Acta Universitatis Cibiniensis

Seria F Chemia 8(2005-2):5-12

Use of Sequential Extraction to Assess Metal Fractionation

in Soils from Bozanta Mare, Romania
Erika-Andrea Konradi1*, T. Frentiu1,
Michaela Ponta1 and E. Cordos
Abstract
Sequential extraction of metals from soil, based on the Tessiers procedure, was
used to evaluate soil contamination in the Bozanta Mare area. The contents of Al, As, Cd,
Cu, Fe, Mn, Pb, Zn in the five fractions (exchangeable, bound to carbonates, bound to iron
and manganese oxide, bound to organic matter and residual) were determined by ICP-AES
and HG-ICP-AES (As). In the bio-available fraction (exchangeable and bound to
carbonates) there were found low amounts of Al, Fe and Mn and relatively high contents
of Cd, Cu, Pb and Zn. The main fractions were that bound to Fe/Mn oxides and the
residual one. Important amounts of Al, As, Cd, Cu and Zn were retained on the organic
matter. An increase of Fe and Mn in soil resulted in an increasing fraction of elements
bound to Fe/Mn oxides. The contents of As, Cd, Cu and Pb in the exchangeable fraction
exceeded the normal values for sensitive soil.
Keywords: sequential extraction procedure; metal partitioning in soil; soil contamination.

I. Introduction
The accumulation of heavy metals in environmental samples represents a potential
risk to human health due to the transfer of these elements to aquatic media, their uptake
1

Babes-Bolyai University, Department of Analytical Chemistry, Str. Arany Janos 11, 400028 ClujNapoca, Romania
*
To whom the correspondence should be addressed.

Erika-Andrea Konradi, T. Frentiu, Michaela Ponta and E. Cordos

by plants and their subsequent introduction into the food chain. The toxicity,
bioavailability and mobility of metals are related to their species [1, 2]. However, the
determination of chemical species is difficult and sometimes impossible. Therefore, the use
of sequential extraction procedures for environmental studies provides an important tool
for the determination of the different chemical forms or ways of binding between trace
metals and soil components [3].
Chemical extraction is employed to assess operationally defined metal fractions,
which can be related to chemical species, as well as to potentially mobile, bioavailable or
ecotoxic phases of a sample. It is generally accepted that the ecological effects of metals
(e.g., their bioavailability, ecotoxicology and risk of groundwater contamination) are
related to such mobile fractions rather than to the total concentration [4].
Chemical fractionation schemes for partitioning trace metals in soils and sediments
have been used extensively since the 70s and are based on three to six fractions [3, 5-7, 9,
10], many of them being slightly modified Tessiers schemes [7].
The objective of this study was to investigate the partitioning of Al, As, Cd, Cu,
Fe, Mn, Pb, Zn in soil collected from Bozanta Mare, a village situated next to the place
where a cyanide spill accident took place four years ago. Determinations were carried out
by inductively coupled plasma atomic emission spectrometry (ICP-AES) or hydridegeneration inductively coupled plasma atomic emission spectrometry (HG-ICP-AES).
Metal contents were compared with the normal values of these elements in soil.
II. Experimental
Sampling
The sampling was done in June 2004 in Bozanta Mare, situated close to the dam
where the cyanide spill, containing a huge quantity of metals, took place in 2000 [4].
Samples were taken from 5 cm depth, using a small shovel, after manual removal of
leaves, roots and stones. Soils were sieved through a 2 mm sieve in order to eliminate
stones and other materials extraneous to soil, then stored in polyethylene bags for transport
to the laboratory. The sieved soils were spread over a polyethylene sheet and air-dried at
room temperature for one week. The residual moisture was removed by heating at 1055 0
C for 3 h. Samples were ground to a fine powder in a tungsten-carbide swing mill for 3
min and sieved through a 90 m sieve. The fraction below 90 m was further
homogenized by mixing in a PVC drum for 1 h, then stored in brown glass bottles at room
temperature until analysis.
Instrumentation
The determinations were carried out using the ICP multi-channel spectrometer
SPECTRO CIROSCCD (Spectro Analytical Instruments Kleve, Germany). For the As
determination a hydride generator (VGA-77, Varian, Australia) was interfaced with the
ICP spectrometer. The optimum conditions to generate arsine were pH 2.000.01 of the
sample with 0.2 % L-cysteine (7 mL min-1), 0.6 % NaBH4 in 0.5 % NaOH as reducing
agent (1 mL min-1) and 0.01 M HCl as carrier (1 mL min-1) [11]. Details about
instrumentation and optimum operating conditions are given in Table 1.
6

