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Communications in Soil Science and Plant Analysis

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Factors Affecting Mehlich III Soil Test Methodologyfor Extractable P

Hamid Shahandeh, Frank M. Hons, Tony L. Provin, John L. Pitt & Jeffrey S.Waskom

To cite this article: Hamid Shahandeh, Frank M. Hons, Tony L. Provin, John L. Pitt& Jeffrey S. Waskom (2017) Factors Affecting Mehlich III Soil Test Methodology forExtractable P, Communications in Soil Science and Plant Analysis, 48:4, 423-438, DOI:10.1080/00103624.2016.1269805

To link to this article: http://dx.doi.org/10.1080/00103624.2016.1269805

Accepted author version posted online: 17Jan 2017.Published online: 17 Jan 2017.

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Factors Affecting Mehlich III Soil Test Methodology for Extractable PHamid Shahandeha, Frank M. Honsa, Tony L. Provina,b, John L. Pitta,b, and Jeffrey S. Waskoma,b

aDepartment of Soil and Crop Sciences, Texas A&M University, College Station, Texas, USA; bTexas A&M AgriLifeExtension Service, College Station, Texas, USA

ABSTRACTAgronomic and environmental testing laboratories in Texas and elsewhere haveadopted Mehlich III (M3) as their official soil test phosphorus (P) methodology.However, M3-P data could be skewed due to non-homogenous soil samples orfailure to follow standard protocol which could influence recommendations orrestrictions. Twelve agricultural soils with a wide range of properties werecollected from across Texas. Exhaustive efforts via multiple methods weremade to prepare homogeneous representative soil samples. The standard M3protocol selected was a 2 g weighed soil sample placed in a 148 ml disposableplastic cup, using a 1:10 soil:M3 solution ratio, shaken on a 200 rpmorbital shakerwith a 2.5 cm throw for exactly 5 min, and filtered through Whatman No. 2 filterpaper. The standard protocol was compared with nine different protocol varia-tions with variables including soil weighing versus scooping, scooping repeat-ability of different technicians, soil sample weight, shaking type, speed and time,different filter papers, and varying soil:extractant ratios. Extent of soilpulverization on M3-P results was also evaluated. Tests were performed in fourreplications for all protocols to assess effects on M3-extractable soil P. Percentrecovery of soil during grinding had no effect on M3-extractable P. Little differ-ence in M3-extractable P was observed between scooping and weighing of 2 gsoil samples. Shaker type had no effect on extractable P in soils with low claycontents, however, increasing shaking speed andusing anorbital shaker resultedin higher extractable P, especially in clayey soils. BothWhatman No. 1 and 2 filterpapers were found suitable for M3-P analyses. Different soil:extractant ratiosresulted in a highly significant influence on the amount of M3-P extracted.However, when ratios were maintained between 1:9 and 1:11, few differencesin extractable P were observed. Using sample weights below 3 g did notsignificantly alter precision or accuracy of results. However, technician variationin scooping of 2 or 5 g soil samples resulted in significant differences in M3-P.

ARTICLE HISTORYReceived 6 September 2016Accepted 8 November 2016

KEYWORDSMehlich III; phosphorus;scooping; shaking time; soil:solution ratio; soil samplesize; soil testing

Introduction

Soil testing is one of the most effective ways of insuring that nutrients are being properly recom-mended at agronomic rates and, thus, surface water bodies and ground water are being protectedthrough this best management practice (BMP) (Sharpley et al. 2006). Both state and federal regulatoryand resource management agencies also rely on agronomic soil testing data as a prerequisite forparticipation in cost-sharing programs, issuance of land use permits, and compliance monitoring(Sharpley et al. 2005). For example, Texas’ initial regulatory requirement for extractable soil phos-phorus (P) from concentrated animal feeding operations (CAFOs) in the early 1990s was a P levelbelow 200 parts per million (ppm) (Keplinger 2001; Texas Commission on Environmental Quality(TCEQ), 2014). However, the regulatory limit of 200 ppm P was applied to all soil test P methodologieseven though different methodologies used by Texas’ and regional soil testing laboratories extractedvarying quantities of P (TCEQ, 2014).

CONTACT Frank M. Hons f-hons@tamu.edu Department of Soil and Crop Sciences, Texas A&M University, MS 2474,College Station, TX 77843-2472, USA.© 2016 Taylor & Francis

COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS2017, VOL. 48, NO. 4, 423–438http://dx.doi.org/10.1080/00103624.2016.1269805

In 1999, TCEQ limited its acceptable P extraction methodology for environmental samples to theMehlich III (M3) method. In January 2004, the Texas A&M AgriLife Extension Service Soil, Water andForage Testing Laboratory (SWFTL) adoptedM3 as its official soil test P method. That same year, TCEQmodified its CAFO rule to limit the soil test P methodology to M3 with P determined by inductivelycoupled plasma optical emission spectrophotometry (ICP-OES Thermo Fisher Scientific, Walman, MA,USA), and the Natural Resources Conservation Service (NRCS) in Texas also drafted rules whichspecified the use of M3 with P analyzed by ICP. As a result, the only recognized soil testing methodfor extractable soil P available to Texas producers participating in NRCS or TCEQ programs is M3 byICP. However, deviation from the standard M3-P protocol may also produce varying results.

