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Characterization of phosphorus fractions in surface horizons of soils from Galicia (N.W. Spain) by 31P NMR spectroscopy
Soil Bid.
Biochem.
Vol. 22, No. I,
(x)38-0717/90
pp. 75-79, 1990
53.00 + 0.00
Copyright @ 1990 Pergamon Press plc
Printed in Great Britain. All rights reserved
CHARACTERIZATION OF PHOSPHORUS FRACTIONS SURFACE HORIZONS OF SOILS FROM GALICIA (N.W. SPAIN) BY 3’P NMR SPECTROSCOPY
IN
F. GIL-S• TRES Depar~amento de Edafologia, Facultad de Farmacia, Santiago de Compostela, Espafia W. ZECH’ and H. G. ALT* ‘Institute
of Soil Science and Soil Geography and *Laboratory of Inorganic Chemistry, University of Bayreuth, Postfach IO 12 51, 8580 Bayreuth, F.R.G. (Accepied 16 June 1989)
NMR spectroscopy was used to study the P fractions in NaOH extracts of surface mineral horizons of soils from Gahcia (N.W. Spain). Mono and djeste~h~phate dominated in all soifs; phosphonates were present under cold and wet conditions. Rankers, Cambisols and Podzols differ in their diester-to-monoester ratio and in the percentage of the NaOH soluble organic P. These results and those for a Rendzina and a Gleysol show a relationship between soil genesis and the forms of organic P present. The relationship between bicarbonate-extractable organic P and the diester content of the soils confirms the nature of labile organic P in these soils and explains the poor correfation between labile organic P and phosphomonoesterase activity.
Suntmary--JiP
fractions in surface horizons of Galician Rankers, Cambisols and Podzols differ due to pedogenesis and to get more information about the poorly-known P cycle in acid soils.
INTRODIJCX’ION
Little work has appeared on the relationships between pedogenesis and soil organic compounds. As for soil inorganic P (Pi), comparisons have hitherto been made on the basis of data obtained by chemical fractionation (Tiessen et al., 1984; Sharpley et a&, 1987; Trasar-Cepeda, 1987). However, fractionation of organic P (P,) in NaOH extracts can now easily be performed using 31P spectroscopy (Newman and Tate, 1980), so it seems of interest to test whether, like chemically-identified P fractions, these spectroscopic P fractions also reflect typical alterations during pedogenesis. The generally acid, highly-organic soils of Galicia (N.W. Spain) are dominantly Rankers, Cambisols and Podzols (Guitian, 1967). Their forms of phosphorus have been aiready quantified using the Hedley ef al. (1982) fractionation method. It was found that organic P dominated over Pi, primary P was almost totally lacking, resin-extractable P concentrations were low, and that the P-distribution was similar in all three soil groups (Traser-Cepeda, 1987). In surface samples of Galician soils, incubation under controlled conditions only affects P fractions extracted with NaOH and ultrasound (Trasar-Cepeda et ai., 1986), i.e. by an extraction technique similar to that normally used for analysis of soil P by NMR. In spite of the generally low solubility of P, compounds in the alkaline extract employed in the NMR method (Tate, 1984; Condron et af., 1985). the method can therefore be applied to these soils in the certainty that it will detect the compounds involved in short-term P transformations. In view of this, we have used ,iP NMR spectroscopy to examine whether the NaOH soluble P
MATERIALS AND METHODS
A total of 20 samples of the surface horizons (O-20cm) of soils from Galicia (N.W. Spain) were studied. Most of the soils belonged to one of the three types that are most common in Galicia, Rankers (7 samples), Cambisols (6 samples) and Podzols (5 samples). Samples of Rend&a and a Gleysol were included as representing extreme situations and for purposes of comparison. All 20 soils underlay heather and fume vegetation. Table I lists the chief physical and chemical properties of the samples as determined using methods described by Guitian-Ojea and Carballas (1976). Extracts for “P NMR analysis were obtained using the method of Newman and Tate (1980). For this purpose 6.7 g of finely ground and air-dried soil were dispersed by ultrasound (200 W, 5 min) in 20 ml 0.5 N NaOH, and the suspension was centrifuged (lO,O~ rev min-‘) for 120 min at 0°C. The resulting su~rnatant was concentrated to ca 2ml under a stream of N, at 4O”C, then 1 ml of D,O was added and the final solution was transferred to NMR tubes 1 cm dia. Proton-decoupled ,‘P spectra were recorded at 20°C on a JEOL FX9OQ Multi-nucleus Fourier transform NMR spectrometer with an observation frequency of 36.2 MHz. Chemical shifts in ppm were measured relative to external I&PO,/D,O. The intensity of the signals was determined by integration. 75
F. GIL-Soram et al.
76
Table I. Physical and chemical uroperties of the surface mineral horizons from soils under studv
c
pH CIK
Rankers (7)** Cambisols f6)*’ Podzols (5)” Rendzina (I)**
N (%)
(%)
Clay (%)
Al,O,
Fe,O, (%)*
(%)’
Max.
Min. Mean Max. Min. Mean Max. Min. Mean Max. Min. Mean Max. Min. Mean Max. Min. Mean
6.30 4.52 3.52
3.60 3.76 2.72
Gleysol ( 1i*;
4.23 4.16 3.20 7.50 4.05
15.88 4.08 8.41 4.60 12.70 3.30
8.18 6.24 8.20 5.19 7.60
1.36 0.60 0.73
0.26 0.30 0.13
0.60 0.46 0.43 0.44 0.53
14 24 t7
9 7 2
10 14 8 11 ;5
1.22 1.93 0.55
0.11 0.51 0.10
0.76 1.38 0.30 1.14 2.06
0.60 1.03 0.40
0.10 0.26 0.04
0.38 0.72 0.20 0.07 0.92
*Extracted with 0.1 N pyrophosphate. **No. of samples in each soil type.
