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Comparison between sequential extraction procedures and

single extractions for metal partitioning in sewage sludge
samples

A. Fernández Alborés, B. Pérez Cid, E. Fernández Gómez and E. Falqué López*

Depto. Quı́mica Analı́tica y Alimentaria, Universidad de Vigo, Facultad de Ciencias de

Ourense, As Lagoas s/n, 32004 Ourense, Spain. E-mail: efalque@uvigo.es
Published on 06 June 2000. Downloaded by Portland State University on 16/09/2014 12:33:32.

Received 13th March 2000, Accepted 10th May 2000

Published on the Web 6th June 2000

The conventional Tessier and BCR sequential extraction methods were applied to metal partitioning in sewage
sludge samples collected from a waste water treatment plant. The results obtained by sequential extraction
methods for Cu, Cr, Ni, Pb and Zn were compared with those estimated from single extractions using identical
operating conditions applied in each individual Tessier and BCR fraction. The metal content in the extracts was
measured by flame atomic absorption spectrometry. In all fractions of the BCR method, and in the three first
fractions of the Tessier method, the results obtained from single extractions were in good agreement with those
obtained using the sequential procedures. The oxidizable fraction of the Tessier method should be leached from
the solid residue of the reducible extraction, since a very low extraction efficiency was obtained for this fraction
when it was directly extracted. The total extractable metal contents obtained by both single and sequential
extractions were similar for all metals in the BCR method and for Cr, Ni and Pb in the Tessier method. The
recoveries obtained ranged from 93.52 to 105.8% in the two studied samples. For Cu and Zn the overall extraction
efficiency of the proposed method was slightly lower than that obtained with the sequential procedure (recoveries
around 90%). The precision of the proposed Tessier and BCR single extraction methods (expressed as RSD) was
lower than 8% for all metals.

Introduction The application of sequential extraction procedures provides

Metal accumulation in soils, sediments and sludge samples
poses an environmental problem concerning possible metal
transfer from these samples to aquatic media as well as their
uptake by plants, thereby including them in the food chain. In
order to estimate the reliable bioavailability of metals and their
potential toxicity it is desirable not only to determine the total
concentration but also the different chemical forms or ways of
binding between trace metals and solid phases of the sample.1
Although the separation of various chemical forms of heavy
metals is very difficult, the use of sequential extraction methods
in this way provides an important approach. Therefore, several
sequential extraction schemes2,3 have been developed to predict
the metal distribution among different fractions, usually
including exchangeable, associated to carbonates, associated to
Fe–Mn oxides (or reducible), associated to organic matter and
sulfides (or oxidizable) and residual.
Despite some limitations associated with the use of sequential
extraction methods4–6 (re-adsorption among phases during the
extraction, poor reagent selectivity, extraction highly affected
by the operating conditions, difficulty of comparing results
from different methods, etc.), they have been widely accepted
for metal fractionation in different samples, in order to predict
the mobility and possible bioavailability of metals in the
environment. A large number of sequential extraction proce-
dures can be found in the literature since the 1970s, many of
which are variants on that proposed by Tessier et al.7 The most
recent sequential extraction method has been developed by the
Community Bureau of Reference (BCR)8 as an attempt to
harmonize different methodologies of the extraction process.
This method has also been widely accepted and applied to metal
fractionation in sediment9–11 and soil9,12–14 samples. Moreover,
in this case, a sediment reference material was prepared and
certified for extractable content of heavy metals.15
relevant environmental information on polluted samples. How-
ever, it is important to emphasize that they are tedious and time
consuming procedures, which are little used for routine
analysis. This problem has also been noted by other researchers,
who have published papers focused on reducing the lengthy
treatment time, and replacing the conventional procedures by
other alternatives, such as microwave heating16–20 and ultra-
sonic shaking.21 Another attractive approach consists in
replacing the sequential stepwise extractions by single step
extractions with the same reagents and operating conditions as
the sequential procedure, but using a separate aliquot of sample
for each reagent. The last option has been proved by Tack et
al.22 with the Tessier method on a sediment sample. However,
it is desirable that more information be obtained from the
literature on this method, establishing the behaviour of other
sequential extraction procedures and other environmental
samples (i.e. soils, sewage sludge, etc.).
Therefore, the main objective of this work was to compare, in
sewage sludge samples, the results obtained using the conven-
tional Tessier and BCR sequential extraction methods with
those achieved from single extractions, employing similar
operating conditions to the corresponding Tessier and BCR
individual steps. The contents of Cu, Cr, Ni, Pb and Zn in the
extracts were measured by flame atomic absorption spectrome-
try (FAAS). The advantages and drawbacks of the two
simplified procedures were considered and the influence of
sample pre-treatment was also evaluated.
Experimental
Instrumentation
Metal determination in the extracts was carried out by means of
a Perkin-Elmer Model 2380 double-beam atomic absorption