In 1984, Mehlich introduced M3 (Mehlich 1984), a modified version of earlier extractants known asMehlich II (Mehlich 1978) and Mehlich I (Mehlich 1973). Mehlich III is a multi-element extractant thatcan be used over a wide range of soil pH to measure macro- and micro-nutrients simultaneously. Manysoil testing laboratories in the eastern, southeastern, andmid-westernUnited States have also adoptedM3as their official agronomic extraction procedure (Eckert andWatson 1996; Elrashidi,Mays, and Lee 2003;Gascho, Gaines, and Plank 1990; Hanlon and Johnson 1984; Lins and Cox 1989; Mylavarapu et al. 2002;Nesse, Grava, and Bloom 1988; Schmisek, Cihacek, and Swenson 1998). More recently, M3 has beenpromoted not only as a reliable agronomic soil test but as an agri-environmental soil test (Khiari et al.2000; Sims et al. 2002; Ziadi and Tran 2008). Sims et al. (2002) recommended M3 as an agri-environ-mental soil P test for the Mid-Atlantic region. Soils with extracted P concentration of ≤50 mg P kg−1 soilwere rated as below optimum, with crop response likely; 51–100mg P kg−1 was optimumwith P responseunlikely; >100mgP kg−1 was assessed as non-limiting to yield with no P recommendation; and >150mgPkg−1 indicated environmental concern where improved Pmanagement should be implemented to reducepotential nonpoint P pollution.

However, tomake reliable P assessments based onM3 as an agri-environmental soil test, the variationin results due to modifications of original protocol for the M3-P procedure should be determined. Wolf,Jones, and Hood (1996) in proficiency testing of soil test methods noted that variability in results may beattributed to: (1) a lack of standardization of test procedures (or standard test procedures not beingfollowed, (2) inherent variability of the test methodology, (3) poor quality control and performance, and(4) errors by laboratory analysts in performing, recording, and/or converting data to appropriate units.The accuracy and precision of M3-extractable soil P within a given laboratory may also be influenced byvariation in sub-sampling for analysis (including both weighing and volumetric methods), shaker type,shaking speed and time, sample size, soil:extractant ratios, type and composition of filter paper used,cleanliness of glassware, calibration standards, and analytical instrument accuracy and performance. SoilP has been shown to be sensitive to several of these sources of variation (Peck 2012).

Modern soil testing laboratories analyze thousands of samples annually, with some exceeding 3million samples. Subsamples of the dry, pulverized soils are either weighed or measured by volume(Gelderman and Mallarino 2012). Since the 1920s, agronomic soil testing laboratories have historicallyrelied on volumetric soil scoops to quantitatively transfer soil into extracting flasks or sample analysiscontainers. Volumetric scooping was proposed by Mehlich (1973) for use with the Mehlich I soil test.Volumetric scooping has the advantage of being rapid, low cost, requires little space and integratesdensity into the sample measurement. It has the potential disadvantage, however, of reduced precisionbetween replicate samples (Peck 2012). Weighing offers the advantage of possible increased precision,but has the disadvantage of requiring more time, greater initial expense for weighing equipment, and alarger work area.

Soil:extractant ratio is another factor that should be considered carefully since it may affect both theextractant concentration and stability, and the amount of re-adsorption of P during extraction (Al-Abbasand Barber 1964; Randal and Grava 1971; Zheng, Simard, and Parent 2003).

These potential differences, as well as alteration of the basic M3 testing protocol by some laboratories,have resulted in regulatory agencies suggesting that multiple M3 testing methods do exist (Miller 2001).The protocols used by agronomic testing laboratories generally have evolved from research at LandGrant Universities, and/or alterations for convenience, speed, economic, and other undocumented

424 H. SHAHANDEH ET AL.

reasons. Regulatory and monitoring agencies especially require assurance that reported data are ofacceptable quality. The overarching challenge is improving reliability and data quality, while minimizingthe impacts of analytical cost on the agricultural and environmental industries.

The primary objectives of this study were to: (1) evaluate intra-laboratory precision of M3-extractable P from <2 mm agronomic soil samples, and (2) determine the effects of several soil,procedural and laboratory factors on M3-extractable soil P.

Materials and methods

Soil collection

Twelve diverse agricultural soils were collected (Table 1). Criteria for selection included: (1) soil texture,(2) geographic location, (3) representativeness of soils in each geographic location, (4) soil pH, and (5)estimated P status of soil. Soil was collected by hand using shovels and was excavated at each site from a2.44 ˟ 2.44 m area to a depth of 0.15 m.

Soil preparation

Soil samples were air dried, and were then initially processed via different methods depending on claycontent and size of air-dried peds. For sandy and friable soil samples, the soil was transferred to a 12 mmscreen where all foreign materials and rocks were removed by hand. Soils with extremely large and hardclods were processed over a 25 ˟ 100mmopening steel grate. Each clodwas inspected for foreignmaterialand rocks prior to being forced through the grate. Following the coarse grate, clayey soils were forcedthrough the 12 mm screen prior to further processing by a custom laboratory DynaCrusher (CustomLaboratory Equipment Inc., Holden, MO, USA). Each soil was then mixed in approximately 350 kgbatches starting with an initial four sub-batches. The mixing was performed in a new 680 kg capacitycement mixer for 30 min for each batch. The individual sub-batches were then split into two and mixed

Table 1. Soil seriesa, vegetation, land use, and some physical and chemical properties of soils used in this study.

