Analytica Chimica Acta 508 (2004) 201–206

Shortened screening method for phosphorus fractionation in sedimentsA complementary approach to the standards, measurements

and testing harmonised protocol

Patricia Pardo, Gemma Rauret, José Fermın López-Sánchez∗

Departament de Qu´ımica Anal´ıtica, Universitat de Barcelona, Mart´ı i Franquès, 1-11. E-08028 Barcelona, Spain

Received 12 March 2003; received in revised form 2 October 2003; accepted 6 November 2003

Abstract

The SMT protocol, a sediment phosphorus fractionation method harmonised and validated in the frame of the standards, measurementsand testing (SMT) programme (European Commission), establishes five fractions of phosphorus according to their extractability. The deter-mination of phosphate extracted is carried out spectrophotometrically. This protocol has been applied to 11 sediments of different origin andcharacteristics and the phosphorus extracted in each fraction was determined not only by UV-Vis spectrophotometry, but also by inductivelycoupled plasma-atomic emission spectrometry. The use of these two determination techniques allowed the differentiation between phospho-rus that was present in the extracts as soluble reactive phosphorus and as total phosphorus. From the comparison of data obtained with bothdetermination techniques a shortened screening method, for a quick evaluation of the magnitude and importance of the fractions given by theSMT protocol, is proposed and validated using two certified reference materials.© 2003 Elsevier B.V. All rights reserved.

Keywords:Phosphorus; Fractionation; Sediment; Water management; Screening procedure; SMT protocol

1. Introduction

To assess the risk of eutrophication in aquatic systemsit is necessary to know not only the total phosphorus con-tent in the sediments but also its distribution among thedifferent sediment phases. For this purpose, chemical frac-tionation methods using extracting agents have been widelyapplied [1–3]. These methods attempt to differentiate thesediment phosphorus pool in the following fractions: labile,associated to Al, Fe and Mn oxides and hydroxides, asso-ciated to Ca, organic and residual[4]. Several compoundsare included in the organic fraction: sugar phosphates, nu-cleotides, humic and fulvic substances, phosphate esters,phosphonates[5,6]. Although some authors have proposedmethods for the fractionation of organic phosphorus in sed-iments[5,7,8], most of them consider the organic phospho-rus in one single fraction, due to the difficulty of separationand identification of these compounds.

∗ Corresponding author. Tel.:+34-93-4029083; fax:+34-93-4021233.E-mail address:fermin.lopez@u.b.edu (J.F. Lopez-Sanchez).

Most procedures attempt to separate at least three ofthe fractions mentioned above (labile, Al/Fe/Mn-bound,Ca-bound, organic and residual). For this purpose, variousreagents are employed (HCl, NaOH, EDTA, NTA. . . ) anddifferent experimental conditions are applied[9–14]. As aresult, the fractions obtained are operationally defined andthe data depend on the experimental conditions[4,15–17].This leads to a lack of comparability of results betweenlaboratories.

In this context, a project was launched in the frame-work of the standards, measurements and testing (SMT)programme (European Commission) which achieved twogoals: (1) the harmonisation of a protocol for the fraction-ation of sediment phosphorus (SMT protocol) and (2) theproduction of a sediment reference material certified forits extractable phosphorus content according to the SMTprotocol (CRM BCR 684). The development of this projecthas been described in detail[18,19].

The SMT protocol, based on the Williams method[20]and modified by Burrus et al.[21], leads to obtainingfive phosphorus fractions: non-apatite inorganic phospho-rus (NAIP), bound to Al, Fe and Mn oxyhydrates; apatite

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2003.11.005

202 P. Pardo et al. / Analytica Chimica Acta 508 (2004) 201–206

phosphorus (AP), bound to Ca; inorganic phosphorus(IP); organic phosphorus (OP) and total phosphorus (TP).Phosphate determination is carried out by UV-Vis spec-trophotometry, using the molybdenum blue method. So theuse of extraction schemes such as the SMT protocol is agood approach to study phosphorus fractionation in sedi-ments. However, these procedures are resource- and time-consuming and it would be useful to have a method thatallows easy screening of the phosphorus distribution insediments.

