LIMNOLOGY
and
OCEANOGRAPHY: METHODS
Limnol. Oceanogr.: Methods 5, 2007, 433–444
© 2007, by the American Society of Limnology and Oceanography, Inc.
Fractionation of sediment phosphorus revisited. I: Fractionation
steps and their biogeochemical basis
Kaarina Lukkari,1 Helinä Hartikainen,2 and Mirja Leivuori1
1
Finnish Institute of Marine Research, Helsinki, Finland
University of Helsinki, Helsinki, Finland
2
Abstract
The aim of this work was to assess the validity of a phosphorus fractionation procedure, introduced by Psenner
and co-workers and modified by Jensen and Thamdrup, in chemical characterization of sediment P. In this procedure, P is separated into 6 pools: loosely bound (and pore water) P, redox-sensitive P (bound to iron and manganese), P bound to oxides of aluminum and nonreducible Fe, calcium-bound P, and mobile and immobile pools
of organic P. The procedure was slightly modified further, and every step of the work is described in detail.
Reproducibility of the method and variation within the extracts obtained at each step were investigated with a
commercial reference material. The validity of the results considered against the theoretical basis of the P fractionation procedure was evaluated in terms of the elemental composition of the separate extracts. The results
showed good reproducibility of the method; variation in amounts of the different P forms in the separate extractions was small (coefficient of variation <15%). In addition, the analysis of selected elements extracted along
with P were reliable enough to deduce the origin of the various P forms. The analytical results for P and the other
elements were in accordance with the general theoretical basis of the fractionation procedure. Despite some shortcomings, the tested procedure, in combination with the laboratory practices and analytical methods described,
gives reliable and valuable results for distinguishing between potentially mobile and immobile P in sediment.
Internal phosphorus (P) loading leading to accelerated
eutrophication of marine systems has made acute the research
on P chemistry in sediments. Studies on the P chemistry in lake
and marine sediments are commonly based on fractionation,
where P compounds are divided into several pools according to
their solubility in or reactivity with various chemical solutions.
Procedures typically begin with dilute extractants, which
remove the most loosely bound P forms, and they proceed
stepwise toward stronger extractants, attacking P forms
more strongly bound to the solid phase (e.g., Ruttenberg 1992).
Acknowledgments
Financial support was received from the Ministry of the Environment,
the Kone Foundation, the Finnish Institute of Marine Research, and the Maj
and Tor Nessling Foundation. We thank the technicians from the laboratory of the Finnish Institute of Marine Research for assistance in the P fractionation studies and the laboratory for Environmental Research, University
of Jyväskylä, for the metal determinations. We are grateful to Professor
Henning Jensen, University of Southern Denmark, for his advice and comments regarding the fractionation procedure. This study was a part of the
research project “Searching efficient protection strategies for the eutrophied Gulf of Finland: the integrated use of research and modeling tools”
(SEGUE) carried out in collaboration with the Finnish Environment Institute
and the University of Helsinki. The project is one section of the Baltic Sea
Research Programme (BIREME) funded by the Academy of Finland.
Solutions may contain anions able to displace phosphate (PO4P) from its adsorption sites through competition, anions able
to alter the chemical state of the adsorption surface, or anions
able to dissolve P-bearing compounds.
A wide variety of extraction procedures for soil and sediment P have been developed, varying with the aim of the
study and the P fractions targeted (for reviews, see Boström et
al. 1982, Van Eck 1982, Pettersson et al. 1988, Ruban et al.
1999). The P forms commonly separated include soluble and
loosely sorbed (labile) PO4-P, redox-sensitive iron (Fe)-bound
P, and P bound to hydrated oxides of (Al) and nonreducible
Fe (surface-bound), calcium (Ca)-bound P (apatite-P), and
organic P. The organic P is sometimes divided into refractory
and labile fractions. In addition to organic P, the refractory
fraction is assumed to contain some inorganic P (e.g., Ruttenberg 1992), possibly as occluded P forms.
Extraction procedures are often poorly explained in the literature, and the theoretical basis of the P fractions separated
using the procedure often remains unclear. In addition, the
procedures are often difficult to follow because the practical
work is inaccurately described. For instance, the determination of PO4-P may be complicated by interference from extractant-derived ions. In this study, a commercial reference material was fractionated using a procedure developed by Psenner
433
Lukkari et al.
Fractionation of phosphorus I
et al. (1984) and modified by Jensen and Thamdrup (1993). To
assess the validity of the procedure in relation to the theoretical basis of the P chemistry, the extract obtained in each fractionation step was also analyzed for other elements. In addition, the repeatability of the analysis and possible chemical
interferences caused by the solute matrices were investigated.
Biogeochemical basis: P forms in sediment
P in mineral lattices—Most terrestrial P ending up in marine
sediments is bound in mineral lattices, primarily in apatite
minerals (Ca5(PO4,CO3)3(F,Cl,OH)). Other P-containing minerals assumed to be present in sediments include brushite
(CaHPO4⋅2H2O), wavellite (Al3(OH)3(PO4)2⋅5H2O), variscite
(AlPO4⋅2H2O), anapaite (Ca3Fe(PO4)3⋅2H2O), strengite (FePO4⋅
2H2O), and vivianite (Fe3P2O8⋅8H2O) (Leckie 1969, Nriagu and
Dell 1974, Lindsay 1979, Psenner et al. 1984, Pettersson et al.
1988, Stumm and Morgan 1996). In soils, P present in wellorganized mineral structures is more or less inert, whereas
other P compounds are subjected to chemical, physical, and
biological transformation processes. When P-containing particles end up in anoxic sea sediment where the environment is
highly reducing and the salinity elevated, they are further
transformed, e.g., reducible metals may go into solution and
carbonate minerals may precipitate in saturated pore waters
(e.g., Berner et al. 1993).
