EFFECT OF INCUBATION AND LIMING ON THE PHOSPHORUS FRACTIONS IN SOIL Armi Kaila University of Helsinki, Department of Agricultural Chemistry Received May 24, 1961 There exists in the soil a dynamic equilibrium between the forms of phosphorus. Rathje (12) emphasized the importance of the equilibrium between hydroxylapatite and sesquioxide phosphates which largely depends on the pH of soil. There is also an equilibrium between the organic and inorganic forms of phosphorus controlled e.g. by the activity of microorganisms. Phosphorus added to the soil in fertilizers may disturb these equilibriums, yet, at a slower or faster rate, it will turn over to that form which is most stable under the conditions of the particular soil. The pro- fitable effect of lime on acid soils is partly attributed to the improvement of phospho- rus conditions either through the mineralization of organic phosphorus compounds or through the rendering phosphorus bound by iron and aluminium complexes more available (1, 6, 13). Which of these ways is the more important one in different ca- ses, has not yet been indisputably demonstrated. The laboratory experiments described in this paper were carried out in order to study the changes in the soil phosphorus resulted from a prolonged incubation, and the role lime is playing in these changes. An attempt was also made to trace the fate of fertilizer phosphorus in these incubation experiments. Material and methods The soil samples were collected from the surface inch of two field trials in which superphosphate and hyperphosphate were compared (8). In the trial on a loam soil superphosphate had been annually applied as a surface dressing at the rate of 200 kg/ ha for three years, in the trial on a silt soil this treatment was repeated in four years. The application of 2 000 kg/ha of hyperphosphate occurred in the trial on the loam isoil four years, and in the trial on the silt soil five years before the sampling. Samples from all the four replicate plots were separately incubated and analyzed. 100 g of air-dry and ground soil was weighed into a glass jar, moistened with distil- led water to the field capacity, and incubated at about 20°C for seven months. Before 3 https://www.c-info.fi/en/info/?token=mXa7MNVJZxSrFu4B.EadZ27uzC5rgftZS6KN2YQ.d9Sy9YJKDEy41vVc4KE4Wm1o1hxlz1GYK5RDd5ES4MlqWHJ6J02MtKRaRj23j67dT9DGwLm1az5MyCAYRRCtW0vVDOIr8CJZ3cwEumLmr417C8zIDZGXT-3gk844lGJfltIHnvp3JwAowcGESKc409s7I99N2hZy 186 the moistening, 1 g of calcium carbonate was added to a second set of samples and thoroughly mixed. At the end of the incubation period the samples were air-dried and ground. The pH-values refer to 1:2.5 water suspension. P soluble in 0.03 N NH 4F-0. 025 N HCI was extracted in the ratio of 1:10 by sha- king for one minute. The acetic acid soluble P was determined by using 0.5 N acid in the ratio of 1:10, the shaking period being half an hour. Phosphorus fractionation was performed by the method introduced by Chang and Jackson (3). Instead of neutral NH 4F-solution a slightly alkaline extractant recommended by Fife (5) was used. For the determination of organic phosphorus two methods were employed. In the acid-alkali extraction procedure 2 g of soil was treated with 50 ml of 4 N H2S0 4 in 100 ml Erlenmeyer flask for 18 hours with occasional stirring during the first hour. After filtration and washing with distilled water to give a leachate of 200 ml the filter paper with soil was transferred to a 300 ml Erlenmeyer flask, 200 ml of 0.5 N NaOH was added, and the suspension was left to stand over night with oc- casional stirring during the first two hours. The thoroughly mixed suspension was centrifuged, or poured into a high glass, and on the following day the clear superna- tant extract was analyzed. For the determinationof the inorganic P extracted, equal amounts of the acid extract and the alkaline extract were mixed, and the dark orga- nic matter precipitated removed by filtering or centrifuging. The destruction of or- ganic matter for the determination of total P extracted was performed either by dry or wet combustion. In the former case, 10 ml of both extracts were mixed and evaporated to dryness with 3mlof 2 N NaNOs , ignited at 550°C, and heated on a boiling water bath for at least two hours in 21 ml of 1 N H 2S04 . The wet combustion was performed with the aid of a mixture containing two parts of 70 % perchloric acid and one part of concentrated