Use of sonication to determine the size distributions of soil particles and organic matter

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1 Use of sonication to determine the size distributions of soil particles and organic matter X. M. Yang, C. F. Drury, W. D. Reynolds, and D. C. MacTavish Greenhouse and Processing Crops Research Center, Agriculture and Agri-Food Canada, Harrow, Ontario, Canada NR 1G ( Received 29 September 8, accepted 6 June 9. Can. J. Soil. Sci. Downloaded from by on 1/4/18 Yang, X. M., Drury, C. F., Reynolds, W. D. and MacTavish, D. C. 9. Use of sonication to determine the size distributions of soil particles and organic matter. Can. J. Soil Sci. 89: Applying ultrasound energy to soil-water suspensions (sonication) is an established method of determining the size distributions of soil primary mineral particles and associated organic matter. The size distributions may vary, however, with sonication input energy and soil type. The objective of this study was to determine the effects of sonication input energy on the size distributions of soil mineral particles and organic matter for a range of soil textures and carbon contents typical of agricultural soils in southwestern Ontario. The soils included a Brookston clay loam, a Brookston clay, a Huron silt loam, a Perth silt loam and a Harrow sandy loam. All soils were under no-tillage management. Nine sonication energies ranging from 5 to 15 J ml 1 were applied to soil-water suspensions (1:4 mass ratio), and the soil particle size distribution results were compared with those obtained using the standard chemical dispersion (pipette) method. The three medium- and coarse-textured soils (Huron, Perth, Harrow) required about 25 J ml 1 for complete dissociation of soil aggregates, while the two fine-textured soils (Brookston) required sonication energies of 675 J ml 1. Increasing sonication energy increased the amount of soil organic carbon (SOC) measured in the clay-size fraction and decreased the amounts in the sand and silt fractions. Therefore, accurate determinations of particle size distribution and SOC contents require an initial assessment of the amount of sonication energy required for the complete dispersion of the particle size fractions. For the Brookston clay loam and Brookston clay soils, 452% less particulate SOC was found in the sand fraction at 75 J ml 1 sonication energy than that obtained using the standard pipette method, indicating particle size reduction by sonication of particle organic matter. It should be noted that the sand-size SOC typically represents a small fraction. Furthermore, sonication had a minor effect on the SOC content of the clay fraction. It was concluded that sonication is a viable technique for determining the size distribution of soil primary mineral particles, as well as the amount of SOC associated with the silt and clay fractions. Key words: Sonication, ultrasound energy, particle size distribution, organic carbon fractionation, clay soil Yang, X. M., Drury, C. F., Reynolds, W. D. et MacTavish, D. C. 9. Recours a` la sonication pour de terminer la granulome trie des particules du sol et la quantite de matie` re organique. Can. J. Soil Sci. 89: L étude des suspensions sol-eau aux ultrasons (sonication) est une me thode bien établie pour de terminer la re partition granulome trique des principales particules mine rales du sol et de la matie` re organique qui y est associée. Cette répartition peut ne anmoins varier avec la puissance des ultrasons et la nature du sol. L e tude devait pre ciser quels effets la puissance des ultrasons a sur la re partition granulome trique des particules mine rales et de la matie` re organique pour divers sols de texture et a` teneur en carbone typiques des sols agricoles du sud-ouest de l Ontario. Ont ainsi e te examine s un loam argileux Brookston, une argile Brookston, un loam limoneux Huron, un loam limoneux Perth et un loam sablonneux Harrow. Les sols avaient tous e té cultive s sans labours. Neuf puissances de sonication allant de 5 à 1 5 J par millilitre ont été applique es aux suspensions sol-eau (ratio 1:4 selon la masse), puis on a compare la répartition granulométrique obtenue à celle e tablie graˆ ce à la me thode normalise e de dispersion chimique (pipette). Les trois sols a` texture moyenne ou grossière (Huron, Perth, Harrow) doivent recevoir environ 25 J par ml d ultrasons pour qu il y ait dissociation comple` te des agrégats, tandis que les deux sols