Environ Monit Assess (2013) 185:6173–6185
DOI 10.1007/s10661-012-3015-0
Mobility and ecological risk assessment of trace metals
in polluted estuarine sediments using a sequential
extraction scheme
F. A. B. Canuto & C. A. B. Garcia & J. P. H. Alves &
E. A. Passos
Received: 14 April 2012 / Accepted: 20 November 2012 / Published online: 7 December 2012
# Springer Science+Business Media Dordrecht 2012
Abstract The three-stage sequential extraction proce-
dure for the fractionation of Cd, Cr, Cu, Ni, Pb, and Zn,
proposed by the Commission of the European
Communities Bureau of Reference, was applied to sed-
iment five samples collected from the Sal estuary,
Sergipe State, northeast Brazil, in September 2009.
The method showed satisfactory recoveries, detection
limits, and standard deviations for determinations of
trace metals in the sediments. Cd and Pb were the metals
most prevalent in the bioavailable fractions (carbonates,
Fe and Mn oxides, organic matter, and sulfides), while
Ni, Zn, Cu, and Cr showed higher percentages in the
inert fraction. The order of mobility of the metals was
Cd (66 %) > Pb (65 %) > Zn (59 %) > Ni (57 %) = Cr
(57 %) > Cu (56 %). Possible toxicity related to these
metals was examined using the risk assessment code,
and by comparing the chemical data with sediment
quality guideline ERL-ERM values. Results obtained
using the two methods were in agreement, and showed
low to medium risk for all metals, indicating that ad-
verse effects on aquatic biota should rarely occur.
Keywords Trace metal . Sediment . Sequential
extraction . Risk assessment code (RAC) . Sediment
quality guidelines (SQG)
F. A. B. Canuto : C. A. B. Garcia : J. P. H. Alves :
E. A. Passos (*)
Laboratório de Química Analítica Ambiental, Departamento
de Química, Universidade Federal de Sergipe,
49.100-000 São Cristóvão, SE, Brazil
e-mail: elisapassos@gmail.com
Introduction
Estuarine and coastal areas are often regions of high
population density and intensive human activity. As a
result, elevated levels of trace metals in sediments can
reflect the impacts of industrial activities and urban
development (Fang et al. 2005). Metal accumulation
in sediments poses an environmental problem due to
possible metal transfer from the sediments to aquatic
media (Filgueiras et al. 2002).
The total sediment concentration of a metal does
not provide information concerning its mobility, avail-
ability, or toxicity (Rauret et al. 1999). Toxicity is
dependent on the amount of metal available for bio-
accumulation (accumulation by biological organisms),
and is determined by those sediment properties that
affect its bioavailability (Ure et al. 1993). Hence, it is
possible for a sediment to be metal-contaminated (to
have a metal content higher than the natural back-
ground), but not manifest any toxic effects, depending
on the geochemical processes that control the avail-
ability of the metal in the sediment (Passos et al. 2011;
Silva et al. 2012).
Sequential extraction is a well-established approach
for the investigation of different forms of trace metals
in soils and sediments. Several sequential fractionation
procedures have been suggested to identify element
distributions in operationally defined pools that in-
clude water-soluble species and recalcitrant forms
immobilized in mineral lattices (Rauret et al. 1999).
Many of these are variants on the scheme proposed by
6174
Tessier et al. (1979). However, it is difficult to com-
pare the results obtained by procedures that employed
different sequences of reagents or different conditions.
For this reason, the Standards, Measurements and
Testing Programme (SM&T), formerly known as the
European Community Bureau of Reference (BCR),
has proposed a sequential extraction procedure in or-
der to harmonize and standardize the different techni-
ques used to fractionate metals in soils and sediments
(Ure et al. 1993).
The BCR protocol fractionates the metals into acid-
soluble/exchangeable, reducible, and oxidizable
phases. The acid-soluble/exchangeable fraction
includes the metals that are precipitated (or co-
precipitated) with carbonate, and is affected by pH
changes. The reducible fraction contains metals asso-
ciated with Fe-Mn oxides, which can be dissolved by
changes in redox potential. In the oxidizable fraction,
metals may be associated, through complexation or
bioaccumulation processes, with various forms of or-
ganic material including living organisms, detritus, or
coatings on mineral particles. In this fraction, the
metals can remain in the sediment for lengthy periods,
with mobilization occurring during decomposition un-
der oxidizing conditions (Filgueiras et al. 2002).
The BCR procedure is one of the most popular
extraction techniques, and has been widely applied to
soils, sediments, and standard reference materials
(Mossop and Davidson 2003; Yuan et al. 2004). The
method is reproducible, and provides good recoveries
when compared to full acid dissolution. Sediment
reference materials (CRM 601 and 701) are available
that are certified for metals extracted by the procedure
(Zemberyov et al. 2006).
Fractionation studies are not only very useful for
determining the degree of association of metals in
sediments, and to what extent the metals may be
remobilized into the environment, but also for distin-
guishing those metals with lithogenic origins from
those with anthropogenic origins (Jain 2004).
According to Rubio et al. (2000), metals with anthro-
pogenic origins are mainly found in the first three
extraction fractions (soluble in acid, associated with
Fe and Mn oxides, and associated with organic matter
and sulfides), while metals with lithogenic origins are
found in the residual fraction obtained in the last stage
of the extraction process. Regions where the metals
are weakly associated with sediments, and show an
affinity for carbonate, have experienced pollution that
Environ Monit Assess (2013) 185:6173–6185
is more recent. Locations where metals are significant-
ly associated with the oxidizable or reducible sediment
fractions are influenced by pollution that is less recent
(Passos et al. 2010).
The risk assessment code (RAC) has been used to
assess environmental risks and estimate possible dam-
age to benthic organisms caused by contaminated
sediments (Arcega-Cabrera et al. 2009; Ishikawa et
al. 2009; Trujillo-Cardenas et al. 2010; Passos et al.
2010; 2011). The RAC considers the metal fraction
that is exchangeable and associated with carbonates.
This fraction is most influenced by human activity,
with the metals being weakly retained and able to
equilibrate with the aqueous phase, hence becoming
more rapidly bioavailable. The RAC classification
scheme defines risk levels as zero, low, medium, high,
and very high, depending on the percentage of metal
found in the exchangeable fraction (Perin et al. 1985;
Ikem and Adisa 2011).
