Development of a harmonised phosphorus extraction procedure and certication of a sediment reference material

V. Ruban,a J. F. Lopez-Sanchez,b P. Pardo,b G. Rauret,b H. Muntauc and Ph. Quevauviller*d

a

Laboratoire Central des Ponts et Chaussees, Division Eau, BP 19, F-44340 Bouguenais, France b Universitat de Barcelona, Departament de Qumica Analtica, Marti i Franques, 1-11, E 08028 Barcelona, Spain c European Commission, Environment Institute, I-21020 Ispra (VA), Italy d European Commission, Standards, Measurements and Testing Programme, 200 rue de la Loi, B-1049 Brussels, Belgium

Received 13th July 2000, Accepted 25th October 2000 First published as an Advance Article on the web 30th November 2000

A harmonised procedure for the determination of the forms of phosphorus in freshwater sediments, developed in the frame of the European Programme, Standards, Measurements and Testing (SMT) has been used for a certication campaign for a reference material. This operationally dened scheme is a good compromise between method performance and reproducibility. Furthermore, the method is rather simple to implement and could be used by water managers on a routine basis. A homogeneous and stable sediment reference material has been prepared and will be available before mid 2001. The so-called SMT protocol, together with the reference material, are useful tools in the eld of water management, especially at a time when quality assurance is of paramount importance in laboratory analyses. Knowledge of the bioavailable forms of phosphorus is important not only for analysis of sediments but also for sludge and soils. Therefore, the SMT protocol could be extended to these materials.

Introduction
Single or sequential extraction procedures are often used in environmental studies in order to assess the mobility and the bioavailability of a given element.13 The determination of specic chemical species is difcult and often hardly possible. Therefore, determination of broader forms or phases dened by their function can be a reasonable compromise, e.g., ``bioavailable'' forms can give sufcient information to achieve a sound environmental policy.4 The proposed methods are operationally dened, related to specic reagents and procedures, i.e., results are interpreted as being related to a specic phase of the sediment (although sensus stricto they are related solely to a chemical procedure). Regarding phosphorus (P), sequential extraction schemes were rst developed for soils and then extended to sediments.5 Many operationally dened schemes are available, allowing the fractionation of the following forms: exchangeable P;67 the fraction associated with Al, Fe and Mn oxides and hydroxides;8 and the fraction in Ca-bound compounds often referred to as apatite P.913 The lack of uniformity in the procedures used did not allow the results to be compared world-wide or the method to be validated (since the results are operationally dened). There is also considerable interest in the certication of reference materials for environmental analysis. However, the usefulness of a certied reference material (CRM) in the validation of an analytical methodology depends on how well the certied values are established.14 In order to improve this situation, the European Commission through the Standards, Measurements and Testing (SMT) programme has launched a collaborative project (SEPHOS, sequential extraction of phosphorus in freshwater sediment), which aimed to: (i) design a harmonised extraction scheme, (ii) test the selected scheme in interlaboratory studies involving expert European laboratories, and (iii) certify the extractable phosphorus content of a sediment CRM.15 The so-called SMT DOI: 10.1039/b005672n

scheme allows the denition of the following forms: NaOHextractable P (NaOH-P; P bound to Al, Fe, Mn oxides or hydroxides), HCl-extractable P (HCl-P; Ca-bound P), organic P (OP), inorganic P (IP), concentrated-HCl P (conc. HCl-P; total P). This paper presents the results of the certication campaign as well as the homogeneity and stability tests carried out on CRM 684, a freshwater sediment.

Origin and preparation of the CRM

The CRM was collected in the Po River (Italy), a large river inuenced by agriculture and industries, large settlement, and extended rural run-off. The collection site is situated at the lower Po River, close to the city of Gorino. The sediment was collected at a depth of 23 m, by means of an INOX grab sampler. The sample was passed through a 2 mm INOX sieve and the fraction less than 2 mm was collected. The sediment was then air-dried at room temperature. The drying process was completed in a drying oven with circulating air, not exceeding 60 C in order to preserve the organic phosphorus compounds. The dried material was passed through a Retsch jaw crusher set at its smallest opening (1 mm) and ground in a Retsch hammer mill equipped with tungsten carbide blades. The ground material was passed through a 90 mm INOX sieve and the fraction less than 90 mm was collected in a specially designed mixing drum. Table 1 gives the composition of the sample. The material was homogenised for 2 weeks. In order to test the bulk homogeneity, ten sub-samples of about 10 g each were taken from the mixing drum and bottled. From each bottle, a pellet was prepared and analysed for a number of major, minor and trace elements. The interbottle and intrabottle variability were measured, special attention was drawn on total P. The homogeneity was good (see results below). J. Environ. Monit., 2001, 3, 121125 121