a` texture fine (Brookston) ne cessitent une sonication de 6 a` 75 J par ml. Quand on augmente la puissance des ultrasons, on accroıˆ t la quantite de carbone organique (CO) quantifiée dans la fraction argileuse et on diminue celle mesure e dans les fractions sableuse et limoneuse. Par conséquent, pour de terminer avec pre cision la re partition granulome trique des particules et la concentration de CO, il faut procéder à une évaluation initiale de la puissance de la sonication requise pour parvenir a` une comple` te dispersion des particules. Dans le loam argileux Brookston et les argiles Brookston, on a de couvert 4 à 52 % moins de CO particulaire dans le sable avec une sonication de 75 J par ml qu avec la technique normalisée de la pipette, signe que les ultrasons re duisent la granulométrie des particules de matie` re organique. Il convient de souligner que le CO de la granulome trie du sable ne constitue habituellement qu une petite fraction. Par ailleurs, la sonication a un léger effet sur la concentration de CO dans la fraction argileuse. Les auteurs en concluent que la sonication est une technique viable pour e tablir la répartition granulome trique des principales particules mine rales du sol ainsi que pour mesurer la quantite de CO associe e au limon et à l argile. Mots clés: Sonication, ultrasons, re partition granulome trique, fractionnement du carbone organique, sol argileux 413 Abbreviations: SOC, soil organic carbon; POM, particulate organic matter; SOM, soil organic matter

2 Can. J. Soil. Sci. Downloaded from by on 1/4/ CANADIAN JOURNAL OF SOIL SCIENCE Soil organic matter (SOM) usually occurs in two forms: (1) chemically/physically bound to primary soil mineral particles in the silt and clay particle size fractions as organo-mineral complexes; and (2) discrete material which is present in the sand-size fraction as particulate organic matter (POM). Particulate organic matter is often measured and characterized to assess the impacts of land management practices on nutrient availability and carbon (C) and nitrogen (N) dynamics (Gregorich et al. 6), whereas organic matter bound to silt and clay is assessed for its ability to store C in soils (Christensen 1996; Hassink 1997). Studies of the nature and distribution of SOM among soil particle sizes (i.e., sand-, silt- and clay-size fractions) requires the dissociation of soil into its primary particles. This is usually accomplished via chemical dispersion or physical techniques (Christensen 1996). Chemical dispersion applies dispersing chemicals (e.g., sodium hexametaphosphate) to dissolve soil aggregates into their primary mineral particles (Christensen 1992), whereas physical dispersion uses ultrasound energy (sonication) to physically disrupt the soil aggregates (Edwards and Bremner 1967; Elliott and Cambardella 1991). Both methods have important advantages and disadvantages. Although chemical dispersion may achieve complete aggregate dispersal and is considered the benchmark against which other methods are validated, the dispersing chemicals can alter SOM chemistry and physiochemical behaviour (Morra et al. 1991; Ladd et al. 1993). Sonication, on the other hand, is considered to maintain the physiochemical integrity of SOM, but the amount of energy required for full aggregate dissociation is still in question as it seems to depend on soil type (Gregorich et al. 1988; Oorts et al. 5; Schmidt et al. 1999a), sonication energy, the soilwater ratio in the sample suspension, and the temperature and gas content of the water (Raine and So 1994). In addition, sonication may alter the original SOM distribution among the mineral particle size fractions, especially the amount of POM associated with the sand fraction (Elliott and Cambardella 1991). Hence, there is a need to determine both the amount of sonication input energy required to fully dissociate soil aggregates and the resulting effects on the distribution of SOM among the mineral particle size fractions. Heavy-textured soils in southwestern Ontario occupy approximately two-thirds of the region s agricultural land base. The productivity of these soils remains substantially below the climatic and crop potential for the region due to poor soil quality, which is related primarily to low SOC and poor physical properties. Therefore, the objectives of this study were:(1) to determine for Brookston soils the minimum sonication energy required for complete dissociation of soil aggregates into their primary