The Sediment Quality Guidelines (SQG) system,
developed by Long and Morgan (1990), has been
adopted as an informal tool to evaluate sediment
chemical data in relation to possible adverse effects
on aquatic biota. ERL (effects range—low) and ERM
(effects range—mean) concentrations have been estab-
lished for eight metals and metalloids (As, Cd, Cr, Cu,
Pb, Ni, Ag, and Zn). These reflect the concentrations
at which toxicity could begin to be observed (ERL),
and above which adverse effects may occur (ERM)
(Long and Morgan 1990; Amin et al. 2009; Garcia et
al. 2011).
The indiscriminate release of untreated domestic
and industrial wastewater constitutes one of the main
factors of degradation of aquatic ecosystems located
near major urban centers and may contribute to the
increase of the concentrations of the trace metals in
these environments (Passos et al. 2005). The study
area—the Sal River estuary is polluted by different
types of industrial and agricultural drainage and do-
mestic wastewater from other resources of metal pol-
lution, such as car washes, pesticides from agricultural
areas, runoff, and so on (Barbier et al. 2010).
The main objectives of the present study were: (a)
to determine the distributions of the trace metals Cd,
Cr, Cu, Ni, Pb, and Zn in surface sediments of the Sal
River estuary (Sergipe State, northeast Brazil), using
the optimized BCR sequential extraction protocol, and
hence to ascertain their mobility; (b) to define the
natural and/or anthropogenic origins of the metals,
Environ Monit Assess (2013) 185:6173–6185
based on contents in the first three sediment fractions;
(c) to perform a risk assessment for each metal, based
on RAC values; and (d) to evaluate possible toxicity,
based on ERL–ERM values.
Materials and methods
Study area
The Sal River, located in Sergipe State, northeast
Brazil, is part of the Sergipe River hydrographic basin,
which covers an area of 3,673 km2, drains approxi-
mately 16.7 % of the State, and has average flows of
560,000 and 70,000 m3 day−1 in the rainy and dry
seasons, respectively. Since the 1990s, there has been
rapid expansion of aquacultural, industrial, and agri-
cultural activities in the region, with simultaneous
developments in civil construction, transportation,
and tourism; such dramatic changes have adversely
affected the water quality in the estuary (Alves et al.
2007; Barbieri et al. 2007).
The study area extended over 8 km, covering the
lower part of the Sal River estuary (Fig. 1). The Sal is
formed after the confluence of the Sergipe river. The
main sources of trace metal contaminants in this area
Fig. 1 Locations of sampling sites in the Sal River estuary
6175
are municipal and industrial wastewaters, agricultural
runoff, and especially metal industries (Barbieri et al.
2007; Garcia et al. 2009).
Sample collection and pre-treatment
Sediment samples were collected during September
2009, at five sites located in industrial, residential,
commercial, and rural zones of the Sal estuary, using
a core sampler composed of cellulose acetate butyrate
(Fig. 1). Site information is provided in Table 1. The
undisturbed top 5 cm of the sediment was sampled and
placed in acid-rinsed polypropylene vessels using a
plastic spatula. Three samples were taken at each
location. The sediments were oven-dried (at 50 °C
for 12 h), homogenized in a porcelain mortar, sieved
(using a 2-mm mesh size), and stored in plastic con-
tainers until the analyses were performed.
Reagents
All reagents were either analytical grade or Suprapur
quality (Merck, Darmstadt, Germany). Ultrapure wa-
ter (18 MΩ cm) was supplied from a Millipore Milli-Q
system. All glassware and plastic utensils were
washed with 10 %v/v nitric acid for 48 h, and rinsed
6176
Table 1 Location, water depth,
temperature, and pH for each Site Longitude (E)
sampling site, and total organic
carbon (TOC) contents of the 1 10°51′50.6″
sediment samples 2 10°52′13.2″
3 10°52′03.8″
4 10°51′43.5″
5 10°52′03.6″
with ultrapure water prior to use. Stock metal standard
solutions (1,000 mgL−1) were prepared from standard
vials (Tritisol, Merck) containing 1.000±0.002 g of
metal in 2 % (v/v) nitric acid. Calibration standards of
each metal were obtained by suitable dilutions of the
stock solutions. Three certified reference materials,
NCS DC 75304 (China river sediment), NCS DC
78301 (China marine sediment), and BCR-701 EUR
19775 (river sediment), plus one reference material,
LKSD-1 CCNRP (Canada lake sediment), were ana-
lyzed for quality control purposes.
Instrumentation
Sediment extractions were conducted using an end-
over-end mechanical shaker (CINTEC CT 712–R). A
centrifuge (Janetzsk T23) was used to obtain the su-
pernatant extracts. PTFE vessels were used throughout
the entire procedure. Additional items of equipment
were a pH meter (Digimed, DM20), a water bath
(Marconi, MA 156), and a digester block (Techal,
TE007A).
Analyses were performed using an atomic absorp-
tion spectrometer (Shimadzu AA6800) equipped with
both flame and graphite furnace atomization, D2 back-
ground correction, and an autosampler. All absorbance
readings were made in triplicate. The operating param-
eters for the individual metals were set as recommen-
ded in the manufacturer’s operating manual. The
following wavelengths (nm) were used: Cd, 228.8;
Cu, 324.8; Cr, 357.9; Ni, 232.0; Pb, 283.3; and Zn,
213.9.
Total metals extraction
For total metals extractions, samples (∼0.5 g) were
digested in closed Teflon vessels with 2 mL of HNO3,
1 mL of HCl, and 4 mL of HF, heated to 140 °C for 2 h.
After cooling, the vessels were opened and heated at
210 °C to complete dryness. The residue was dissolved
Environ Monit Assess (2013) 185:6173–6185
Latitude (N) Water depth (m) Temperature (°C) pH TOC (%)
37°05′0.6″ 5.60 26.5 7.79 1.24
37°03′45.8″ 11.4 27.5 7.69 3.49
37°03′41.2″ 5.90 27.3 7.70 1.19
37°03′0.5″ 3.30 27.0 7.89 0.82
37°02′12.3″ 14.0 27.3 7.97 2.52
in 10 mL of 0.5 molL−1 HCl, and the final volume was
made up to 50 mL (Passos et al. 2010). The solutions
were stored in polyethylene flasks for later determina-
tion of metals using either flame or graphite furnace
atomic absorption spectrometry.