Soil numberSeries% Sand

County% Clay Classification pH Vegetation/Land use

1 Orelia San Patricio Typic Argiustolls/Tilled Harvested corn69 23 6.1

2 Lake Charles Liberty Typic Paleuderts/Bahiagrass Pasture40 26 5.5

3 Darco Smith Grossarenic Paleusdults/Bahiagrass Pasture82 4 4.8

4 Hidalgo Hidalgo Typic Calciustolls/Tilled Sugarcane60 28 8.0

5 Amarillo Bailey Aridic Paleustalfs Tilled corn88 6 6.8

6 Pullman Deaf Smith Torrertic Paleustolls/Tilled Harvested wheat46 20 7.6

7 Tillman Wilbarger Vertic Paleustalf/Bermudagrass Pasture26 22 5.5

8 Branyon Williamson Udic Haplusterts/Tilled Harvested wheat32 36 8.1

9 Burleson Williamson Udic Haplusterts Tilled corn23 35 6.4

10 Ships Burleson Chromic Hapluderts/Tilled Harvested corn11 45 8.1

11 Windthorst Erath Udic Paleustalf/Bermudagrass Pasture71 9 8.0

12 Hockley Harris Plinthic Paleudalfs/Bermudagrass Pasture77 9 6.0

aSeries as mapped by the United States Department of Agriculture – National Resources Conservation Service (USDA-NRCS) in eachcounty.

COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 425

with other split sub-batches until a minimum of 24 sub-batch mixings were performed. The mixed soilswere then stored in poly-Supersaks, with approximately 110 L of each soil being transferred to a sealablepoly-container for use during the study. Seven liters of each soil was then removed from each containerand further processed using a plate mill set to 0.5 mm spacing. This further processing was to insure asuniform soil as possible to assess the impact of laboratory analysis methods on M3-P results. The 7 L ofeach soil were then mixed in a new small format mortar mixer and stored in two, 4 L poly containers.

Each of the 12 soils was analyzed for pH (2:1/DI water:soil), extractable P by M-3, and soil textureusing the settling hydrometer method outlined by Day (1965) (Table 1).

Mehlich III protocol

The M3 extractant is composed of 0.2 M acetic acid, −0.25 M ammonium nitrate (NH4NO3), −0.015 Mammonium fluoride (NH4 F), −0. 013 M nitric acid (HNO3), −0.001 M ethylenediaminetetraacetic acid(EDTA) with pH adjusted to 2.5 plus or minus 0.1 pH units (Mehlich 1984). For the purpose of thisstudy, the standard protocol from which all results were compared was a 2 g weighed sample placed in a148 ml disposable plastic cup with 20 ml of M3 extractant, shaken on an orbital shaker with a 2.5 cmthrow at 200 revolutions per minute (rpm) for exactly 5 min (min), and filtered throughWhatman No. 2filter equivalent cellulose filter paper. Extractant P concentrations were determined by a Spectro GenesisICP (SPECTRO Analytical Instruments, Kleve, Germany) operating at 1200 W in radial view. Fourreplications of each soil were used with the standard M3-P methodology as well as samples extractedusing modifications of the M3-P protocol (Table 2). The original M3 procedure (Mehlich 1984) used a2.5 g scooped sample, 1:10 soil:extractant ratio, shaking for 5 min with 4 cm throw at 200 rpm, filtrationthrough medium porosity paper, and colorimetric analysis.

Soil recovery study

Due to texture and soil condition when sampled and the use of a soil crusher like DynaCrusher, somesoils may receive sub-optimal pulverization (Munter 1988). Multiple grab samples of the pulverized soilwere collected fromOrelia, Lake Charles, Pullman, Tillman, Burleson, and Ships soils and were used for asoil recovery impact study. The six soils selected for this portion of the study were dominated by clay andclay loam textures (Table 1). Soil that was initially retained by the sieve was returned to the DynaCrusheruntil 100% recovery was achieved using three additional grindings/recoveries. Easily recoverable soil wasdefined as the soil passing the sieve after initial processing by the DynaCrusher. All remaining stock soilfrom samples selected for the recovery study and that from all other soils were further pulverized usingan AgVise Soil Pulverizer (AgVise Laboratories, Benson, MN, USA) fitted with a shaker sieve maintainedin near new condition.

Table 2. Description of soil test procedures for evaluation of Mehlich III-extractable P in study soils.

Soil test number Soil test protocol Description of soil subtest

1 Scooping vs. weighing Based on 2 g sample2 Shaking speed x and 1.25x (x = 200 epm or rpm)3 Filter paper Whatman No. 1 and No. 2 papers4 Shaker type Inline vs. orbital5 Soil:solution ratio 1:7, 1:8, 1:9, 1:10, 1:11, 1:126 Sample size 1, 2, 3, 4, and 5 g samples7 Shaking time 5, 6, 7, 8, and 9 min8 Scooping repeatability Four technicians using 2 g scoop9 Scooping repeatability Four technicians using 5 g scoop

426 H. SHAHANDEH ET AL.

Scooping versus weighing modification

Most agronomic and environmental soil testing laboratories utilize volumetric sample scoops totransfer known amounts of soil from a bulk pulverized sample to the extracting cup or flask (Peck2012). Common scoop sizes are 1, 2, 5, and 10 g, based on the assumption of a ground soil sampledensity of 1.10 g cm−3 (Peck 2012). This density will change depending on sample pulverizationfineness. Results from 2 g scooped samples were compared with those from 2 g weighed samples(Table 2). A single technician was instructed to scoop each sample, strike the scoop’s metal shaftthree times with a 15 cm metal spatula, and scrape off the excess sample volume by running the flatedge of spatula perpendicular across the top of the scoop. These scoops are designed to provide anopen flat surface, with laboratory technicians forcing the scoop through the soil perpendicular to thesoil surface and twisting the scoop upward. Rapping the scoop multiple times with a striker isintended to compact the sample, and the technician then levels the surface using a simple scrapingsweep across the surface of the scoop (Tucker 1984). Each soil sample was scooped four times. Theweighing protocol involved weighing 2 g of each soil using a two-digit top-loading balance and fourreplicates for each soil. Samples were then extracted and analyzed according to the above-describedstandard M3 protocol.

