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Speciation of Heavy Metals in Soils

and Sediments. An Account of the
Improvement and Harmonization of
Extraction Techniques Undertaken
Under the Auspices of the BCR of
the Commission of the European
Communities
a b c b
A. M. Ure , Ph. Quevauviller , H. Muntau & B. Griepink
a
Dept of pure and applied chemistry, University of Strathclyde,
295 Cathedral St., UK-Glasgow, G1 1XL
b
Commission of the European Communities, Community Bureau
of Reference (BCR), 200 rue de la Loi, B-1049, Brussels
c
Commission of the European Communities, Joint Research
Centre, I-21020, Ispra, Varese
Version of record first published: 24 Sep 2006.

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of Heavy Metals in Soils and Sediments. An Account of the Improvement and Harmonization
of Extraction Techniques Undertaken Under the Auspices of the BCR of the Commission of the
European Communities, International Journal of Environmental Analytical Chemistry, 51:1-4,
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SPECIATION OF HEAVY METALS IN SOILS

AND SEDIMENTS. AN ACCOUNT OF THE
IMPROVEMENT AND HARMONIZATION OF
EXTRACTION TECHNIQUES UNDERTAKEN
UNDER THE AUSPICES OF THE BCR OF THE
COMMISSION OF THE EUROPEAN
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COMMUNITIES

A.M. U E * , PH. QUEVAUVILLERt, H. MUNTAUS and B. GRIEPINKt

*Dept ofpure and applied chemistq University of Strathclyde, 295 Cathedral St.
UK-Glasgow, GI 1AZ tComrnission of the European Communities, Community Bureau
of Reference (BCR), 200 rue de la Loi, 8-1049 Brussels #Commissionof the European
Communities,Joint Research Centre, I-21 020 Ispra (Varese)
(Received in finalform, I7 July 1992)

An account is presented ofa series of investigationsand collaborative studies, initiatedby BCR,on current methods
of metal speciation by extraction of soils and sediments with chemical reagents. It was established by extensive
consultation with European experts that the diverse procedures used could be harmonized into agreed methods.
These methods, including both single extractant and sequential extraction procedures were subjected to collabora-
tive, interlaboratory trials and the results, presented briefly here, showed that it was both possible and desirable
that reference soils and sediments, characterised by certified values for extractable contents, be prepared. As a
consequenceof these studiestwo soils have been prepared and will shortlybe the subject of interlaboratoryanalysis
with a view to certification of their EDTA and acetic acid extractable contents of some heavy metals. Following
this workshop a feasibility study of the agreed sequential extraction procedure will, it is believed, shortly lead to
certification of sediments for contents extractable by a defined sequential extraction procedure.

KEY WORDS: Speciation, heavy metals, extractable contents, single extraction methods, sequential extraction
methods, soils, sediments, reference materials, BCR.

INTRODUCTION

Some thirty years ago, the Macaulay Institute for Soil Research in Aberdeen was approached
by NIST, then the National Bureau of Standards,as to the feasibility ofpreparing a reference
soil certified for its extractable contents as distinct from its total contents. In view of A.
135
 136 A. M.URE et al.

Ure’s opinion then that the difficulties of preparation, stability and analysis were too great,
it is ironic that he should now be describing the approach to doing precisely that. The
difficulties foreseen then were real enough but the power of the collaborative approach
adopted by BCR in these current studies is evidenced by the considerable progress now
made in overcoming them.
An initial study of the literature on the speciation of metals in soils and sediments, mainly
by chemical extraction procedures, and a consultation with European experts was carried
out by the first author on behalf of BCR. The objectives of this study were to determine
whether or not there was a need for a BCR involvement in improving and harmonizing the
methodology for speciation in these materials. This study was discussed at a meeting of
representatives of leading European soil and sediment laboratories with BCR in Brussels in
1987 and the following recommendationswere made for action under the auspices of BCR
1) that an interlaboratorytrial analysis of soil and sediment extracts be carried out using
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both single and sequential extraction procedures, but mainly single extraction for soil and
sequential extraction for sediment,
2) the elements to be Cd, Cr, Cu, Ni, Pb and Zn
3) the soil to be a sludge-amended,<2 mm, air-dried unground soil,
4) the sediment to be selected from materials available from the CEC Joint Research
Centre in Ispra, Italy
5 ) a long and a short sequential extraction scheme be tried,
6) provided that adequatemethodologieswere available or were made so by these studies,
there was both a need and a demand for soil and sediment reference materials whose
extractable contents were certified by such extraction procedure.