sulphuric acid. The difference of the totaland inor- ganic P extracted represents the organic P. In the ignition method 2 g of air-dry soil was ignited for 1 hour at 550 °C. Both the ignited residue and a 2 g lot of untreated soil were then extracted with 200 ml of 0.2 N H 2S04 for two hours. The difference between the P extracted from the ig- nited and the unignited soil is taken to represent the organic P of the soil. Since the acid-alkali extraction method probably gives too low results, while the values obtained by the ignition method are likely to be too high, an average of the figures was supposed to be the most reliable estimation of soil organic P. Results The effect of incubation and liming on the reaction of the experimental soils is illustrated by the data inTable 1. Both soils are slightly acid, the loam soil somewhat more acid than the silt soil. A marked drop in the pH-values is caused by incubation, while liming has been able to increase the pH-values so much that even at the end of the incubation the soils are neutral. The P test values, also recorded in Table 1, reveal the effect of phosphate fer- tilizers in the degree that could be expected: the treatment with superphosphate is 187 Table. 1. pH and phosphorus test values in the originaland incubated soil samples Loam soil Silt soil 0 Super Hyper 0 Super Hyper Original 5.7 5.6 5.7 5.9 5.7 5.9 Incubated 5.0 5.0 5.1 5.3 5.1 5.3 Incubated with lime 7.0 7.2 7.2 7.3 7.3 7.3 L.S.D. at 5 % 0.1 0.1 P ppm extracted by 0.03 N NH.F-0.025 N HCI Original 22 62 33 14 39 23 Incubated 36 80 59 24 57 37 incubated with lime 32 65 41 25 51 34 L.S.D. at 5 % 8 9 P ppm extracted by 0.5 N acetic acid Original 8 16 96 12 17 101 Incubated 8 16 85 12 18 99 Incubated with lime 8 20 55 12 24 69 L.S.D. at 5 % 23 29 not apparent in the acetic acid values, while it is evident from the results of the acid fluoride extraction. The latter numbers also show the treatment with hyper- phosphate, although to a far less degree than do the corresponding figures for the acetic acid soluble P. The incubation has increased the acid fluoride soluble P in all the samples, but the treatment with lime has, particularly in the loam soil, reduced this increase. The amount of acetic acid soluble P has not been changed by the in- cubation. The fact that acetic acid has not dissolved more P from the limed samples may be attributed to the neutralizing effect of lime on the acid: the pH of the extract of the limed samples was about 3.5 while that of the unlimed samples kept at about pH 3.1. On the basis of these test values it might be concluded that the incubation has probably somewhat improved the availability of phosphorus in these samples. No positive effect of lime is apparent. The results of the P fractionation give a more thorough picture of the changes brought about by the incubation. The data obtained for the samples from the un- treated plots are reported in Table 2. In both soils the incubation has caused an in- crease in the inorganic P extracted by ammonium fluoride and sodium hydroxide. No changes in the amount of acid soluble P has occurred. The increase in the inor- ganic P extracted is most likely due to the mineralization of organic P, since a signi- ficant decrease in this form is found. The effect of lime is most evident in the reducing of the accumulation of the mineralized P as the alkali-soluble form. Only in the loam soil it has, to some degree, increased the amount of acid-soluble P, in the silt 188 Table 2. P fractions in the original and incubated samples of untreated soil (Expressed as P ppm) Increased by Incubated P fractions Original Incubated ... ~ incubationwith lime incubation . .. lime + lime Loam soil Inorganic P extracted by NH.CI 1 2 3 1* 2** I** NH.F 33 45 45 12** 12* 0 NaOH 95 112 99 17** 4 -13*** H 2SO, 264 264 274 0 10*** 10** Total 393 423 421 30* 28* -2 Organic P 358 332 333 -26*** -25*** 1 Silt soil Inorganic P extracted by NH.CI 3 2 5-1 2** 3** NH 4 F 18 33 38 15** 20** s** NaOH 69 89 75 20** 6** -14*** HjSO, 341 344 346 3 5 2 Total 431 468 464 37** 33** -4 Organic P 307 285 273 -22** -34*** -12 soil a slight increase in the fluoride-soluble fraction may be detected. It is of interest to find out that lime did not exert any effect on the mineralization of organic P in these soils. The P fractions of the soil from the plots treated with superphosphate (Table 3) have undergone changes similar to those found in