Shaking type and speed modification

Two shaker types were used along with two speeds for each shaker (Table 2). An inline shaker with2.5 cm throw was set at 200 and 250 evolutions per minute (epm). The use of the inline shaker requiredthe use of 100 ml Erlenmeyer flasks to prevent loss of the extractant/soil mixture from the extractionvessels. An orbital shaker with 2.5 cm throw was also operated at both 200 and 250 rpm. Soil P wasextracted from four replicates of all collected soils using all combinations of shaker type and speed andthe standard M3 procedure.

Filter paper modification

Filter paper retention capacity of fines and its influence on M3 soil test P was evaluated usingWhatman No. 1 and No. 2 filter papers in four replicates with each collected soil (Table 2).Whatman No. 1 filters have a mean retention of 11 µm and larger, while Whatman No. 2 filtersretain 8 µm and larger particles (Whatman 2016). All other parameters remained the same as thestandard M3 protocol.

Soil:solution ratio modification

The soil:solution ratio modification was designed to evaluate the impact of equilibrium betweenextracting solution and soil on the absolute concentrations of P extracted by M3. Four replicates ofeach soil were extracted using six extracting ratios including 1:7, 1:8, 1:9, 1:10, 1:11, and 1:12(Table 2). All other parameters remained the same as the standard M3 protocol.

Sample size modification

The mass of soil sample extracted is normally considered a factor in both precision and accuracy ofanalysis. Five sample weights of soil were studied and included 1, 2, 3, 4, and 5 g while maintaining a1:10 soil:extractant ratio (Table 2). Four replicates of each soil were weighed to within 0.05 g of targetweight. All other parameters for the standard M3 protocol were followed.

COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 427

Shaking time modification

The concentration of P extracted from soil is often related to shaking time, especially when shakingtimes are less than 15 min (Rezaian et al. 1992). Shaking times studied were 5, 6, 7, 8, and 9 min ofshaking using four replicates of each soil (Table 2). All other M3 parameters remained constant.

Technician volumetric and mass repeatability

This portion of the study explored the precision and accuracy associated with both scooping andweighing 2 and 5 g soil samples by four different laboratory technicians (Table 2). Four replicates ofeach soil were used and all other parameters remained constant.

Statistical analyses

The effect of different procedural modifications on M3-P were tested using Proc Means in SAS 9.2(SAS Institute 2008) at P < 0.05. Means of significant effects were separated using Fisher’s protectedleast significant difference (LSD).

Results and discussion

Factors of importance to soil sample preparation

Soil sample recoverySoil P concentrations for the 6 clay and clay loam soils upon initial pulverization and from threeadditional recoveries to approximate 100% are given in Table 3. The percent of total soil recoveredfollowing the first grinding ranged from 54.0% to 74.9% and that following the second grindingranged from 63.7% to 83.3%, while total recovery ranged from 81.8% to 91.6% after the thirdgrinding. By the fourth pulverization, 100% recovery was achieved for the six soils studied. Althoughconsiderable difference was noted in percent soil recovered following the successive pulverizations,no significant differences in extractable P were observed between the various recovered fractionswithin soil series (Table 3). The hypothesis was that if poor soil recovery during pulverizationoccurred, results for extracted P might potentially be altered, with skewing possibly resulting fromdifferences in soil texture (Table 1). Soil test laboratories commonly discard any remaining samplethat does not initially pass the sieve upon first pulverization, with analytical results determined onthe initial recovered soil (Kauffman and Gardner 1976). Standard soil analysis requires that soils bepulverized or crushed to pass a 2.0 mm screen. However, it has been suggested that for better resultsand analytical homogeneity, coarse textured soils (sandy loams, loamy sands) should be pulverizedand screened to pass 1.0 mm, with medium and fine textured soils (loams, silt loams, clay loams,clays) screened to pass 0.8 mm or finer (Gavlak, Horneck, and Miller 2005). Zbíral (2006), however,reported no differences in M3-P between <2 mm and <0.15 mm soil samples.

While our study found no significant difference in extracted P based on soil recovery, thesefindings should not be construed as to suggest that laboratories should not attempt to recover nearly100% of the <2 mm fraction. The North American Proficiency Testing program (NAPT) protocol forsample pulverization was used in this experiment (Miller 2001). The Western States LaboratoryProficiency Testing Program found intra-laboratory precision/error up to 25% on 2 mm pulverizedand mixed samples (TCEQ 2014). As a result, the program recommended finer grinding of samples.Upon grinding to approximately 0.45 mm, the intra-laboratory error decreased to less than 2%.Effect of the degree of soil pulverization on M3-P may need to be examined more closely ifdifferences in soil test results occur (Peck and Soltanpour 1990).

428 H. SHAHANDEH ET AL.

Factors of importance to Mehlich III soil P extraction

The statistical significance of the various protocol modifications (Table 2) influencing M3-extrac-table P were determined (Table 4). The most influential factors were sample size, soil:solution ratio,technician repeatability, especially when using 5 g soil scoops, and shaking time. The least significantinfluence was observed with filter paper retentive capacity followed by shaking speed, shaker type,and scooping versus weighing of soil samples.