Acta Universitatis Cibiniensis

Seria F Chemia 8(2005-2):5-12

Table 1. Instrumentation and operating conditions for the SPECTRO CIROSCCD atomic
emission spectrometer.
Generator:
Free running 27.12 MHz operated at 1400 W.
Plasma torch
Inductively coupled plasma, axial viewing; torch position (mm):
X= -3.9; Y=+3.6; Z=+2.6.
Argon flow rates:
Outer gas 12 L min-1.
Intermediate gas 0.6 L min-1.
Nebulizer gas 1 L min-1.
Sample introduction
system

Optics

Detector
Data processing

Wavelength/nm

Pneumatic nebulization with 4 channel peristaltic pump, K2 crossflow nebulizer, double pass Scott type spray chamber
sample uptake rate: 2 mL min-1
flushing time: 40 s
delay time: 20 s
160 800 nm double-grating Paschen-Runge multi-channel
spectrometer
chamber filled with Ar
22 CCD detectors
Smart Analyzer Software
Al
396.1

As
193.7

Cd
Cu
228.8 324.7

Fe
259.9

Mn
279.5

Pb
283.3

Zn
213.8

Reagents and stock solutions

All used reagents were of purris p.a. quality (Flucka, Germany). Stock solutions of
1000 g mL-1 As and Cd and 4000 g mL-1 Cu, Zn, Pb, Fe, Mn and Al were prepared
starting from oxide (As) or metallic powder. A multi-element standard solution of (g mL1
) 50 As, 20 Cd, 160 Pb, 200 Cu, Zn, Al and 400 Fe, Mn was prepared, using the single
elemental solutions. This solution was used to select the wavelength in order to analyze the
samples by the multi-elemental standard addition method. All dilutions were made with 2
% (v/v) HNO3.
The following extraction solutions were prepared: (a) 1.0 M MgCl2 solution
adjusted to a pH of 7.0 with NaOH; (b) 1.0 M NaAc solution adjusted to a pH of 5.0 with
HAc; (c) 0.04 M NH2OHHCl in 25% HAc solution; (d) 0.02 M HNO3 /30 % H2O2
solution adjusted to a pH of 2.0; 3.2 M NH4Ac/20 % HNO3 solution; (e) aqua regia
(HCl+HNO3 : 3+1).
Summary of the sequential extraction procedure (SEP)
The sequential extraction procedure used was similar to that of Tessier et al [7] and
adapted by Beck and Sneddon [8] for sediments. According to the Tessiers procedure, the
SEP operationally groups heavy metals into the following five fractions:
7

Erika-Andrea Konradi, T. Frentiu, Michaela Ponta and E. Cordos

F.1.

Soluble and exchangeable

The soluble and exchangeable metals from soil were extracted with 20 mL of a 1.0
M MgCl2 solution adjusted to a pH of 7.0 by mechanical shaking for 1 h at 20 oC.
F.2.
Bound to carbonates
The carbonates in the residue from the previous step were extracted with 20 mL of
a 1.0 M NaAc solution adjusted to a pH of 5.0 with HAc by continuously shaking
for 4 h at room temperature.
F.3.
Bound to iron and manganese oxides
Metals bound to iron and manganese oxides were extracted from the residue of the
second extraction by shaking with 50 mL of a 0.04 M NH2OHHCl/25 % HAc
solution. The extraction was performed at 963 oC for 5.5 h.
F.4.
Bound to organic matter
Metals bound to organic matter were extracted by pouring 7.5 mL of a 0.02 M
HNO3 solution and 12.5 mL of a 30 % H2O2 solution adjusted to a pH of 2.0 onto
the residue from F3, then providing continuous agitation for 2 h at the temperature
of 85 oC. An additional volume of 7.5 mL of the 30 % H2O2 solution adjusted to a
pH of 2.0 is then added, while maintaining continuous agitation and a temperature
of 85 oC for another 3 h. This solution was then cooled to room temperature. An
aliquot of 12.5 mL of a 3.2 M NH4Ac/ 20 % HNO3 solution was added and shaken
for 30 min.
F.5.
Bound to the soil matrix (residual fraction)
The residue from F4 was quantitatively transferred into a digestion vessel and
metals were dissolved in aqua regia using 7 mL of 10 M HCl and 2.3 mL of 15.8
M HNO3. The temperature of the reaction mixture was slowly risen until reflux
conditions and maintained for 2 h.
Three parallel extractions were carried out, in each case using 2.5000 g soil. All
solid/liquid separations were performed by centrifuging, at 5000 rpm for 15 min. The
supernatant was removed and analyzed for metals. The residue was washed with 20 mL of
de-ionized water. After centrifugation for 15 min the supernatant was discarded. All
dilutions were made to 50 mL with 2 % (v/v) HNO3. For each fraction a blank was
subjected to the same procedure.
III. Results and discussions
The element contents determined in the five fractions (average of 3 parallel
samples) following the SEP are shown in Table 2, while the distribution among the
fractions is presented in Fig. 1. a, and b. The data show a preferential distribution of
elements among fractions.