P on particle surfaces—A common chemical P species in the
pH range (usually 5–8) of aquatic environment and sediments
is orthophosphate anion, commonly HPO42–. Orthophosphate (PO4-P) can be bound by Al and Fe in hydrated oxides
or on the edges of mineral lattices by a specific ligand
exchange reaction (anion penetration). In this reaction, water
(H2O) or hydroxide (OH–) ligand in the metal cation coordination sphere is replaced by PO4-P anion (Hingston et al.
1967, 1972, 1974). White (1980) has compiled several feasible
reaction mechanisms. PO4-P bound with single coordinate
bond (monodentate binding) is considered labile and available to terrestrial plants. PO4-P may also form 2 bonds with 1
metal cation (bidentate binding), or bond to 2 metal cations
(binuclear binding) forming a more stable bond. The bonding
pattern depends on the P concentration and may change
during sorption or desorption processes (Hingston et al.
1974). Furthermore, PO4-P can be replaced by competing
anions, such as anions of weak organic and inorganic acids
(e.g., OH–, arsenate HAsO4–, and fluoride F–, see Ryden et al.
1987). At high pH, which allows the silicic acid to dissociate
(pKa 9.5), also H3SiO4– may replace PO4-P (Scheffer and Scheffer 1984). Fine clay particles are high in Al and Fe oxides with
large reactive surface area for P-binding. Because they settle
in the water column only slowly, they efficiently transport
P with water currents.
P in organic matter—In organic matter, P can be a structural
part of the organic molecule or be bound to the organic matter
via metal cations, e.g., Fe3+, Al3+, and manganese Mn4+ (Schnitzer
1969, Steinberg and Muenster 1985). The most abundant
434
organic P forms in sediments are orthophosphate monoesters
and diesters (e.g., DNA, phospholipids; Ingall et al. 1990). Also
phosphosaccharides (e.g., ATP) and inositol-hexaphosphate
(phytate) have been reported (e.g., Uhlmann et al. 1990, De
Groot and Golterman 1993). Phosphonates, which are found
in many sediments, have a strong direct bond between P and
carbon (C). They are resistant to acid and base hydrolysis and
possibly also to bacterial attack (Ingall et al. 1990, Carman et
al. 2000). Lake sediments are also rich in polyphosphates synthesized and stored as inorganic granules in microbial cells
(Hupfer et al. 1995). Availability of P in organic matter to producers depends on the size and stability of the molecules
against hydrolysis and degradation processes, on microbial
activity and on the environmental conditions (e.g., pH and
redox-potential). Degradation processes in the water column
and sediment first remove the most unstable compounds,
enriching the deposited and buried material with C (Froelich et
al. 1982, Berner et al. 1993). Some of the P remains trapped in
large, highly complex molecules of humic matter, and is
removed from the short-term nutrient cycle. Organically
bound P is gradually transformed to authigenic mineral P in
biogeochemical processes (Pettersson et al. 1988).
The traditional classification of humic matter is based on
its solubility. According to the prevailing concept, humic
compounds can retain P through their complexed metal
cations. Fulvic acids (FAs) represent a fraction soluble in both
alkali and acid; humic acids (HAs) are soluble in alkali but
insoluble in acid, and the insoluble part is called humin. HAs
are usually of larger molecular size and higher C content than
FAs (Ishiwatari 1985). Humin can be divided into stable
residue and a hydrolysable fraction (Vandenbroucke et al.
1985), and despite the refractory nature of humin, oligocarbophilic bacteria have been reported to grow on it (Steinberg
and Muenster 1985). FAs have been shown to support algal
growth, with effect on the seasonal fluctuation of their
molecular size fraction. A fraction of larger molecular size has
been supposed to degrade more slowly and, in addition, to
preferentially form complexes with metal cations (Steinberg
and Muenster 1985, Vandenbroucke et al. 1985). Metal-FA
complexes are able to react with PO4-P via the metal to form
FA-metal-phosphate (Schnitzer 1969).
P in sediment pore water—P may occur in pore water in dissolved form or as fine particulate inorganic and organic P. The
form of P in the porewater depends on oxygen (O2) conditions
as well as on pH and elemental composition and other properties of the sediment. If the near bottom water and pore water
in the sediment surface layer contain dissolved O2 or nitrate
(NO3–), oxidized forms of Fe compounds can bind dissolved
PO4-P (Mortimer 1941 and 1942, Hupfer and Uhlman 1991).
Under anoxic conditions, Fe compounds are reduced and febound P occurs in the pore water in dissolved form. In this
case, the P is susceptible to diffusion and able to participate in
chemical precipitation reactions with metal cations and mineral phases (Leckie 1969, Stumm and Morgan 1996).
Lukkari et al.
Fractionation of phosphorus I
Fig. 1. Scheme of the P fractionation procedure (modified from Jensen and Thamdrup 1993).
Materials and procedures
Sample material—The sediment material for this study was the
commercial reference material BCR-684, 5 P fractions of which
have been certified according to the extraction procedure of the
European Commission’s Standards, Measurements and Testing
Programme (SMT). The procedure (Ruban et al. 1999, 2001) is a
modification of the procedure of Williams et al. (1976). The
material originates from Ca-rich, fine-grained sediment from the
agriculturally and industrially loaded River Po, Italy. According
to the certification report, it was collected from a water depth of
2–3 m, sieved (<2 mm and <90 µm), air- and oven-dried (<60°C),
and carefully homogenized. Certified values for the SMT procedure are 550 ± 22 mg kg–1 (18 µmol P g–1) for 1 M sodium hydrox-
ide (NaOH) extractable P, 536 ± 28 mg kg–1 (17 µmol P g–1) for the
following 1 M hydrochloric acid (HCl) extractable P, and 1373 ±
35 mg kg–1 (44 µmol P g–1) for the separately determined concentrated HCl-extractable P (after calcination). Certified values
for inorganic and organic P in separate extractions are 1113 ± 24
and 209 ± 9 mg kg–1 (36 and 7 µmol P g–1), respectively.