SPECIATION DEFINITION

One of the first tasks was the clarification of the term “speciation” whose use had become
widespread with little attempt to define its meaning I . Chemical speciation, in the context
of soils and sediments was defined in the past as:
1) the process of identifying and quantifying the different defined species, forms or
phases present in a material. The “species” are further defined:
a) functionally,
b) operationally or
c) as specific compounds or oxidation states of an element.
2) speciation can also be defined in a similar way as the description of the amounts and
kinds of species, forms and phases present.
The “species” can be defined, as above,
a) functionally, as for example as “plant-available species” as “mobile forms” or as
“exchangeable cations”, or defined,
b) operationally,by the procedures, reagents or extractantsused to isolate them; examples
include the physical isolation of a soil solution or a particle size fraction, or the use of acid
ammonium oxalate to extract metals associated with “moderately reducible” soil or sedi-
ment components.
 SPECIATION OF HEAVY METALS 137

More recently, the term “speciation” has been defined as the determination of a specific
form (monoatomic or molecular) or configuration in which an element can occur or to a
’.
distinct group of atoms consistently present in different matrices In this paper, the term
speciation will be used although it is understood that the term “extractable trace metals”
related to a defined reagent should be used instead.

SINGLE EXTRACT ANTS-FUNCTIONALLY DEFINED SPECIATION

Speciation,in the functionallydefined sense, of plant-available species, or at least of species

that are correlated with plant content or uptake, has been widely used in agriculture long
before the term speciationwas invented. Its role was mainly in the prediction and assessment
of trace element deficiency or toxicity in crops or in animals eating them by the analysis of
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soil extracts.
A large number of single extractants for soils have been evolved. These have mostly been
empirically derived but validated by field experiments correlating plant contents with
extractable soil contents. These include water (hot) for B, EDTA and DPTA for Cu and Zn,
acetic acid for Co and Ni, mixed ammonium acetateEDTA for Cu and Zn, ammonium
acetate for Mo, and weak neutral salt solutions such as calcium chloride, sodium and
ammonium nitrate for Cd and Pb. While there are very many other extractants, perhaps
mention should be made of the soil solution, or sediment pore water, itself which could be
included in this category but which suffers, like the weak neutral salt extracting solutions,
from analytical difficulties because of the low element concentrations present.
While it is apparent that many of these extractants quantify species that correlate with
the plant-available forms they tend to be element-or crop-specific. In addition significant
methodological variations exist within nominally identical procedures as they are applied
in different laboratoriesand countries. Consultation with expert opinion in Europe indicated
that the most generally acceptable of these functionally defined extractants were:
a) EDTA 0.05 moVl or DTPA 0.005 moyl with similar roles but with EDTA preferred
as it extracted greater amounts and was simpler to prepare and use,
b) ammonium acetate 1 mom at pH 7 and
c) calcium chloride 0.05 moV1.
It was considered, too, that a standardisation procedure for these reagents could readily
be agreed.

OPERATIONALLY DEFINED SPECIATION

Operationallydefined speciation is often, and conveniently, equated with the quantification

of the elements in a specific phase of a soil or sediment, despite the fact that the phase may
be ill-defined or the procedure insufficiently specific. Single extractants can be used, as was
the case for functionally defined speciation, or the procedure can be made more selective
by employing several extractants in a defined sequence. Some of these sequential extraction
procedures are discussed later.
 138 A. M.URE et al.

TEMPORAL STABILITY OF EXTRACTABLE CONTENTS

One of the major hurdles to be surmounted before the preparation of a reference material
certified for extractable trace metal contents could be contemplated is the question of their
temporal stability. This problem has been studied by Salomons and Scheltens 4 , who
repeated, in 1987,the sequentialextraction and analysis of freshwater sediments first carried
out in 1975. While those authors were somewhat disappointed in that significant changes
did occur in the measured species concentrations after 12 years, it is likely that the temporal
stability was sufficient for the sediments to be characterisedon each occasion in such a way
that decisions on the management or use of the sediments would be unchanged.
A test of the stability of the extractable contents of a single soil was carried for EDTA,
acetic acid, ammonium acetate and calcium chloride extracts and the results are shown in
Table 1. It can be seen that, with the possible exception of Cr, the EDTA extractable contents
are stable over both 1 and 3 year intervals within about 10% for the elements Cd, Cu, Fe,
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Mn, Ni, Pb and Zn. For acetic acid extracts the 1 year changes for Cr, Fe, Mn and Zn are
poorer than for EDTA but for Cd, Cu, Ni and Pb the extracts are reasonably stable. For
ammonium acetate and calcium chloride extracts, however, the results, apart from Cd and
Ni in ammonium acetate, are useless. This failure in ammonium acetate and calcium
chloride, however, may not be related to temporal instability but, more probably, to the fact

Table 1 Temporal stability of soil extracts. Temporal stability over 1 year and 3 years of
EDTA-extractable contents of sludge-amendedsoil.