Calculation of the limit of detection (LOD) for each
metal was based on the expression 3 s/b, where s is the
standard deviation of the blank, and b is the slope of
the calibration graph (Miller and Miller 1993). Ten
extraction procedure blanks were analyzed. The LOD
values took into account the use of 1 g portions of
sample in the extractions, and any necessary dilutions.
The calibration curve correlation coefficients (r) were
better than 0.998 for all elements studied. The results
showed that there was no significant analytical con-
tamination, and that LOD values varied from 0.002
(Cd) to 0.100 (Pb)μgg−1.
The efficiency of the sequential extraction method
was determined by analysis of four replicates of river
sediment (NCS DC 75304/China) and marine sedi-
ment (NCS DC 78301/China) certified reference mate-
rials. The recoveries of the metals from the reference
materials were in the range 92–106 %.
Partial metals extraction
Partial metal concentrations were determined using US
EPA Method 200.8. Samples (ca. 1 g) were treated in
closed Teflon vessels using 4 mL of (1+1) HNO3 solu-
tion and 10 mL of (1+5) HCl solution. The mixtures
were heated at 95 °C for 30 min. Following extraction,
the samples were filtered using quantitative filter paper,
transferred to 50-mL volumetric flasks, and the volumes
were made up using ultra-pure water. The solutions were
stored in polyethylene flasks prior to the determination
of metals using flame or graphite furnace atomic absorp-
tion spectrometry (Garcia et al. 2009).
Ten blank solutions were prepared in the same way
as samples during the digestion procedure, and for
quality control purposes a lake sediment reference
Environ Monit Assess (2013) 185:6173–6185 6177

material (LKSD-1 CCNRP/Canada) was analyzed
alongside the samples. LOD values (in μgg−1) varied
from 0.001 (Cd) to 0.080 (Pb). Recoveries of the trace
metals were in the range 97–103 %.
Sequential extraction procedure
The optimized BCR sequential extraction procedure
was applied, in quadruplicate, to 1.00 g portions of dried
sediment, using the reagents and experimental condi-
tions briefly summarized in Table 2. Details of the
optimized BCR extraction procedure can be found else-
where (Passos et al. 2010, 2011). This extraction proce-
dure fractionates metals into: (a) an acid soluble fraction
(F1) consisting of exchangeable metals, and metals
bound to carbonates, which are easily released to the
water column; (b) a reducible fraction (F2) which con-
sists of metals bound to iron and manganese oxyhydr-
oxides, which can be released when redox conditions
change; and (c) an oxidizable fraction (F3) that repre-
sents the metal bound to organic matter and sulfides,
which can be released under oxidizing conditions.
The optimized BCR protocol was used to obtain
information on the non-residual speciation of the metals.
The fourth fraction, obtained by digestion of the residue
from the third fraction extraction, provides an indication
of the contents of metals associated with the residual
components of the sediment matrix (Rauret et al. 1999).
A mixture of concentrated acids (as described in Total
metals extraction section) was used to leach all the
metals remaining in the residue (Passos et al. 2010).
For each extraction stage, the limits of detection
were determined from the standard calibration curves
of each element. The LOD values varied from
0.001 μgg−1 (Cd, first fraction) to 0.100 μgg−1 (Pb,
residual fraction). The accuracy of the analytical pro-
cedures for metal determination in the different
extracts was checked by analysis of four replicates of
certified sediment reference material (BCR-701 EUR
19775). The BCR-701 analysis was performed as part
of the batch, at the same time as the sample analyses.
The measured contents of the elements were compared
with the certified extractable contents of Cd, Cr, Cu,
Ni, Pb, and Zn. Recoveries of the trace metals varied
from 85 to 117 % for the first fraction (F1), from 93 to
105 % for the second fraction (F2), from 95 to 114 %
for the third fraction (F3), and from 111 to 115 % for
the residual fraction (R).
Statistical analysis
One-way analysis of variance and Tukey’s multiple
comparison test were employed (using Origin v. 7.0)
to test for significant differences (p≤0.05) between the
concentrations of metals in the different sediments. An
α value of 0.05 was adopted as the critical level for all
statistical testing (giving a 95 % confidence level).
Results and discussion
Partitioning of trace metals in the sediments
Chemical fractionation is very useful for distinguish-
ing metals with lithogenic origins from those with
anthropogenic origins. Metals from anthropogenic
sources are mainly contained in the earlier extractions,
while those from lithogenic sources are present in the
residual fraction (Rubio et al. 2000; Passos et al.
2010).