P fractionation procedure—The extraction procedure used in
this study principally follows the 5-step procedure described
in Jensen and Thamdrup (1993), with slight modifications
(Fig. 1). This procedure was chosen because it emphasizes the
redox-sensitive and organic P forms, which are interesting
pools with respect to the liberation or burial of P in sediment.
In addition, the sediments intended to be studied are known
to be poor in calcium carbonate (CaCO3) but rich in humic
435
Lukkari et al.
Fractionation of phosphorus I
matter and, consequently, in organic P. As for P release from
this kind of sediments, it is useful to distinguish between the
more labile and recalcitrant organic P fractions. Jensen and
Thamdrup (1993) modified the procedure of Psenner et al.
(1984) replacing 1 M ammonium chloride (NH4Cl) in the first
step with 0.46 M sodium chloride (NaCl) to avoid the dissolution of Ca-bound P (Hieltjes and Lijklema 1980, Pettersson et
al. 1988, Jensen and Thamdrup 1993) and to mimic the composition of marine water. It is likely, however, that in noncalcareous sediments Ca in the 1st extraction step originates
from exchangeable reserves on particle surfaces. Jensen and
Thamdrup added rinsing steps to the scheme to minimize tailing, i.e., the transfer of P left in the interstitial water to the following pool. In addition, the molarity of 0.1 instead of 1.0 was
chosen for NaOH. The more dilute NaOH is expected more
sensitively to extract the easily hydrolysable organic P compounds than does 1.0 M NaOH (e.g., Hupfer et al. 1995).
In the 1st step, the sediment sample is extracted with 0.46 M
NaCl for 1 h to extract loosely adsorbed and pore water P (presented in results as NaCl-iP). In the 2nd step, the sediment
residue from the previous step is extracted with 0.11 M
sodium dithionite (Na2S2O4 ) in 0.11 M sodium bicarbonate
(NaHCO3) buffer (pH 7.0) to separate the redox-sensitive fraction of P bound to hydrated oxides (NaBD-iP), mainly those of
Fe. The use of dithionite without simultaneous addition of
bicarbonate causes a steep decline in pH (Mehra and Jackson
1960). Extraction for 1 h is followed by 2 NaBD rinses to complete the reduction and to minimize tailing. In the 3rd step,
0.1 M NaOH is used to extract the rest of the P bound to
oxides of Fe, as well as that bound by Al oxides (NaOH-iP), and
also a marked part of the organic P compounds (nonreactive P
or NRP). This 18-h extraction is followed by 1 rinse with 0.1 M
NaOH. In the 4th step, 1-h extraction with 0.5 M HCl is used
to extract Ca-bound P, mainly from apatite minerals (HCl-iP).
Steps 1 to 4 all end with 15-min rinsing with 0.46 M NaCl.
Extractants and the rinsing solutions used in each step are
combined into 1 sample.
Sediment residue from step 4 is moved quantitatively to
tared ceramic vials, with use of 10–20 mL of MilliQ water to
rinse the extraction tubes; it is dried for 24 h at 105°C and
weighed. The last step of Jensen and Thamdrup’s (1993) procedure was modified to determine residual or recalcitrant
organic P: instead of the combusted (2 h at 520°C) sediment
residues being boiled in 0.5 M HCl (10 min), they were combusted for 2 h at 550°C in ceramic vials, cooled, weighed, and
removed quantitatively back to the extraction tubes. The vials
were carefully rinsed with portions of the extraction solution
to be used in the last step, the rest of the 1 M HCl extractant
was added to the tubes, and the sediment residues were
extracted for 16 h. This step, based on determination of total
and organic P (Saunders et al. 1955, Aspila et al. 1976), is used
in the sediment P extraction procedure (SEDEX) described by
Ruttenberg (1992) and has been evaluated as one benefit of
the SEDEX procedure (Ruban et al. 2001).
436
Extraction—Three replicates of 0.500 g dry weight (DW) of
the reference material were carried out through the procedure
to reveal the variation between replicates. The same amount
(50.0 mL) of each extractant was used, giving the sediment dry
matter (DM) to solution ratio of 1:100 instead of the 1:25 (fresh
sediment) used by Jensen and Thamdrup (1993). This sedimentto-solution ratio was used in this study to ensure that in no step
would saturation limit the liberation of P from the solid phase.
A similar dry sediment weight-to-extractant volume ratio of
1:100 has been used, e.g., in fractionation procedures by Ruttenberg (1992) and Hieltjes and Lijklema (1980). Higher volumes of extractants also ensured that enough supernatant was
received from each step for determination of P and other elements. The same volumes (50.0 mL) of extractant and rinsing
solution were used in all steps, added with an automatic dispenser. All extraction steps described were carried out at room
temperature using 100-mL polypropylene (PP) tubes with snapon caps and an orbital shaker table (400 rpm). After each extraction and rinsing step, samples were centrifuged for 15 min
(4000 rpm, room temperature) and the supernatant was immediately poured into PP sample bottles.
Extraction tubes, sample bottles, and ceramic vials were
rinsed with 10-fold diluted 65% nitric acid (HNO3) and a few
times with MilliQ-water before use. All reagents used,
throughout the procedure, were of p.a. grade (only trace impurities of Fe were mentioned for the Na2S2O4 reagent). Blank
samples (2 or 3 replicates) were run in each set of samples to
find any contamination originating from chemicals, equipment, or working practices. PO4-P concentrations in the blank
samples were usually negligible.