EDTA PERCENTAGE CHANGE PERCENTAGE CHANGE

1989-I990 1987-I990

Cd - 3.5 + 19
Cr -31 - 2.8
cu + 5.0 + 2.6
Fe + 0.2 -
Mn - 8.7 - 0.3
Ni + 5.2 -

Pb + 6.5 - 12.8
Zn + 14 - 3.0
Temporal stability over 1 year of acetic acid, ammonium acetate and calcium chloride ex-
tractable contents of sludge-amended soil.

PERCENTAGE 1989-1990 HOAc NH4OAc CaCl2

% % %

Cd + 1.4 + 7.5 + 118

Cr + 36 + 101
cu +21 + 71 + 158
Fe - 18 + 131 -
Mn + 63 + 242 + 1120
Ni + 10 - 11 + 53
Pb + 1.9 - 19 -
Zn + 19 - 27 + 100
 SPECIATION OF HEAVY METALS 139

that the solution concentrationsmeasured are too low for reliable determinationby the FAAS
of ICPOES methods used.

PRELIMINARY TRIAL OF SEQUENTIAL EXTRACTION

Four laboratories,
(a) The Technical University of Hamburg-Harburg,Germany, (U. Farstner, M. Kersten);
(b) The Institute for Soil Fertility, Haren, The Netherlands, (W. Salomons, M.N. Kerdijk
and S. Scheltens);
(c) The Central Highways Laboratory, Bougenais, France, (D. Robbe);
(d) C.E.C. Joint Research Centre, Ispra, Italy, (H. Muntau and M. Van Son),
took part in an interlaboratory trial of 3 different sequential extraction schemes with 7
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sediments and 1 soil supplied by the Joint Research Centre in Ispra.

The sequential extraction methods were:
(I) the modified Tessier procedure of FBrstner 6,
(11) the short method of Salomons and FBrstner and ’
’,
(111) that of Meguellati shown in Table 2,3, and 4.
Laboratories (a) and (d) used method (I), laboratory (b) used method (11) and laboratory
(c) method (111). In order to make comparisons of the three different methods, M.N. Kerdijk
evolved a method of grouping steps from the different methods to produce fractions that
were directly comparable. Because the method of determining the residual phase was
different in the three methods, Kerdijk’s procedure was modified so that the residual phase
was calculated from the total content. The comparable Fractions 1,2 and 3 thus obtained are
shown in Tables 2, 3 and 4 and an example of the bar charts used to display the results is

Table 2 Method ( I ) Modified tessier scheme.

STEP EXTRACTANT PHASE .!XTRACTED*

I MgC12 I moM EXCHANGEABLE

PH 7
2 NaOAc I moVl CARBONATE
PH 5
3 NH2OH.HCL 0.04 mow FeMn OXIDE
25% HOAC
4 H202 8.8 mOUI/HN03 ORGANIC + SULPHIDE
NaOAc EXTRACT
5 HF/HC104 RES1DUAL:SILICATE

FRACTION I = STEP 1 + STEP 2

(EXCHANGEABLE +CARBONATE)
FRACTION 2 = STEP 3 + STEP 4
(REDUCIBLE FeMn OXIDE + ORG/SULPHIDE)
FRACTION 3 = TOTAL - STEPS 1 + 2 + 3 + 4 (RESIDUAL)
(TOTAL = METHOD 111 STEP 5 )
* Reagents may also extract phases partly; this table just refers to commonly used names
of “forms” and does not have any scientifically proven meaning.
 140 A. M. URE et al.

Table 3 Method (11) Short method of Salomons and F6rstner.