Table 3 provides the extract contents of Cd, Cr, Cu,
Ni, Pb, and Zn (as μgg−1, on a dry mass basis) for
each step of the BCR sequential extraction procedure,
the sum of contents for four fractions (ΣF1+F2+F3+

Table 2 Summary of the optimized BCR sequential extraction protocol

Step Fraction Reagents/conditions Extracted phases

1 F1: exchangeable and Acetic acid (0.11 mol/L); pH 2.85; 16 h Soluble forms, exchangeable metals,
acid soluble and metals bound to carbonates
2 F2: reducible Hydroxyl ammonium chloride (0.5 mol/L); pH 1.5 Metals bound to iron and manganese
oxyhydroxides
3 F3: oxidizable Hydrogen peroxide (8.8 mol/L); 2 h at 85 °C; Metals bound to iron and manganese
ammonium acetate (1 M) at pH 2.0 oxyhydroxides
4 R: residuala Acid mixture: HCl/HNO3/HF Metals bound in lithogenic minerals
a
Digestion of the residual material is not specified in the BCR protocol
6178 Environ Monit Assess (2013) 185:6173–6185

Table 3 Average concentrations of Cd, Cr, Cu, Ni, Pb, and Zn in different fractions of surface sediments from the Sal River estuary (n=4)

Cd Cr Cu Ni Pb Zn

Fraction 1 (μgg−1) 1 0.04 0.42 0.71 1.56 1.35 5.09

2 0.07 2.10 2.07 2.55 3.33 6.04
3 0.05 1.03 0.70 0.69 0.75 3.48
4 0.06 1.68 0.75 0.76 1.03 3.36
5 0.07 1.99 1.51 2.25 2.40 6.83
Mean±SD % 15±1 7±2 8±1 14±2 12±2 12±3
1 0.05 2.16 1.60 1.21 2.34 7.90
2 0.06 7.75 3.22 2.01 3.67 11.5
Fraction 2 (μgg−1) 3 0.04 2.88 1.11 0.78 1.51 4.04
4 0.06 3.86 1.49 0.84 1.76 6.32
5 0.06 5.46 2.99 2.45 4.06 12.1
Mean ± SD % 14±1 22±2 14±1 13±1 19±2 21±2
1 0.12 2.72 3.75 3.11 4.40 10.8
2 0.18 11.1 8.91 4.75 6.97 16.1
−1
Fraction 3 (μgg ) 3 0.11 3.34 2.65 1.41 2.62 5.23
4 0.15 4.80 2.77 2.16 3.35 6.61
5 0.17 6.67 7.77 5.20 5.80 13.6
Mean ± SD % 37±3 28±2 34±4 30±2 34±2 26±1
1 0.10 4.07 4.86 4.23 4.64 18.5
2 0.15 17.0 11.4 6.78 8.35 24.1
Residual (μgg−1) 3 0.12 5.14 2.75 2.25 2.90 7.32
4 0.13 6.26 4.69 2.79 3.30 10.2
5 0.13 12.6 9.21 7.34 5.21 24.3
Mean ± SD % 32±2 43±4 44±4 43±1 35±3 41±3
1 66 57 55 58 64 56
ΣF1+F2+F3(%) 2 67 55 55 58 63 58
3 59 59 62 56 63 63
4 66 62 52 57 65 62
5 70 53 62 57 70 57
Mean ± SD % 66±4 57±4 56±5 57±1 65±5 59±5
1 0.32 9.37 10.9 10.1 12.7 42.3
ΣF1+F2+F3+R (μgg−1) 2 0.46 37.9 25.6 16.1 22.3 57.7
3 0.34 12.4 7.22 5.13 7.80 20.1
4 0.41 16.6 9.70 6.56 9.40 26.4
5 0.43 26.7 19.7 17.2 17.5 56.8
Mean % 100 100 100 100 100 100
1 0.32 9.28 11.5 8.54 10.8 41.3
Total metal (μgg−1) 2 0.46 39.9 26.8 15.6 20.7 51.7
3 0.29 10.8 6.55 4.55 6.80 17.3
4 0.34 14.3 8.31 5.58 8.10 23.5
5 0.41 23.5 19.1 14.9 17.6 53.4
Recovery ± SD % 109±10 108±9 104±9 114±6 111±8 110±5
Environ Monit Assess (2013) 185:6173–6185 6179

R), the sum of contents for three fractions (ΣF1+F2+ The oxidizable (F3) and residual (R) fractions were
F3), and the total metal contents. The standard devia- generally the largest, and the environmentally mobile
tions of the four replicates ranged from 1 to 10 %, (F1) and reducible (F2) fractions the least significant.
indicating good repeatability. This can be seen in Fig. 2, which illustrates the metal

Fig. 2 Fractionation of a Cd, b Cr, c Cu, d Ni, e Pb, and f Zn in sediments from the Sal River estuary
6180
percentages extracted in each fraction, for the five
different sediments. The percentage of metal extracted
was calculated from the ratio between the concentra-
tion of the element in each fraction, and the sum of
concentrations in all fractions. The metal distribution
in the sediment fractions was similar along the estuary,
indicating that the elements had similar origins and
deposition mechanisms.
According to the results obtained for Cd fraction-
ation (Table 3, Fig. 2), the oxidizable fraction was the
most abundant (37 % of the total content), followed by
the residual (32 %), acid soluble (15 %), and reducible
(14 %) fractions. Cd was the metal with the greatest
percentage in the labile fraction. The metals in this
fraction are weakly bound with the sediment, so it is
therefore considered to be the most unstable and reac-
tive phase, which has greater potential for adverse
effects on the aquatic environment than the other
fractions (Li et al. 2007). Other studies have also
reported that in sediments Cd is associated with the
labile fraction (Jain 2004; Cuong and Obbard 2006;
Passos et al. 2010; Rodrigues et al. 2010).
Cr and Zn were found mainly in association with the
residual (44 and 41 %, respectively) and oxidizable (28
and 26 %, respectively) fractions, followed by the re-
ducible (22 and 21 %, respectively) and acid soluble (7
and 12 %, respectively) fractions. Of all the metals, Cr
and Zn showed the greatest percentages in the reducible
fraction (>20 % of the total content), where the elements
are associated with manganese and iron oxides and
hydroxides. Significant associations between trace met-
als and Fe and Mn oxides and hydroxides in sediments
have been found for regions that receive discharges of
industrial effluents (Relic et al. 2005; Sakan et al. 2007;
Li et al. 2007; Kirkelund et al. 2010).
Ni and Pb were found mainly in association with the
residual (43 and 35 %, respectively) and oxidizable (30
and 34 %, respectively) fractions, followed by the reduc-
ible (13 and 19 %, respectively) and acid soluble (14 and
12 %, respectively) fractions. These metals showed high
contents (greater than 30 %) in the oxidizable fraction. In
this sediment fraction, the metals are strongly com-
plexed, and are released following either degradation of
organic matter, or oxidation of sulfides to sulfates
(Passos et al. 2011). This is consistent with the results
reported by other investigators (Yuan et al. 2004; Morillo
et al. 2004; Cuong and Obbard 2006; Passos et al. 2010).
Finally, for Cu the residual fraction was predominant
(44 % of the total content), followed by the oxidizable
Environ Monit Assess (2013) 185:6173–6185
(34 %), reducible (14 %), and acid soluble (8 %) frac-
tions. Cu showed a greater content in the residual frac-
tion compared to the other metals. In this fraction, the
metals are bound to silicates and are therefore unavail-
able to the aquatic system (Passos et al. 2010). The inert
fraction of this metal in the sediments was probably
derived from natural sources, with the high percentage
being indicative of low levels of pollution (Cuong and
Obbard 2006). Hence, Cu was less available to aquatic
fauna, and less likely to enter the food chain than the
other metals. The results obtained here are in agreement
with those reported in the literature (Jain 2004; Ren-
Ying et al. 2007; Passos et al. 2011), where Cu was also
mainly found in the residual fraction.