mineral particles and determine whether this minimum sonication energy varies with soil texture; (2) to evaluate the impact of sonication on the distribution of SOM among the particle size fractions in Brookston soils; and (3) to evaluate sonication as a technique for isolating the POM fraction from Brookston soils. MATERIALS AND METHODS Soils Two fine-textured soils and three coarse- and mediumtexture soil samples (- to 1-cm depth) were collected from no-tillage fields in southwestern Ontario (Table 1). Eight soil cores were collected at three locations in each field using T samplers, and these were composited. Each composite sample was air dried, visible crop residues and roots were removed and then it was thoroughly mixed and passed through a 2-mm sieve. The soils were maintained and utilized in an air-dried state, but soil masses were determined on an oven-dried (68C) basis to account for texture- and organic matter-induced variations in air-dry soil water content. Sonication Method The apparatus and procedures used in this study were similar to those used by others (Gregorich et al. 1988; Raine and So 1994; Schmidt et al. 1999b). A probe-type sonicator (Cole-Parmer, Montreal, QC) with temperature controller was used. When the sample suspension reached a pre-set temperature (328C), the sonicator probe switched off automatically and did not restart until the suspension temperature dropped to 38C. The sonicator also used a pulse:non-pulse ratio of a 3:1 to prevent excessive localized temperatures in the sample suspension, and the sample was housed in an ice bath to further ensure that the sample temperature was maintained below 328C. The operating time of the probe was monitored so that the actual amount of energy delivered to each sample could be controlled. Since the actual energy output of a sonicator can sometimes differ from the displayed energy output (Schmidt et al. 1999a), the sonicator used in this study was calibrated by measuring temperature changes produced by sonicating a given mass of water for a specified period of time, as described in Oorts et al. (5). There is some evidence that sonicator calibration can be affected by the formation of cavitation bubbles, which may change the amount of energy applied to pure water relative to the amount of energy applied to soilwater suspensions (Crum and Fowlkes 1986). However, Atchley and Crum (1988) found that the concentration of cavitation bubbles tended to be inversely related to the soil-water ratio. Although Edwards and Bremner (1967) noted that the dispersion of the soil could decrease with increasing soil-water ratio, other researchers found that dispersion efficiency was either not affected by soil-water ratio, or increased with increasing soil-water ratio (Hinds and Lowe 198; Raine and So 1994). We used 1:4 ratio of soil mass to water mass, which is comparable to the ratios used by many others (Christensen 1992).

3 YANG ET AL. * SOIL PARTICLES AND ORGANIC MATTER 415 Table 1. Sample descriptions Sample number and soil series name Sample location Cropping Organic C content (g C kg 1 ) Total N content (g N kg 1 ) Sand (g kg 1 ) Texture Silt Clay 1. Brookston clay loam 42813?N 82845?W C-S z Brookston clay 42813?N 82845?W BG Huron silt loam 43887?N 81823?W C-S-WW Harrow sandy loam 428?N 82854?W C-S Perth silt loam 43831?N 8181?W C-S-WW z Cgrain corn, Ssoybean, WWwinter wheat, BGbluegrass sod. Can. J. Soil. Sci. Downloaded from by on 1/4/18 Sonication was performed on a soil suspension of g soil (oven-dry equivalent) and 8 ml distilled water (1:4 mass ratio) in a 25-mL glass beaker and the sonication probe tip was submersed 17 mm into the suspension. Three replicate suspensions were analyzed for each soil sample. The samples were treated with nine energy levels ranging from 5 to 15 J ml 1 soil suspension (Table 2). The dispersed soil suspensions were separated into three size fractions: B.2 mm (clay), B.53 mm (claysilt), and.53 to 2 mm (sand). The sand fraction was separated using distilled water to jet-wash all fine particles in soil suspension through a.53-mm sieve until the elutriant solution exiting the sieve was clear. This typically required between 5 and 7 ml of distilled water. The elutriant (containing silt and clay) was then transferred to a 1-mL sedimentation cylinder, thoroughly suspended by agitation, and a subsample collected by siphoning out a 25 ml aliquot immediately after mixing. The clay fraction (B.2 mm) was collected by pipetting 