Pretreatment and analysis of extracts—Extracts from each step
were divided into 2 portions, and 1 of them was vacuum filtered [Nuclepore polycarbonate (PC) membranes, pore size
0.4 µm]. Both portions were preserved with 4.5 M sulfuric acid
(H2SO4), except the acidic extracts from steps 4 and 5. PC
membranes were rinsed with a few milliliters of the extract
before filtration, and the filtrate used for rinsing was discarded. Comparison of the pure extracts obtained from each
step revealed that neither PC membranes nor filtration equipment caused PO4-P contamination. pH of the extracts was
adjusted to 2 with H2SO4 to keep the sample preserved, to keep
the metals of interest in dissolved form, and to obtain a suitable molarity of H2SO4 in the sample for optimal color development in PO4-P determination (Koroleff 1983). Dissolved
inorganic P (DIP, referred to later as –iP) was analyzed in the
filtered samples by the molybdate-blue method of Murphy
and Riley (1962) modified by Koroleff (1983). For total P (TP),
the nonfiltered, carefully mixed sample was digested with acid
persulfate (autoclaving time 1 h; Koroleff 1983). PO4-P (for
both DIP and TP) was measured at 880 nm using a UV-VIS
spectrophotometer (Genesys 10uv Thermo Spectronic)
equipped with flow injection cuvette (cell length 50 mm).
Organic P, referred to as NRP, was calculated as the difference
between TP and DIP.
Lukkari et al.
NaBD extracts (step 2) were bubbled carefully with compressed air (purity 99.5%) in a fume hood to remove dithionite
(which decomposes to thiosulfate and hydrogen sulfite ions)
before acid addition. Dithionite interferes with the reducing
phosphomolybdate complex and disturbs spectrophotometric
analysis of PO4-P (e.g., Ruttenberg 1992, Jensen and Thamdrup
1993). The aeration time needed to remove dithionite from a
given volume of the extract was tested by bubbling air into
NaBD solutions with known PO4-P concentrations for different
times and by comparing PO4-P concentrations of the test solutions with the concentration of a reference solution prepared in
MilliQ-water. Owing to oxidation of Fe compounds, the NaBD
extracts turned brownish during aeration, but quickly became
colorless after acid addition (pH 2). In a few cases where aeration
was not complete, a white precipitate of sulfur (see also Jensen
and Thamdrup 1993, Paludan and Jensen 1995) formed in the
sample after addition of phosphomolybdate reagent containing
H2SO4. This occurs because thiosulfate in acid solutions readily
decomposes to give elemental sulfur and hydrogen sulfite. In
these cases, the original sample was aerated again, the precipitate
was allowed to settle (overnight, 5°C), and a subsample was
taken from the upper part of the sample for DIP analysis. However, careful oxidation of the NaBD extract before acid addition
allowed use of the same treatment as for the other extracts.
Addition of H2SO4 to the NaOH extracts caused precipitation of HAs dissolved in the base solution. To separate lowmolecular-weight (LMW, particulates <0.4 µm) and highmolecular-weight (HWM, >0.4 µm) NRP, the sample was first
carefully mixed and split into 2 portions. One portion was
acidified for TP analysis and the other was filtered before acid
addition. Brownish precipitate also formed in the filtered sample after acid addition, suggesting the presence of LMW humic
acids in addition to dissolved FAs. In the filtered extract, the
precipitated humic matter was allowed to settle (usually
overnight at 5°C), and a subsample from the upper part of the
extract was analyzed for DIP. TP of the filtered extract (TPdiss)
was determined (after careful mixing) to calculate the amount
of LMW-NRP originally in dissolved (<0.4 µm) form in the
NaOH extract. NRP in the whole NaOH extract, including
nondissolved (>0.4 µm) forms, was calculated as the difference
between TP in the nonfiltered sample and DIP. HMW-NRP, in
turn, was calculated as the difference between NRP and LMWNRP.
Separation of NaOH-extractable organic P (i.e., NRP) on the
basis of its dissolution or particle size differs from the step
described in Jensen and Thamdrup (1993) and Paludan and
Jensen (1995). NRP extracted to 0.1 M NaOH in P fractionation methods has been considered, among other things, as P
originating from organisms, including polyphosphates
(Jensen and Thamdrup 1993), leachable organic P (Jensen et
al. 1995), moderately resistant organic P (Olila et al. 1995),
and extractable biogenic P assumed to be autochthonous in
origin (Penn et al. 1995). Composition of NaOH-NRP and
bioavailability of P in the NRP pool are not fully known. In
Fractionation of phosphorus I
addition to PO4-P, [31P]NMR studies have revealed the presence
of at least orthophosphate mono- and diesters and polyphosphates in NaOH extraction (Uhlman et al. 1990, Oluyedyn et
al. 1991, Hupfer et al. 1995). Despite differing concepts in the
literature (Stewart and Wetzel 1982, Pant et al. 2002, Young et
al. 2005), we can assume that the LMW part of the NaOHextractable NRP is more available than the HMW part. HMWNRP, still in particulate form in 0.1 M NaOH solution after
18-h extraction, is assumed to be of low availability (Vandenbroucke et al. 1985) and treated in this study as refractory P
(see below). Particle size-based separation of organic P is justified by earlier findings that low-molecular-weight dissolved
organic matter (DOM) in coastal seawater is used primarily by
heterotrophic bacteria (Ogura 1975).
Extracts from step 3 were slightly colored (brownish). However, because of the high concentration of P, they were always
diluted at least 10-fold before DIP or TP analysis. Background
absorbance was checked by analyzing diluted extracts using
only ascorbic acid reductant without molybdate reagent. The
dilution was found to have eliminated any possible interference by the color. A brown precipitate in TP and TPdiss samples
disappeared during 1-h autoclaving in acid persulfate digestion
and thus did not disturb the spectrophotometric analysis of TP.
For DIP and TP analysis, subsamples of acidic extracts containing HCl (steps 4 and 5) were diluted 10-fold in step 4 and
20-fold in step 5 (chosen on the basis of results obtained from
matrix effect tests, see below), and pH of the diluted extract
was raised (with 1 M NaOH) to the same level as in the other
extracts. TP determinations of unfiltered samples from steps 4
and 5 were done a couple of times, but TP in the extract from
step 5 was always almost the same as DIP. In some cases, TP
was even slightly smaller than DIP and the same occasionally
held true for the NaBD solution, probably owing to inaccuracy
of the analytical method.