Their interpretation is based on literature assignments (Newman and Tate, 1980; Zech et ai., 1985). The extracts were analysed for total P (= P,) by calcination and inorganic P (= Pi) according to Saunders and Williams (1955). Organic P (= P,) was calculated by difference. Total C in the extract was determined according to Guitian-Ojea and Carballas (1976). Total soil P was determined with HF and HCIQ. Bicarbonate-extractable P was measured in all samples according to Olsen et al., (1954) modified by Hedley et al. (1982). In all cases, P was determined calorimetrically using the Molybdenum Blue method of Murphy and Riley (1962). RESULTS
Rankers
In the surface horizons of the Rankers studied, soil P, ranges from 388 to 1505 pg g-’ (mean 678 pgg-I). NaOH and ultrasound treatment extracted a mean 262 pg g-r (Table 2), so that the proportion of P, subjected to 31P NMR analysis was 36%. According to chemical analysis, 74% of the extracted P was of organic and only 26% of inorganic origin. This is in agreement with the “P NMR data (Table 3), which reveal 30% of P extracted to be orrhophosphate and 70% to be organic. The 3iP NMR spectra of these soils generally exhibited high background noise and considerable
overlapping between the monoester and inorganic ~r~hophosphate regions (Fig. 1). The extended spectra show a complex monoester region near 5.6 ppm, generally exhibiting several peaks, perhaps due to the presence of more than one type of monoesterphosphate (Condron er al., 1985). The diester region is equally complex. The average proportion of diester (11%) was less than the average proportion of monoester (45%). Signals due to pyrophosphate and polyphosphates were lacking in all Ranker samples. Phosphonate signals only appeared in the spectrum of a pseudo-alpine Ranker collected at an altitude of 1700 m, in which they made up 17% of total extracted P. The presence of phosphonates in the soi! devefoped under the coldest and wettest climate support the hypothesis of Newman and Tate (1980) that C-P compounds accumulate in cold, wet, acid soils, possibly as the result of low bacterial activity. The spectra of all the Rankers exhibited resonances between 4.5 and 3.0 ppm that may reflect the presence of hydrolysis products of phosphoglycerides like ChoIinephosphate (3.5 ppm) and e~anola~nephosphate (4.2 ppm), or even of simple sugarphosphates (fate and Newman, 1982). Such signals are only absent in the spectrum for a surface horizon from one Ranker developed at sea level and with a pH 6.5. Cambisols
The Cambisoi group was more homogeneous than the Rankers in chemical analysis. Total P contents
Table 2. Total soil P contents and P cantent in the NaOH extracts as determined chemically Total soil P (ggg-‘) Rankers Cambisols Podzols Rendzina Gleysol
Extraction (%)
Total extracted P (Icgg-‘)
Max.
Min.
Mean
Max.
Min.
1505 692 1159
388 396 60
678
764 319 657
102 134 31
591 330*** 697 991
Mean 262 193 189.” 59 408
Max.
Min.
St 70 65
18 24 50
P, in extract (%)+ Mean
Max.
36a** 91 40a 88 56b 75 8 41
Min.
Mean
51 71 55
74ab 82a 67b 86 68
lPercentage of total extracted P. **Means with the same fetter are not si~ifi~ntly different (P = 0.01). ***If one anomalous sample is excluded, the mean total soil P content is 123 pg 8-l and the mean P content of the extract 72 pgg-‘. Table 3. Average P distribution in the NaOH extracts as determined by integration of “P NMR signals Orrhophosphate I%) Rankers Cambisols Podaols Rendzina Gleysol
30a 22a 29a 22 I5
Total P, (%) 70a 78a 7la 78 85
Monoesters (%) 4.5(65)a* 61 (78)b 49 f69)a 26 (33) 58 (68)
Diesters (%)
Others** (%)
1I (16)a’ 9(12)a 19 (27)a 29 (38) 27 (32)
14 (20)a* 8UO)a 3 (4)s 23 (29)
*Values expressed as percentage of P, extracted. **Include phosphonates, polyphosphates and others not identified compounds. Means with the same letter are not significantly different, P = 0.01.
Diester/ monoester 0.25a 0. tbb 0.42~ 1.11 0.46
77
Soil genesis and P, l20
l15
llO
mm
.!i
0
-5
-10
background noise, the monoester and diester regions being simpler and the fact that the Cambisols only occasionally exhibit signals in the regions corresponding to cholinephosphate, ethanolaminephosphate and simple sugarphosphates.
-15
a*
h
54
RANJiL
Podzols
Mean P, content of the Podzol-A horizons was 33Opg g-l. NaOH and ultrasound treatment extracted 56% of P, (Table 2), a significantly higher percentage than for Rankers and Cambisols. The Podzol extracts also differed form those of the latter soil groups in regard to the proportion of P, to Pi (67% to 33% respectively), and the P, extracted made up an average 48% of the total soil P,, an appreciably larger proportion than for Rankers and Cambisols. In their spectra, both the monoester and diester signals are complex with a large number of peaks, probably due to disturbances by paramagnetic substances. In spite of this, it is generally clear that compared with Rankers and Cambisols, the diesterto-monoester ratio rose to 0.42. Almost all the soils of the Podzol group exhibited cholinephosphate and ethanolaminephosphate signals; for one sample (Podzol over quarzites), the strong cholinephosphate signal at 3.2 ppm represents 24% of the P, content of the extract, ca 30 pg g-’ of the whole horizon. Rendzina
NaOH and ultrasound extracted only 8% of P, in the Rendzina Ah (Table 2), low extraction is due to lack of removal of CO,Ca by and before NaOH extraction. Its “P NMR spectrum reveals large proportions of diesters (29%) and unidentified organic phosphates (23%) while monoesters account only 26%. The typical feature of this soil is its high diester-to-monoester ratio of 1.11 (Table 3).