25 ml of the remaining suspension at the appropriate depth, time and temperature following a standard particle size distribution protocol (Kroetsch and Wang 8). The three size fractions were oven-dried at 68C, weighed, and then stored for future analysis. Table 2. Sample mass recovery at various sonication energies Chemical Dispersion Method To assess the effectiveness of sonication for particle size determinations, the soil samples were also processed using a standardized chemical dispersion method (Kroetsch and Wang 8). Briefly, -g samples (oven-dry equivalent) were weighed into 25 ml jars (I-Chem TM, tall wide-mouth clear type III glass) and 5-mL distilled water was added. Then, organic matter was removed by slowly adding 1 ml of 3% by volume H 2 O 2 to the sample while it was continuously stirred on a hotplate. Complete organic matter removal was judged when the sample became colourless and effervescence stopped; organic-matter-enriched samples often required 1 ml of 3% H 2 O 2 to achieve complete organic matter removal. To complete the dispersion of the aggregates, ml of sodium hexametaphosphate dispersant (37.5 g L 1 concentration) was added to samples and distilled water was added to make the volume up to 15 ml. The samples were then agitated overnight using a rotary-shaker set at 6 rotations per minute. The agitated suspension was poured through a.53-mm sieve to separate out the sand fraction, and the remainder collected in a 1-mL sedimentation cylinder. The siltclay fraction was obtained by subsampling the sedimentation cylinder (immediately after agitation), and the clay fraction was Energy level (J ml 1 ) Brookston clay loam Brookston clay Huron silt loam Harrow sand loam Perth silt loam Mass recovery (%) (.79) z 99.2 (.47) 96.4 (.72) 99. (.13) 98.8 (3.9) (.41) 99.6 (.4) y 98.4 (2.47) 99.6 (1.4) (1.34) 98. (.23) 96.5 (.84) 99.1 (1.97) 99.1 (1.7) (1.81) 99.2 (1.62) 99.3 (1.31) 98.9 (1.79) 98.8 (1.51) (2.1) 98.5 (1.27) (1.23) 98. (.27) 97.2 (1.84) 99.6 (2.94) (.94) 97.6 (.62) 99.3 (.49) 98.1 (2.8) (.9) 98. (.76) (1.47) 99.8 (.38) 98.6 (1.) 1.1 (1.76) mean 98.7 (.81) 98.7 (.81) 97.7 (1.46) 98.7 (.39) 99.3 (.49) z Standard error (n3). y No data.

4 Can. J. Soil. Sci. Downloaded from by on 1/4/ CANADIAN JOURNAL OF SOIL SCIENCE collected by siphoning out an aliquot of the suspension at the appropriate depth and time based on Stokes law (Kroetsch and Wang 8). The summation of particle sample weights collected relative to the total initial weight sample was used to determine sample recovery rates. As a reference for macro-organic matter in the sandsized fraction collected from the samples after sonication, carbon in the POM fraction of two Brookston soils was determined using the method of Christensen (1992) and Gregorich and Ellert (1993). Organic Carbon Determination The carbon contents in both the bulk soil and the sand-, silt- and clay-size fractions were determined for the Brookston soils (Table 1). Total carbon was determined using a LECO CN- analyzer (LECO Corporation, St. Joseph, MI), and water-soluble carbon was obtained using a Shimadzu TOC 55-A carbon analyzer (Shimadzu, Japan). As the soils were slightly acidic and free of mineral carbonates, total carbon was equivalent to SOC. The SOC in the bulk soil and sand- and clay-size fractions were measured directly using the above analyzers, while the SOC in the silt fraction was calculated by mass balance using: C Si (M SiCl )(C SiCl ) (M Cl )(C Cl ) 1 (1) (M Si ) where C Si is the carbon content (g C kg 1 ) in the silt fraction, M SiCl and C SiCl are, respectively, mass% of particles and organic carbon content of the siltclay fraction, M Cl and C Cl are, respectively, the mass% of particles and organic carbon content of the clay fraction, and M Si is the mass% of particles in the silt fraction. RESULTS AND DISCUSSION Sample Recovery The five samples were ultrasonically fractionated into sand (.532 mm), silt (..53 mm) and clay (B.2 mm) using 59 sonication energies (Fig. 1, Table 2). Sample recovery after sonication ranged from 96.4 to 11.1%, with Perth soil producing the greatest mean recovery among energy levels (99.3%), where the Huron soil had the lowest mean recovery (97.7%) (Table 2). These sample recovery rates are similar to those obtained in other sonication studies (Gregorich et al. 1988; Oorts et al. 5), and a little greater than the recovery rates typically obtained using the chemical dispersion method [94. to 96.4% (Gregorich et al. 