DOI: 10.1039/b001983f Analyst, 2000, 125, 1353–1357 1353

This journal is © The Royal Society of Chemistry 2000
 View Article Online

spectrometer. Hollow cathode lamps were used as radiation
source. Resonance lines employed were 324.8, 357.9, 230.0,
217.0 and 213.9 nm for Cu, Cr, Ni, Pb and Zn, respectively.
Lamp intensity and bandpass width were used according to the
manufacturer’s recommendations. The air–acetylene flow rate
was 11–1 l min21 for all the elements, except for Cr where a
more reducing flame was required (11–2 l min21). A domestic
cutter (Model Nevir) was used for fast reduction of sample
particle size. A centrifuge (Kubota 5100) was used for complete
separation of the extracts. A Crison 2000 pH-meter was used for
pH adjustment of the extracts.
mm). The reagents and operating conditions for these methods
are summarized in Table 1. The extraction was carried out in 50
ml polyethylene tubes, which were also used for centrifugation
to minimize the possible loss of solid in the centrifuge–washing
steps. After each extraction step the supernatant liquid was
separated from the solid phase by centrifugation at 2500 rpm for
5 min. It was then decanted into polyethylene vessels and stored
at 4 °C before analysis. The remaining residue was washed with
10 ml of doubly distilled water, with shaking for 15 min, and the
washings were discarded after centrifugation.

Single extractions
Reagents
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Single extractions were carried out by employing a separate

The extractant solutions (listed in the protocol of the Tessier and
BCR schemes) were prepared from analytical-reagent grade
reagents (Merck). Stock metal solutions (1000 mg ml21) for
atomic absorption spectrometry were obtained by dissolving the
pure metal (for Cu and Zn) or the appropriate salts (for Pb, Ni
and Cr) and making up to a corresponding volume with
ultrapure water. Standard calibration solutions were made daily,
by appropriate dilution of the stock solutions. The hydrox-
ylammonium chloride was prepared prior to use. Dilute nitric
acid and ammonia solutions were used for pH adjustments.
Sample collection and pre-treatment
Sewage sludge samples were collected in polyethylene contain-
ers from an urban waste water treatment plant located in the
town of Ourense (Spain). The samples were dried at 110 °C in
a heater until a constant weight was achieved, after which one
part of the sample was ground using an agate mortar and the
other using a Nevir cutter equipped with metallic blades. Then,
the sample was sieved using a nylon-fibre sieve and the sludge
with a particle size < 70 mm was separated, homogenized and
stored in polyethylene bottles in a desiccator at room tem-
perature. Both the Tessier and BCR sequential extraction
procedures were applied to sludge samples prepared using both
the mortar and Nevir cutter, in order to establish the effect of the
latter on the extractability of metals.
Sequential extraction schemes
The Tessier7 and BCR8 sequential extraction methods were
applied, in triplicate, to 2 g of pre-treated sludge sample ( < 70
aliquot of sample (2 g) for each individual reagent and using the
same operating conditions listed in Table 1. Centrifugation and
storage of extracts was performed as in the sequential extraction
schemes. However, in this case, the solid residue was also
rejected. The main advantage of this proposed procedure is that
all fractions can be simultaneously extracted and, consequently,
it supplies faster results than the sequential methods. In contrast,
a larger sample amount is needed, which does not pose an
additional problem when environmental samples are being
studied.
With the exception of the first fraction of the two methods,
the metal content corresponding to each individual fraction was
calculated by subtracting the results obtained in two consecutive
stages. For example, to estimate the ‘carbonate fraction’ in the
Tessier method from single extractions, the amounts of metal
extracted with MgCl2 were subtracted from those extracted with
the NaOAc reagent. The other fractions were obtained in a
similar way. In all cases, metal recovery was calculated as the
following ratio: [metal content using single extraction/metal
content using sequential extraction] 3 100.
Results and discussion
Effect of sample pre-treatment on the extraction efficiency
The two sequential extraction procedures (Tessier and BCR)
were applied to pre-treated sewage sludge sample (A) following
two different procedures: using an agate mortar and cutting with
a domestic Nevir cutter. The extracts obtained were analysed for
Cu, Cr, Ni, Pb and Zn. The results of this study, expressed in mg