Acta Universitatis Cibiniensis

Seria F Chemia 8(2005-2):5-12

Table 2. Fractions of elements in soil following the sequential extraction procedure (m=3).
Al
As
Cd
Cu
Fe
Mn
Pb
Zn
Sample Fraction

Soil 1

Soil 2.

Exchangeable
Carbonates
Fe/Mn oxides
Organic
Residual
Sum of
fractions
Exchangeable
Carbonates
Fe/Mn oxides
Organic
Residual
Sum of
fractions

16.3
23.1
349
3810
3962
8160

<1
3.8
11.7
6.18
23.4
45.2

15.5
37.7
1280
590
96.1
2020

6.2
3.9
50
8.2
111
179

Concentration (mg Kg-1)

<0.02*
111
15.1
1.8
12.1
88.9
2.6
110
19649
0.9
154
198
0.8
34.8
2235
6.1
422
22186
2.1
19.2
47.8
1.2
0.8
71.1

70.8
70.2
3433
339
451
4364

27.8
13
24890
2240
4845
32015

<0.2*
120
5006
308
111
5545

23.1
52.6
175
35.3
76.2
362

23
405
1322
228
149
2127

25
225
6892
250
139
7531

35.6
15.3
185
21.1
28.6
286

47.5
2545
36533
587
16462
56174

Concentration corresponding to the detection limit expressed as dry mass

Bio-available species. The exchangeable and bond to carbonates species,

corresponding to the first two fractions, are generally called bio-available, as they
exhibit a mobility relative to the environment and are potentially available for plants.
Results show that the amounts of Al, Fe and Mn in bio-avaliable form are very low,
namely below 1 % Al, 1 % Fe and 3 % Mn in soil 1 and around 2 % Al, less than 1 % Fe
and 3 % Mn in soil 2. The bio-available As ranges between 5 and 10 % and in the first soil,
is exclusively bound to carbonates. In both samples, 30 % of Cd and 20 % of Pb are in bioavailable form. Cadmium is mainly bound to carbonates, while Pb is between 6-12 % as
exchangeable species and 5-15 % associated to carbonates. Relatively high proportions of
Cu (30%) and Zn (20 %) are bio-available in the first soil and much less in the second
sample (3 % Cu; 5 % Zn).
Species bound to Fe-Mn oxides. Logically, Fe and Mn occur mainly in the oxide
fraction, in similar proportions in both samples (77-89 % Fe and more than 90 % Mn).
Arsenic and Zn bond to oxides occur in the same proportion in both soils, around 27 % and
65 % respectively. The fraction of Al, As, Cd, Cu and Pb associated with oxides is much
higher in the second soil, especially in the case of Al. The fraction of elements bound to
Fe/Mn oxides in soil 2 is the most important.

Erika-Andrea Konradi, T. Frentiu, Michaela Ponta and E. Cordos

Extraction degree

100%
80%

Rezidual

60%

Organics
Fe and Mn oxides

40%

Carbonates

20%

Exchangeable

0%
Al

As

Cd

Cu

Fe

Mn

Pb

Zn

Element

Extraction degree

100%
80%

Rezidual

60%

Organics
Fe and Mn oxides

40%

Carbonates
Exchangeable

20%
0%
Al

As

Cd

Cu

Fe

Mn

Pb

Zn

Element

Fig. 1. Partitioning of metals in soil; a. soil 1, b. soil 2.

Species bound to organic matter. Elements are generally bound in a greater
proportion to organic matter in soil 1 compared to soil 2, excepting Fe. High amounts of Al
(up to 47 %) and Cu (up to 36 %) and also As, Cd and Zn are bound to the organic matter.
Species in residual fraction. The proportion of As (above 50 %) in the residual
form of is much higher than the other elements, while the weight of Mn in this fraction is
relatively low (up to 2 %), in both samples. The residual fractions of Al, Cd, Pb are higher
in soil 1 than in soil 2, while Cu, Fe and Zn show the reverse.
In summary, Cd, Fe, Mn, Pb and Zn are present mainly in the oxides fraction, Al is
found as residual or bound to Fe/Mn oxides, Cu is bound to oxides and organic matter,
while the main fraction for As is the residual. For all elements, an increase of the fraction
bound to Fe/Mn oxides occurs with an increase of Fe and Mn content in soil.
10