An internal check on the sequential extraction proce-
dure was performed by comparing the sum of the four
fractions (Σacid-soluble + reducible + oxidizable +
residual) with the total measured concentrations. The
recovery of the sequential extraction was calculated as
follows: recovery = [(ΣF1+F2+F3+R)/full digestion]×
100. The results are given in Table 3. There was good
agreement between the sums of the fractions and the
total metal contents, with recoveries of between 104 and
114 %, which is within the range of other reports
(Martin et al. 1998; Li et al. 2007; Yuan et al. 2004).
Identification of natural and anthropogenic sources
Chemical fractionation differentiates metals of natural
origin from those derived from anthropogenic sources
(Passos et al. 2010). Elevated concentrations of metals
in the residual fraction indicate that sediments are
relatively unpolluted, and that the elements derive
mainly from lithogenic origins (Chandra Sekhar et al.
2003). Anthropogenic metals are predominantly found
in the most labile sediment fractions (carbonates, Fe
and Mn oxides, organic matter, and sulfides), which
are vulnerable to small changes in environmental con-
ditions, such as those caused by human activity
(Pempkowiak et al. 1999).
Table 3 lists the percentages of each metal extracted
in the most labile fractions (ΣF1+F2+F3). The order
of mobility (from most to least bioavailable) was: Cd
(66 %) > Pb (65 %) > Zn (59 %) > Ni (57 %) = Cr
(57 %) > Cu (56 %).
Cd and Pb showed the greatest amounts in the
bioavailable fractions, with 15 % (Cd) and 34 % (Pb)
of the total contents in the first and third fractions,
respectively. It has been found previously that Cd was
Environ Monit Assess (2013) 185:6173–6185
weakly adsorbed (retained on the sediment surface by
weak electrostatic interaction), and that the release of
Pb under oxidizing conditions could be explained by
binding to a mineral fraction (such as sulfides) that
was unstable under oxidizing conditions (Jain 2004;
Cuong and Obbard 2006; Passos et al. 2010). Ni, Zn,
Cu, and Cr showed higher percentages in the inert
fraction. Similar results have been reported previously
for these metals (Yuan et al. 2004; Ren-Ying et al.
2007; Passos et al. 2011).
Lithogenic metals are predominantly found in the
residual sediment fraction, where high percentages
indicate that the sediments are relatively unpolluted.
Elevated concentrations of metals in the most labile
sediment fractions are indicative of pollution, and that
the elements derive mainly from anthropogenic sour-
ces. Hence, the significant amount of metals in the
most bioavailable fractions probably resulted from the
presence of anthropogenic material (Passos et al.
2010). The Sal estuary receives discharges from mu-
nicipal and industrial wastewaters, agricultural runoff,
and particularly from metal industries (Garcia et al.
2011). These effluents contribute to the inputs of met-
als into the region’s aquatic systems.
Environmental implications
The fractionation of metals is of critical importance for
their potential toxicity and mobility (Maiz et al. 2000).
Fig. 3 Risk assessment
codes (RAC) for Cd, Cr, Cu,
Ni, Pb, and Zn in surface
sediments from the Sal
River estuary
6181
The fractions that are most influenced by human ac-
tivity include the exchangeable and carbonate-bound
fractions, in which metals are weakly retained and can
become more bioavailable due to equilibration with
the aqueous phase (Rath et al. 2009). The reactivity of
sediments was evaluated by applying the criteria of the
risk assessment code.
The RAC is a scale used to assess potential mobil-
ity and risk, based on the percentage of exchangeable
and carbonate-bound metal in the sediment (Perin et
al. 1985; Jain 2004; Ghrefat and Yusuf 2006; Karak et
al. 2011). Sediments can be classified as presenting no
risk (<1 %), low risk (1–10 %), medium risk (11–
30 %), high risk (31–50 %), and very high risk
(>50 %) to the ecosystem (Ikem and Adisa 2011;
Perin et al. 1985; Jain 2004).
Figure 3 illustrates the results of the risk factor
analysis, with values for the six trace metals given as
percentages for the fraction soluble in acid (%F1). In
general, the sediments showed low to medium risk for
all metals. The code as applied to the present study
revealed that 13–16 % of Cd, 4–10 % of Cr, 7–10 % of
Cu, 12–16 % of Ni, 10–15 % of Pb, and 10–17 % of
Zn were present in exchangeable fractions. Cr and Cu
showed a low risk at most of the sites (RAC <10 %),
while Cd, Ni, Pb, and Zn showed a medium risk (RAC
>10 %). Small variations in environmental conditions,
such as pH or salinity, could therefore increase avail-
ability of the elements to the aquatic system.
6182
In an earlier study in the same region (Garcia et al.
2011), it was shown that sediments from sites 2 and 5,
located in areas directly influenced by effluent inputs,
were vulnerable to enrichment by anthropogenic met-
als. Concentrations of Cd exceeded the probable nat-
ural background of the region, which was indicative of
enrichment. There was no evidence of anthropogenic
pollution for the other metals studied; however, in the
earlier work total elemental concentrations were used
to calculate possible enrichment. It is acknowledged
that the total concentration of an element only reflects
the amount stored in the system, and provides no
information concerning availability.
Similar studies conducted by Passos et al. (2010),
using sediments from the Poxim River (northeast
Brazil), which is highly polluted due to discharges of
domestic and industrial effluents, showed that the
sediments generally presented a medium risk for Cd,
Cr, Cu, Ni, Pb, and Zn. RAC values greater than 11 %
indicated a substantial risk of metal mobilization from
sediments across the entire study region. For the same
locations, Zn showed a high to very high risk (RAC
>30 %), while Ni showed a low risk (RAC <10 %).