RENDZINA
+20
+15
*lo
.s
pm
0
-5
-10
-15
Glqysol
Fig. 1. Selected )‘P NMR spectra of surface mineral horizons from representative Galician soils.
ranged from ca 400 to 700 pgg-’ with a mean of 591 pg g-l (Table 2), and NaOH extracted 40% of P,. The extracted P was slightly more organic than in the NaOH-extract from the Rankers, 82% being organic and only 18% inorganic. The “P NMR spectra of these soils are very similar to those of the Rankers though with less overlapping between the monoester and orthophosphate regions. Integration again confirms the results from the chemical analysis (Tables 2 and 3). Diesters make up an average of 9% of the extracted P, a slightly smaller figure than for Rankers. The main differences with respect to the Rankers consist in the extended spectra having less
The Ah-horizon of the Gleysol and its NaOHextract had large P, contents, 991 and 408 pgg-’ (= 41%), respectively (Table 2), NaOH extracted about 15% Pi and 85% P,. Like the Gleysols of the Bavarian Alps, the diester peak was remarkably sharp (Zech et al., 1986). The diester-to-monoester ratio was very similar to the average for the Podzols. DISCUSSION
Due to high stabilization (Brannon and Sommers, 1985) P, compounds seem to be poorly extractable with NaOH and ultrasounds. But since the percentages of P, extracted were very similar to the percentages of carbon extracted, the C-to-P, ratios of the whole horizons were nevertheless relatively close
Table 4. Mean C- and P,-values of the NaOH extract and the C/P, ratio in soils and NaOH extracts Soil C extracted (%)
Rankers Cambisols Podzols Rcndzina Gleysol
Soil P. extracted (%)
C/P. in soils
C/P, in extracts
MU.
Min.
Mean
Max.
Min.
Mean
Max.
Min.
Mean
Max.
Min.
Mean
60 52 42
13 29 31
35a 38a 3Sa 18 36
49 44 67
10 23 37
31a 32a 48b 8 39
213
86 121 115
154a ISla 738b 81 106
243 213 759
84 69 92
175a I70a 557b 180 98
Means with the same letter are not significantly different (P = 0.01).
78
F. GIL-S• TRESet ol.
to those of the extracts (Table 4). Only from the Podzols was a significantly higher proportion of P, extracted than C, possibly due either to much of the organic matter being unhumified (GuitianOjea and Carballas, 1968b) or to the dominance of diesters over monoesters. This suggests that for the purposes of studying the distribution of P,, the NaOH-extracted organic matter may be considered as representative of the total organic matter content. It was seen above that in regard to the distribution of P,, the soils of each group have certain common features. These chiefly involve their monoester and diester contents, since the presence of phosphonates and polyphosphates appears to depend largely on microclimatic factors (Newman and Tate, 1982). Comparison of the monoester and diester contents of the various groups suggests certain relationships between the distribution of P, and the evolution of the soils. In Gahcia, Rankers are young soils with a low rate of carbon mineralization, though those developed over granite quickly form insoluble complexes due to the action of the Al oxides derived from rapid weathering of plagioclases by the Galician climate (Carballas, 1982). “P NMR analysis of these soils showed that on average, 65% of the P, extracted was stabilized as monoesters while only 16% was incorporated in diesters (Table 3). The chemical complexity of the pool of monoester and diester components, which is evidenced by the large number of significant peaks in the corresponding spectral regions, shows that the humic substances of these soils have undergone little degradation; this conclusion is supported by the high proportion of uncharacterized P, compounds (almost 20%), which seem likely to be intermediate products of the composition of plant remains. Galician Cambisols are usually Humic Cambisols. occasionally with an allochthonous surface horizon (Guitian-Ojea and Carballas, 1968a). Accordingly. their organic matter is more humidified than in Rankers and is highly stabilized by Al as in Andosols (Carballas, 1982). This is reflected by the distribution of their P contents: the proportion of monoesters is greater than in Rankers, diesters make up only 12% of the organic P extracted (Table 3) and, consequently, the average diester-to-monoester ratio falls from 0.25 in Rankers to 0.16 in Cambisols. The simplification of the P, compounds is likewise reflected by the reduction in the number of peaks in the monoester and diester regions of the NMR spectra and by the almost complete disappearance of intermediate signals. Podzols are developed in Galicia under cold, wet conditions, over quarzites and sandstones that are poor in bases, Fe and Al (Guitian-Ojea and Carballas, 1968b). The agents capable of stabilizing humic substances (clay and extractable Fe and Al oxides) are accordingly extremely scarce in surface horizons of these soils (Table l), also biological activity probably is depressed because the high C-toN ratio of 19 in the A horizons (Table 1). These characteristics explain both the ease with which phosphorus can be extracted from these soils (on average, nearly half the total soil P, was extracted) and the
high diester-to-monoester ratio associated with the accumulation of P, as diesters which made up a third of the P, extracted (Table 3). Although Rendzinas and Gleysols were represented in this study by just one sample each, the spectra obtained were in agreement with the physicochemical environments in which these soils developed. The very small quantity of P, extracted from the Rendzina may reflect the insolubility of calcium phytates in alkaline media, which should explain the joint dominance of the diesters and uncharacterized P compounds over monoesters in the extract. The particular Rendzina studied is biologically very active (Acea, 1985) but its high content of available P, may be possibly inhibit enzymatic hydrolysis of P, compounds (Tate, 1984) and so favour their accumulation in the soil. The proportion of P, extracted from the Gleysol was similar to that obtained from Rankers, Cambisols and Podzols. The hydromorphous nature of this soil results in the loss of P, (possibly through the solubilization of insoluble iron phosphates in a reducing medium) and the reduction of biological activity during water saturation, which leads to the persistence of diesters and a diester-tomonoester ratio slightly greater than the average for Podzols. It is quite generally agreed that bicarbonateextractable P, is the most rapidly mineralizable fraction of the total P, content of the soil (Harrison, 1982; Tiessen et al., 1984). Various experiments (Bowman and Cole. 1978; Tiessen and Stewart, 1983; Tate, 1984) suggest that bicarbonate-extractable P, mainly consists of diesters, though it has also been simply attributed a non-phytate nature (Condron et al., 1985; Jones and Blomfield, 1982; Brannon and Sommers, 1985). For the soils studied here. Figure 2 shows the correlation between the bicarbonateextractable P, and the diester and non-monoester contents of the NaOH extracts. Though the correlation is high in both cases, the slope of almost unity for the diesters suggests that these are perhaps the basis source. The correlation is in either case important for the P cycle in these soils, since it means that the mineralization of this forms of P, depends
. ,*, , , , , , , , , , , , , , 0
100 Extract-PO
200
?I00
(+g g-l)
Fig. 2. Correlation between bicarbonate P,, and extractable P, (0, diester P, full line y =0.99 x + 13.2; *, total P, minus monoester P. broken line Y = 0.52 x + 14.01.