Total NRP concentration was calculated as the sum of NRP
pools from steps 1 to 3, the LMW-NRP in NaOH extraction
forming the major part. HMW-NRP, assumed to be slowly
degradable (see above), was summed with the refractory
organic P (Res-P) in step 5. When extracted P fractions are
roughly characterized as immobile and mobile forms (Jensen et
al. 1995), the mobile pool here is considered to include NaCliP, NaBD-ip, and the LMW-NRP from the NaOH extraction
together with the NRP obtained in the first 2 steps. The rest of
the fractions form the immobile pool: HCl-iP, Res-P, the HMWNRP, and the inorganic P in the NaOH extraction (NaOH-iP).
Total dissolved elements—Part of the filtered and acidified
extracts (mainly from steps 1 to 4) was separated for determinations of total dissolved (Tdiss) Fe, Mn, Al, Ca, magnesium (Mg),
silicon (Si), and P. Measurements were made with ICP-OES in
the laboratory of the Institute for Environmental Research at
the University of Jyväskylä. Detection limits were 10 mg L–1 for
Fe, Mn, and Al and 20 mg L–1 for the rest of the elements. Standards for ICP-OES determinations were prepared in NaCl acidified to pH 2 with H2SO4.
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Lukkari et al.
Fractionation of phosphorus I
Total analysis of sediment—Total P, as well as total Fe, Mn,
Al, Ca, and Mg, was determined with ICP-AES (TJA-25,
Thermo Jarrell Ash) after combined fluoric acid (HF), aqua
regia, and boric acid (H3BO3) digestion in a CEM-205
microwave oven. The technique follows the accredited
method (testing laboratory T40 accredited by the Centre of
Metrology and Accreditation) used for determination of Al,
Mn, and Fe in the FIMR laboratory (method modified from
Loring and Rantala 1992, more detailed description in
Leivuori 2000). Reference materials used for sediment total
analysis were Buffalo River sediment 8704 (not certified for P)
and MESS-3 (NRCC, National Research Council of Canada;
informative value for P only). In these materials, the recovery
of P varied between 96 and 112% and that of the other elements between 88% (Ca, Fe) and 108% (Al). Detection limit
for TPsed was 5 µg g–1 (0.16 µmol g–1).
Table 1. Average concentrations (µmol P g–1 DW) and statistical parameters of various P components in different extracts of
BCR-684.
Extract
NaCl
438
Average
SD
CV%
Min
Max
iP
TP
NRP
2.2
2.7
0.5
0.2
0.2
0.1
9.2
8.2
15.8
1.7
2.3
0.4
2.6
3.2
0.6
iP
TP
NRP
19.4
20.3
1.0
0.7
1.0
0.8
3.5
4.9
81.4
18.5
17.0
0
21.2
22.0
2.9
iP
TPdiss
NRPdiss
TP
NRP
iP
TP (n = 18)
NRP (n = 18)
1.4
3.2
1.9
3.9
2.5
10.2
10.6
0.4
0.2
0.4
0.4
0.3
0.3
0.3
0.5
0.4
14.2
13.9
20.9
7.9
11.8
3.2
5.1
92.1
1.0
2.0
0.6
3.5
2.1
9.7
9.2
0
1.8
4.1
2.4
4.5
3.2
10.6
11.7
1.1
Res-P
4.9
4.0
48.6
42.1
0.4
1.1
2.7
1.1
7.7
26.8
5.5
2.7
4.3
2.5
45.7
39.5
6.2
6.7
50.9
44.8
NaBD
NaOH
Assessment
Testing of the matrix effect—Possible analytical errors (e.g.,
bending of the standard curve) in the spectrophotometrical
PO4-P analysis, caused by the use of different extract matrices
in the various steps, were assessed with 3 replicate sets of
determinations for DIP and TP. The tests were made by preparing 6 PO4-P standard solutions (0, 0.5, 1.0, 5.0, 10.0, and
20.0 µmol PO4-P L–1) for standard curves and 3 PO4-P control
solutions (0.42, 4.20, and 10.6 µmol PO4-P L–1) for test samples
in each of 5 diluted extract matrices. Extract matrix here means
a solution containing exactly the same proportion of extraction and rinsing solutions as used in an extraction step, taking
into account acidification (used for preservation) and dilution
before PO4-P analysis. Standard and control solutions of the
same PO4-P concentrations were also prepared in MilliQ water
(acidified) for comparison of the results. In testing the matrix
effect, the significance of differences between the control samples prepared in water and in the different extract matrices was
tested with the t test for paired samples. P < 0.050 was considered as a significant difference in the average values.
Analytical error due to the different extract matrices was
found to be negligible after dilution (data not shown). The PO4P concentrations of the different control solutions, calculated
on the basis of standard curves prepared in either MilliQ water
or various extract matrices, did not differ statistically. Thus standards prepared in acidified MilliQ water were routinely used for
DIP and TP analysis of all extracts. Because the P concentrations
in our extracts were high enough to allow dilution, modifications of molybdate reagents, as made by Zhang et al. (2004) to
overcome the analytical uncertainties, were not needed. Dilutions of extracts fixed to avoid disturbance of the matrix were
also found to be suitable to keep the PO4-P concentrations in
each extract of the reference material sample within the concentration range of the 6 PO4-P standards.