Passos et al. (2011) identified low to medium risks for
Cd, Cr, Cu, Ni, Pb, and Zn in sediments from the
Sergipe River (also in northeast Brazil). In an environ-
mental risk assessment of metals in sediments from
lakes formed by the Cambé stream (Paraná State,
Brazil), Ishikawa et al. (2009) identified low to medi-
um risks for Cu and Pb, and a very high risk for Zn.
Chemical fractionation of metals in Lake Chapala
sediments (Mexico) showed that Cd, Pb, and Cr were
present in the exchangeable and carbonate fractions,
and that there was a very high risk of pollution of the
water column due to metal transfer (Trujillo-Cardenas
et al. 2010). Similar results were reported by Arcega-
Table 4 Partial metals concentrations in surface sediments from the Sal River Estuary (mean ± standard deviation, n=3)
Station Cd (μgg−1) Cu (μgg−1) Cr (μgg−1)
1 0.10±0.02 11.4±0.2 9.28±0.1
2 0.08±0.01 26.8±0.3 39.9±0.2
3 0.04±0.01 6.55±0.1 10.8±0.1
4 0.06±0.01 8.31±0.1 14.2±0.2
5 0.11±0.02 19.1±0.2 23.5±0.2
ER-L 1.20 34.0 81.0
ER-M 9.60 270 370
ER-L effects range low, ER-M effects range mean (Long and Morgan 1990)
Environ Monit Assess (2013) 185:6173–6185
Cabrera et al. (2009) for the mine-impacted Taxco
River in Mexico. Bioavailable Pb percentages greater
than 50 % in the river bed sediments indicated a very
high potential environmental risk, according to the
RAC.
ERL–ERM predictions
The ERL (effects range—low) and ERM (effects
range—mean) concentrations presented in the
Sediment Quality Guide, developed by Long and
Morgan (1990), have been adopted as an informal tool
to evaluate sediment chemical data in relation to pos-
sible adverse effects on aquatic biota. ERL and ERM
values indicate concentrations at which toxicity could
start to be observed, and above which adverse effects
might occur, respectively (Long and Morgan 1990;
Amin et al. 2009; Garcia et al. 2011). The SQG criteria
were derived and validated using data of test of toxic-
ity in sediments only for coastal regions of the USA
(Long and Morgan 1990), but despite this limitation
have been used to interpret data for sediments from
various global regions (Birch and Taylor 2002; Muniz
et al. 2004; Mora et al. 2004; Hyun et al. 2007; Gomes
et al. 2010).
In order to allow assessment of toxicity based on
SQG criteria, partial trace metal concentrations were
determined following extraction using nitric and
hydrochloric acids, which is a procedure compatible
with that used in the development of the SQG. Table 4
shows the partial concentrations obtained for each
metal, together with the respective ERL and ERM
values (Long and Morgan 1990).
The ranges of metal concentrations found were (μg
g−1): 0.04–0.11 (Cd); 6.55–26.8 (Cr); 9.28–39.9 (Cu);
4.55–15.6 (Ni); 6.78–20.7 (Pb); and 17.3–53.4 (Zn).
Ni (μgg−1) Pb (μgg−1) Zn (μgg−1)
8.54±0.1 10.8±0.1 41.3±0.5
15.6±0.2 20.7±0.2 51.7±0.5
4.55±0.1 6.78±0.1 17.3±0.2
5.58±0.1 8.14±0.1 23.4±0.3
14.9±0.2 17.6±0.2 53.4±0.6
20.9 46.7 150
51.6 218 410
Environ Monit Assess (2013) 185:6173–6185
For all the metals studied, the values were well below
the ERL, indicating that adverse effects on aquatic
biota should rarely occur. Ni showed concentrations
nearest to the ERL (20.9 μgg−1) at sites 2 (15.6 μgg−1)
and 5 (14.9 μgg−1), however the ability of ERL to
predict adverse effects is lower for Ni than for the
other metals (Long and Morgan 1990).
It must be emphasized that SQG criteria should be
used with caution, as there cannot be any guarantee of
a complete absence of toxicity at concentrations lower
than the ERL, or that samples where the ERM is
exceeded must necessarily be toxic, particularly con-
sidering that the SQG were not specifically developed
for the biogeochemistry of the region under study. For
greater confidence, it is important that the results
obtained here should be validated using toxicity tests
(Garcia et al. 2009).
Conclusions
Sequential extraction procedures enable evaluation of
the different chemical forms of metals present in sedi-
ments. For over three decades, these techniques have
been applied to environmental matrices including soils,
sediments, atmospheric particulates, and sewage sludge.
Fractionation of Cd, Cr, Cu, Ni, Pb, and Zn in
sediments was performed for samples collected from
five locations in the Sal River estuary. A significant
amount of Cd was present in the soluble fraction,
while the greatest percentages of Cr and Zn were in
the reducible fraction. Ni and Pb showed high contents
in the oxidizable fraction, and Cu was present in the
residual fraction.
The mobility of the metals was in the order Cd > Pb >
Zn > Ni = Cr > Cu. Cd and Pb, preferentially associated
with the more labile fractions (exchangeable, reducible,
and oxidizable), could be used as indicators for inputs
from anthropogenic sources, while Zn, Ni, Cr, and Cu
were associated to a greater extent with the inert frac-
tion, indicative of natural origins.
All of the sediment samples showed evidence of
anthropogenic enrichment, so that the metal concentra-
tions measured were not representative of natural con-
ditions in this estuarine region. Around 40 % of the metal
content was present in the residual fraction, similar to the
proportion found for contaminated regions worldwide.
Risk assessment code evaluation indicated low to
medium environmental risk for all metals in most
6183
samples. The results suggested that fluctuations in
pH or salinity might cause mobilization of metals from
the sediments to the water column. Possible metal-
related toxicity was investigated by comparing sedi-
ment chemical data with ERL–ERM values. It was
found that adverse effects on aquatic biota should
rarely occur. Despite agreement between the two
methods, further validation of these results should be
undertaken using toxicity tests in benthic organisms.
Acknowledgments The authors thank Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and
Fundação de Apoio à Pesquisa e à Inovação Tecnológica do
Estado de Sergipe (FAPITEC) for studentships. We are also
grateful to the Environmental Analytical Chemistry Laboratory
of Sergipe Federal University for technical support.