Table 1 Operating conditions required in the conventonal Tessier and BCR sequential extraction methods

Shaking time
Stage Fraction Reagent and temperature

Tessier method—
1 Exchangeable 8 ml 1 mol dm23 MgCl2 (pH = 7) 1 h at 25 °C
2 Associated to carbonates 8 ml 1 mol dm23 NaOAc (pH = 5) 5 h at 25 °C
3 Associated to Fe–Mn oxides (or reducible) 20 ml 0.04 mol dm23 NH2OH·HCl in 25% m/v HOAc 6 h at 96 °C
4 Associated to organic matter (or oxidizable) 3 ml 0.02 mol dm23 HNO3 5 ml 30% m/v H2O2 2 h at 85 °C
+
3 ml 30% m/v H2O2 3 h at 85 °C
+
5 ml 3.2 mol dm23 NH4OAc 30 min at 25 °C
5 Residual HNO3–HCl–HF 26 min
BCR method—
1 Acid-soluble (e.g. carbonates) 20 ml 0.11 mol dm23 HOAc 16 h at 25 °C
2 Reducible (e.g. Fe–Mn oxides) 20 ml 0.1 mol dm23 NH2OH·HCl (pH = 2) 16 h at 25 °C
3 Oxidizable (e.g. organic matter) 5 ml 30% m/v H2O2 (Evaporation) 1 h at 25 °C 1 h at 85 °C
+
5 ml 30% m/v H2O2 (Evaporation) 1 h at 85 °C
+
25 ml 1 mol dm23 NH4OAc 16 h at 25 °C
4 Residual HNO3–HCl–HF 26 min

1354 Analyst, 2000, 125, 1353–1357

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g21, for the two methods are shown in the partitioning patterns
for Fig. 1.
According to the results, it is possible to conclude that
contamination from the metallic blades of the cutter was
undetectable for all metals in the extractable fractions of the
Tessier and BCR procedures. A similar behaviour was also
observed for Cu, Ni, Pb and Zn in the residual fraction. In this
case, the Cr content obtained using the metallic cutter was
considerably higher than that obtained using the agate mortar.
This means that the increasing amount of Cr formed on the
blades of the cutter in the residual fraction did not improve the
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extractable content of this element in the sample. In contrast,
when the total metal content is required the agate mortar should
be used.
Metal partitioning using the conventional Tessier and
BCR methods and that estimated from single extractions
Extractable metal contents in sludge samples A and B, applying
both the conventional Tessier method and single extractions, are
shown in Table 2. By analogy, the results corresponding to the
BCR method are summarized in Table 3. In both cases, all data
are reported as mean values (mg g21) ± standard deviation of
three separate extractions.

Tessier method. The exchangeable fraction constitutes the

Fig. 1 Partitioning patterns corresponding to the Tessier (a) and BCR (b)
methods in a sewage sludge sample. M = Agate mortar; C = domestic
cutter.
first step of the Tessier sequential method and consequently it is
always directly extracted. In this fraction all metals were
quantified except for Cr and Pb in sample A and Cr in sample
B.
In the carbonates associated fraction, the metal content
estimated from single extraction was in good agreement with
that obtained using the sequential procedure. Hence, in the two
studied samples, the recoveries ranged from 94.31 to 102.8%
for all metals, except for Cr. The last element was not apparent
at a quantifiable level in this fraction.
The reducible fraction estimated from single extraction
displayed, for most of the metals, similar results to those
obtained using the sequential procedure. The recoveries ob-
tained vaired between 94.31 and 98.27% for Cr, Ni, Pb and Zn
in sample A and between 99.69 and 102.4% for Ni, Pb and Zn
in sample B. Cr was not detectable in the latter sample. In
relation to Cu, a similar behaviour was found for this element in
the two samples. It was more highly leached using the
sequential procedure than using the single extraction method. In
both cases, a negative concentration was obtained for this
element when the proposed approach was employed.
The oxidizable fraction was also evaluated by single
extraction. These results have not been included in Table 2
since, for all metals, the extraction efficiency of the sequential
procedure was considerably higher than that obtained using the
simplified approach. The same effect was also noted by Tack et
al.22 in sediment samples. By taking into account that, except
for Cu, the reducible fraction could be estimated using the
proposed method it should be possible to evaluate the
oxidizable fraction from the remaining residue after the
NH2OH·HCl individual extraction. The results obtained for the
oxidizable fraction following the proposed two-step procedure
are shown in Table 2. The recoveries obtained for all metals