In this paper, the SMT protocol is applied to 11 sedimentsof different origin and characteristics. The phosphoruscontent in each fraction was determined by both UV-Visspectrophotometry and inductively coupled plasama-atomicemission spectrometry (ICP-AES). The latter was cho-sen because it offers complementary information to thatobtained spectrophotometrically, since the phosphorus de-termined by UV-Vis is the soluble reactive phosphorus,assimilated to inorganic orthophosphate, and ICP-AESdetermines all the phosphorus present in the solution. Com-parison of data obtained by both techniques allows us topropose a simplified method to evaluate the phosphoruspartitioning when the SMT protocol is applied.

This proposed method consists in performing two of thefive extractions established by the SMT protocol (AP andNAIP steps) and determining the extracted phosphorus byboth UV-Vis and ICP-AES. The three remaining fractions(TP, IP and OP) were then estimated from combining the re-sults obtained in the AP and NAIP steps using the followingrelationships:

TPS = APICP + NAIPICP

IPS = APUV-Vis + NAIPUV-Vis

OPS = TPS − IPS

The application of this methodology to two sediment ref-erence materials (CRM BCR 601 and CRM BCR 684) gavea good approximation to the results obtained when the SMTprotocol was applied.

2. Experimental

2.1. Apparatus

A single-beam Helios Gamma spectrophotometer (Uni-cam) was used for UV-Vis spectrophotometric measure-ments of phosphate.

A Thermo Jarrell Ash Model 25 ICP-AE spectrometerequipped with a vacuum system was used for phosphorusdetermination.

X-ray fluorescence (XRF) measurements to determinewhether the major components were carried out using aPhillips PW-1400 X-ray spectrometer with Rh and Au ex-citation tubes.

A NA 2100 Protein Thermo Quest SA elemental anal-yser equipped with a flash combustion furnace, a Porapakchromatographic column and a thermal conductivity detec-tor was used for organic carbon determination. Samples wereweighed using a Mettler micro-balance and tin capsules.

2.2. Reagents

Standard stock solutions of 1000 and 250 mg P l−1 wereprepared from the anhydrous K2HPO4 (Suprapur®, Merck).Diluted working solutions were prepared daily from these.

The reagents used to prepare the extraction solutions wereof Suprapur® (Merck) or Baker Instra Analyzed® (JT Baker)quality.

The following reagents were used for the spectropho-tometric determination of phosphorus: 96% H2SO4(Suprapur®), K(SbO)C4H8O6·1/2 H2O (puris. Merck),(NH4)6Mo7O24·4H2O and L(+)-ascorbic acid (both p.a.,Merck).

All solutions were prepared using double deionised water(USF Purelab Plus 18.3 M� cm−1 resistivity) and all glass-ware was cleaned three times with double deionised waterafter being soaked in HNO3 (10%, v/v) overnight.

2.3. Samples

Samples from S2 to S34 came from the Joint ResearchCentre (Ispra, Italy). These samples were air-dried andsieved to 90�m. They are lake sediments, except for S20,which is a reservoir sediment. Samples from B3/10/88 toT5/12/85 were collected in the Barcelona area (Spain),air-dried at room temperature, desegregated and sievedto 63�m. They are river sediments. CRM BCR 684 is ariver sediment whose extractable phosphorus contents ap-plying the SMT protocol are certified. CRM BCR 601 isa lake sediment sample, but the phosphorus content is notcertified.

2.4. Procedures

2.4.1. Determination of major componentsThe major components of all samples were determined

by XRF. Organic C was determined by an elemental anal-yser with a thermal conductivity detector (EA/TCD) afteracidic attack of the samples[22]. The aluminium, calcium,iron, phosphorus, silicate and organic carbon contents of thesamples used in this work are shown inTable 1.

2.4.2. SMT protocolThe protocol for obtaining the different phosphorus frac-

tions is shown inFig. 1. In this scheme three leachingprocedures are applied to separate samples. An extraction(16 h) using 1 mol l−1 NaOH is performed to remove theextractable P. The residue of this extraction is extracted(16 h) with 1 mol l−1 HCl (apatite P, associated with car-bonates), and an aliquot of the 1 mol l−1 NaOH extract is

P. Pardo et al. / Analytica Chimica Acta 508 (2004) 201–206 203

Table 1Major components of sediment samples (%)