Step Extractant Phase Extracted*

1 NH20H.HCL 0.1 moVl ACID REDUCIBLE

2 HH202 8.8 moVI; NaOAc EXT. ORGANIC
3 HFMC104 RES1DUAL:SILICATE

FRACTION 1 = STEP 1
FRACTION 2 = STEP 2
FRACTION 3 = TOTAL - STEPS (1 + 2)
* Reagents may also extract phases partly; this table just refers to commonly used names
of “forms” and does not have any scientifically proven meaning.

given for one of the sediments in Figure 1 (reproduced from ref. lo. With the exception of
one sediment the results show, as in Figure 1, that the fractional contents by the different
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procedures are in good enough agreement for the sediments to be sufficiently well charac-
terized for decisions to be made on the management or use of the sediments. There are
however serious failures in detail and it can be concluded that, while the results are
promising, improvements are necessary if reference material certification were to be
attempted. Furthermore, any future study must use a single well specified procedure.

INTERLABORATORYTRIAL OF SOIL EXTRACTION

A sewage sludge-amended soil from Great Billings Sewage farm, Northampton(by courtesy
of Anglian Water) was collected, air-dried, sieved and some 12 kg of the <2 mm soil
homogenised and bottled in 100 g lots for an interlaboratory trial analysis. The large range
of particle sizes in the < 2 mm soil material normally used for extraction necessitates the
use of large (5-20 g) subsamplesto ensure representative sampling of the bulk material for
extraction and analysis. This posed the not trivial problem of homogenisation of the bulk
material and the determinationby experiment of the minimum sample size required for each
extraction and analysis and the verification that each sample bottle was statistically repre-
sentative of the whole.
Homogenisation of the bulk soil was achieved by rolling it in a polyethylene bag. The

Table 4 Method (111) Meguellatti.

Step Extractant Phase Extractant

BaC12 1 moM, pH 7 EXCHANGEABLE

H202 8.8 moV1, HNo3 ORGANIC + SULPHIDE
NaOAc 1 mom PH 5 CARBONATE
NH2OH.HCIO.1 moVl Fe/Mn OXIDE
25% NaOAc
5 ASIUHFMCI RESIDUAL:SILICATE

FRACTION 1 = STEPS (1 + 3 + 4)
FRACTION 2 = STEP 2
FRACTION 3 = TOTAL - STEPS (1 + 2 + 3 + 4)
* Reagents may also extract phases partly; this table just refers to commonly used names
of “forms” and does not have any scientifically proven meaning.
 SPECIATION OF HEAVY METALS 141
ca Z”

oao- I

064 -

0.6 -

032.

016-

O W - Ir
-1-B
A C D
A B C
-2- FR*CTIQ*

zn
D A
-3-
B C D

ERACTW

Pb
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Figure 1 Bar-chart comparison of Fractions 1,2 and 3 (and total contents) calculated for three different sequential
extraction procedures, Methods (I), (11) and (Ill), applied to sediments I , 2, 3, 4, 6, 7 and 8. Results for four
laboratories A, B, C and D are presented. Laboratories A and D used Method (I), B used Method (111) and C used
Method (11).

whole material was subsampled into 100 bottles, each containing approximately 100 g of
soil, by coning and quartering. It was determined that a 5 g subsample, taken from each
bottle by coning and quartering the whole contents of the bottle was representative of the
whole material. By analysing the EDTA 0.05 moYl and the ammonium acetate 1 moVl
extractable trace metal soil contents from a number of subsamples taken from different
bottles and the same number from a single bottle the “between bottle” and the “within bottle”
CVs respectively were obtained as shown in Table 5. Only a small decrease in reproduc-
 142 A. M. URE et al.

Table 5 Homogeneity test on Great Billings Soil.

EDTA 0.05 moln NH4OAc I moln

Between bottle Within bottle Between bottle Within bottle
Mean cv Mean CV Mean CV Mean CV
PgkJ % P€!k % Pgk % Pgk 96