the untreated samples. Only the effect of lime on the increase of the acid-soluble fraction and the decrease in the al- kali-soluble fraction seems to be more marked than in the untreated soils. In the results obtained for the samples treated with hyperphosphate (Table 4) some new features may be observed. The incubation has brought about in both soils a marked decrease in the acid-soluble P with a corresponding higher increase in the fluoride- and alkali-soluble fractions. Apparently, lime has prevented the turning over of acid-soluble P to the alkali-soluble form, and the organic P minerali- zed is in the limed samples mainly accumulated in the fraction which is extracted by ammonium fluoride or in the acid soluble form. Even in the samples from the plots treated with superphosphate or hyperphos- phate, the effect of lime on the mineralization of organic phosphorus appears to be negligible. In all the cases incubation with lime has increased the low amount of P extracted by the first treatment, or by ammonium chloride. In the loam soil, also incubation without lime has improved the solubility of P in this extractant. The closest estimate of the changes in the fertilizer phosphorus which may be obtained on the basis of this material, is the difference between the corresponding 189 Table 3. P fractions in the original and incubated samples of soil treated with superphosphate (Expressed as P ppm) Increased by Incubated P fractions Original Incubated ... .. incubationwith lime incubation , .. lime + lime Loam soil Inorganic P extracted by NH 4 CI 3 5 8 2** 5* 3* NH.F 91 103 105 12** 14** 2 NaOH 138 157 136 19** - 2 -21** H.2 SO, 262 260 273 -2 11 13** To al 494 525 522 31* 28* - 3 Organic P 370 343 340 -27*** -30*** - 3 Silt soil Inorganic P extracted by NH.CI 4 5 10 1 6** s*** NH 4F 57 73 76 16** 19*** 3 NaOH 110 132 110 22*** 0 -22** HjSO, 349 344 355 - 5* 6 11*** Total 520 554 551 34*** 31*** - 3 Organic P 299 272 271 -27* -28** - 1 Table 4. P fractions in the original and incubated samples of soil treated with hyperphosphate (Expressed as P pmm) Increased by Incubated " P fractions Original Incubated .... incubationwith lime incubation .. limelime -f- Loam soil Inorganic P extracted by NH.CI 2 3 4 1 *** 2* 1 NH,F 54 82 66 28** 12** -16* NaOH 118 139 111 21*** - 7** -28** H,SO, 486 468 505 -18** 19* 37*** Total 660 692 686 32* 26*** - 6 Organic P 388 356 360 -32*** -28** 4 Silt soil Inorganic P extracted by NH.CI 3 4 6 1 3* 2** NH,F 35 54 54 19*** 17** - 2 NaOH 94 119 91 25*** - 3 -28*** HjSO, 538 515 539 -23** 1 24*** Total 670 692 688 22** 18* - 4 Organic P 290 267 269 -23** -21** 2 190 results for the samples from the treated and untreated plots. The tracing of the fate of the applied phosphorus in this way is not theoretically sound, owing to the fact that the samples originated from field trials in which the uptake of phosphorus by plants was not equal from the treated and untreated plots. Yet, in the lack of any- thing better this approach may be employed to get some idea of the distribution of fertilizer phosphorus in these soils. The differences in the various fractions of inorganic phosphorus in the samples treated with superphosphate and in the untreated samples are the following: I' ppm Loam soil Silt soil extracted Original Incubated Incubated Original Incubated Incubated by with lime with lime NH 4 CI 2 3 5 13 5 NH.F 58 58 60 39 40 38 NaOH 43 45 37 41 43 35 H 2 SO, 0 0 0 8 0 9 In the unincubated samples of the loam soil the treatment of superphosphate seems to have increased the fractions soluble in ammonium fluoride and sodium hydroxide. The incubation has not caused any changes in the amount of these frac- tions, and this holds trae also with the incubation with lime, since the decrease in the alkah soluble fraction is not statistically significant. The »superphosphate-phospho- rus» in the silt soil also accumulated in the fluoride- and alkali-soluble fractions, and no changes in these forms can be detected due to the incubation or the incubation with lime. In this soil the incubation seems to have decreased the low amount of acid-soluble phosphorus which is assumed to originate from the treatment with su- perphosphate. Incubation with lime has prevented this decrease. The easily soluble part of »superphosphate- phosphorus» was increased in both soils as a result of the incubation with lime. The corresponding differences in the values for the samples