Soil gem :sis and P,
more on phosphodiesterases than of phosphomonoesterases. The rapid stabilization of monoesters in acid media and the inability of phophomonoesterases to dephosphorilate them once stabilized (McGill and Cole, 1981) would explain the poor correlation found by Trasar-Cepeda and Gil-Sotres (1987) between bicarbonate-extractable P, and soil phosphatase activity. Acknowledgements-This work was supported by a research grant awarded to F. Gil-Sotres by the Ministerio de Educacion y Ciencia of Spain and by the Deutsche Forschungsgemeinschaft, F.R.G. REFERENCES
Acea Ma. J. (1985) Esrudio de la poblacicin microbiana en melos de la zona humeda (Universidad de Santiago, Ed.). Santiago de Compostela, Spain. Bowman R. A. and Cole C. V. (1978) An exploratory method for fractionation of organic phosphorus from grassland soils. Soil Science 125, 95-101. Brannon Ch. A. and Sommers L. E. (1985) Stability and mineralization of organic phosphorus incorporated into model humic polymers. Soil Biology & Biochemistry 17, 221-227. Carballas M. (1982) Estudio de la genesis de1 Ranker Atlrinfico (Universidad de Santiago, Ed.). Santiago de Compostela, Spain. Condron L. M., Goh K. M. and Newman R. H. (1985) Nature and distribution of soil phosphorus as revealed by a sequential extraction method followed by a 3 lP nuclear magnetic resonance analysis. Journal of Soil Science 36, 199-207.
Guitian F. (1967) Suelos de la zona humeda espaftola. I. Tipos principales y sus relaciones geneticas. Anales de Edafologia y Agrobiologia 26, 1369-I 378. Guitian-Ojea F. and Carballas T. (1968a) Suelos de la zona humeda espafiola. III Ranker Atlantico. Anales de Edafologia y Agrobiologia 27, 57-73.
Guitian-Ojea F. and Carballas T. (1968b) Suelos de la zona hirmeda espatiola. IV Podzoles. Anales de Edafologia y Agrobiologia 27, 741-78 1. Guitian-Ojea F. and Carballas T. (1976) Tecnicas de analisis dp suelos (Pica sacro, Ed.). Santiago de Compostela, Spain. Harrison A. F. (1982) Labile organic phosphorus mineralization in relationship to soil properties. Soil Biology & Biochemistry 14, 343-35 1. Hedley M. J., Stewart J. W. B. and Chauhan B. S. (1982) Changes in inorganic and organic soil phosphorus fractions induced by cultivation practices and by laboratory incubations. Soil Science Society of America Journal 46, 970-976.
79
Jones 0. L. and Blomfield S. M. (1982) Macromolecular organic phosphorus in decomposing plants and in pasture soils. Soil Bioloav & Biochemistrv 14. 145-151. McGill W. B. and-Cole C. V. (1981) Comparative aspects of cycling of organic C, N, S and P though soil organic matter. Geoderma 26, 287-309. Murphy J. and Riley J. P. (1962) A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27,31-36. Newman R. H. and Tate K. R. (1980) Soil nhosnhorus characterization by 31P nuclear magnetic resonance. Communications in Soil Science and Plant Analysis 11, 835-842.
Olsen S. R., Cole C. V., Watanabe F. S. and Dean L. A. (1954) Estimation of available phosphorus in soils by extraction with sodium bicarbonate. USDA Circular 939.
Saunders W. H. H. and Williams E. G. (1955) Observations on the determination of available phosphorus in soils. Journal of Soil Science 6. 254-267.
Sharpley A. N., Tiessen H. and Cole C. V. (1987) Soil phosphorus forms extracted by soils tests as a function of pedogenesis. Soil Science Society of America Journal 51, 362-365. Tate K. R. (1984) Biological Transformation of P in soil. Plant and Soil 76, 245-256.
Tate K. R. and Newman R. H. (1982) Phosphorus fractions of a chnosequence of soils in New Zealand tussock grassland. Soil Biology & Biochemistry 14, 191-196. Tiessen H. and Stewart J. W. B. (1983) The Biogeochemistry of Soil Phosphorus. In Planefury Ecology (D. E. Caldwell, J. A. Brieley and C. L. Brieley, Eds), pp. 463-472. Van Nostrand Reinhold. Tiessen H., Stewart J. W. B. and Cole C. V. (1984) Pathways of phosphorus transformations in soils of differing pedogenesis. Soil Science Society of America Journal 48, 854-858.
M’. C. (1987) Estudio de la variucion y contenido del P organico en suelos gallegos (Universidad
Trasar-Cepeda
de Santiago, Ed.).Santiago de Compostela, Spain. Trasar-Ceneda Ma. C.. Gil-Sotres F. and Guitian-Oiea F. (1986) Transformaciones del fosforo en suelos gallegos sometidos a incubation. Anales de Edafologia y Agrobiologia 45, 3 I l-326.
Trasar-Cepeda M’. C. and Gil-Sotres F. (1987) Phosphatase activity in acid high organic matter soils in Galicia (NW Spainj. Soil Biology &Biochemistry 19, 281-287. Zech W.. Alt H. G.. Zucker A. and Koeel I. (1985) 31P NMR spectroscopic investigations of-NaOH extracts from soils with different land use in Yucatan (Mexico). Zeirschrifi Pflanzenerdhr. Bodenk. 148, 626-632. Zech W., Alt H. G., Haumaier L. and Blasek R. (1986) Characterization of phosphorus fractions in mountain soils of the Bavarian Alps by 31P N.M.R. spectroscopy. Zeitschrift Pflanzenerniihr. Bodenk. 150, 119-123.