This journal is # The Royal Society of Chemistry 2001

Table 1 Composition of CRM 684 Si Mean (%) sa (%) RSDb (%)

a

Al 8.1 0.11 1.36

Ca 5.6 0.06 1.07

Fe 4.8 0.07 1.46

P 0.126 0.002 1.59

Org. C 2.7 0.04 1.48

Table 2 Within- and between-bottle variances resulting from the homogeneity study (between-bottle, n~20; within-bottle, n~10) Within-bottle RSD (%) Between-bottle RSD (%) 1 NaOH-P HCl-P IP OP Conc. HCl-P 2.5 2.7 1.6 1.9 1.5 2.2 2.7 1.6 2.9 2.1 2 4.2 3.8 1.4 2.6 1.6 3 1.7 3.3 1.5 3.1 1.7 4 1.8 1.9 1.0 2.0 2.0

23.7 0.30 1.27

s~standard deviation. bRSD~relative standard deviation.

Analytical techniques
The determination of major and trace elements was carried out by X-ray uorescence spectrometry using a Siemens sequential spectrometer. Organic carbon was determined by applying both C, H, N elemental analysis and Wasthoff combustion analysis. Calibration was performed using CRMs. The SMT protocol is described in the Appendix, further details are given elsewhere.15 This operationally dened scheme comprises ve steps i.e., NaOH-P (#P bound to Al, Fe and Mn oxides and hydroxides), HCl-P (#P associated with Ca), OP, IP, and conc. HCl-P (#total P). In this procedure, NaOH and HCl are used as extractants and an intake of 200 mg of sediment is necessary for the extraction. For each form of P, all laboratories made ve independent replicates on two different days and from two different bottles. All 15 laboratories used spectrophotometry, based on the Murphy and Riley spectrophotometric method16 as the nal detection method for the phosphorus extracted by the SMT procedure. Although the Williams scheme is an operationally dened extraction scheme, biotests were carried out (on Scenedesmus quadricauda) in order to test the bioavailability of NAIP (nonapatite inorganic phosphorus), the fraction bound to iron oxides and which is called NaOH-P in the SMT protocol. Therefore, the SMT protocol can be considered as a valuable tool in the estimation of the available P fraction in a sediment. All the reagents used were of analytical reagent grade. Suprapure KH2PO4 was used to prepare standard solutions and suprapure NaOH and HCl were used for calibration. The glassware and plasticware were soaked in 0.3% HCl and rinsed with de-ionised water. A quality control procedure was applied throughout the different steps from sampling, to preparation and analysis. The data were treated using TEDI software (Tractament Estadistic de Dades Interlaboratori, Statistical Treatment of Intercomparison Data) supplied by the University of Barcelona and designed by A. Padro. This treatment was used to calculate the mean values and the standard deviations of each of the laboratory sets, which were submitted to a series of statistical tests, e.g., the Nalimov test to detect outlying values in the population of results and the Cochran test to detect outlying values in the laboratory variances.

when applying the two-tailed F-test at a signicance level of 0.05 (Table 3) showed no difference between the within- and between-bottle homogeneity variances.