Cd 21.4 2.5 20.9 0.4 2.36 3.8 2.44 2.2

Cr 9.9 2.4 9.7 0.4 1.36 13 1.66 5.4
cu 134 2.4 132 1 .o 5.32 6.0 5.76 1.6
Fe I580 2.7 1580 0.5 6.98 52 9.64 2.4
Mn 87.0 2.4 86.5 0.6 12.7 2.9 13.0 1.2
Ni 13.5 1.4 13.1 1.8 0.88 9.5 0.86 6.4
Pb 205 1.8 202 1.3 0.53 13 0.62 11
Zn 409 1.7 417 0.7 12.0 6.3 13.0 0.9
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ibility is seen on going from “within bottle” to “between bottle” CVs and in both cases they
are acceptably low for all the elements tested in the two extracts. As both temperature of
extraction and the vigour of the mechanical shaking process lo could be expected to affect
the amount of metal extracted, limits for both of these parameters were prescribed in the
detailed protocol for extraction and analysis issued to participating laboratories along with
the soil sample. As a check on each laboratory’s calibration, reference solutions containing
6 analytes,Cd, Cr, Cu, Ni, Pb and Zn were issued with the soil and analysed with the extracts.
Three types of single extractant were used, EDTA 0.05 moY1, acetic acid 0.43 moVl and
ammonium acetate 1 moYl at pH 7.
The overall mean of the individual laboratory mean extractable contents for six elements
in the three extractants are shown in Table 6. It can be concluded that, with the exception
of Cr, the CVs for the contents in the EDTA extracts are acceptably low (ca +/- 10%) and
that for this, or a similarly contaminated soil, certification of EDTA extractable contents is
practicable. While for acetic acid extracts the CVs for Ni and Pb are a little higher than for
the EDTA extracts, it is likely that certification of acetic acid extractable contents is also
feasible. For ammonium acetate extraction only Cd shows acceptable precision and it must
be concluded that certification of ammonium acetate extractable contents is not yet feasible
in this or similar soils without significant improvement in the methodology. This failure
is almost certainly due to the low element contents extracted by this reagent, as will be
discussed briefly later.

Table 6 Overall means of laboratory mean extractable contents (mgkg air dry soil).

EDTA EXTRACTS
Cd Cr cu Ni Pb Zn
Mean 23.1 8.05 162 16.3 255 492
cv Yo 9.2 25.88 8.5 13.0 11.8 5.8
ACETIC ACID EXTRACTS
Mean 19.3 26.1 29.2 15.7 3.36 522
cv % 7.5 8.2 10.0 18.1 24.6 6.9
AMMONIUM ACETATE
Mean 3.43 1.39 5.65 1.42 2.21 18.4
cv % 10.9 40.6 23.4 22.5 26.8 22.5
 SPECIATION OF HEAVY METALS 143

30
T T
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1 2 3 4 5 6 7 8 91011121517181921232527

Lab. codes

Lab. means Corr. means MEAN

between lab

Figure 2 EDTA extractable Cd content in soil (rngkg dry mass)lab. means: means of five replicate determina-
tions (means of individual laboratories).

It was a notable feature of this trial, and the sediment sequential extraction study carried
out at the same time, that calibration by the individual laboratories was frequently in error,
often by lo%, and sometimes by extremely large factors, as revealed for example by the
range of the value of the factors, F2, required by the analysis of the reference calibrant, to
normalize all the laboratory results. These factors ranged, in the case of EDTA extract
analysis, from 0.41 to 3.66 instead of the reference value of 1.O. While this calibration error
did not significantly effect the overall mean of laboratory means, in individual cases its
correction by the F2 factor made rejected values acceptable. This is illustrated for Cd in the
EDTA extracts by Figure 2 lo.

INTERLABORATORY TRIAL OF SEQUENTIAL EXTRACTION OF SEDIMENT

For this trial, run concurrently with the above soil extraction trial, a simple 3-step extraction
’,
procedure, evolved from that of Salomons and F6rstner and outlined in Table 7, was used.
The results obtained in this trial again indicatedthe importanceof checking the laboratory
calibration, as systematicerrors of 10% from this cause were commonly found and, in a few
 144 A. M. URE et 01.

Table 7 Three-step sequential extraction procedure for the sediment trial.

Step I 0.5 g sediment extracted for 5 hours with 20 ml of acetic acid 0.1 1
mobl, centrifuged and supematant decanted for analysis by AAS or
ICPOES
Step 2 Residue from step 1 extracted overnight (16 hours) with 20 ml of
hydmxylammoniumchloride (NH2OH.HCI0.1 mom) acidified with
nitric acid to pH 2, centrifuged and the supematant decanted for
analysis
Step 3 Residue h m step 2 treated twice with 10 ml of hydrogen peroxide
8.8 movl and the dry residue extracted overnight with 50 ml of
ammonium acetate 1 mom adjusted to pH 5 with acetic acid. The
supernatant, separated by centrifugation, is retained for analysis

cases, major, and quite unacceptable, systematicerrors were revealed by the calibrant check
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analyses.The results for the overall mean of the laboratory mean extractable contents found
in each of the steps are summarised in Table 8.
For only 6 of the overall step means, out of a total of 18, are the CVs less than 20%. for
Cd the CVs lay between 11 and 20%,for Cr, between 14 and 32%,for Cu all steps had CVs
of about 20% while for Zn the CVs fell between 10 and 25%. For Ni and Pb the results were
much poorer, with CVs between 26 and 45% and between 50 and 80% respectively.