Table 2 Results obtained for sludge samples A and B (expressed in mg g21) using both the conventional Tessier method and single extractions

Exchangeable fraction Associated to carbonates fraction Associated to Fe–Mn fraction Associated to organic matter fractiona

Sequential Single Recovery Sequential Single Recovery Sequential Single Recovery Sequential Single Recovery
extraction extraction (%) extraction extraction (%) extraction extraction (%) extraction extraction (%)

Sample A—
Cu 16.90 ± 0.11 16.90 ± 0.11 100.0 9.84 ± 0.12 9.28 ± 0.56 94.31 2.12 ± 0.16 218.37 ± 0.62 b 286.6 ± 4.3 292.8 ± 3.3 102.2
Cr NDc ND — ND ND — 1.79 ± 0.11 1.75 ± 0.10 97.76 3.35 ± 0.21 3.40 ± 0.22 101.6
Ni 2.61 ± 0.09 2.61 ± 0.09 100.0 1.37 ± 0.09 1.36 ± 0.10 99.27 4.04 ± 0.00 3.97 ± 0.13 98.27 3.93 ± 0.33 4.08 ± 0.30 103.8
Pb ND ND — 3.06 ± 0.23 3.10 ± 0.24 101.3 41.69 ± 0.45 40.09 ± 0.81 96.16 9.49 ± 0.6 8.98 ± 0.52 94.63
Zn 29.84 ± 0.20 29.84 ± 0.20 100.0 77.13 ± 0.48 74.60 ± 1.08 96.72 488.1 ± 9.5 460.3 ± 13.1 94.31 84.83 ± 1.00 90.44 ± 6.56 106.6

Sample B—
Cu 18.72 ± 0.02 18.72 ± 0.02 100.0 10.54 ± 0.16 10.20 ± 0.29 96.77 12.62 ± 0.57 213.25 ± 0.77 b 396.2 ± 0.3 393.9 ± 2.9 99.35
Cr ND ND — ND ND — ND ND — 6.70 ± 0.18 6.59 ± 0.28 98.36
Ni 2.31 ± 0.01 2.31 ± 0.01 100.0 2.58 ± 0.09 2.50 ± 0.170 96.90 3.28 ± 0.24 3.36 ± 0.20 102.4 7.11 ± 0.49 7.12 ± 0.15 100.1
Pb 3.40 ± 0.17 3.40 ± 0.17 100.0 5.69 ± 0.18 5.85 ± 0.40 102.8 22.12 ± 0.92 22.40 ± 0.42 101.3 1.55 ± 0.12 1.52 ± 0.09 98.06
Zn 39.31 ± 0.87 39.31 ± 0.87 100.0 54.77 ± 0.50 53.38 ± 1.02 97.46 546.7 ± 2.5 545.1 ± 5.4 99.69 131.2 ± 3.4 131.3 ± 0.9 100.0
aThis fraction was obtained from the remaining residue after the extraction of the reducible fraction in a separate sub-sample. b Negative recovery value was
obtained. c ND = Not determined.