Sediment Al2O3 CaO Fe2O3 P2O5 SiO2 Corg

S2 6.98 3.34 4.47 0.99 24.34 21.91S10A 9.35 2.21 3.79 0.53 42.61 17.53S20 15.04 0.85 5.76 0.24 58.71 5.11S22 6.04 30.04 2.47 0.13 24.64 2.36S23 12.58 14.01 6.14 0.19 39.26 1.44S24 6.18 27.89 2.32 0.22 28.28 4.23S34 0.17 22.45 30.55 3.44 2.33 0.20B3/10/88 8.68 25.75 3.27 1.19 27.42 4.28B3/12/86 0.66 36.67 15.29 2.03 5.96 0.51B6/12/86 11.04 11.93 4.42 3.35 26.75 15.15T5/12/85 9.39 14.17 9.66 1.16 31.26 11.41

CRM BCR 601[23] 13.93 5.73 7.27 0.88 48.78 5.67CRM BCR 684 13.38 7.00 6.43 0.73 46.09 2.41

treated (16 h) with 3.5 mol l−1 HCl (non-apatite inorganicphosphorus, NAIP, the forms associated with oxides andhydroxides of Fe, Al, or Mn). In a separate sample, an ex-traction (16 h) using 1 mol l−1 HCl is performed to removeinorganic phosphorus. The residue from this extraction isplaced in a porcelain crucible and calcined in a furnace for3 h at 450◦C. After this, the residue is extracted (16 h) againwith 1 mol l−1 HCl to remove the phosphorus associatedwith the organic matter of the sediment. To obtain the totalP, after the calcination of a separate sample (3 h, 450◦C), asimple extraction (16 h) with 3.5 mol l−1 HCl is performed.For all cases, phosphate was determined in the extractsby following the spectrophotometric procedure describedbelow. Detailed experimental conditions are described in[18,19].

Fig. 1. The SMT protocol.

2.4.3. Determination proceduresSpectrophotometric determination of phosphate in all ex-

tracts was carried out using the molybdenum blue methodproposed by Murphy and Riley[24], as described by Watan-abe and Olsen[25]. Measurements were made at 882.0 nm.The calibration method used was external calibration, sinceprevious validation studies[26] demonstrated that matrixmatching was not necessary.

For ICP-AES measurements, (1+ 9) dilution of the ex-tracts was required to minimise matrix effects, mainly fromthe extractant. The calibration method used was external cal-ibration (with matrix matching when necessary), accordingto the results obtained in validation studies[26]. The emis-sion line used for phosphorus was 178.0287 nm.

3. Results and Discussion

Samples were selected in order to obtain a pool of sam-ples representative of different sediment compositions (i.e.calcareous, siliceous and organic rich). The SMT protocolwas applied to the samples and phosphorus was deter-mined in the extracts using both determination techniquesfollowing the procedures previously described. The resultsobtained are summarised inTable 2; they are the mean ofthree independent replicates carried out in different days. Itcan be observed that in some cases the phosphorus contentsdetermined by ICP-AES are higher than those determinedspectrophotometrically. To confirm this a Student’s t-testwas applied, after a previous study of the variances for eachset of results using a F-test (significance level,α = 0.05).The results of the application of the t-test show that for

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Table 2Results (in mg P kg−1) of the application of the SMT protocol to the sediment samples (mean± S.D.; n = 3)

