.8 5.8 12.9 12.0 13.2 12.0

0.7 1.1 1.0 1.0 2.6 1.1 0.0 2.8 1.8 0.0 1.3 1.2 1.0

88.9 85.4 82.0 79.2 76.0 79.8 68.1 88.3 88.7 85.9 85.4 83.9 85.0

11.1 14.6 18.0 21.8 24.0 20.2 31.9 11.7 11.3 14.1 14.6 16.1 15.0

0.051 0.055 0.059 0.050 0.066 0.053 0.060 0.048 0.046 0.053 0.060 0.061 0.063

0.045 0.037 0.032 0.035 0.030 0.032 0.030 0.033 0.031 0.032 0.030 0.025 0.030

88 67 54 70 45 60 50 69 67 60 50 41 48

77.5 (5.36)

2.48 (1.43)

2.71 (0.79)

2.28 (1.45)

13.8 (6.62)

1.20 (0.82)

82.8 (5.89)

17.3 (5.95)

0.056 (0.001)

0.032 (0.004)

59.2 (13.0)

a OrthoP, orthophosphate; pyroP, pyrophosphate; polyP, polyphosphate; phon, phosphonates; mono, orthophosphate monoesters; di, orthophosphate diesters; NMR Pi, inorganic P from adding percentages of inorganic P forms (orthophosphate, pyrophosphate, and polyphosphate); NMR Po, inorganic P from adding percentages of organic P forms (phosphonates and orthophosphate monoesters and diesters); SRP, soluble reactive P. b P forms used for spiking are described in the text.

TABLE 4. Relative Distribution of P in Particulate P (PP) Forms in Unspiked Water Samples and in Water Samples Spiked with Various P Compounds before Filtrationa spike compdb

orthoP (%)

pyroP (%)

polyP (%)

phon (%)

mono (%)

di (%)

NMR Pi (%)

NMR Po (%)

total P (Mg g-1)

no spike no spike no spike no spike no spike no spike no spike mean (std dev)

69.2 69.8 72.0 70.1 75.1 69.7 77.3 71.9 (3.15)

5.9 5.5 3.0 2.9 2.9 2.8 3.4 3.77 (1.34)

2.0 2.1 2.5 2.8 2.5 2.3 2.0 2.31 (0.30)

1.5 1.4 1.5 2.1 1.5 2.3 2.1 1.77 (0.38)

18.9 15.8 20.0 20.6 14.1 21.2 14.2 17.8 (3.06)

2.5 5.4 1.0 1.5 3.9 1.7 1.0 2.43 (1.66)

77.1 77.4 77.5 75.8 80.5 74.8 82.7 78.0 (2.73)

22.9 22.6 22.5 24.2 19.5 25.2 17.3 22.0 (2.73)

0.202 0.199 0.188 0.227 0.201 0.209 0.231 0.208 (0.016)

70.2 66.2 57.8 69.5 69.0 71.6

4.8 5.7 3.9 4.7 5.3 4.3

2.0 3.6 2.0 2.1 1.8 3.8

2.0 2.4 1.5 2.6 1.2 2.1

18.1 19.8 32.8 15.8 17.5 16.6

2.9 2.3 2.0 5.3 5.3 1.6

77.0 75.5 63.7 76.3 76.1 79.7

23.0 24.5 36.3 23.7 23.9 20.3

0.238 0.256 0.264 0.219 0.231 0.268

AEP G6P IHP DNA pyroP polyP a

Abbreviations are as defined in Table 3, footnote a.

b

P forms used for spiking are described in the text.

lyophilized in NaOH-EDTA, there were peaks at 6.0 (29%), 4.7 (22%), 4.5 (41%), 4.4 (8%), and 4.0 ppm (1%), and when g6P was lyophilized in water and then lyophilized in NaOHEDTA, peaks were present at 6.0 (41%), 4.7 (53%), 4.5 (5%), and 4.0 ppm (1%). When samples were neutralized before lyophilization in NaOH-EDTA, peaks were seen at 6.0 (2%), 4.8 (14%), 4.8 (70%), and 4.5 ppm (14%), with no differences when the sample was lyophilized in water first. These results highlight chemical shift sensitivity to pH and salts and indicate that lyophilization in NaOH-EDTA can hydrolyze P compounds, which can be minimized by neutralizing samples before lyophilization. This orthophosphate release from P compounds is surprising, as Turner et al. (24) detected no changes in orthophosphate when lyophilized NaOH-EDTA extracts of soil were spiked with a wide range of P compounds. However, they did not lyophilize any compounds; just the soil extracts prior to spiking. It is also important to note that the P compounds used in this degradation study were selected because they were inexpensive and dissolved easily in water; they may not represent the versions of these compounds found in nature. Polyphosphates have been reported in lyophilized NaOH-EDTA extracts of soils, plant litter, and aquatic samples (e.g., ref 7878

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10). Degradation of polyphosphates has been reported in lyophilized NaOH-EDTA extracts of manure (34), but only from increasing the NaOH concentration. It is more than likely that natural compounds such as polyphosphates are more complex in nature and are stabilized in different ways, such as through cations, than are these commercially available compounds. And neutralization of compounds with high metal concentrations may introduce changes by altering the solubility of metals. Further tests are required. (4) P Spiked in Natural River Water. Spiking before or after filtration produced few changes for most DP compounds (Table 3, Supporting Information Figure 3). The exception was IHP: spiking after filtration resulted in almost twice as much P in the orthophosphate monoesters as spiking before filtration. This suggests that IHP is lost to particulates, and this is supported by the PP results (Table 4, Supporting Information Figure 4). This is not surprising, as IHP is known to readily adsorb to surfaces in soils (32). Most of the IHP peaks were present in all DP and PP samples, including those not spiked with IHP. Although we made no attempt to determine colloidal P in our samples, it may comprise a high proportion of the DP (2, 3) and is likely to account for the IHP in DP samples.