Scooping versus weighingFour soils (Orelia, Amarillo, Tillman, and Hockley) exhibited significantly higher M3-P in scoopedversus weighed samples (Table 5). All these soils, except Tillman, had relatively high sand contents(Table 1). Only the Branyon soil, with relatively high clay content, had significantly greater M3-P inweighed versus scooped samples. Burleson and Ships soils that have higher clay contents also tended tohave greater M3-P in weighed samples, but differences were not significant. Across all soils, sample

Table 3. Effect of degree of pulverization on soil recovery and its influence on mean Mehlich III-P concentration in selected soilswith higher clay contents.

Soil, pulverizationSoil recovered(% ± SD)

P concentration(mg P kg−1) Soil, pulverization

Soil recovered(% ±SD)

P concentration(mg P kg−1)

Orelia, 1st 74.9 ± 2.9 72.1aa Lake Charles, 1st 55.9 ± 4.6 13.9a2nd 83.3 ± 4.6 72.1a 2nd 73.3 ± 4.9 13.9a3rd 91.6 ± 4.9 73.6a 3rd 86.6 ± 2.8 13.8a4th 100.0 ± 2.8 72.2a 4th 100.0 ± 0.9 13.9aAverage 72.1 13.9Pullman, 1st 72.8 ± 9.4 655.6a Tillman, 1st 54.0 ± 2.8 37.1a2nd 81.9 ± 23.7 652.4a 2nd 69.3 ± 1.9 37.3a3rd 90.9 ± 4.5 652.4a 3rd 84.7 ± 2.4 34.4a4th 100.0 ± 8.6 645.3a 4th 100.0 ± 0.9 34.3aAverage 651.5 35.7Burleson, 1st 59.1 ± 1.3 70.0a Ships, 1st 56.9 ± 1.9 44.2a2nd 72.7 ± 1.3 69.9a 2nd 63.7 ± 1.9 43.8a3rd 86.4 ± 2.1 71.1a 3rd 81.8 ± 2.6 44.5a4th 100.0 ± 1.3 69.1a 4th 100.0 ± 2.2 44.3aAverage 70.1 44.2

1st indicates initital soil grinding, 2nd is additional grinding of soil retained on the sieve following the first grinding, 3rd is grindingof soil retained on the sieve after the second grinding, and 4th is grinding of soil retained on the sieve following the thirdgrinding.

aMeans followed by the same letter within soil are not significantly different according to Fisher’s protected LSD at P < 0.05%.

Table 4. Significance of Mehlich-III protocol modifications on extractable P for each soil studied.

Soil test protocol

SoilScoop vs.weigh (1)a

Shakingspeed (2)

Filterpaper (3)

Shakertype (4)

Soil:Solutionratio (5)

Samplesize (6)

Shakingtime (7)

Repeat2 g (8)

Repeat5 g (9)

Orelia * ns ns ns * ** * * *Lake Charles ns ns ns ns ** * * * *Darco * ns ns ns * * * * **Hidalgo ns * ns * ** * * * *Amarillo * ns ns ns * * ns * **Pullman ns ns ns ns ns ** * ns **Tillman ns ns ns ns * * ns * *Branyon ns * ns * ** * * ns *Burleson ns * ns * ** * * ns *Ships ns * ns ns *** ** * * nsWindthorst * ns ns ns * * ns * **Hockley * ns ns ns * * * * **

Significant at *P < 0.05, **0.01, or ***0.001, respectively.aNumbers in parentheses refer to the specific soil test protocols in Table 2.

COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 429

scooping resulted in significantly greater M3-P levels, but much of this difference occurred fromAmarillo and Pullman soils that had very high extractable P.

Vaughan (1993) found that soil test P was affected by the type of soil and grinding process and theweight of soil scooped. He explained the difference by the fact that finer soil particles in finer texturedsoils could pack more closely than those in soils with coarser particles. Additionally, the fineness ofsample pulverization may also cause significant differences in the ability of the sample to compact(Soltanpour, Khan, and Schwab 1979). The historic assumption is that the soil density will be approxi-mately 1.25 g cm−3, but this may vary with texture and extent of pulverization.

Soil:solution ratioSoil:solution ratio significantly affectedM3-P for all soils except Pullman (Table 4; Figure 1). Others havealso shown that changing the soil:solution ratio altered extracted P concentrations, since it may affectboth the stability of the extractant and the amount of re-adsorption of P during extraction (Al-Abbas andBarber 1964). These authors reported that the amount of P extracted increased with a widening of thesoil:extractant ratio. Similar results were found in our experiment, especially for more clayey soils(Figure 1). For example, increasing the soil:solution ratio from 1:7 to 1:12 in three more clayey soils,Branyon, Burleson, and Ships, resulted in approximately 55%, 27%, and 71% increases in extractable P,respectively. Coarser soils showed less difference in extractable P with increasing soil:solution ratio.Clayey soils exhibited the lowest extractable P at 1:7 ratio, with P increasing as the ratio widened to 1:10.Extractable P in soils generally did not significantly increase beyond the 1:10 ratio, except for the Shipssoil. Higher clay content in Branyon and Burleson soils may not have influenced extractable P because ofhigher soil P saturation possibly masking the effect of soil texture on the amount of re-adsorption of Pduring extraction (Zheng, Simard, and Parent 2003).