from the plots treated with hyperphosphate and from the untreated plots are the following: P ppm Loam soil Silt soil extracted Original Incubated Incubated Original Incubated Incubated by with lime with lime NH 4 CI 1110 2 1 NH.F 21 37 21 17 21 16 NaOH 23 27 12 25 30 16 H2 SO, 222 204 231 197 171 193 As could be expected, in both soils the hyperphosphate phosphorus is accumu- lated mainly in the acid-soluble fraction. Yet, there seems to be some »fertilizer phosphorus» also in the ammonium fluoride-soluble and the alkali-soluble fractions. Provided, these differences in the phosphorus content of the soil treated with hyper- phosphate and the untreated soil really represent fertilizer phosphorus, this would mean that during the four or five years of contact with the soil in the field, some apa- tite has been disolved. 191 Incubation has brought about a significant decrease in the acid-soluble »fertilizer phosphorus» with an increase in the values for the fluoride-soluble phosphorus. The presence of lime has prevented this reaction, and there also seems to be less »fertilizer phosphorus» in the alkali-soluble fraction. In every case, results of this kind of cal- culation must be treated with caution. Discussion In the present incubation experiments liming did not exert any effect on the mineralization of organic phosphorus: the turning over of organic phosphorus into inorganic forms was equally high in samples incubated with or without lime. One possible reason for this result may be found in the fact that neither of the experimen- tal soils was very acid, the pH values being 5.7 and 5.9 respectively. The incubation increased the acidity to pH 5.0 and 5.3, but incubation with hme could keep the pH values as high as 7.0 and 7.3, respectively. These differences in the reaction and calcium carbonate content of the samples incubated without or with lime were reflected in the changes observed in the frac- tions of inorganic phosphorus. In the unlimed samples the phosphorus released from the organic compounds apparently was accumulated in the fractions extracted by ammonium fluoride and sodium hydroxide, or fractions which are supposed to rep- resent aluminium bound and iron bound phosphorus, respectively. Yet, the former fraction may also contain dicalcium phosphate (9), and the latter one phosphorus disssolved from aluminium complex and adsorbed by iron oxide (2). When the samp- les were incubated with lime, the mineralized phosphorus mainly tended to enhance the ammonium-fluoride soluble fraction. In one case also an increase in the acid- soluble fraction was detected. This holds true both in regard to the samples from untreated plots and from the plots treated with superphosphate. In the samples originating from the plots to which hyperphosphate was applied some years before the sampling, the incubation with its increase in the acidity of the soil resulted in a considerable increase in the ammoniumfluoride and sodium hydro- xide soluble phosphorus accounted not only to the mineralisation of organic phos- phorus but also to a marked decrease in the acid-soluble fraction. This suggests that a part of the apatite of the hyperphosphate was dissolved and turned over to fluoride- and alkali-soluble forms. Liming prevented also in these samples the accumulation of the mineralized phosphorus as the alkali-soluble form, and it kept the acid-soluble fraction from decreasing. An attempt to calculate on the basis of the results obtained the amounts of fertilizer phosphorus in various fractions could not, of course, lead to very reliable results. It seemed that incubation with or without lime did not change the distri- bution of the »superphosphate-phosphorus» which mainly occurred in the fractions extractable by ammonium fluoride or sodium hydroxide. The incubation without lime appears to have reduced the acid-soluble fraction of hyperphosphate phospho- rus and caused some increase in the fluoride-soluble forms. The phosphorus test values obtained by the extraction with 0.03 N NH4F 0.025 N HCI were in accordance with the picture found by the fractionations. If 192 these values are supposed to indicate the availability of phosphorus to plants, it must be concluded that liming did not in any case improve the availability more than the incubation did. The test values for acetic acid soluble phosphorus proved to be ra- ther worthless as the indicator of the phosphorus conditions of the