References
Alves, J. P. H., Passos, E. A., & Garcia, C. A. B. (2007). Metals
and acid volatile sulfide in sediment cores from the Sergipe
River Estuary, Northeast, Brazil. Journal of the Brazilian
Chemical Society, 18, 748–758.
Amin, B., Ismail, A., Arshad, A., Yap, C. K., & Kamarudin, M.
S. (2009). Anthropogenic impacts on heavy metal concen-
trations in the coastal sediments of Dumai, Indonesia.
Environmental Monitoring and Assessment, 148, 291–305.
Arcega-Cabrera, F., Armienta, M. A., Daesslé, L. W., Castillo-
Blum, S. E., Talavera, O., & Dótor, A. (2009). Variations of
Pb in a mine-impacted tropical river, Taxco, Mexico: use of
geochemical, isotopic and statistical tools. Applied Geo-
chemistry, 24, 162–171.
Barbier, E., Passos, E. A., Aragão, K. A. S., Santos, D. B., &
Garcia, C. A. B. (2010). Assessment of trace metal levels
in catfish (Cathorops spixii) from Sal River Estuary,
Aracaju, State of Sergipe, Northeastern Brazil. Water
Environment Research, 82, 2301–2305.
Barbieri, E., Garcia, C. A. B., Passos, E. A., Aragão, K. A. S., &
Alves, J. P. H. (2007). Heavy metal concentration in tissues
of Puffinus gravis sampled on the Brazilian Coast. Revista
Brasileira de Ornitologia, 15, 69–72.
Birch, G. F., & Taylor, S. E. (2002). Assessment of possible
sediment toxicity of contaminated sediments in Port
Jackson, Sydney, Australia. Hydrobiology, 472, 19–27.
Chandra Sekhar, K., Chary, N. S., Kamala, C. T., Suman Raj, D.
S., & Sreenivasa Rao, A. (2003). Fractionation studies and
bioaccumulation of sediment-bound heavy metals in
Kolleru lake by edible fish. Environmental International,
29, 1001–1008.
Cuong, D., & Obbard, J. P. (2006). Metal speciation in coastal
marine sediments from Singapore using a modified BCR-
sequential extraction procedure. Applied Geochemistry, 21,
1335–1346.
Fang, T., Li, X., & Zhang, G. (2005). Acid volatile sulfide and
simultaneously extracted metals in the sediment cores of
6184
the Pearl River Estuary, South China. Ecotoxicology and
Environmental Safety, 61, 420–431.
Filgueiras, A. V., Lavilla, I., & Bendicho, C. (2002). Chemical
sequential extraction for metal partitioning in environmen-
tal solid samples. Journal of Environmental Monitoring, 4,
832–857.
Garcia, C. A. B., Barreto, M. S., Passos, E. A., & Alves, J. P. H.
(2009). Regional geochemical baselines and controlling
factors for trace metals in sediments from the Poxim River,
Northeast Brazil. Journal of the Brazilian Chemical Soci-
ety, 20, 1334–1342.
Garcia, C. A. B., Passos, E. A., & Alves, J. P. H. (2011).
Assessment of trace metals pollution in estuarine sediments
using SEM-AVS and ERM-ERL predictions. Environmen-
tal Monitoring and Assessment, 181, 385–397.
Ghrefat, H., & Yusuf, N. (2006). Assessing Mn, Fe, Cu, Zn, and
Cd pollution in bottom sediments of Wadi Al-Arab Dam,
Jordan. Chemosphere, 65, 2114–2121.
Gomes, M. V. T., Costa, A. S., Garcia, C. A. B., Passos, E. A., &
Alves, J. P. H. (2010). Concentrações e associações geo-
químicas de Pb e Zn em sedimentos do rio São Francisco
impactados por rejeitos da produção industrial de zinco.
Quimica Nova, 33, 2088–2092 (In Portuguese).
Hyun, S., Lee, C., Lee, T., & Choi, J. (2007). Anthropogenic
contributions to heavy metal distributions in the surface
sediments of Masan Bay, Korea. Marine Pollution Bulletin,
54, 1059–1068.
Ikem, A., & Adisa, S. (2011). Runoff effect on eutrophic lake
water quality and heavy metal distribution in recent littoral
sediment. Chemosphere, 82, 259–267.
Ishikawa, D. N., Noale, R. Z., Ohe, T. H. K., Souza, E. B. R.,
Scarmínio, I. S., Barreto, W. J., & Barreto, S. R. G. (2009).
Avaliation of the environmental risk by metal distribution
in sediments from lakes formed by Cambé Stream at
Londrina. Quimica Nova, 32, 1744–1449.
Jain, C. K. (2004). Metal fractionation study on bed sediments
of River Yamuna, India. Water Research, 38, 569–578.
Karak, T., Abollino, O., Bhattacharyya, P., Das, K. K., & Paul,
R. K. (2011). Fractionation and speciation of arsenic in
three tea gardens soil profiles and distribution of As in
different parts of tea plant (Camellia sinensis L.). Chemo-
sphere, 85, 948–960.
Kirkelund, G. M., Ottosen, L. M., & Villumsen, A. (2010).
Investigations of Cu, Pb and Zn partitioning by sequential
extraction in harbour sediments after electrodialytic reme-
diation. Chemosphere, 79, 997–1002.
Li, Q., Wu, Z., Chu, B., Zhang, N., Cai, S., & Fang, J. (2007).
Heavy metals in coastal wetland sediments of the Pearl River
Estuary, China. Environmental Pollution, 149, 158–164.
Long, E. R., & Morgan, L. G. (1990). The potential for biolog-
ical effects of sediment-sorbed contaminantes tested in the
Naticional Status and Trends Program. NOAA Technical
Memorandum NOS OMA 52 (p. 346). Seattle: National
Oceanic an Atmospheric Administration.
Maiz, I., Arambarri, I., Garcia, R., & Millan, E. (2000). Evalu-
ation of heavy metal availability in polluted soils by two
sequential extraction procedures using factor analysis. En-
vironmental Pollution, 110, 3–9.
Martin, R., Sanchez, D. M., & Gutierrez, A. M. (1998). Sequential
extraction of U, Th, Ce, La and some heavy metals in sedi-
ments from Ortigas river, Spain. Talanta, 46, 1115–1121.