Table 8 Sediment sequential extraction summary (Overall mean of means m&kg dry
sediment).

CADMIUM
Step O/A MEAN STD DE V cv %
1 7.18 0.81 11.3
2 3.41 0.63 18.5
3 1.03 0.196 20.1
CHROMIUM
1 1.36 0.20 14.7
2 3.29 1.07 32.5
3 76.3 10.4 13.6
COPPER
1 3.69 0.76 20.6
2 3.13 1.96 20.1
3 63.4 13.2 20.8
NICKEL
1 9.76 4.36 44.7
2 5.79 1.54 26.6
3 10.2 3.32 32.5
LEAD
1 5.06 2.50 49.4
2 11.0 8.87 80.6
3 6.93 4.78 69.0
ZINC
1 262 35.1 13.4
2 140 34.2 24.4
3 89.7 9.14 10.2
 SPECIATION OF HEAVY METALS 145

Table 9 Extract solution concentrations(mg/l) for sediment steps 1 to 3 and for EDTA,
acetic acid and ammonium acetate extracts of soil, calculated From mean of laboratory
means.

Elemeni Siep Sediment EDTA soil HOAc soil Amm. Ac. soil

Cd 1 0.18 4.6 0.48 0.21

2 0.08
3 0.01
Cr 1 0.03 I .6 0.65 0.09
2 0.08
3 0.76
cu 1 0.09 32.4 0.73 0.35
2 0.08
3 0.63
Ni I 0.24 0.4 0.4 0.09
2 0.14
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3 0.10
Pb 1 0.12 51 0.08 0.14
2 0.28
3 0.07
Zn 1 6.6 98 13 1.2
2 3.5
3 0.9

It can be concluded that the results are much poorer than the results obtained for the
EDTA and the acetic acid extracts obtained in the soil trial above and were somewhat poorer
even than the inadequate ammonium acetate soil extract results.
While there may be several reasons for this failure in the sediment trial, the principal
cause of the poor precision of analysis lies in the very low concentrations found in the
extracts of this sediment as shown in Table 9 lo, concentrations in many cases too close to
the technique detection limits for reliable determination. The general conclusions can be
drawn that for certification of sediment contents obtained by this sequential extraction
procedure more sensitive analytical techniques are required, and perhaps, even then, a
sediment more contaminated by heavy metals will need to be used.

CONCLUSION

The collaborative studies outlined here, and presented in more detail elsewhere [ 101, have
succeeded in harmonizing the extractants and procedures for chemical speciation of ele-
ments in soils and sediments. The agreed procedural protocols have allowed BCR to
commence the process of preparing reference soils for certification of their extractable
contents with a good prospect of success in the near future. A similar stage in the preparation
of sediments certified for metal contents extracted by a sequential extraction procedure
should shortly be reached ' I .
Both single and sequential extraction schemes were critically examined by the partici-
pating laboratoriesand amended in order to design common procedures which could be used
throughout the EC Member States; these procedures are given in Annex lo.
 146 A. M. URE et a/.

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3. S. Hetland, 1. Martinsen, B. Radzuk and Y . Thomassen. Anal. Sci, 7,1029-1032 (1991).
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Heidelberg, (CEP Consultants, Edinburgh, 1983) pp. 1090-1093.
9. S. J. Adams and B. J. Alloway, Environ. Technol. Letts., 9,695-702 (1988).
10. A. Ure, Ph. Quevauviller,H. Muntau and B. Griepink, Euroreport, (BCR, Brussels, 1992) in press.
11. Ph. Quevauviller, A. Ure, H. Muntau and B. Griepink, this issue.
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 SPECIATION OF HEAVY METALS 147

ANNEX 1

Single extraction procedure for soil analysis

Extractable contents of the following trace metals shall be determined: Cd, Cr, Cu, Ni, Pb
and Zn using 0.05 moVl EDTA and 0.43 moVl acetic acid.
The extraction shall be performed in 250 ml pre-cleaned borosilicate glass, polypropyl-
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ene or PTFE bottles using an end-over-end shaker. All laboratory glassware shall be cleaned
with HC1, rinsed with distilled water, cleaned with 0.05 moVl EDTA and rinsed again with
distilled water.
Extractants shall be prepared according to the following procedure:

1) 0.05 moVl EDTA shall be prepared as an ammonium salt solution by adding in a fume
cupboard 146.12 + - 0.05g of EDTA free acid to 800 + - 20 ml distilled water and partially
dissolved by stirring in 130 + - 5 ml of saturated ammonia solution (prepared by bubbling
ammonia gas into distilled water). The addition of ammonia shall be continued until all the
EDTA has dissolved. The obtained solution shall be filtered through a filter paper ofporosity
1.4 to 2.0 pm (capable of retaining particles of 8.0 pm size) into a 10 litre polyethylene
container and diluted with water to 9.0 + - 0.5 1. The pH shall be adjusted to 7.00 + - 0.05
by addition of a few drops of either ammonia or hydrochloric acid as appropriate. The
solution shall thereforebe diluted with distilled water to 10.0 + - 0.1 1,well mixed and stored
in stoppered polyethylene container.
2) 0.43 moVl acetic acid shall be prepared by adding in a fume cupboard 250 + - 2 ml of
redistilled glacial acetic acid to about 5 litres of distilled water in a 10 1 polyethylene
container. The solution shall be diluted with distilled water to 10 1 volume, well mixed and
stored in a stoppered polyethylene container.

Extraction shall be batch-wise (e.g. shaking), followed by centrifugation according to

the following procedure:

1) a 5 g soil sample shall be transferred to an extraction bottle in which 50 ml of 0.05

moyl EDTA will be added. The obtained mixture shall be shaken on an end-over-end shaker
operating at 30 revolutions per minute for 1 hour in a room at 20 "C.
2) a 5 g soil sample shall be transferred to an extraction bottle to which 200 ml of 0.43
moVl acetic acid will be added. The mixture shall be mixed by shaking in an end-over-end
shaker as described above for 16 hours (e.g. overnight) in a room at 20 "C.

The temperature of the room shall be measured at the beginning and at the end of the
extraction as well as the temperature of the extracting solution in the bottle at the end of the
 148 A. M.URE et al.

shaking period. The extracts shall be immediately filtered through a filter paper (porosity
0.4 to 1.1 pm capable of retaining particles of 2.7 pm size) previously rinsed with 0.05 moVl
EDTA followed by distilled water. The filtrates shall be collected in polyethylene bottles.
Blank extractions (i.e. without soil) shall be carried out for each set of analysis using the
same reagents as described above.
The sample for analysis should be taken as it is. Before a bottle is opened it should be
manually shaken for 5 min to rehomogenisethe content. The results should be corrected for
dry mass: this correction must be performed on a separate portion of 1 g taken at the same
time from the same bottle by drying in an oven at 105 + - 2 "Cfor 2-3 hours until constant
mass is attained (successive weighings should not differ by more than 1 mg).
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 SPECIATION OF HEAVY METALS 149

ANNEX 2

Protocol for the sequential extraction scheme for sediment analysis

APPARATUS
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All laboratory-ware shall be of borosilicate glass, polyethylene, polypropylene or PTFE,

except for the centrifuge tubes, which will be of borosilicate glass of PTFE.
Clean vessels in contact with samples or reagents with €€NO34 moVl (overnight) and
rinse with distilled water. Determine the blank as follows: to one vessel from each batch,
taken through the cleaning procedure, add 40 ml of acetic acid (solution A, see below).
Analyse this blank solution along with the sample solutions from step 1 described below.
Use a mechanical shaker, preferably of the horizontal rotary or the end-over-end type, at a
speed of 30 rpm and record the speed. Carry out the centrifugation at 1500 G.

REAGENTS

Water

Glass-distilledwater is normally suitable; simple de-ionised water may contain organically

complexed metals and should not be used. Analyse a sample of distilled water with each
batch of step 1 extracts.

Solution A (acetic acid 0.I 1 mol/l)

Add in a fume-cupboard, 25 + - 0.2 ml of redistilled glacial acetic acid (or for example
Analar or Suprapur grade acetic acid without distillation) to about 0.5 1 of distilled water in
a 1 1 polyethylene bottle and make up to 1 1 with distilled water. Make up 250 ml of this
solution (acetic acid 0.43 moM) with distilled water to 1 1 to obtain an acetic acid solution
of 0.1 1 moV1. Analyse a sample of each batch of solution A.
 150 A. M.URE et al.