Testing of repeatability—Because of the different extraction
procedure, the P fractions separated in this study are not fully
comparable with the P fractions certified for BCR-684, but
Component
HCl (0.5m)
HCl (1 m)
Total NRP
TPsed (n = 18)
TPextr
Concentrations were measured with a spectrophotometer. n = 20 unless
otherwise noted.
since homogeneity of the reference material is ensured, differences in the results of replicate analysis can be attributed to
the method. Assessment of the repeatability of the method
was based on the basic statistical parameters calculated from
the fractionation results obtained with 20 replicates extracted
in separate sets of samples. A coefficient of variation (CV%)
<15 was considered to indicate good repeatability. This rather
stringent limit was chosen because it was the percentage of
error practically never exceeded when measured and calculated PO4-P concentrations of the control solutions were compared. Differences between the fractionation results of the reference material obtained in separate extraction rounds were
tested by calculating average values, standard deviations (SD),
and CV% values of 20 replicates.
The CV% values obtained for 20 replicate samples showed
the reproducibility of the method to be good (Tables 1 and 2)
for the spectrophotometrically analyzed P fractions. CV% was
>15% only for NRP fractions calculated as the difference
between TP and DIP. In ICP-OES analysis (Table 2), CV% for
TPdiss slightly exceeded 15% only in the NaOH extraction. The
reproducibility of determinations of co-occurring elements in
BCR-684 was slightly worse. Differences between SDs and
mean concentrations were smallest in steps 4 and 5, where
CV% exceeded 15% only for TdissCa. In NaCl the concentra-
Lukkari et al.
Fractionation of phosphorus I
Table 2. Average concentrations (µmol g–1 DW) and statistical
parameters of various total dissolved elements in different extracts
of BCR-684 measured by ICP-OES.
Extract
Component
Average Stdev CV% Min Max
NaCl
TdissMg (n = 18)
TdissSi
TdissAl
TdissCa
TdissP
TdissMn
TdissFe
75.2
2.9
0.4
137
2.6
0.1
0.2
4.1
0.5
0.2
17.0
0.3
0
0.2
5.4
16.1
44.7
12.4
12.1
32.7
80.0
68.3
2.2
0
123
1.9
0.1
0
83.1
4.3
0.9
195
3.3
0.2
0.8
TdissMg (n = 18)
TdissSi
TdissAl
TdissCa
TdissP
TdissMn
TdissFe
21.5
19.8
1.1
279
20.3
7.0
97.5
1.4
1.1
0.4
67.9
2.1
0.4
36.8
6.6
5.5
38.8
24.3
10.4
5.6
37.7
17.9
17.9
0.5
142
16.8
6.4
37.4
25.0
21.8
2.0
388
26.6
8.3
158
TdissMg (n = 19)
TdissSi
TdissAl
TdissCa
TdissP
TdissMn
TdissFe
1.5
84.4
34.5
38.4
2.8
0.1
5.2
1.8
12.4
4.3
5.9
0.4
0.1
5.0
120
14.7
12.4
15.4
15.9
143
97.1
0
62.8
29.5
28.8
2.3
0
1.2
6.7
112
47.6
55.5
4.1
0.6
17.4
TdissMg (n = 19)
TdissSi
TdissAl
TdissCa
TdissP
TdissMn
TdissFe
150
47.1
62.5
714
11.7
5.5
156
7.9
5.0
4.0
165
1.0
0.4
30.3
5.3
10.7
6.4
23.1
8.5
7.4
19.4
133
40.8
56.0
208
10.4
4.7
98.3
164
57.9
69.3
874
14.4
6.4
209
TdissMg
TdissSi
TdissAl
TdissCa
TdissP
TdissMn
TdissFe
405
421
633
4.7
5.8
2.1
298
32.0
8.3
32.5
1.0
0.3
0.1
20.6
7.9
2.0
5.1
20.5
5.8
6.4
6.9
355
411
587
3.4
5.4
2.0
270
452
433
690
6.3
6.4
2.4
332
NaBD
NaOH
HCl
Res (n = 6)
n = 20 unless otherwise noted.
tions of TdissFe, TdissMn, and TdissAl, and in NaOH the concentration of TdissMn, were too small (<0.5 µmol g–1) to have any
marked effect on the results. However, CV% of TdissFe was as
much as 38% in NaBD and 97% in NaOH. If 15% is considered
as the critical CV%, both TdissAl and TdissCa exceeded it in
NaBD, and TdissMg exceeded it in NaOH extraction.
Analytical reliability and repeatability—Analytical accuracy
and reproducibility (Tables 1 and 2) of the procedure seem to
be satisfactory for purposes of quantification of the various
sediment P forms. Even though our solid-to-solution ratio differed from that used by Jensen and Thamdrup (1993), the P
concentration in extracts was high enough to allow sample
dilution. This, in turn, enabled the use of phosphate standards
and controls prepared in acidified MilliQ water, which
speeded up the laboratory work. Extraction of 20 replicates of
dry reference material samples showed good reproducibility in
separating P forms. CV% was worst in NRP, probably owing to
the cumulative errors in the various analytical steps used in
the calculation. In addition, particulate material present in the
acidified NaOH extract might cause variation between subsamples separated for DIP and TP analysis.
Spectrophotometrical measurement of TP after acid persulfate digestion gave results comparable to ICP-OES measurement
of TPdiss. However, ICP-OES analysis cannot be used alone for
determination of organic P, because the required filtration of
the NaOH extract removes the HMW-NRP fraction, which in
our modification is considered to represent a part of the immobile organic P. On the other hand, part of the NaOH-extractable
organic P compounds are resistant to acid and base hydrolysis,
and may not be totally degraded with acid persulfate digestion
(Ingall et al. 1990, Monaghan and Ruttenberg 1999). One weakness of the analytical methods is the hydrolysation of the most
sensitive organic P compounds and consequent overestimation
of inorganic PO4-P in the DIP analysis when using the molybdate-blue method and reagent containing H2SO4 (e.g., Koroleff
1983, Baldwin 2003). Except for elements occurring in very low
concentrations, deviation in the results for co-occurring elements (Table 2) was low, even though the reference material was
certified for separate P forms only. Thus, amounts of co-occurring elements can be reliably used in assessing the relevance of
the fractionation procedure. The highest CV% values (TdissFe
in NaBD and NaOH) might be due to fine iron particulates
(<0.4 µm) present in the extract (Hens 1999).