Soil texture has previously been reported as an important soil property influencing extractable P. Forexample, in more clayey soils the highest P desorption occurred at the lowest soil:solution ratio when Pbuffering capacity was measured (Zheng, Simard, and Parent 2003). For the Olsen (Olsen et al. 1954)sodium bicarbonate (NaHCO3) soil test method, the critical extractable P concentration resulting in a Pfertilizer recommendation to improve plant growth is based on soil texture. For sandy, loamy, and claysoil, the critical Olsen P levels are 4, 8, and 12 mg kg−1, respectively (Olsen et al. 1954), while with theammoniumbicarbonate–diethylenetriamine pentaacetic acid (AB-DTPA) test, a narrower soil:extractantratio is recommended for clayey soil (Soltanpour, Khan, and Schwab 1979). Authors have argued that forclayey soils, lower soil:extractant ratios would extract less P because of smaller diffusion coefficients inthese soils, and that widening the soil:solution ratio could at least partly eliminate the soil texturaldiffusion effect (Soltanpour and Delgado 2002; Sorn-Srivichai et al. 1988; Olsen et al. 1954).

Table 5. Mean Mehlich III-extractable P for 2 g weighed versus scooped soil samples.

Soil series Weighed Scooped P soil testing ratinga

mg P kg−1

Orelia 76.0ab (2.7c) 81.3b (1.8) HighLake Charles 10.5a (0.8) 10.5a (0.4) LowDarco 17.4a (0.9) 19.6a (0.7) LowHidalgo 51.8a (1.1) 54.1a (1.3) HighAmarillo 403.7a (15.0) 492.3b (32.1) Very highPullman 706.6a (19.8) 740.3a (32.1) Very highTillman 36.4a (1.4) 37.5b (0.5) ModerateBranyon 86.0b (1.0) 82.8a (1.0) HighBurleson 71.5a (1.4) 69.9a (1.7) HighShips 48.5a (1.30) 46.5a (1.7) ModerateWindthorst 30.2a (0.8) 30.4a (0.2) ModerateHockley 41.2a (1.8) 44.6b (1.3) ModerateMean 131.7a 142.5b

aTexas A&M AgriLife Extension Soil, Water, and Forage Testing Lab soil test P ratings.bMeans within soil followed by the same letter are not significantly different according to Fisher’s protected LSD at P < 0.05.cValues in parentheses indicate one standard deviation.

430 H. SHAHANDEH ET AL.

Two of the clayey soils used in our study were also calcareous (Branyon and Ships). Randal andGrava (1971) found that with calcareous soils, extractable P could be greatly increased by wideningthe soil:solution ratio and that at lower ratios, there was a significant and inverse relationshipbetween the amount of P extracted and CEC. These authors suggested that in calcareous soilswith large amounts of extractable P, wider soil:extractant ratios be used. Mehlich (1978) alsosuggested that to achieve a selective extractability of P in calcareous soils, a wider soil:solutionratio be used. The two calcareous clayey soils in our study showed the greatest proportional increasein extractable P with increasing soil:solution ratio as previously noted.

4(Hidalgo) 8(Branyon) 9(Burleson) 10(Ships)

M3-

P (

mg

P k

g-1)

0

20

40

60

80

Soil Number (Series)

M3-

P (

mg

P k

g-1)

0

20

40

60

80

100

Soil : Solution RatioSandier Soils

More Clayey Soils

1(Orelia) 3(Darco) 11(Windthorst) 12(Hockley)

M3-P Critical-Limit

1:71:81:91:101:111:12

Figure 1. Influence of soil:solution ratio on extractable Mehlich III-P (M3-P) in sandier and more clayey soils studied (bars representone standard error).

COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 431

Some authors have also reported that M3 to some extent is not suitable for calcareous soils becausesoluble P may be precipitated by calcium fluoride (CaF2), a product of the reaction between ammoniumfluoride (NH4F) and calcium carbonate (CaCO3) (Fixen and Grove 1990; Iatrou et al. 2014; Jones 1990;Smillie and Syers 1972). However, Tran et al. (1990) suggested that theM3 extractant was less vigorous indissolving Ca–P compounds and was less affected by carbonates in moderately calcareous soils becauseof its buffering, with M3-P on these soils showing good correlation with Olsen P.

In general, widening soil:solution ratios beyond 1:10 could increase extractable P to levels that mightresult in incorrect conclusions and recommendations. For example, using a 1:12 ratiowould have resulted inM3-P levels that suggested an unlikely crop response to P fertilizer addition for Ships soil, whereas extractedP levels for the 1:10 ratio for this soil would have triggered a recommendation for P addition (Figure 1).

Sample sizeIncreasing sample mass of the extracted sample is traditionally considered an easy method of improvingprecision. While a 2 g sample is the traditional sample size for most laboratories using the M3 extractantfollowed by ICP analysis, some laboratories extract 1 g samples. Laboratories using 1 g samples aregenerally limited to areas dominated by very sandy soils (Peck 2012). In our study for most of the soilsused, M3-P tended to increase as soil weight increased from 1 to 3 g, but then decreased for 4, andespecially 5 g samples, particularly with more clayey soils (Figure 2). The reduction in extracted P for 5 gsamples may be associated with the inability of orbital shakers with a 2.5 cm throw to keep the entiremass of sample in an agitated mode, as we observed a portion of the soils remaining in the bottom of theextraction cups in small wetted clumps. Khiari et al. (1999) reported that extractable M3-P decreasedacross 24 soils as sample weight increased between 1 and 5 g. However, in our experiment, extractable Pgenerally increased for 1–3 g samples, then declined with a further increase in sample weight (Figure 2).