Acta Universitatis Cibiniensis

Seria F Chemia 8(2005-2):5-12

Table 3 presents the exchangeable fraction of several elements in soil from

Bozanta Mare area in comparison with the normal/alert/intervention levels (mg Kg-1) in
sensitive soil. Only the exchangeable fraction was considered in this comparison, as it is
the most mobile form under normal conditions.
Table 3. Exchangeable fraction of several elements in soil from Bozanta Mare area
compared to normal/alert/intervention levels (N/A/I) in mg Kg-1 in sensitive soil*
Element
As
Cd
Cu
Mn
Pb
Zn
Soil 1

111

23.1

23.0

Soil 2
N/A/I

6.2

2.1

70.8

25

35.6

47.5

5/15/25

1/3/5

20/100/200 900/1500/250 20/50/100 100/300/600

0

Monitorul Oficial, 303/bis/06.11.1997

In the case of As, Cd and Pb, whose compounds are known to be toxic, the
amounts of exchangeable species exceed the normal values in soil but are not over the alert
limits. However, continued monitoring is recommended. If only the exchangeable
fractions of Mn and Zn are considered, one can say that the normal levels for sensitive soil
are not exceeded. Only the level of Cu is close or slightly over the alert limit.
Generally, when the pH becomes lower as a result of acid rain in the area, the
exchangeable fraction could increase on the account of the higher mobility of the elements
bound to carbonates.
Data in Table 2 and Table 3 show that, four years after the cyanide spill accident,
the total content of metals in soil from Bozanta Mare is still high in comparison with the
normal level in soil.
IV. Conclusions
This study has shown that soil composition and physicochemical characteristics
strongly influence metal partitioning in soil. Elements are mainly bound to Fe/Mn oxides
and are present in the exchangeable fraction in only small amounts, except for Cu and to a
slight extent As and Cd. The amount in this oxide fraction increases with the increase of Fe
and Mn contents in soil. The organic matter has an important influence on the mobility of
several species. Thus, elements like Al, As, Cd, Cu and Zn can be retained on organic
matter. Metals immobilized on the organic matter in soil become less mobile and less bioavailable which results in a decrease of the exchangeable fraction. A significant proportion
of elements is present in residual form. The cyanide spill accident that occurred close to
Bozanta Mare village in 2000 was recognized as having long-term consequences for the
ecosystem. Consequently, a periodical evaluation of the soil status is necessary in the area.

11

Erika-Andrea Konradi, T. Frentiu, Michaela Ponta and E. Cordos

V. References
1. Li, B., Wang, Q., Huang, B., Li, S., Anal. Sci., 2001, 17, 1561.
2. Quevauviller, P., Rauret, G., Muntau, H., Ure, A. M., Rubio, R., Lopez-Sanchez, J. F.,
Fiedler, H. D., Griepink, B., Fresenius J. Anal. Chem., 1994, 349, 808.
3. Rauret, G., Lopez-Sanchez, J. F., Sahuquillo, A., Rubio, R., Davidson, C., Ure, A.,
Quevauviller, P., J. Environ. Monit., 1999, 1, 57.
4. Cordos, E., Rautiu, R., Roman, C., Ponta, M., Frentiu, T., Sarkany, A., Fodorpataki,
L., Macalik, K., McCormick, C., Weiss, D., European Journal of Mineral Processing
and Environmental Protection, 2003, 3, 324.
5. Han, F. X., Banin, A., Kingery, W. L., Triplett, G. B., Zhou, L. X., Zheng, S. J., Ding,
W. X., Advances in Environ. Research, 2003, 8, 113.
6. Ianni, C., Ruggieri, N., Rivaro, P., Frache, R., Anal. Sci., 2001, 17, 1273.
7. Tessier, A., Campbell, P. G. C., Bisson, M., Anal. Chem., 1979, 7, 844.
8. Beck, J. N., Sneddon, J., Microchem. Journal, 2000, 66, 73.
9. Navas, A., Lindhorfer, H., Environ. Int, 2003, 29, 61.
10. Hlavay, J., Prohaska, T., Weisz, M., Wenzel, W., Stingeder, G.J., Pure Appl. Chem,
2004, 76, 415.
11. Cordos, E., Frentiu, T., Ponta, M., Marginean, I., Abraham, B., Roman, C., Chem.
Spec. Bioavailab., 2005, in press.

12