Biochem.
Vol. 22, No. I,
(x)38-0717/90
pp. 75-79, 1990
53.00 + 0.00
Copyright @ 1990 Pergamon Press plc
Printed in Great Britain. All rights reserved
CHARACTERIZATION OF PHOSPHORUS FRACTIONS SURFACE HORIZONS OF SOILS FROM GALICIA (N.W. SPAIN) BY 3’P NMR SPECTROSCOPY
IN
F. GIL-S• TRES Depar~amento de Edafologia, Facultad de Farmacia, Santiago de Compostela, Espafia W. ZECH’ and H. G. ALT* ‘Institute
of Soil Science and Soil Geography and *Laboratory of Inorganic Chemistry, University of Bayreuth, Postfach IO 12 51, 8580 Bayreuth, F.R.G. (Accepied 16 June 1989)
NMR spectroscopy was used to study the P fractions in NaOH extracts of surface mineral horizons of soils from Gahcia (N.W. Spain). Mono and djeste~h~phate dominated in all soifs; phosphonates were present under cold and wet conditions. Rankers, Cambisols and Podzols differ in their diester-to-monoester ratio and in the percentage of the NaOH soluble organic P. These results and those for a Rendzina and a Gleysol show a relationship between soil genesis and the forms of organic P present. The relationship between bicarbonate-extractable organic P and the diester content of the soils confirms the nature of labile organic P in these soils and explains the poor correfation between labile organic P and phosphomonoesterase activity.
Suntmary--JiP
fractions in surface horizons of Galician Rankers, Cambisols and Podzols differ due to pedogenesis and to get more information about the poorly-known P cycle in acid soils.
INTRODIJCX’ION
Little work has appeared on the relationships between pedogenesis and soil organic compounds. As for soil inorganic P (Pi), comparisons have hitherto been made on the basis of data obtained by chemical fractionation (Tiessen et al., 1984; Sharpley et a&, 1987; Trasar-Cepeda, 1987). However, fractionation of organic P (P,) in NaOH extracts can now easily be performed using 31P spectroscopy (Newman and Tate, 1980), so it seems of interest to test whether, like chemically-identified P fractions, these spectroscopic P fractions also reflect typical alterations during pedogenesis. The generally acid, highly-organic soils of Galicia (N.W. Spain) are dominantly Rankers, Cambisols and Podzols (Guitian, 1967). Their forms of phosphorus have been aiready quantified using the Hedley ef al. (1982) fractionation method. It was found that organic P dominated over Pi, primary P was almost totally lacking, resin-extractable P concentrations were low, and that the P-distribution was similar in all three soil groups (Traser-Cepeda, 1987). In surface samples of Galician soils, incubation under controlled conditions only affects P fractions extracted with NaOH and ultrasound (Trasar-Cepeda et ai., 1986), i.e. by an extraction technique similar to that normally used for analysis of soil P by NMR. In spite of the generally low solubility of P, compounds in the alkaline extract employed in the NMR method (Tate, 1984; Condron et af., 1985). the method can therefore be applied to these soils in the certainty that it will detect the compounds involved in short-term P transformations. In view of this, we have used ,iP NMR spectroscopy to examine whether the NaOH soluble P
MATERIALS AND METHODS
A total of 20 samples of the surface horizons (O-20cm) of soils from Galicia (N.W. Spain) were studied. Most of the soils belonged to one of the three types that are most common in Galicia, Rankers (7 samples), Cambisols (6 samples) and Podzols (5 samples). Samples of Rend&a and a Gleysol were included as representing extreme situations and for purposes of comparison. All 20 soils underlay heather and fume vegetation. Table I lists the chief physical and chemical properties of the samples as determined using methods described by Guitian-Ojea and Carballas (1976). Extracts for “P NMR analysis were obtained using the method of Newman and Tate (1980). For this purpose 6.7 g of finely ground and air-dried soil were dispersed by ultrasound (200 W, 5 min) in 20 ml 0.5 N NaOH, and the suspension was centrifuged (lO,O~ rev min-‘) for 120 min at 0°C. The resulting su~rnatant was concentrated to ca 2ml under a stream of N, at 4O”C, then 1 ml of D,O was added and the final solution was transferred to NMR tubes 1 cm dia. Proton-decoupled ,‘P spectra were recorded at 20°C on a JEOL FX9OQ Multi-nucleus Fourier transform NMR spectrometer with an observation frequency of 36.2 MHz. Chemical shifts in ppm were measured relative to external I&PO,/D,O. The intensity of the signals was determined by integration. 75
F. GIL-Soram et al.
76
Table I. Physical and chemical uroperties of the surface mineral horizons from soils under studv
c
pH CIK
Rankers (7)** Cambisols f6)*’ Podzols (5)” Rendzina (I)**
N (%)
(%)
Clay (%)
Al,O,
Fe,O, (%)*
(%)’
Max.
Min. Mean Max. Min. Mean Max. Min. Mean Max. Min. Mean Max. Min. Mean Max. Min. Mean
6.30 4.52 3.52
3.60 3.76 2.72
Gleysol ( 1i*;
4.23 4.16 3.20 7.50 4.05
15.88 4.08 8.41 4.60 12.70 3.30
8.18 6.24 8.20 5.19 7.60
1.36 0.60 0.73
0.26 0.30 0.13
0.60 0.46 0.43 0.44 0.53
14 24 t7
9 7 2
10 14 8 11 ;5
1.22 1.93 0.55
0.11 0.51 0.10
0.76 1.38 0.30 1.14 2.06
0.60 1.03 0.40
0.10 0.26 0.04
0.38 0.72 0.20 0.07 0.92
*Extracted with 0.1 N pyrophosphate. **No. of samples in each soil type.