TP IP OP AP NAIP

S2 ICP-AES 4162± 76 3205± 65 1027± 76 540± 69 3282± 166UV-Vis 4242 ± 33 2786± 30 906± 10 509± 15 2231± 23

S10A ICP-AES 2349± 34 1627± 52 678± 39 415± 27 1502± 56UV-Vis 2266 ± 21 1392± 19 611± 12 319± 20 1029± 18

S20 ICP-AES 835± 5 483± 14 283± 14 266± 20 489± 56UV-Vis 938 ± 1 486± 6 306± 6 263± 6 219± 3

S22 ICP-AES 518± 36 419± 53 124± 25 340± 35 120± 36UV-Vis 537 ± 5 380± 11 95± 9 293± 8 93 ± 5

S23 ICP-AES 584± 16 437± 39 136± 10 334± 14 329± 11UV-Vis 662 ± 7 485± 3 139± 2 347± 6 92 ± 1

S24 ICP-AES 692± 40 520± 18 163± 6 358± 25 306±74UV-Vis 766 ± 2 532± 5 159± 4 352± 7 136± 1

S34 ICP-AES 537± 44 404± 14 113± 9 295± 27 67± 9UV-Vis 622 ± 7 449± 1 121± 7 332± 6 104± 2

B3/10/88 ICP-AES 3866± 73 3282± 71 305± 21 2293± 104 1174± 35UV-Vis 3974 ± 45 3383± 215 266± 21 2146± 44 1007±47

B3/12/86 ICP-AES 1980± 146 1757± 71 146± 9 1188± 53 298± 35UV-Vis 1915 ± 51 1483± 33 127± 7 1126± 38 329± 33

B6/12/86 ICP-AES 14169± 214 13156± 39 854± 23 5516± 92 7589± 113UV-Vis 13639± 745 12107± 246 778± 3 4929± 192 6949± 269

T5/12/85 ICP-AES 4798± 69 4152± 89 370± 18 2201± 43 1917± 96UV-Vis 4166 ± 43 3752± 91 332± 8 1980± 47 1657± 11

most of the samples the results obtained by ICP-AES aresignificantly higher than those obtained by UV-Vis, espe-cially for the NAIP, AP and OP fractions. Since UV-Vismainly determines inorganic orthophosphate and ICP-AESdetermines all forms of phosphorus present in the extract,when the ICP-AES value was significantly higher, otherphosphorus forms (mainly organic) were considered tobe present in the extracts. These phosphorous forms wereco-extracted with the inorganic orthophosphate althoughthey were not determined when following the SMT pro-tocol, in which UV-Vis determination is mandatory. This

Table 3Estimated values (in mg kg−1) for total, inorganic and organic fractions according to the proposed screening method

TPS IPS OPS

AP(ICP) + NAIP(ICP)a AP(UV-Vis) + NAIP(UV-Vis)

a TPS − IPSa

S2 3822± 180 (4.7) 2740± 27 (1.0) 1082± 182 (16.8)S10A 1917± 62 (3.2) 1348± 27 (2.0) 569± 68 (11.9)S20 755± 59 (7.9) 482± 7 (1.4) 273± 60 (21.9)S22 460± 50 (11) 386± 9 (2.4) 74± 51 (69.0)S23 663± 18 (2.7) 439± 6 (1.4) 224± 19 (8.4)S24 664± 78 (12) 488± 7 (1.4) 176± 78 (44.6)S34 362± 28 (7.7) 436± 6 (1.4) NAB3/10/88 3467± 110 (3.2) 3153± 64 (2.0) 314± 127 (40.5)B3/12/86 1486± 63 (4.2) 1455± 50 (3.4) NAB6/12/86 13105± 146 (1.1) 11878± 330 (2.8) 1227± 361 (29.4)T5/12/85 4118± 105 (2.5) 3637± 48 (1.3) 481± 116 (24.1)

NA: Calculation not applicable as the TPS is not significantly higher than the IPS estimated value. Values in parentheses are relative standard deviation in %.a Indicates screening procedure.

co-extraction was specially relevant in the NAIP fraction,in which the use of NaOH as extractant can solubilise partof the organic matter, whereas extractions with HCl couldlead to the release of acid-soluble organic forms in the otherfractions. Most of the sediment samples showed significantdifferences between UV-Vis and ICP-AES measurementsin at least one of the inorganic fractions, whereas significantdifferences were also observed in the TP and IP fractionsextracted from samples with a high organic matter content.

Based on the differences observed between the UV-Visand ICP-AES measurements, a rapid screening method to

P. Pardo et al. / Analytica Chimica Acta 508 (2004) 201–206 205

Table 4Application of the screening method to the sediments certified reference materials BCR 601 and BCR 684

Samples RM BCR 601 CRM BCR 684

Applied procedure SMT Screening Certified values SMT Screening

Determination technique UV-Visa UV-Visa ICP-AES UV-Vis UV-Visa UV-Visa ICP-AES

AP 1733± 17 1733± 17 1899± 97 536± 28 498± 20 498± 20 490± 18NAIP 1392± 52 1392± 52 1716± 17 550± 21 579± 19 579± 19 581± 20