Stability study
The stability of the extractable phosphorus content was tested to determine the suitability of this kind of material as a candidate reference material. Sample bottles were stored at z4, z20 and z40 C during a period of 12 months starting in December 1998 and the extractable phosphorus contents were determined (in six replicates) after 1, 3, 6, and 12 months. Any change in the content of an analyte with time indicates an instability provided that a good long-term analytical reproducibility is obtained. Instability would be detected by comparing the contents of different analytes in samples stored at different temperatures with those stored at a low temperature, at the different stages of analysis. The samples stored at z4 C were used as the references for the samples stored at z20 and z40 C. Table 4 gives the ratios (RT, where T denotes the temperature of storage in C) of the mean values (XT) of six measurements made at both z20 and z40 C, and the mean value (X4) from six determinations made at the same time on samples stored at a temperature of z4 C: RT ~XT =X4 The uncertainty UT has been obtained from the RSD of six measurements made at each temperature: UT ~(RSD2 zRSD4 )1=2 .RT =100 T For ideal stability, the ratios RT should be 1. In practice, however, there are some random variations due to the error on the measurement. As can be seen from Table 4, the ratio RT is close to 1 for all the forms of P extracted ; at z20 C 0.94vRTv1.03 and at z40 C 0.96vRTv1.02. The uncertainty UT is small, generally less than 0.03. The variations of RT with time are slightly higher for HCl-P. Note that the stability of another sample, rich in organic matter, had previously been tested using the SMT protocol and proved satisfactory.18

Homogeneity study
One of the prime conditions for acceptance of a candidate reference material is for it to be homogeneous. The betweenbottle homogeneity of the extractable P content was veried by the application of the extraction procedure on sub-samples taken from 20 bottles selected at random from the total set. The within-bottle homogeneity was assessed by 10 replicate determinations on the well mixed content of one bottle.17 The relative standard deviation (RSD) and the total uncertainty (URSD) are presented in Table 2. Between-bottle RSDs are low, ranging from 1.5% for conc. HCl-P to 2.7% for HClP. For the within-bottle homogeneity, RSDs are also very low. The lowest values are for IP (11.6%), the highest for HCl-P (1.93.8%) and NaOH-P (1.74.2%). RSDs obtained for the between-bottle homogeneity are sometimes lower than those for the within-bottle homogeneity but this is not systematic. Furthermore, the values obtained 122 J. Environ. Monit., 2001, 3, 121125

Table 3 F-test used for the homogeneity study F experimental (F-test for comparison of variances) BW1 NaOH-P HCl-P IP OP Conc. HCl-P 1.303 1.046 1.025 2.204 1.991 BW2 2.769 2.095 1.361 1.782 1.277 BW3 2.113 1.567 1.107 2.570 1.308 BW4 1.887 1.919 2.813 1.042 1.877

Critical values for a two-tailed test (P~0.05): F(19.9)~3.68; F(9.19)~2.88.

Table 4 Stability tests on sediment CRM 684 Fraction NaOH-P Time/month 1 3 6 12 1 3 6 12 1 3 6 12 1 3 6 12 1 3 6 12 R20U20 1.030.02 1.010.01 0.980.02 0.980.01 0.980.03 1.000.02 0.940.02 0.990.01 0.980.01 1.010.01 0.980.01 1.020.01 0.970.03 1.000.03 0.980.01 0.980.02 1.000.02 1.010.01 1.010.01 1.030.02 R40U40 1.010.02 1.000.03 1.000.01 0.940.01 1.010.03 1.020.02 0.970.04 1.010.01 0.970.02 1.010.01 0.990.01 1.010.01 0.980.03 1.000.02 0.960.01 0.930.02 1.000.02 1.000.01 1.010.01 1.000.02