Their interpretation is based on literature assignments (Newman and Tate, 1980; Zech et ai., 1985). The extracts were analysed for total P (= P,) by calcination and inorganic P (= Pi) according to Saunders and Williams (1955). Organic P (= P,) was calculated by difference. Total C in the extract was determined according to Guitian-Ojea and Carballas (1976). Total soil P was determined with HF and HCIQ. Bicarbonate-extractable P was measured in all samples according to Olsen et al., (1954) modified by Hedley et al. (1982). In all cases, P was determined calorimetrically using the Molybdenum Blue method of Murphy and Riley (1962). RESULTS
Rankers
In the surface horizons of the Rankers studied, soil P, ranges from 388 to 1505 pg g-’ (mean 678 pgg-I). NaOH and ultrasound treatment extracted a mean 262 pg g-r (Table 2), so that the proportion of P, subjected to 31P NMR analysis was 36%. According to chemical analysis, 74% of the extracted P was of organic and only 26% of inorganic origin. This is in agreement with the “P NMR data (Table 3), which reveal 30% of P extracted to be orrhophosphate and 70% to be organic. The 3iP NMR spectra of these soils generally exhibited high background noise and considerable
overlapping between the monoester and inorganic ~r~hophosphate regions (Fig. 1). The extended spectra show a complex monoester region near 5.6 ppm, generally exhibiting several peaks, perhaps due to the presence of more than one type of monoesterphosphate (Condron er al., 1985). The diester region is equally complex. The average proportion of diester (11%) was less than the average proportion of monoester (45%). Signals due to pyrophosphate and polyphosphates were lacking in all Ranker samples. Phosphonate signals only appeared in the spectrum of a pseudo-alpine Ranker collected at an altitude of 1700 m, in which they made up 17% of total extracted P. The presence of phosphonates in the soi! devefoped under the coldest and wettest climate support the hypothesis of Newman and Tate (1980) that C-P compounds accumulate in cold, wet, acid soils, possibly as the result of low bacterial activity. The spectra of all the Rankers exhibited resonances between 4.5 and 3.0 ppm that may reflect the presence of hydrolysis products of phosphoglycerides like ChoIinephosphate (3.5 ppm) and e~anola~nephosphate (4.2 ppm), or even of simple sugarphosphates (fate and Newman, 1982). Such signals are only absent in the spectrum for a surface horizon from one Ranker developed at sea level and with a pH 6.5. Cambisols
The Cambisoi group was more homogeneous than the Rankers in chemical analysis. Total P contents
Table 2. Total soil P contents and P cantent in the NaOH extracts as determined chemically Total soil P (ggg-‘) Rankers Cambisols Podzols Rendzina Gleysol
Extraction (%)
Total extracted P (Icgg-‘)
Max.
Min.
Mean
Max.
Min.
1505 692 1159
388 396 60
678
764 319 657
102 134 31
591 330*** 697 991
Mean 262 193 189.” 59 408
Max.
Min.
St 70 65
18 24 50
P, in extract (%)+ Mean
Max.
36a** 91 40a 88 56b 75 8 41
Min.
Mean
51 71 55
74ab 82a 67b 86 68
lPercentage of total extracted P. **Means with the same fetter are not si~ifi~ntly different (P = 0.01). ***If one anomalous sample is excluded, the mean total soil P content is 123 pg 8-l and the mean P content of the extract 72 pgg-‘. Table 3. Average P distribution in the NaOH extracts as determined by integration of “P NMR signals Orrhophosphate I%) Rankers Cambisols Podaols Rendzina Gleysol
30a 22a 29a 22 I5
Total P, (%) 70a 78a 7la 78 85
Monoesters (%) 4.5(65)a* 61 (78)b 49 f69)a 26 (33) 58 (68)
Diesters (%)
Others** (%)
1I (16)a’ 9(12)a 19 (27)a 29 (38) 27 (32)
14 (20)a* 8UO)a 3 (4)s 23 (29)
*Values expressed as percentage of P, extracted. **Include phosphonates, polyphosphates and others not identified compounds. Means with the same letter are not significantly different, P = 0.01.
Diester/ monoester 0.25a 0. tbb 0.42~ 1.11 0.46
77
Soil genesis and P, l20
l15
llO
mm
.!i
0
-5
-10
background noise, the monoester and diester regions being simpler and the fact that the Cambisols only occasionally exhibit signals in the regions corresponding to cholinephosphate, ethanolaminephosphate and simple sugarphosphates.
-15
a*
h
54
RANJiL
Podzols
Mean P, content of the Podzol-A horizons was 33Opg g-l. NaOH and ultrasound treatment extracted 56% of P, (Table 2), a significantly higher percentage than for Rankers and Cambisols. The Podzol extracts also differed form those of the latter soil groups in regard to the proportion of P, to Pi (67% to 33% respectively), and the P, extracted made up an average 48% of the total soil P,, an appreciably larger proportion than for Rankers and Cambisols. In their spectra, both the monoester and diester signals are complex with a large number of peaks, probably due to disturbances by paramagnetic substances. In spite of this, it is generally clear that compared with Rankers and Cambisols, the diesterto-monoester ratio rose to 0.42. Almost all the soils of the Podzol group exhibited cholinephosphate and ethanolaminephosphate signals; for one sample (Podzol over quarzites), the strong cholinephosphate signal at 3.2 ppm represents 24% of the P, content of the extract, ca 30 pg g-’ of the whole horizon. Rendzina
NaOH and ultrasound extracted only 8% of P, in the Rendzina Ah (Table 2), low extraction is due to lack of removal of CO,Ca by and before NaOH extraction. Its “P NMR spectrum reveals large proportions of diesters (29%) and unidentified organic phosphates (23%) while monoesters account only 26%. The typical feature of this soil is its high diester-to-monoester ratio of 1.11 (Table 3).