TPTPS = AP(ICP) + NAIP(ICP)

b 3833± 18 3615± 98 1373± 35 1337± 13 1071± 26

IPIPS = AP(UV-Vis) + NAIP(UV-Vis)

b 3180± 39 3125± 55 1113± 24 1118± 11 1077± 28

OPOPS = TPS − IPSb 440 ± 21 490 ± 113 209 ± 9 203± 3 N.Ac

Results are given as the mean± S.D., in mg kg−1 (n = 3). Values initalics are those obtained by calculation in the screening method.a Values for both procedures are the same as the screening method uses the AP and NAIP fractions of the SMT protocol.b Expression used for calculation of TP, IP and OP fractions in the screening method.c Direct calculation not applicable as TPS is not significantly higher than the IPS estimated value.

estimate phosphorus partitioning was designed. This short-ened method is based on performing the AP and NAIP ex-tractions according to the SMT method and then measuringthe extracted amounts of phosphorus by both determinationtechniques and, finally, estimating the three remaining frac-tions using the previously defined expresions (seeSection 1).According to the SMT protocol, the sum of AP and NAIPcontents determined spectrophotometrically yields a goodestimation of phosphorus extracted in the IP fraction,whereas the total phosphorus was estimated as the sum of thephosphorus extracted in these fractions but now determinedby ICP-AES. Finally, organic phosphorus was estimated bythe difference between the values for total and inorganicphosphorus previously obtained.Table 3shows the estimatedvalues for TP, IP and OP according to the rapid screeningmethod. The uncertainty of the estimated values was deter-mined by combination of the corresponding variances. Whencomparing the values obtained with the proposed (TPS, IPS

and OPS) and the SMT procedures the following relation-ships were obtained: TPS = 0.969× TP− 171,r = 0.999;IPS = 0.899× IP+15,r = 0.999: OPS = 1.157×OP−16,r = 0.950. It can be observed that the proposed screeningmethod yields data that are quite well correlated with thoseobtained when using the SMT procedure for the TP andIP fractions, whereas for the OP fraction the correlation isslightly poorer. From the slopes of these relationships it canbe inferred that, in general, the TP and IP fractions are un-derestimated (−4 and−10%, respectively) whereas the OPfraction is overestimated (+16%), although the agreementbetween the two sets of data was considered acceptable forscreening purposes. The main drawback of the proposedmethod is that the estimation of the OP fraction is basedon the fact that the ICP-AES data in the AP and NAIPfractions should be significantly higher than those obtainedspectrophotometrically. On the contrary the uncertainty as-sociated with the estimated value for the OP fraction could

be quite important. However, the proposed method gavesatisfactory values for 75% of the samples used in thisstudy.

In order to validate the proposed procedure, the rapidscreening method and the SMT protocol were applied to thesediment certified reference materials BCR 601 and BCR684. The results obtained are summarised inTable 4. As canbe observed, a good estimation of the experimental valueswas obtained for the TP and IP fractions for both referencesamples, although an appropriate estimation for the OPfraction was only possible for BCR 601. For BCR 684, ex-perimental data showed no significant differences betweenICP-AES and UV-Vis for the AP and NAIP fractions andneither of the estimated values showed such differences, sothe estimation of the OP fraction was not possible. On theother hand, it is important to remark that the results ob-tained for sediment BCR 684 showed that the material canbe misused if this reference sample is used to validate theprocedure using ICP-AES as the determination technique.

4. Conclusions

A shortened screening method is proposed to estimatephosphorus partitioning according to SMT protocol. Themethod implies the existence of significant differences be-tween the results obtained from ICP-AES and UV-Vis de-termination of phosphorus in the AP and the NAIP fractionsof the SMT protocol and it was applicable for most (75%) ofthe studied sediment samples. The rapid screening methodwas also applied to two sediment reference materials andthe estimated values obtained showed acceptable agreementwith those obtained by the SMT validated procedure, al-though when more accurate results for the OP fraction areneeded the application of the SMT protocol is mandatory.Moreover, the application of the proposed procedure means

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a reduction of the time of analysis and resources in relationto the SMT procedure. This increases three-fold the samplethroughput that facilitates the application of the procedureon a routine basis in laboratories performing environmentalmonitoring tasks.

Acknowledgements

The authors thank the Joint Research Centre (Ispra,Italy) for supplying some sediment samples and the ServeisCientıfico-Tècnics of the Universitat of Barcelona forthe sample characterisation. The authors also thank CEC(project SMT4-CT96-2087) and DGICYT (project PB95-0844-A) for the financial support to carry out this study.

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