Discussion
As can be seen from Table 3, no difference was detected between the within- and the between-homogeneity variances using an F-test. Therefore, the material was considered to be homogeneous. There was also no signicant difference in the RT values, the ratio being close to 1, which showed the stability of the material both at 20 and 40 C. The CRM is homogeneous and stable and can be used for the certication campaign. Before starting the statistical discussion a technical discussion was carried out to make sure that all laboratories strictly followed the SMT extraction protocol. A critical point that was stressed was the necessary calibration using extracting solutions (or external calibration with cross-check of calibrants in the extracting solutions). All laboratories used colorimetry as the nal method of determination. A wavelength of 880 nm was originally specied in the protocol. However, two laboratories used a wavelength of 700 nm and did not observe any difference with the other results based on a 800 nm wavelength. Although it is recognised that the choice of wavelength (700 or 880 nm) has an effect on the performance characteristics of the colorimetric method, in particular, its sensitivity, it had no detectable effect on the between-laboratory agreement (i.e., no detectable bias was observed for one particular wavelength). Therefore it was decided to accept both wavelengths in the analytical protocol. The temperature of extraction was also discussed. A temperature of 211 C was required in the original protocol. It was pointed out that several laboratories had lower or higher temperatures without their results being affected. Therefore, the revised protocol was made more exible and now stipulates a temperature of 213 C. Regarding the certication campaign, the estimates of the within-laboratory standard deviation (sW) and the betweenlaboratory deviation (sB), as derived from the one-way ANOVA, demonstrated that the sB was not signicant. For reasons of uniformity, it was decided to base the certication on the laboratory means rather than on all individual results. The half-width of the 95% CI of the mean of the data set means was adopted as the uncertainty. For Cochran and Nalimov tests, a value is called an ``outlier'' when the hypothesis that it belongs to the population of results considered can be rejected with a risk of error of 0.01. The criterion was adopted that an outlier of variance would be eliminated only if the standard error of the mean (si/dni) of the set exceeded the standard deviation of the distribution of all laboratory means. The statistical evaluation of the results was carried out in order to ensure that the population of results accepted for

HCl-P

IP

OP

Conc. HCl-P

Certication campaign
The sets of results were submitted to the following statistical tests: (i) a KolmogorovSmirnovLilliefors test, to assess the conformity of the distributions of individual results and of laboratory means to normal distributions; (ii) a Nalimov test, to detect ``outlying'' values in the population of individual results and in the population of laboratory means; (iii) a Barlett test to assess the overall consistency of the variance values obtained by the participating laboratories; (iv) a Cochran test to detect outlying values in the laboratory variances (s2 ); and i (v) a one-way analysis of variance (ANOVA; F-test) to compare and estimate the between- and within-laboratory components of the overall variance of all individual results. A summary of the statistical data, as obtained from computing (HOSTAN software, SMT), is given in Table 5. The sets of results found acceptable on statistical grounds were represented in the form of bar charts in which the length of a bar corresponded to the 95% condence interval of the mean of laboratory means. The certied values were calculated as the arithmetic means of laboratory means (taking into account the number of sets accepted for certication after both statistical and technical scrutiny). This value is featured as a vertical dotted line on the bar graphs; its uncertainty is given by the half-width of the 95% condence interval of the mean of laboratory means. Fig. 1 gives an example of a bar graph for IP.
Table 5 Summary of statistical data for extracts of CRM 684 (mg kg21)a

NaOH-P Number of data sets Number of accepted replicates All data sets compatible two by two? (Scheffe's multiple t-test) Outlying data sets? (Dixon test, Nalimov t-test and Grubbs test) Outlying variances? (Cochran test) Means of means sW sB Between-data s signicant? (Snedecor test) Variances homogeneous s of means Data sets means normally distributed? (KolmogorovSmirnovLilliefors test) 95% CI of the mean of means
a

HCl-P 14 70 Yes No No 536 17 47 No Yes 48 Yes 28

IP 15 75 Yes No No 1113 26 41 No No 43 Yes 24

OP 14 70 Yes No No 209 6 14 No Yes 15 Yes 8

Conc. HCl-P 15 75 Yes No No 1373 27 61 No No 62 Yes 34

12 60 Yes No No 550 17 32 No No 33 Yes 21

CI, condence interval; sW within-laboratory and sB between-laboratory standard deviations, respectively.

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of sediments but also for sludge and soils, the SMT protocol could be extended to these materials. New research will be launched in this direction.

Availability of CRM 684

CRM 684 will be available by the middle of 2001 from the Institute for Reference Materials and Measurements (IRMM), Retieseweg, B-2440 Geel, Belgium (Fax: z32 14 590406; E-mail: bcr.sales@irmm.jrc.be). Further information on other available CRMs can be obtained from the IRMM Website at http://www.irmm.jrc.be/mrm.html.