Chemical composition of the extracts—The assessment of the
validity of the method was based on the chemical composition of extracts obtained in fractionation of 3 replicates of the
reference material simultaneously (Figs. 2, 3, and 4).
P e x t r a c t e d i n d i ff e r ent steps. When total P extracted in
different steps was measured spectrophotometrically (TPextr), it
amounted to 40.9 µmol P g–1 DW (Figs. 2 and 3), but when it
was measured by ICP-OES (Figs. 2 and 4) as TdissP, it was
slightly greater, amounting to 43.1 µmol g–1 DW. The NaOH-iP
(4%) and NaCl-iP (6%) represented minor portions of TPextr. In
NaOH extraction, ICP-OES analysis yielded 1.0 µmol P g–1 DW
lower concentration than spectrophotometric analysis. Total
NRP constituted 10% of TPextr, and recalcitrant organic P in the
residual fraction (Res-P) 12%; thus, the portion of total
organic P was 22%. Half of the total NRP originated in the
NaOH extraction (mostly in the <0.4-µm fraction) and 27%
from HCl extraction in step 4. No measurable NRP was found
in NaBD extraction. HCl-iP formed 25% and NaBD-iP almost
half (48%) of the TPextr.
439
Lukkari et al.
Fractionation of phosphorus I
Fig. 2. Chemical composition of fractionation extracts of BCR-684. Concentrations of the measured components are presented as µmol g–1 DW on
the y-axis. Solid lines on top of each bar represent SD of 3 replicate samples.
Total elements extracted in different steps. Of the total Fe
extracted (560 µmol g–1 DW), 51% was in the residual fraction
and 28% in the NaBD fraction (Figs. 2 and 4). HCl in step 4
extracted 20% of Fe and NaOH only 1%. NaCl-TdissFe was almost
under the detection limit. The portion of total Mn extracted
(14.0 µmol g–1 DW) was biggest in NaBD (46%), followed by 0.5 M
HCl (38%). The residual fraction contained 15% of the TdissMn,
and NaOH and NaCl extracted only minor amounts of it (1%
and 0.5%, respectively). Up to 85% of total extracted Al (684 µmol
g–1 DW) was in the residual fraction, and 9% was in the HCl
pool. NaOH extracted 5% of Al and NaBD only 0.2%. NaClTdissAl was barely above the detection limit.
For the earth alkaline metals, HCl in step 4 extracted 72%
of the total Ca (1133 µmol g–1 DW) and from the residual pool
(step 5) only 0.3%. Portions of 11% and 13% of total Ca were
extracted from the NaCl and NaBD pools, respectively, and
NaOH extracted 4% of this. More than half of extractable Mg
(660 µmol g–1 DW) was left in the residual fraction (63%), and
23% was extracted in previous HCl extraction. NaCl extracted
11% of total Mg, NaBD 3%, and the concentration of NaOHTdissMg was under the detection limit.
The largest portion of total extracted Si (585 µmol g–1 DW)
was in the residual fraction (74%), and NaOH extracted up to
14% of this. The portion of TdissSi extracted with HCl (step 4)
was 9%, and only 3% and 0.4% were extracted with NaBD and
NaCl, respectively.
Total element concentrations in the r e f e r e n c e m a t e r i a l .
HF/aqua regia/H3BO3 digestion of the BCR-684 reference material and subsequent ICP-AES analysis gave the following concentrations (in µmol g–1 DW): 48.6 P, 402 Fe, 2.7 Mn, and 2747
Al. Extracted sediment residues (also 3 replicates) were
digested and analyzed for total elements in another set of samples. Concentration of P left in the sediment residue after the
whole fractionation scheme was only 0.1 µmol g–1 DW (below
the detection limit), and the concentrations of TFe and TMn
were 13.8 and 0.1 µmol g–1 DW, respectively.
Extract composition in relation to biogeochemical basis
of the method. The amount of loosely bound P (NaCl-iP) was
high (Figs. 2 and 3) relative to the amount of P usually
extracted with neutral salts from sea and lake sediments
(Jensen and Thamdrup 1993, Koski-Vähälä et al. 2001). This
indicates low PO4-P sorption capacity of the material. Relatively large quantities of Ca2+ and Mg2+ (Fig. 2) extracted by
NaCl were in exchangeable form and replaced by Na+ ions.
The high content of Ca in the NaBD extract (Fig. 2) can be
attributed to exchangeable Ca2+ left on the particle surfaces
and to Ca2+ on the carbonates. Cation exchange is based on
equilibrium reactions, and several treatments with neutral salt
Fig. 3. Percentage of fractionated P forms of the total extractable P in BCR-684.
440
Lukkari et al.
Fig. 4. Percentage of dissolved total elements of their total extractable
concentrations in different fractionation extracts of BCR-684.
are needed to replace all exchangeable cations on the surface.
In their study with carbonate sediments, Jensen et al. (1998)
suggested that NaBD extracts some P from surfaces of CaCO3
particles, even though Ca dissolved in NaBD was low. They
also concluded that the specificity of the NaBD step for Febound P decreased with increasing CaCO3 content. Part of P in
the NaBD-extractable pool might originate in the Ca-bound
form and could cause overestimation of the content of Febound P (Uhlmann et al. 1990). Because the reference material
was collected from shallow water and dried (Ruban et al.
2001), it was well oxidized. A large part (~40%) of NaBD-iP of
TPextr can be explained by binding of P to oxidized forms of Fe.