Shaking timeThe influence of shaking time has been reported to have a dramatic influence on the extractability of soilP (Rezaian et al. 1992). In contrast, however, the results of our experiment showed shaking time to be lesssignificant than sample size, soil:solution ratio, and other factors (Table 4). Quantities of M3-P extractedwere somewhat variable between 5 and 7 min, but generally showed either little difference or a decliningtrend with increasing time, especially with coarser soils (Figure 3). With 9 min shaking time, however,M3-P tended to significantly decrease. The probable cause of this decline may be precipitation of Pcompounds in the slurry matrix with increasing time (Kowalenko 2008).

A 5 min shaking time for M3-P has been suggested by a number of researchers (Grava 1975; vanLierop 1988; Rezaian et al. 1992). Van Lierop (1988) showed that NH4F, a 5 min extraction time, and a1:10 soil:extractant ratio were necessary for optimizing the relationship between P concentration andshaking time in acidic soils, but to a lesser extent in alkaline and calcareous soils. Rezaian et al. (1992)conducted an experiment using a shaking time range of 1–29 min at soil solution ratios of 1:8 to1:22 forFlorida soils. These authors showed no optimum shaking time for any soil:solution ratio, but concludedthat a 5 min shaking time and 1:10 soil:solution ratio was an acceptable protocol for Florida soils tominimize analytical time, chemical waste, and costs. Rezaian et al. (1992) assessed M3-P based on theresults of both P and potassium (K) analysis and concluded that no specific combination of soil:solutionratio and shaking time was found that would justify its selection over that of the published values of 1:10and shaking time of 5 min. Tran et al. (1990) also suggested that among acid extractants, M3-P was themost appropriate and economical procedure for different kinds of soil because of its capacity tosimultaneously extract other soil nutrients.

Several recent studies on M3-P (Iatrou et al. 2014; Ostatek-Boczynski and Lee-Steere 2012; Paz-Ferreiro, Vázquez, and De Abreu 2012) also utilized 5 min shaking times. Zbíral (2006), however, used10min. Based on our study, laboratories should not attempt to shake batches of samples greater than canbe fully dispensed, shaken, and filtered in 8 min. Since P reactions will be occurring anytime theextractant is in contact with the soil, accurate, consistent, and expedient sample processing is requiredduring this relatively short time frame.

432 H. SHAHANDEH ET AL.

Shaker type and speedOnlyHidalgo, Branyon, and Burleson soils showed a significant difference in extracted P based on shakertype, with all three exhibiting greater P with orbital versus inline shaking (Table 6). Clay content and pHdid not appear to be controlling factors as clay and pH for these soils ranged from 28% to 45% and 6.4 to8.1, respectively (Table 1). Relative increases in extracted P for orbital versus inline shaking for these soilsranged from 6.5% for the Burleson soil to 17.8% for Branyon. Inline shaking at higher speed extractedless P in two soils and more in another two, while the same comparison with orbital shaking resulted in asimilar result, but with different soils (Table 6). Grava (1975) found that shaker speed significantlyaffected test results depending on type of extraction vessel and shaker used and reported that shaking

4(Hidalgo) 8(Branyon) 9(Burleson) 10(Ships)

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1(Orelia) 3(Darco) 11(Windthorst) 12(Hockley)

M3-P Critical-Limit

Figure 2. Influence of soil sample size on extractable Mehlich III-P (M3-P) in sandier and more clayey soils studied (bars representone standard error).

COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 433

speeds of 160–240 rpm had little effect on amount of P extracted using 50 ml Erlenmeyer flasks, but hadan effect withWheaton bottles. Stirring has sometimes been used in place of shaking for mixing soil withextracting solution, however, Agbooia and Omueti (1980) reported shaking to be a more precisemethod.

Filter paper typeTwo common filter paper specifications used by laboratories includeWhatmanNo. 1 andNo. 2.WhatmanNo. 1 is a coarser paper with higher flow rate than the more traditional Whatman No. 2, thus providingfaster filtration and quicker sample throughput. Concern has been expressed as to the potential for small

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1(Oreila) 3(Darco) 11(Windthorst) 12(Hockley)

4(Hidalgo) 8(Branyon) 9(Burleson) 10(Ships)

M3-P Critical-Limit

Figure 3. Influence of sample shaking time on extractable Mehlich III-P (M3-P) in sandier and more clayey soils studied (barsrepresent one standard error).

434 H. SHAHANDEH ET AL.

particulates passing through the coarser filter and potentially skewing results (Grava et al. 1980). However,we observed no significant differences in M3-P between filter paper types for any soil (Table 4).

Technician repeatabilityWhen consistent soil extraction and analytical procedures are followed, common soil test P methodsshould provide reproducible values (Kleinman et al. 2001). However, we found significant technicianbias for M3-extractable P (Tables 4, 7). Of all factors studied, technician scooping resulted in 16 ofthe possible 24 maximum and minimum M3-P values for the 12 soils studied (Table 7). Technicianscooping of 2 g samples gave 7 out of 12 maximum extracted P values, while technician scooped 5 gsamples gave 9 of the 12 minimum P levels. Technician scooping of 2 g samples consistently resultedin greater M3-P than scooped 5 g samples. Bias in sample scooping may result from a host of factorsincluding: (1) how hard technicians strike the scoop handle, (2) the location where technicians strikethe scoop handle, (3) the force with which the technician pulls the scoop through the soil box, (4) theangle at which the technician pulls the scoop through the soil box, and (5) the angle at which thetechnician scrapes the excess soil from the scoop.