Analyst, 2000, 125, 1353–1357 1355

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ranged from 94.61 to 106.6% in sample A and from 98.06 to
100% in sample B. Hence, it is possible to conclude that the
two-step simplified procedure provides similar results to the
four-step Tessier method.
BCR method. Likewise, the exchangeable fraction in the
Tessier method (the acid-soluble fraction) is the first extractable
fraction of the BCR method and is also always extracted using
an individual sub-sample of the two studied samples; all metals
were quantified in this fraction, except for Cr and Pb.
In the reducible fraction, Zn was the only detectable element
when the sequential procedure was employed. However, it was
leached in a small amount when it was estimated from single
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account the specific treatment with H2O2 in both cases. Thus,
the treatment with H2O2, in the BCR method, could be
considered more effective than that employed in the Tessier
method (shaking for 5 h at 85 °C), since it is carried out twice
with complete evaporation of the reagent. For this reason, the
direct oxidizable fraction of the BCR method can be considered
representative of acid-soluble plus reducible plus oxidizable
metals, but this is not the case in the Tessier method.
Total extractable metal contents obtained from the sequential
Tessier and BCR methods together with those evaluated by
single extractions are shown in Table 4. As can be observed, in
the Tessier method the overall metal content leached from both
compared extraction procedures was equivalent. The recoveries
obtained for all metals ranged from 95.29 to 101.3% in the two

extractions, so the results were considerably lower than those

obtained using the conventional procedure. In relation to Cu,
this element was not extracted at quantifiable levels employing
the sequential procedure. Moreover, negative concentration
values were found in the two studied samples using the
simplified procedure, which means that this element was poorly
extracted when the treatment with NH2OH·HCl was carried out
on an individual sub-sample. In contrast, similar results were
obtained for Cr,Ni and Pb from both the compared methods. On
comparing these results with those previously reported from the
same fraction of the Tessier method, it is possible to conclude
that better results were obtained for the reducible Tessier
fraction. The different behaviour of the direct reducible fraction
in the two methods (Tessier and BCR) could be attributed to the
distinct composition of the extracting agent employed in each
case. Hence, whereas in the Tessier method the hydroxylamine
is mixed with HOAc (25% m/v), this is not the case in the BCR
method. The addition of acetic acid to the hydroxylamine could
increase the extraction efficiency of the latter, and consequently
allow the overall extraction of the exchangeable, carbonate-
bound and reducible metals when the two first fractions were
not previously leached using the sequential procedure. In
contrast, the extraction efficiency of hydroxylamine alone in the
BCR method was worse.
Unlike the Tessier method, the oxidizable BCR fraction
estimated from single extractions provides, for all metals,
equivalent results to those obtained using the sequential
procedure. The recoveries achieved varied between 93.58 and
100.5% in sample A and between 94.44 and 108.2% in sample
B. In this fraction the opposite effect to that previously found in
the reducible fraction was observed. In this case, the BCR
fraction can be directly leached; however, the Tessier fraction
must be estimated from the solid residue of the reducible
fraction (two-step procedure), given that a low extraction
efficiency was obtained when it was leached directly. In our
opinion, these peculiar results can be explained by taking into
studied samples. For the BCR method, sequential and single
extractions also supplied a similar overall extractable content
for Cr, Ni and Pb (recoveries between 93.52 and 105.9%) in the
two samples. For Cu and Zn, the global extraction achieved with
the simplified procedure was slightly lower than that provided
by the conventional method (recoveries around 90%), except
for Zn in sample B, where the recovery achieved was close to
100%. According to the above, it is possible to state that the
environmental information obtained from the single and
sequential extraction methods is virtually the same.
The precision of the proposed single extraction approach was
evaluated in terms of RSD (n = 3). The values obtained were
similar to those provided by the Tessier and BCR sequential
extraction methods and always lower than 8% for all metals in
the two studied samples.
The total metal content in the two studied sewage sludge
samples (A, B) was determined by using a microwave
procedure optimized in previous work.23 The results obtained
for sample A were 442.74 ± 4.35, 21.55 ± 3.17, 37.32 ± 2.64,
396.39 ± 12.07 and 882.44 ± 7.19 for Cu, Cr, Ni, Pb and Zn,
respectively. The corresponding results obtained for the same
elements in sample B were 480.95 ± 28.36, 37.47 ± 6.98, 44.00
± 1.19, 490.17 ± 1.82 and 1065.99 ± 20.34. In all cases, the
results are expressed as mean values (mg g21) ± standard
deviation of three separate determinations.
Conclusions
The application of sequential extraction methods to environ-
mental samples provides relevant information about possible
toxicity when they are discharged into the environment. The
long treatment time required in these procedures could be
shortened by replacing the sequential treatment by single
extractions.