1988)]. Particle Size Distribution Increasing ultrasonic energy decreased sand and silt content and increased clay content (Fig. 1), which is consistent with other studies (Gregorich et al. 1988; Raine and So 1994; Roscoe et al. ). Assuming that the clay content obtained via chemical dispersion represents complete particle separation, it appears that complete dissociation of aggregates required a sonication energy of 6 to 75 J ml 1 for the fine textured soils (Brookston clay loam and Brookston clay), and about 25 J ml 1 for the medium- and coarse-textured soils (Perth silt loam, Huron silt loam and Harrow sandy loam) (Fig. 1). The amount of energy required to separate the particle size fractions in the coarse-textured soils (:25 J ml 1 ) is similar to the energy levels reported by Oorts et al. (5) for soils with B15 g kg 1 of clay content. However, the sonication energies required for the fine soils (675 J ml 1 ) are somewhat greater than the ]5 J ml 1 found by Gregorich et al. (1988) for a soil type with g kg 1 clay, but much less than the 18 J ml 1 found by De Cesare et al. () to disperse a clay loam soil with 186 gkg 1 clay. Notwithstanding the variability, it is still clear that the sonication energy input required for complete dissociation of aggregates increases with increasing clay content. It should also be noted that application of 15 J ml 1 sonication energy to our samples yielded only marginally greater clay content and slightly less silt content than the 6 to 75 J ml 1 energies (Fig. 1), which is consistent with Gregorich et al. (1988), who concluded that increasing clay content with greater sonication energies was not the result of sonic fragmentation of sand and silt particles. Organic Carbon Distribution Averaged over the Brookston clay and Brookston clay loam, increasing sonication energy from 5 to 15 J ml 1 caused an approximately 7% decrease in SOC concentration in the sand fraction, 1% decrease in the silt fraction, and 3% increase in the clay fraction (Table 3). Hence, increasing sonication energy caused a shift of SOC from the sand- and silt-size fractions to the clay fraction, which is consistent with the results of both Gregorich et al. (1988) and Roscoe et al. (). These changes in SOC distribution correlated well with the changes in particle size distribution up to the optimum sonication input energy (675 J ml 1 ), but then diverged for the greatest input energy (15 J ml 1 ) (Table 3, Fig. 2). This indicates that for sonication energies up to the optimum energy (i.e., 675 JmL 1 ), the observed SOC changes resulted from the progressive dissociation of soil aggregates, but at the greatest input energy some sonic diminution of SOC particles occurred. Particulate Organic Matter The standard protocol for determining the SOC of the particulate organic matter associated with the sand-size fraction (i.e., the POM fraction) includes initial chemical dispersion of aggregates (e.g., mild shaking of bulk soil in a sodium hexametaphosphate solution), and then determining the SOC content of the material retained

5 YANG ET AL. * SOIL PARTICLES AND ORGANIC MATTER Brookston clay loam Can. J. Soil. Sci. Downloaded from by on 1/4/18 Particle size fraction (%) Huron silt loam Perth silt loam Sonication Energy (Joule ml -1 ) on a.53-mm sieve (Christensen 1992; Gregorich and Ellert 1993). The resulting SOC contents were 18.5 g C kg 1 sand for Brookston clay loam and 19.8 g Ckg 1 sand for Brookston clay, which were greater than the sand fraction SOC obtained via sonication (at Brookston clay Harrow sand loam Sand Silt Clay Clay - H 2 O 2 pipette method Fig. 1. The amount of sand-, silt-, and clay-size particles recovered by sonication at various input energies. The dotted lines indicate the amount of clay recovered using the standard chemical dispersion method (Kroetsch and Wang 8). 75 J ml 1 ) by about 61 and 16%, respectively (Fig. 3). Decreased POM in the sand-size fraction of the Brookston clay soil was likely accumulated in the clay-size fraction. The discrepancy likely represents sonic diminution of the POM into silt and clay sizes Table 3. Soil organic carbon (SOC) concentrations associated with the particle size fraction as a function of sonication energy Brookston clay loam Brookston clay Energy level Sand Silt Clay Whole soil z Sand Silt Clay Whole soil z (J ml 1 ) (g C kg 1 ) S.D z Calculated using weight percent particle size fraction (Fig. 1) and the corresponding SOC concentration (Fig. 2).