Solution B (hydroxylamine hydrochloride or hydroxyammoniumchloride 0.I mol/l

Dissolve 6.95 g of hydroxylamine hydrochloride in 900 ml of distilled water. Acidify with

HN03to pH 2 and make up to 1 1with distilled water. Prepare this solution on the same day
as the extraction is carried out. Analyse a sample of each batch of solution B.

Solution C (hydrogenperoxide solution 300 mg/g, i.e. 8.8 moll!)

Use the H202 as supplied by the manufacturer, i.e. acid-stabilized to pH 2-3. Analyse a
sample of solution C.

Solution D (ammonium acetate I mol/l)

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Dissolve 77.088of ammonium acetate in 900 ml of distilled water, adjust to pH 2 with HNG
and make up to 1 1 with distilled water. Analyse a sample of each batch of solution D.

SEQUENTIAL EXTRACTION PROCEDURE

Determine the extractable contents of the following trace metals, Cd, Cr, Cu, Ni, Pb and Zn
using the procedure below.
Cany out all extractions on the sediment as received in the glass bottle.
Before subsampling the sediment, with a suitable plastic (see apparatus above) spatula,
shake the contents of the sample bottle, with the PTFE ball supplied in the bottle, for 3
minutes.
Dry a separate 1 g sample of the sediment in a layer of about 1 mm depth in an oven at
105 "C for 2 hours and weigh. From this a correction "to dry mass" is obtained and applied
to all analytical values reported (quantity per g dry sediment).
Perform the extractionsby shaking in a mechanical shaker at 20 + -2 "C. The temperature
of the room shall be measured at the start and at the end of the extraction procedures.
Perform the sequential extraction procedure according to the steps described below:

Step 1 Add 40 ml of solution A to 1 g of sediment (as received) in a 100ml centrifuge tube

and extract by shaking for 16 hours at ambient temperature (overnight). No delay should
occur between the addition of the extractant solution and the beginning of the shaking.
Separate the extract from the solid residue by centrifugation and decantation of the
supernatant liquid into a high pressure polyethylene container. Stopper the container and
analyse the extract immediately or store it at 4 "C prior to analysis. Wash the residue by
adding 20 ml of distilled water, shaking for 15 minutes and centrifuging. Decant the
supernatant and discard, taking care not to discard any of the solid residue.
Break the "cake" obtained upon centrifugation by using a vibrating rod prior to the next
step.
 SPECIATION OF HEAVY METALS 151

Step 2 Add 40 ml of solution B to the residue from step 1 in the centrifuge tube and extract
by shaking for 16 hours at ambient temperature (overnight). No delay should occur between
the addition of the extractant solution and the beginning of the shaking. Separate the extract
from the solid residue by centrifugation and decantation as in step 1. Retain the extract in a
stoppered polyethylene tube, as before, for analysis. Wash the residue by adding 20 ml of
distilled water, shaking for 15 minutes, and centrifuging. Decant the supernatant liquid and
discard, taking care to avoid discarding any of the solid residue. Retain the residue for step
3.
Break the “cake” obtained upon centrihgation by using a vibrating rod prior to the next
step.

Step 3 Add carefully, in small aliquots to avoid losses due to violent reaction, 10 ml of
solution C to the residue in the centrifuge tube. Cover the vessel with a watch glass and
digest at room temperature for 1 hour with occasional manual shaking. Continue the
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digestion for 1 hour at 85 “C and reduce the volume to a few ml by further heating of the
uncovered vessel in a steam bath or equivalent.
Add a further aliquot of 10 ml of solution C. Heat the covered vessel again to 85 “C and
digest for 1 hour. Remove the cover and reduce the volume of the liquid to a few ml.
Add 50 ml of extracting solution D to the cool moist residue and shake for 16 hours at
ambient temperature (overnight). No delay should occur between the addition of the
extractant solution and the beginning of the shaking. Separate the extract by centrifugation
and decant into a high pressure polyethylene tube. Stopper and retain as before for analysis.

IMPORTANT:

- The calibrant solutions should be made up with the appropriate extracting solutions.
Interlaboratoryconsistencyof calibration will be assessed by the analysis by the participants
of simulated extracts.
- The total elemental content (HF method) will be determined by specialist laboratories.

- With each batch of extractions a blank sample (i.e. a vessel with no sediment) shall be
carried through the complete procedure.