RENDZINA
+20
+15
*lo
.s
pm
0
-5
-10
-15
Glqysol
Fig. 1. Selected )‘P NMR spectra of surface mineral horizons from representative Galician soils.
ranged from ca 400 to 700 pgg-’ with a mean of 591 pg g-l (Table 2), and NaOH extracted 40% of P,. The extracted P was slightly more organic than in the NaOH-extract from the Rankers, 82% being organic and only 18% inorganic. The “P NMR spectra of these soils are very similar to those of the Rankers though with less overlapping between the monoester and orthophosphate regions. Integration again confirms the results from the chemical analysis (Tables 2 and 3). Diesters make up an average of 9% of the extracted P, a slightly smaller figure than for Rankers. The main differences with respect to the Rankers consist in the extended spectra having less
The Ah-horizon of the Gleysol and its NaOHextract had large P, contents, 991 and 408 pgg-’ (= 41%), respectively (Table 2), NaOH extracted about 15% Pi and 85% P,. Like the Gleysols of the Bavarian Alps, the diester peak was remarkably sharp (Zech et al., 1986). The diester-to-monoester ratio was very similar to the average for the Podzols. DISCUSSION
Due to high stabilization (Brannon and Sommers, 1985) P, compounds seem to be poorly extractable with NaOH and ultrasounds. But since the percentages of P, extracted were very similar to the percentages of carbon extracted, the C-to-P, ratios of the whole horizons were nevertheless relatively close
Table 4. Mean C- and P,-values of the NaOH extract and the C/P, ratio in soils and NaOH extracts Soil C extracted (%)
Rankers Cambisols Podzols Rcndzina Gleysol
Soil P. extracted (%)
C/P. in soils
C/P, in extracts
MU.
Min.
Mean
Max.
Min.
Mean
Max.
Min.
Mean
Max.
Min.
Mean
60 52 42
13 29 31
35a 38a 3Sa 18 36
49 44 67
10 23 37
31a 32a 48b 8 39
213
86 121 115
154a ISla 738b 81 106
243 213 759
84 69 92
175a I70a 557b 180 98
Means with the same letter are not significantly different (P = 0.01).
78
F. GIL-S• TRESet ol.
to those of the extracts (Table 4). Only from the Podzols was a significantly higher proportion of P, extracted than C, possibly due either to much of the organic matter being unhumified (GuitianOjea and Carballas, 1968b) or to the dominance of diesters over monoesters. This suggests that for the purposes of studying the distribution of P,, the NaOH-extracted organic matter may be considered as representative of the total organic matter content. It was seen above that in regard to the distribution of P,, the soils of each group have certain common features. These chiefly involve their monoester and diester contents, since the presence of phosphonates and polyphosphates appears to depend largely on microclimatic factors (Newman and Tate, 1982). Comparison of the monoester and diester contents of the various groups suggests certain relationships between the distribution of P, and the evolution of the soils. In Gahcia, Rankers are young soils with a low rate of carbon mineralization, though those developed over granite quickly form insoluble complexes due to the action of the Al oxides derived from rapid weathering of plagioclases by the Galician climate (Carballas, 1982). “P NMR analysis of these soils showed that on average, 65% of the P, extracted was stabilized as monoesters while only 16% was incorporated in diesters (Table 3). The chemical complexity of the pool of monoester and diester components, which is evidenced by the large number of significant peaks in the corresponding spectral regions, shows that the humic substances of these soils have undergone little degradation; this conclusion is supported by the high proportion of uncharacterized P, compounds (almost 20%), which seem likely to be intermediate products of the composition of plant remains. Galician Cambisols are usually Humic Cambisols. occasionally with an allochthonous surface horizon (Guitian-Ojea and Carballas, 1968a). Accordingly. their organic matter is more humidified than in Rankers and is highly stabilized by Al as in Andosols (Carballas, 1982). This is reflected by the distribution of their P contents: the proportion of monoesters is greater than in Rankers, diesters make up only 12% of the organic P extracted (Table 3) and, consequently, the average diester-to-monoester ratio falls from 0.25 in Rankers to 0.16 in Cambisols. The simplification of the P, compounds is likewise reflected by the reduction in the number of peaks in the monoester and diester regions of the NMR spectra and by the almost complete disappearance of intermediate signals. Podzols are developed in Galicia under cold, wet conditions, over quarzites and sandstones that are poor in bases, Fe and Al (Guitian-Ojea and Carballas, 1968b). The agents capable of stabilizing humic substances (clay and extractable Fe and Al oxides) are accordingly extremely scarce in surface horizons of these soils (Table l), also biological activity probably is depressed because the high C-toN ratio of 19 in the A horizons (Table 1). These characteristics explain both the ease with which phosphorus can be extracted from these soils (on average, nearly half the total soil P, was extracted) and the
high diester-to-monoester ratio associated with the accumulation of P, as diesters which made up a third of the P, extracted (Table 3). Although Rendzinas and Gleysols were represented in this study by just one sample each, the spectra obtained were in agreement with the physicochemical environments in which these soils developed. The very small quantity of P, extracted from the Rendzina may reflect the insolubility of calcium phytates in alkaline media, which should explain the joint dominance of the diesters and uncharacterized P compounds over monoesters in the extract. The particular Rendzina studied is biologically very active (Acea, 1985) but its high content of available P, may be possibly inhibit enzymatic hydrolysis of P, compounds (Tate, 1984) and so favour their accumulation in the soil. The proportion of P, extracted from the Gleysol was similar to that obtained from Rankers, Cambisols and Podzols. The hydromorphous nature of this soil results in the loss of P, (possibly through the solubilization of insoluble iron phosphates in a reducing medium) and the reduction of biological activity during water saturation, which leads to the persistence of diesters and a diester-tomonoester ratio slightly greater than the average for Podzols. It is quite generally agreed that bicarbonateextractable P, is the most rapidly mineralizable fraction of the total P, content of the soil (Harrison, 1982; Tiessen et al., 1984). Various experiments (Bowman and Cole. 1978; Tiessen and Stewart, 1983; Tate, 1984) suggest that bicarbonate-extractable P, mainly consists of diesters, though it has also been simply attributed a non-phytate nature (Condron et al., 1985; Jones and Blomfield, 1982; Brannon and Sommers, 1985). For the soils studied here. Figure 2 shows the correlation between the bicarbonateextractable P, and the diester and non-monoester contents of the NaOH extracts. Though the correlation is high in both cases, the slope of almost unity for the diesters suggests that these are perhaps the basis source. The correlation is in either case important for the P cycle in these soils, since it means that the mineralization of this forms of P, depends
. ,*, , , , , , , , , , , , , , 0
100 Extract-PO
200
?I00
(+g g-l)
Fig. 2. Correlation between bicarbonate P,, and extractable P, (0, diester P, full line y =0.99 x + 13.2; *, total P, minus monoester P. broken line Y = 0.52 x + 14.01.