6 418 CANADIAN JOURNAL OF SOIL SCIENCE 1 8 Sample 1, Brookston clay loam (NT) e d c bc bc b ab ab a (Amelung and Zech 1999; Schmidt et al. 1999b; Oorts et al. 5), and implies as a consequence, that sonication can substantially underestimate the POM content of a soil. Can. J. Soil. Sci. Downloaded from by on 1/4/18 SOC distribution in size fraction (%) a a ab b b b c c c a b c c c de de de e a b bc cd cd cd d d e Sonication Energy (J ml -1 ) C in sand C in silt C in clay Sample 2, Brookston Clay (SOD) c c bc ab ab ab ab a a a a a a a a a a a Fig. 2. The distribution of soil organic carbon among particle size fractions as a function of sonication energy for the Brookston clay loam and Brookston clay. Values in the same size fraction of the same soil followed by the same letter(s) are not significantly different according to LSD test, P5.5. C concentration (g C kg -1 sand) Chem. Dispersion Sonication a Soluble Organic Carbon Content Sonication energy did not exhibit an appreciable or consistent influence on the amounts of water-soluble carbon in Brookston soils (Fig. 4). Note also that the among-energy standard deviations were only 1.32 and 1.14 mg C kg 1 for the Brookston clay loam and Brookston clay, respectively, which are less than.1% of total SOC in the samples. Hence, sonication energy had a negligible effect on the concentration of water soluble organic carbon. CONCLUSIONS Using a standard chemical dispersion method as reference, sonic dissociation of soil aggregates in a 1:4 soilwater suspension increased with increasing sonication energy, and complete dissociation into the sand, silt and clay-size fractions occurred at an energy of 6 to 75 J ml 1 for the fine-textured soils (a Brookston clay loam and a Brookston clay), and at an energy of about 25 J ml 1 for the medium- and coarse-textured soils (Huron, Perth and Harrow). Increasing sonication energy broke up SOC, especially sand-sized POM, and this material was captured in the clay fraction. This had little effect on the SOC content of the clay, however, as the amount of sand-sized SOC was small relative to the clay SOC. Sonication had no appreciable effect on the amount of water-soluble carbon in the soils. It appears that carefully calibrated sonication is a viable technique for determining both soil particle size distribution and the amount of SOC in the clay fraction. The sonic input energy required for full separation of Proportion of POM-C in whole soil SOC (%) Soil 1 Soil 2 Soil 1 Soil Chem. Dispersion Sonication b Fig. 3. Amount (a) and proportion (b) of soil organic carbon in the sand particle size fraction of the Brookston clay loam and Brookston clay obtained by chemical dispersion (Gregorich and Ellert 1993) and sonication at 75 J ml 1 energy input. Soil 1 Brookston clay loam, Soil 2Brookston clay.

7 YANG ET AL. * SOIL PARTICLES AND ORGANIC MATTER 419 Can. J. Soil. Sci. Downloaded from by on 1/4/18 Water soluble carbon (mg C kg -1 ) Brookston clay loam (mean=1.7, S.D.=1.32) Brookston clay (mean=11., S.D.=1.14) Sonication Energy (Joule ml -1 ) Fig. 4. Amount of water-soluble carbon recovered in the Brookston clay loam and the Brookston clay as a function of sonication energy. soil particle fractions varies with soil texture. Sonication underestimates the amount of soil POM relative to chemical dispersion method. Based on our present study and the literature, we believe there is no one universal power setting for all soils, as a setting that is high enough for all soil textures may be excessive for some sandier soils. Hence, it is necessary to pre-determine the optimum sonic energy input required for any given soil type before running the assay on all of the samples. If researchers only want to determine the particle size of soil, then the traditional sodium hexametaphosphate dispersion and sedimentation methods would be preferred, as they do not take as much time to perform the analysis and do not require the more costly equipment (sonicator). However, the sonification method is required when researchers also wish to characterize the organic matter associated with the particle size fractions as the sodium hexametaphosphate dispersing agent may change the quality of soil organic matter (Ladd et al. 1993). ACKNOWLEDGEMENT We are grateful to the AAFC Peer Review Program for funding this research. We also thank Vic Bernyk and Wayne Calder for providing expert technical assistance. Amelung, W. and Zech, W Minimisation of organic matter disruption during particle-size fractionation of grassland epipedons. Geoderma 92:7385. Atchley, A. A. and Crum, L. A Acoustic cavitation and bubble dynamics. Pages 164 in K. S. Suslick, ed. 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