Acknowledgement
This project was carried out under EC contract no. SMT4CT96-2087 and was co-ordinated by the Laboratoire Central des Ponts et Chausse es. The following laboratories partici pated in the interlaboratory studies: Bundesamt und Forschungszentrum fu r Landwirtschaft, Vienna (Austria); CEMAGREF (France); Geological Survey of Finland, Espoo (Finland); Institut National de Recherche Agronomique, Villenave d'Ornon (France); Joint Research Centre, Environment Institute, Ispra (Italy); Laboratoire Central des Ponts et Chausse es, Bouguenais (France); Macaulay Institute for Land Use Research, Aberdeen (UK); Universitat de Barcelona, Departament de Qu mica Anal tica (Spain); Universidad de Cordoba (Spain); University of Gent (Belgium); Universita t Hamburg (Germany); Univer sidad de Huelva (Spain); University of Lisboa (Portugal); Universite Montpellier I (France); University of Uppsala, Erken laboratory (Sweden); University of Wageningen (The Netherlands).

Fig. 1 CRM 684. An example of a bar graph for IP.

certication had a normal distribution before the 95% CI was calculated. This was true in all cases (KolmogorovSmirnov Lilliefors tests). In addition there were no outlying values (Nalimov). The set of variances was often not homogeneous (three cases of ve), which was due to the different repeatability and reproducibility of the method as applied by the different laboratories. A few laboratories were withdrawn: for NaOH-P, 3 labs; for HCl-P, 1 lab; and for OP, 1 lab. All the labs were accepted for IP and conc. HCl-P.

Certied values
The certied values (unweighted mean of P accepted sets of results) and their uncertainties (half-width of the 95% CIs) are given in Table 6 as mass fractions of the respective extracts obtained at the different steps (in mg kg21 dry mass).

Appendix: SMT protocol

A NaOH-extractable P and HCl-extractable P (1) Weigh 200 mg of dry sediment in a centrifuge tube. It is important to keep the sediment : volume ratio constant. 200 mg of sediment is the minimum required. (2) Add with a pipette, 20 ml of 1 M NaOH. (3) Cover the tube and stir overnight (16 h). Thorough mixing is necessary, the sediment must be kept in suspension (use, e.g., a magnetic stirrer, a shaker table). (4) Centrifuge at 2000g for 15 min. a NaOH-P. (1) Collect the extract. (2) Set apart (with a pipette) 10 ml of the extract in a test-tube. (3) Add 4 ml of 3.5 M HCl. (4) Stir energetically for 20 s and let stand overnight (16 h). Cover the tube. (5) A brown precipitate appears and progressively settles. Centrifuge at 200g for 15 min. (6) NaOH-P is determined in the supernatant. b HCl-P. (1) Wash the cake of the previous centrifugation (A-4) with 12 ml of 1 M NaCl. Stir for 5 min. (2) Centrifuge at 2000g for 15 min, discard the supernatant. (3) Repeat b-1 and b-2 once. (4) Add with a pipette, 20 ml of 1 M HCl. (5) Cover the tube and stir overnight (16 h). (6) Centrifuge at 2000g for 15 min. (7) HCl-P is determined in the extract. B Concentrated HCl-extractable P (1) Weigh 200 mg of dry sediment in a porcelain crucible. (2) Calcine at 450 C for 3 h. (3) Pour the cool ash into a centrifuge tube. (4) Add 20 ml of 3.5 M HCl with a pipette. HCl can be added directly to the crucible to ease the transfer of the ash. (5) Cover the tube and stir overnight (16 h). (6) Centrifuge at

Follow-up
A homogeneous and stable sediment reference material for the certication of extractable phosphorus has been prepared and will be available before the middle of 2001 under the reference number CRM 684. This will allow laboratories to check and improve their results in this eld. The harmonised SMT protocol, together with the reference material are useful tools in the eld of water management, especially at a time when quality assurance is of paramount importance in laboratory analytical work. Finally, the results of the certication campaign carried out in the frame of the SEPHOS European project (EC Contract SMT4-CT96-2087) are promising. Since the knowledge of the bioavailable forms of P are important not only for the analysis
Table 6 Certied values of extractable contents of phosphorus in CRM 684 Certied value/mg kg21 NaOH-P HCl-P IP OP Conc. HCl-P 550 536 1113 209 1373 Uncertainty/ mg kg21 21 28 24 9 35 P (number of data sets) 12 14 15 14 15