Soil gem :sis and P,
more on phosphodiesterases than of phosphomonoesterases. The rapid stabilization of monoesters in acid media and the inability of phophomonoesterases to dephosphorilate them once stabilized (McGill and Cole, 1981) would explain the poor correlation found by Trasar-Cepeda and Gil-Sotres (1987) between bicarbonate-extractable P, and soil phosphatase activity. Acknowledgements-This work was supported by a research grant awarded to F. Gil-Sotres by the Ministerio de Educacion y Ciencia of Spain and by the Deutsche Forschungsgemeinschaft, F.R.G. REFERENCES
Acea Ma. J. (1985) Esrudio de la poblacicin microbiana en melos de la zona humeda (Universidad de Santiago, Ed.). Santiago de Compostela, Spain. Bowman R. A. and Cole C. V. (1978) An exploratory method for fractionation of organic phosphorus from grassland soils. Soil Science 125, 95-101. Brannon Ch. A. and Sommers L. E. (1985) Stability and mineralization of organic phosphorus incorporated into model humic polymers. Soil Biology & Biochemistry 17, 221-227. Carballas M. (1982) Estudio de la genesis de1 Ranker Atlrinfico (Universidad de Santiago, Ed.). Santiago de Compostela, Spain. Condron L. M., Goh K. M. and Newman R. H. (1985) Nature and distribution of soil phosphorus as revealed by a sequential extraction method followed by a 3 lP nuclear magnetic resonance analysis. Journal of Soil Science 36, 199-207.
Guitian F. (1967) Suelos de la zona humeda espaftola. I. Tipos principales y sus relaciones geneticas. Anales de Edafologia y Agrobiologia 26, 1369-I 378. Guitian-Ojea F. and Carballas T. (1968a) Suelos de la zona humeda espafiola. III Ranker Atlantico. Anales de Edafologia y Agrobiologia 27, 57-73.
Guitian-Ojea F. and Carballas T. (1968b) Suelos de la zona hirmeda espatiola. IV Podzoles. Anales de Edafologia y Agrobiologia 27, 741-78 1. Guitian-Ojea F. and Carballas T. (1976) Tecnicas de analisis dp suelos (Pica sacro, Ed.). Santiago de Compostela, Spain. Harrison A. F. (1982) Labile organic phosphorus mineralization in relationship to soil properties. Soil Biology & Biochemistry 14, 343-35 1. Hedley M. J., Stewart J. W. B. and Chauhan B. S. (1982) Changes in inorganic and organic soil phosphorus fractions induced by cultivation practices and by laboratory incubations. Soil Science Society of America Journal 46, 970-976.
79
Jones 0. L. and Blomfield S. M. (1982) Macromolecular organic phosphorus in decomposing plants and in pasture soils. Soil Bioloav & Biochemistrv 14. 145-151. McGill W. B. and-Cole C. V. (1981) Comparative aspects of cycling of organic C, N, S and P though soil organic matter. Geoderma 26, 287-309. Murphy J. and Riley J. P. (1962) A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27,31-36. Newman R. H. and Tate K. R. (1980) Soil nhosnhorus characterization by 31P nuclear magnetic resonance. Communications in Soil Science and Plant Analysis 11, 835-842.
Olsen S. R., Cole C. V., Watanabe F. S. and Dean L. A. (1954) Estimation of available phosphorus in soils by extraction with sodium bicarbonate. USDA Circular 939.
Saunders W. H. H. and Williams E. G. (1955) Observations on the determination of available phosphorus in soils. Journal of Soil Science 6. 254-267.
Sharpley A. N., Tiessen H. and Cole C. V. (1987) Soil phosphorus forms extracted by soils tests as a function of pedogenesis. Soil Science Society of America Journal 51, 362-365. Tate K. R. (1984) Biological Transformation of P in soil. Plant and Soil 76, 245-256.
Tate K. R. and Newman R. H. (1982) Phosphorus fractions of a chnosequence of soils in New Zealand tussock grassland. Soil Biology & Biochemistry 14, 191-196. Tiessen H. and Stewart J. W. B. (1983) The Biogeochemistry of Soil Phosphorus. In Planefury Ecology (D. E. Caldwell, J. A. Brieley and C. L. Brieley, Eds), pp. 463-472. Van Nostrand Reinhold. Tiessen H., Stewart J. W. B. and Cole C. V. (1984) Pathways of phosphorus transformations in soils of differing pedogenesis. Soil Science Society of America Journal 48, 854-858.
M’. C. (1987) Estudio de la variucion y contenido del P organico en suelos gallegos (Universidad
Trasar-Cepeda
de Santiago, Ed.).Santiago de Compostela, Spain. Trasar-Ceneda Ma. C.. Gil-Sotres F. and Guitian-Oiea F. (1986) Transformaciones del fosforo en suelos gallegos sometidos a incubation. Anales de Edafologia y Agrobiologia 45, 3 I l-326.
Trasar-Cepeda M’. C. and Gil-Sotres F. (1987) Phosphatase activity in acid high organic matter soils in Galicia (NW Spainj. Soil Biology &Biochemistry 19, 281-287. Zech W.. Alt H. G.. Zucker A. and Koeel I. (1985) 31P NMR spectroscopic investigations of-NaOH extracts from soils with different land use in Yucatan (Mexico). Zeirschrifi Pflanzenerdhr. Bodenk. 148, 626-632. Zech W., Alt H. G., Haumaier L. and Blasek R. (1986) Characterization of phosphorus fractions in mountain soils of the Bavarian Alps by 31P N.M.R. spectroscopy. Zeitschrift Pflanzenerniihr. Bodenk. 150, 119-123.