This explanation is further supported by the high content of
TdissFe in NaBD extraction. Also Mn, being a reducible metal,
was mainly extracted in this fraction. The content of Al not
susceptible to redox reactions was low (in NaBD). These findings support the assumption that NaBD is selective in distinguishing between the reducible and nonreducible metals. The
same was concluded in earlier studies (Chang and Jackson
1957, Psenner et al. 1984, Psenner and Pucsko 1988). The
release of Si in NaBD suggests that, because of the similarity of
silicate and phosphate structures and their binding properties,
some Si was present in the Fe-bound form. Si in Fe-bound
form was found in lake sediments by Hartikainen et al. (1996).
P extracted in NaOH was mainly organic. NRP in NaOH
formed the major part of the total NRP extracted during the
procedure (Table 1). Most of it was in dissolved (<0.4 µm) form,
suggesting that the LMW organic matter was rich in P. This suggestion is supported by earlier findings that FAs contain 2 to 5
times as much P as HAs (e.g., Rashid 1985). Only a minor
amount of P in NaOH was inorganic, as also reported earlier
(e.g., by Jensen and Thamdrup 1993). This inorganic P was
Fractionation of phosphorus I
probably bound by Al oxides, which were extracted in high
quantities in this fraction (Fig. 2). The concentration of dissolved Al is known to increase with pH owing to the formation
of aluminate anions (e.g., Lindsay 1979). Part of the inorganic P
extracted in NaOH might be bound by nonreducible Fe oxides
present in small amount, and some of it might be bound to Al
and Fe complexed with humic material (Schnitzer 1969). NaOH
extracted large amounts of Si (Fig. 2), and evidently the main
part of it was biogenic. Part of the Si in NaOH could be inorganic and bound onto Al oxides with the same mechanism as P.
Dissolution of Ca in NaOH may also indicate its biogenic origin.
The large proportion of P in the HCl (step 4) and in other
immobile fractions (Fig. 3) underlines the irrelevance of total
P when assessing the risk of internal P loading. If the assumption is true that some Ca-bound P is dissolved in the NaBD
fraction (Pettersson et al. 1988, Jensen and Thamdrup 1993),
the proportion of HCl-P would be even greater. HCl is assumed
to extract P from the surfaces of apatite minerals and dissolve
some other Ca-containing minerals. The very high Ca/P ratio
in the HCl extracts (Fig. 2) suggests that carbonates may be
dominant over apatite. Simultaneous dissolution of Mg points
to the presence of some dolomite. The calcareous nature of the
reference material might cause an artifact. Benzing and
Richardson (2005) have confirmed the proposal of De Groot
and Golterman (1990) that the use of NaOH before HCl in a
fractionation sequence overestimates HCl-P in calcareous
material. The reason for this is that Ca is dissolved, and Aland Fe-bound P is transferred from the NaOH step. It is likely
that the Al and Fe extracted in HCl were originally central
cations exposed to extractant on the edges of mineral lattices.
P in the residual fraction is presumed to be mostly organic
(Saunders et al. 1955, Aspila et al. 1976). However, this fraction may also contain some inorganic P (Ruttenberg 1992),
originating, perhaps, from occluded P, especially if the sediment is high in Fe and Mn.
Concomitant dissolution of Si with Al, Mg, and Fe in the
residual fraction (Fig. 2) provides evidence of disruption of lattice minerals. The high TdissCa in HCl (step 4) compared with
that in the residual fraction supports the previous conclusion of
the calcareous nature of the reference material. Negligible concentration of P left in the extracted sediment residue after total
digestion reveals that the fractionation method was efficient in
P removal. However, the P concentration obtained in the total
digestion of the sample exceeded the sum of P extracted in separate fractions, indicating that some P was lost during the multiphase extraction procedure.
Comparison of the P forms separated here by the Jensen and
Thamdrup (1993) procedure with the certified P forms (the
SMT method, Ruban et al. 2001) for the reference material is
complicated by the differences in the extractants and the
extraction protocols (sequential extraction versus independent
steps). Some comparisons can be made, however. The total
extractable P obtained in our fractionation (41 µmol g–1 DW) is
close to the certified value for concentrated HCl-extractable P
441
Lukkari et al.
Fractionation of phosphorus I
(44 µmol g–1 DW). Furthermore, our sum of the inorganic fractions (NaCl-, NaBD-, NaOH- and HCl-iP amounting to 33 µmol
g–1 DW) and the total organic P (total NRP and Res-P amounting to 9 µmol g–1 DW) is close to the certified inorganic P
(36 µmol g–1 DW) and organic P (7 µmol g–1 DW). The agreement indicates that the 2 procedures with their different steps
nevertheless result in a closely similar outcome. The European
Commission’s Standards, Measurements and Testing Programme cited easy laboratory work and good reproducibility as
the advantages of the SMT procedure (Ruban et al. 1999, 2001).
However, the Jensen and Thamdrup (1993) procedure provides
more detailed information on P forms of interest in risk assessment of internal P loading: that is, on mobile and immobile
organic P and redox-sensitive P. Moreover, reagents are more
dilute, disturbing the material less and thus giving more reliable estimates for the bioavailability of sediment P.
Discussion
The P fractionation procedure tested, with the described
analytical practices, appears to give a reliable characterization
of the different binding forms of P in sediments. Results for P
and co-occurring elements obtained in extractions of commercial reference material provided good evidence for the
validity of the fractionation scheme. The sequential and multiphase nature of the procedure easily results in some loss of
the sample material, and careful laboratory work is required to
obtain repeatable results.
With the many types of naturally occurring P classified via
only a few extraction steps, chemical fractionation methods
cannot be more than an approximate approach. Still, determination of the chemical character of sediment P allows a general estimate of the portion of sediment P that, under certain
environmental conditions, can become available for primary
production in marine environment. At the same time, sequential extraction provides useful information on the proportions
and forms of P that do not participate in the nutrient cycle but
are buried with deposited material.
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Submitted 4 July 2006
Revised 23 August 2007
Accepted 11 September 2007