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2000g for 15 min. (7) Collect the extract in a test-tube for the determination of concentrated HCl-P C Inorganic and organic P IP. (1) Weigh 200 mg of dry sediment in a centrifuge tube. (2) Add with a pipette, 20 ml of 1 M HCl. (3) Cover the tube and stir overnight (16 h). (4) Centrifuge at 2000g for 15 min. (5) Collect the extract in a test-tube for IP determination. b OP. (1) Add 12 ml demineralised water to wash the residue. Stir for 5 min. (2) Centrifuge at 2000g for 15 min, discard the supernatant. (3) Repeat a-1 and a-2 once. (4) Let the residue dry (in the tubes) in a ventilated drying cupboard at 80 C. Put the tubes in an ultrasonic bath for 10 s and transfer to a porcelain crucible. (5) Calcine at 450 C for 3 h. (6) Pour the cool ash into the centrifuge tube. (7) Add 20 ml of 1 M HCl with a pipette. HCl can be added directly to the crucible to ease the transfer of the ash. (8) Cover the tube and stir overnight (16 h). (9) Centrifuge at 2000g for 15 min. (10) Collect the extract in a test-tube for OP determination. Calculation The concentration, C, in mg g21 (dry weight) is: C~ SV 103 m () (A1)

References
1 A. Ure, Ph. Quevauviller, H. Muntau and B. Griepink, Int. J. Environ. Anal. Chem., 1993, 51, 135. 2 A. Ure, Ph. Quevauviller, H. Muntau and B. Griepink, EURReport 14763 EN, European Commission, Brussels, 1993, 86 pp. 3 G. Rauret, J. F. Lopez-Sanchez, A. Sahuquillo, R. Rubio, C. Davidson, A. Ure and Ph. Quevauviller, J. Environ. Monit., 1999, 1, 57. 4 Ph. Quevauviller, A. Ure, H. Muntau and B. Griepink, Int. J. Environ. Anal. Chem., 1993, 51, 129. 5 S. C. Chang and M. L. Jackson, Soil Sci., 1957, 84, 133. 6 A. H. M. Hieltjes and L. Lijklema, J. Environ. Qual., 1980, 3, 405. 7 K. C. Ruttenberg, Limnol. Oceanogr., 1992, 7, 1460. 8 R. Psenner and R. Puckso, Archiv. Hydrobiol., 1988, 30, 43. 9 J. D. H. Williams, J. K. Syers, S. S. Shukla, R. F. Harris and D. E. Armstrong, Environ. Sci. Technol., 1971, 11, 1113. 10 J. D. H. Williams, J.-M. Jaquet and R. L. Thomas, J. Fish. Res. Bd. Can., 1976, 33, 413. 11 J. D. H. Williams, T. Mayer and J. O. Nriagu, Soil Sci. Soc. Am. J., 1980, 44, 462. 12 H. L. Golterman, Hydrobiologia, 1982, 92, 683. 13 H. L. Golterman, Hydrobiologia, 1996, 5, 1. 14 A. Sahuquillo, J. F. Lopez-Sanchez, R. Rubio, G. Rauret, R. P. Thomas, C. M. Davidson and A. Ure, Anal. Chim. Acta, 1999, 382, 317. 15 V. Ruban, J. F. Lopez-Sanchez, P. Pardo, G. Rauret, H. Muntau and Ph. Quevauviller, J. Environ. Monit., 1999, 1, 51. 16 J. Murphy and J. P. Riley, Anal. Chim. Acta, 1962, 27, 31. 17 J. F. Lopez-Sanchez, A. Sahuquillo, H. D. Fiedler, R. Rubio, G. Rauret, H. Muntau and Ph. Quevauviller, Analyst, 1998, 123, 1675. 18 P. Pardo, J. F. Lopez Sanchez, G. Rauret, V. Ruban, H. Muntau and Ph. Quevauviller, Analyst, 1999, 124, 407.

with: S = P concentration in the extract (IP, OP, HCl-P) in mg l21; V = volume of reagent used for extraction (20 ml); and m = mass of the test sample (200 mg dry weight). For NaOH-P: C~ S 14V 104 m () (A2)

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