There is considerable variability in total DP. In fact, the unspiked sample had higher total DP concentrations than some of the spiked samples. Unfortunately, we assumed low sample variability and did not measure TP in each sample before and after spiking, and so we are unable to estimate the recovery of each P compound. For most compounds, spiking resulted in a detectable increase in the relative percentage of P in the region of the spectrum in which the added compound should be visible, with the exception of DNA due to the low added P. It is interesting that changes could be detected, given that total DP in these samples was 56 µg of P L-1, while the P added from compounds other than DNA ranged from 0.85 to 6.53 µg of P L-1. This suggests that the technique is sensitive to very small changes in P. Spiking with AEP increased the peak at 21 ppm from about 1% to 3% when spiked before or after filtration for DP and produced no noticeable change in PP. As observed in the initial river samples, a number of other small peaks are also present in the phosphonate region. The g6P spike produced an increase in the 4.9-ppm peak for DP (from 1-2% to 6%) and no change in PP. In samples not spiked with IHP, IHP was 3-5% in DP and 10-16% in PP, which is consistent with levels detected in the July RI samples. In samples spiked with IHP before filtration, IHP was 8.3% in DP and 31% in PP; spiking after filtration resulted in 26% of P as IHP in DP. Spiking with pyroP after filtration increased the -3.9-ppm peak in DP to 5% from 1-2% for all samples except the unspiked control sample (for which pyroP was also 5.4%) and the samples spiked with polyP before (4.7%) or after (3.0%) filtration. For PP, the relative percentage of P in the -3.9 ppm peak was fairly constant, at 3-6%, regardless of treatment. Spiking with polyP did not increase the relative percentage of the peaks at -3.5 or -20 ppm (both 1-2% for DP and PP) but affected the -3.9-ppm peak as noted above. It is interesting to note that while polyP added as spike appears to have been degraded, other polyP peaks were present in all samples. This again suggests that natural polyphosphates may be stabilized in some way. The average dry weight of particulates recovered from RI samples for the spiking study was 0.58 g. This is higher than the original July RI sample, is comparable to the FP sample, and reflects seasonal variation. The average P recovery from extracting PP for the spike study was 88%, which was higher than for the original RI and FP samples. This may also reflect seasonal changes in material. Extraction of lyophilized PP removed 85-90% of TP, which is comparable to the original RI and FP samples and is higher than for the lyophilized spiked DI sample, which stuck to the container more than did the river samples. Despite differences in particulate weights, the relative distribution of P compounds for the unspiked February RI sample is similar to that of the July RI sample, with the exception of polyphosphates, which were detected in the February sample but not the July sample. The DP TP concentration of the February sample is comparable to that from July. However, TP in particulates is lower in February (0.2 mg g-1) than in July (0.9 mg g-1). In the February RI samples (Table 3), SRP comprised 41-88% of the TP, which was comparable to the July RI sample. The SRP concentration should not be affected by degradation, because it was determined on samples that were not lyophilized. In all but the unspiked control, the SRP percentage of TP was lower than that of orthophosphate or of total Pi determined by NMR. This has been reported elsewhere (35) and suggests that calculating Po by the difference between TP and SRP will overestimate Po. However, in light of the degradation observed in spiked compounds, it is also possible that NMR is overestimating Pi; further investigation is warranted. Advances in NMR instrumentation have improved spectral resolution relative to earlier studies (19). As Fe was present

in all of our natural river waters extracts, lanthanide shift reagents were not required (19). The 4.32-s delay time should be adequate for full relaxation (10, 33). Peak areas were calculated by integration across the entire spectrum, and the presence of peaks was determined by a combination of the peak-picking subroutine of the NMR processing software, changes in the integral corresponding to apparent peaks, and researcher judgment. However, the signal-to-noise ratio in the phosphonate and polyphosphate regions is low, and we may have overestimated peak areas in these regions. It is difficult to determine potential error for peak areas, but presumably it is higher for smaller peaks (e.g., phosphonates) than for prominent peaks (e.g., orthophosphate). The standard deviations from the six unspiked PP samples give a good measure of variability, although this reflects both natural and analytical variability. It is probably best to assume an error of (3-5% for orthophosphate and orthophosphate monoesters and (1-2% for the other regions.

Acknowledgments We thank Dr. Corey Liu (Stanford Magnetic Resonance Laboratory) and Dr. Guangchao Li (ICP analysis). This work was supported by NSF award 0136105.

Supporting Information Available Spectra for the spiking and degradation trials. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Received for review August 1, 2006. Revised manuscript received September 6, 2006. Accepted September 27, 2006. ES061843E