Different technicians scooping 2 or 5 g samples made the most difference in the amount of M3-extractable P among various soil test protocols used in this experiment. For example, for Orelia soil, thestandard M3 protocol for a 2 g weighed sample resulted in 76.0 mg P kg−1 (Table 5), while technicianscooping repeatability for the same soil ranged from 48.9 mg P kg−1 for 5 g scooped samples to 92.8 mg Pkg−1 for 2 g scooped samples, respectively (Table 7). These data suggested that to properly use volumetricsampling (scooping), continuing training may be needed for technicians assigned this task. Techniciandifferences have previously been found to be an important source of volume weight variability (Glenn1983). Therefore, periodic assessment of the performance of individual analysts will likely be requiredthrough use of check or reference “blind” and “double blind” samples (Garfield 1991).

Conclusions

The analysis of potential intra- and inter-laboratory methodological differences for M3-P showed thatnot following a standardized protocol could potentially significantly affect extractable soil P, therebyaltering recommendations for agronomic and environmental samples. However, our results suggestedthatM3 for extractable P, when performed by properly trained technicians following a standard protocol,was a reproducible test for the assessment of soil P. The relative percent recovery of soil during

Table 6. Effect of shaking speed and shaker type on Mehlich III-extractable P.

Inline Inline Orbital Orbital

200 epm 250 epm 200 rpm 250 rpm Inline Orbital

Shaking speed Shaker type

Soil series mg P kg−1

Orelia 80.1aa 82.4a 70.7a 78.4b 70.0ab 69.7aLake Charles 12.1b 9.3a 11.5a 10.5a 10.9a 10.7aDarco 18.4a 17.1a 17.2a 16.3a 16.8a 17.5aHidalgo 49.5a 48.7a 50.5a 55.8b 48.3a 52.1bAmarillo 453.8a 416.0a 397.0a 380.0a 418.5a 408.1aPullman 656.1a 664.3a 704.9b 643.4a 695.9a 695.7aTillman 34.6a 36.1a 39.3b 33.8a 35.6a 34.2aBranyon 76.2a 76.2a 82.7a 82.3a 69.7a 82.1bBurleson 68.2a 76.3b 71.5a 72.1a 67.7a 72.1bShips 34.9a 40.9b 47.3a 48.9a 41.1a 42.3aWindthorst 35.1b 30.2a 30.7a 29.8a 31.3a 29.8aHockley 36.8a 37.4a 39.8a 40.7a 39.6a 41.5a

aMeans within soil and shaker type followed by the same letter are not significantly different by Fisher’s protected LSD at P < 0.05.bOverall means for shaker type within soil followed by the same letter are not significantly different by Fisher’s protected LSD atP < 0.05.

COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 435

pulverization had no effect on M3-extractable P. Both Whatman No. 1 and 2 filter papers were suitablefor M3-P analysis. Shaker type generally had minimal effect on M3-extractable P, while shaking speedexhibited limited and inconsistent effects. Soil:extractant ratio very significantly influenced M3-extrac-table P. However, when ratios were kept between 1:9 and 1:11 (method standard of 1:10), minimaldifferences in extractable P were observed. Soil sample size used for extraction did not significantly alterprecision of P analyses. However, if sample size is increased beyond 4 g, extractable P may decrease asmany orbital laboratory shakers may be unable to adequately maintain a moving slurry of the soil andextractant. Shaking of samples during extraction slightly beyond the 5 min shaking time had limitedinfluence on M3-P. However, significantly extending shaking time to 9 min resulted in reduced Pextraction. Little difference in M3-P was noted between weighed and scooped 2 g samples. However,technician variation in scooping of soil samples resulted in significant differences in M3-P.

Funding

This research was partially funded by a grant from the Texas State Soil and Water Conservation Board.

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Table 7. Method factors resulting in maximum and minimum Mehlich III-P values for the soils studied.

Soil series Maximum Minimum

mg P kg−1 mg P kg−1

Orelia 92.8a (Technician, 2 g scoop) 48.9a (Technician, 5 g scoop)Lake Charles 13.5 (Shaking time, 8 min) 7.8 (Technician, 5 g scoop)Darco 22.5 (Shaking time, 7 min) 13.5 (Technician, 5 g scoop)Hidalgo 58.3 (Technician, 2 g scoop) 36.1 (Technician, 5 g scoop)Amarillo 526.3 (Technician, 2 g scoop) 363.4 (Technician, 5 g scoop)Pullman 756.3 (Technician, 2 g scoop) 530.9 (Technician, 5 g scoop)Tillman 40.5 (Shaking time, 8 min) 24.3 (Technician, 5 g scoop)Branyon 93.5 (Shaking time, 7 min) 50.0 (Soil solution ratio, 1:7)Burleson 78.2 (Technician, 2 g scoop) 50.2 (Soil solution ratio, 1:7)Ships 53.0 (Soil solution ratio, 1:12) 30.2 (Soil solution ratio, 1:7)Windthorst 38.5 (Technician, 2 g scoop) 23.2 (Technician, 5 g scoop)Hockley 46.8 (Technician, 2 g scoop) 31.7 (Technician, 5 g scoop)

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