present soil samp- les. The results of these experiments are mostly in accordance with what has been previously published (4, 7, 10, 11). Only the complete failure of liming to increase the mineralization of organic phosphorus was somewhat surprising. Also the appa- rent accumulation of mineralized organic phosphorus into the ammonium-fluoride soluble fraction in the limed soils deserves attention. It may be taken to indicate either that aluminium oxide and hydroxide is able to bind phosphate also in the presence of calcium carbonate, or that this fraction may contain e.g. dicalcium phos- phate. Summary Samples from two field trials were incubated at 20°C for seven months with or without an addition of 1 per cent CaC03 . Both the samples of loam soil and silt soil originated from the surface inch of plots treated with no phosphate, superphosphate or hyperphosphate, resp. It was found that liming did not in any case increase the amount of organic phosphorus mineralized during the incubation. Its effect was observed in the distri- bution of this phosphorus in the various fractions of inorganic phosphorus. In the distinctly acid samples which were incubated without lime the mineralized phospho- rus seemed to accumulate as the ammonium fluoride-soluble and alkali-soluble forms, while in the neutral samples incubated with lime an increase only in the for- mer fraction was detected. When the samples from the hyperphosphate plots were incubated without lime, apparently some apatite of the fertilizer was dissolved and sorbed as the ammonium fluoride soluble or alkali-soluble forms. No decrease in the acid-soluble fraction of these samples incubated with lime did occur. REFERENCES (1) Askinasi, D. L. & Jarussow, S. S. 1930. Kalkung als Faktor der Phosphorsäuremobilisation in Podsolboden. Zeitschr. f. Pflanzenern. Düng. u. Bodenk. A 15: 218 233. (2) Aung Khin & Leeper, G. W. 1960. Modifications in Chang and Jackson’s procedure for fractiona- ting soil phosphorus. Agrochimica IV; 246 254. (3) Chang, S. C. & Jackson, M. L, 1957. Fractionation of soil phosphorus. Soil Sei. 84: 133—144. (4) 1958.Soil phosphorus fractions in some representative soils. J. Soil Sei. 9: 109—119. (5) Fife, C. V. 1959. An evaluation of ammonium fluoride as a selective extractant for aluminum-bound soil phosphate: 11. Preliminary studies on soils. Soil Sei 87: 83 88. (6) Ghani, M. O. & Aleem, S. A. 1942. Effect of liming on the transformation of phosphorus in acid soils. Indian J. Agric. Sei. 12: 873 882. (7) Hsu, P. H. & Jackson, M. L. 1960. Inorganic phosphate transformations by chemical weathering in soils as influenced by pH. Soil Sei. 90: 16 24. 193 (8) Hänninen, P. & Kaila, A. 1960 Field trials on the store dressing with rock phosphate. J. Sei. Agr. Soc. Finland 32: 107-117. (9) Kaila, A. 1961. Fertilizer phosphorus in some Finnish soils. Ibid 33; 131—139. (10) Laverty, J. C. & Mclean. E, O. 1961. Factors affecting yields and uptake of phosphorus by dif- ferent crops: 3. Kinds of phosphate native, applied, and formed. Soil Sei. 91: 166 171 (11) Pratt, P. F. & Shoemaker, H. E. 1955. Acid- and alkali-soluble phosphorus in relation to soil reaction. Ibid 80; 381 389. (12) Rathje, W. 1942. Zur Kenntnis der Phosphate. IV. Mitteilung: Das Phosphat-Gleichgewicht im Boden. Bodenk. u. Pflanzenern. 28; 129 159. (13) Sauerlandt, W. 1936. Untersuchungen über die Salpeterbildung und die Umsetzungen der Phos- phorsäure unter dem Einfluss von Kalkdüngungund dem Kalkgehalt der Böden. Zeitschr. f. Pflanzenern. Düng. u. Bodenk. 45: 129—153. SELOSTUS MUHITUKSEN JA KALKITUKSEN VAIKUTUKSESTA MAAN FOSFORIIN Armi Kaila Yliopiston maanviljelyskemian laitos, Helsinki Tutkimuksessa selostetaan kahden superfosfaatin ja hienofosfaatin vertailukokeen maanäytteiden rauhituksessa saatuja tuloksia. Todettiin, ettei kalkitus kummassakaan maassa lisännyt muhituksessa mineraloituvan orgaanisen fosforin määrää. Sen sijaan se vaikutti tämän fosforin sitoutumismuotoon: kun kalkitsematta muhitetuissa ja selvästi happamissa näytteissä vapautuva fosfori lisäsi sekä ammo- niumfluoridiin että emäkseen liukenevaa fosforia, kalkituissa näytteissä havaittiin vain edellisen frak- tion kasvaneen. Hienofosfaattiruutujen näytteitä muhitettaessa ilman kalkkia liukeni ilmeisesti lannoit- teen apatiittia, joka pidättyi ammoniumfluoridiin ja emäkseen liukenevana fosforina. Kalkituissa näyt- teissä ei havaittu happoonliukenevan fraktion pienentyvän.