Table 3 Results obtained for sludge samples A and B (expressed in mg g21) using both the conventional BCR method and single extractions

Acid-soluble fraction Reducible fraction Oxidizable fraction

Sequential Single Recovery Sequential Single Recovery Sequential Single Recovery

extraction extraction (%) extraction extraction (%) extraction extraction (%)

Sample A—
Cu 22.01 ± 0.58 22.01 ± 0.58 100.0 NDa 214.82 ± 0.65 b 269.9 ± 1.5 256.1 ± 1.9 94.89
Cr ND ND — ND ND — 7.79 ± 0.39 7.29 ± 0.42 93.58
Ni 2.89 ± 0.22 2.89 ± 0.22 100.0 ND 0 ± 0.43 — 7.93 ± 0.65 7.97 ± 0.62 100.5
Pb ND ND — ND ND — 15.12 ± 0.34 14.67 ± 1.00 97.02
Zn 34.12 ± 0.69 34.12 ± 0.69 100.0 64.39 ± 0.82 7.09 ± 0.40 9.08 474.7 ± 3.4 469.2 ± 2.5 98.84
Sample B—
Cu 25.77 ± 0.32 25.77 ± 0.32 100.0 ND 219.08 ± 0.34 b 366.8 ± 8.74 346.4 ± 5.17 94.44
Cr ND ND — ND ND — 9.38 ± 0.74 9.92 ± 0.49 105.7
Ni 2.90 ± 0.21 2.90 ± 0.21 100.0 ND 0.04 ± 0.23 — 7.86 ± 0.40 7.59 ± 0.49 96.56
Pb ND ND — ND ND — 1.62 ± 0.43 1.63 ± 0.38 100.6
Zn 40.33 ± 1.04 40.33 ± 1.04 100.0 47.43 ± 1.17 21.96 ± 2.64 b 617.5 ± 14.9 668.2 ± 3.64 108.2
a ND = Not determined. b Negative recovery value was obtained.

1356 Analyst, 2000, 125, 1353–1357

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Table 4 Total extractable metal content obtained for sludge samples A and B by the conventional Tessier/BCR methods and using single extractions

Tessier method/mg g21 BCR method/mg g21

Sequentiala Singlea Recoveryc Sequentialb Singleb Recoveryc

extraction extraction (%) extraction extraction (%)

Sample A—
Cu 315.5 ± 4.2 300.6 ± 3.4 95.29 291.9 ± 1.6 263.3 ± 2.1 90.19
Cr 5.14 ± 0.22 5.15 ± 0.24 100.19 7.79 ± 0.39 7.29 ± 0.42 93.58
Ni 11.95 ± 0.35 12.02 ± 0.35 100.58 10.82 ± 0.69 10.86 ± 0.82 100.4
Pb 54.24 ± 0.78 52.17 ± 0.99 96.18 15.12 ± 0.34 14.67 ± 1.00 97.02
Zn 679.9 ± 9.6 655.2 ± 14.7 96.36 573.2 ± 3.6 510.4 ± 2.71 89.04
Sample B—
Cu 538.1 ± 0.7 408.9 ± 3.0 93.36 392.6 ± 8.7 353.1 ± 5.2 89.84
Cr 6.7 ± 0.18 6.59 ± 0.28 98.36 9.38 ± 0.74 9.92 ± 0.49 105.8
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Ni 15.28 ± 0.55 15.29 ± 0.56 100.1 10.76 ± 0.45 10.53 ± 0.48 97.86
Pb 32.76 ± 1.04 33.17 ± 0.52 101.3 1.62 ± 0.10 1.63 ± 0.09 100.6
Zn 771.9 ± 4.34 769.1 ± 6.85 99.62 705.3 ± 15.0 706.3 ± 3.8 100.2
a Sum of metals extracted in the four stages of the Tessier method (expressed in mg g21). b Sum of metals extracted in the three stages of the BCR method

(expressed in mg g21). c Recovery is calculated using the following ratio: [total metal content extracted by the sequential procedure/total metal content
extracted using the proposed single extraction method] 3 100.

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