Environ Monit Assess (2013) 185:6173–6185
Miller J.C., & Miller J.M. (1993). Statististc for anaytical chem-
istry. Printed in Great Britain, 11–120.
Mora, S., Sheikholeslami, M. R., Wyse, E., Azemard, S., &
Cassi, R. (2004). An assessment of metal contamination
in coastal sediment of the Caspian Sea. Marine Pollution
Bulletin, 48, 61–77.
Morillo, J., Usero, J., & Gracia, I. (2004). Heavy metal distri-
bution in marine sediments from the southwest coast of
Spain. Chemosphere, 55, 431–442.
Mossop, K. F., & Davidson, C. M. (2003). Comparison of
original and modified BCR sequential extraction proce-
dures for the fractionation of copper, iron, lead, manganese
and zinc in soils and sediments. Analytica Chimica Acta,
478, 111–118.
Muniz, P., Danulat, E., Yannicelli, B., Garcia-Alonso, J.,
Medina, G., & Bícego, M. C. (2004). Assessment of con-
tamination by heavy metals and petroleum hydrocarbons in
sediments of Montivedeo Harbour (Uruguay). Environ-
mental International, 29, 1019–1028.
Passos, E. A., Barbieri, E., Garcia, C. A. B., & Alves, J. P. H.
(2005). Contaminação das águas do estuário do Rio do Sal,
Sergipe e Poxim. O Mundo da Saúde, 29, 226–237 (In
Portuguese).
Passos, E. A., Alves, J. C., Santos, I. S., Alves, J. P. H., Garcia,
C. A. B., & Costa, A. C. S. (2010). Assessment of trace
metals contamination in estuarine sediments using a se-
quential extraction technique and principal component
analysis. Microchemical Journal, 96, 50–57.
Passos, E. A., Alves, J. P. H., Garcia, C. A. B., & Costa, A. C. S.
(2011). Metal fractionation in sediments of the Sergipe
River, Northeast. Brazil. Journal of the Brazilian Chemical
Society, 22, 828–835.
Pempkowiak, J., Sikora, A., & Biernacka, E. (1999). Speciation
of heavy metals in marine sediments vs their bioaccumu-
lation by mussels. Chemosphere, 39, 313–321.
Perin, G., Craboledda, L., Lucchese, M., Cirillo, R., Dotta, L.,
Zanette, M., & Orio, A. (1985). Heavy metals speciation in
the sediments of Northern Adriatic Sea—a new approach
for environmental toxicity determination. In: Lekkas, T.D.
(Ed.), Heavy Metal in the Environment 2, (pp. 454–456).
Edinburg: CEP Consultants.
Rath, P., Panda, U. C., Bhatta, D., & Sahu, K. C. (2009). Use of
sequential leaching, mineralogy, morphology and multivar-
iate statistical technique for quantifying metal pollution in
highly polluted aquatic sediments—a case study: Brahmani
and Nandira Rivers, India. Journal of Hazardous Materi-
als, 163, 632–644.
Rauret, G., Lopez-Sanchez, J. F., Sahuquillo, A., Rubio, R.,
Davidson, C., Ure, A., & Quevauviller, P. (1999). Improve-
ment of the BCR three step sequential extraction procedure
prior to the certification of new sediment and soil reference
materials. Journal of Environmental Monitoring, 1, 57–61.
Relic, D., Dordevic, D., Popovic, A., & Blagojevic, T. (2005).
Speciations of trace metals in the Danube alluvial sedi-
ments within an oil refinery. Environmental International,
31, 661–669.
Ren-Ying, L., Hao, Y., Zhi-Gaol, Z., Jun-Jiel, L., Xiao-Hua, S., &
Fengl, J. (2007). Fractionation of heavy metals in sediments
from Dianchi Lake, China. Pedosphere, 17, 265–272.
Rodrigues, S. M., Henriques, B., Coimbra, J., Ferreira, S. E.,
Pereira, M. E., & Duarte, A. C. (2010). Water-soluble
Environ Monit Assess (2013) 185:6173–6185
fraction of mercury, arsenic and other potentially toxic
elements in highly contaminated sediments and soils. Che-
mosphere, 78, 1301–1312.
Rubio, B., Nombela, M. A., & Vilas, F. (2000). Geochemistry of
major and trace elements in sediments of the Ria de Vigo
(NW Spain): an assessment of metal pollution. Marine
Pollution Bulletin, 40, 968–980.
Sakan, S., Gržetić, I., & Đorđević, D. (2007). Distribution and
fractionation of heavy metals in the Tisa (Tisza) River
sediments. Environmental Science and Pollution Research
International, 14, 229–236.
Silva, A. F., Lima, G. R. S., Alves, J. C. A., Santos, S. H.,
Garcia, C. A. B., Alves, J. P. H., Araújo, R. G. O., &
Passos, E. A. (2012). Evaluation of trace metal levels in
surface sediments of the Sergipe River Hydrographic
Basin, Northeast Brazil. Journal of the Brazilian Chemical
Society, 23, 1669–1679.
Tessier, A., Campbell, P. G. C., & Bisson, M. (1979). Sequential
extraction procedure for the speciation of particulate trace
elements. Analytical Chemistry, 51, 844–851.
6185
Trujillo-Cardenas, J. L., Saucedo-Torres, N. P., Del Valle, P. F.
Z., & Donato, R. (2010). Speciation and sources of toxic
matals in sedimetn of lake Chapala, Mexico. Journal of the
Mexican Chemical Society, 54, 79–87.
(1993). Speciation of heavy-metals in soils and sediments—an
account of the improvement and harmonization of extrac-
tion techniques undertaken under the auspices of the BCR
of the Commission-of-the-European-Communities. Inter-
national Journal of Environmental Analytical Chemistry,
51, 135–151.
Yuan, C. G., Shi, J. B., He, B., Liu, J. F., Liang, L. N., &
Jiang, G. B. (2004). Speciation of heavy metals in
marine sediments from the East China Sea by ICP-
MS with sequential extraction. Environmental Interna-
tional, 30, 769–783.
Zemberyov, M., Bartekov, J., & Hagarov, I. (2006). The utiliza-
tion of modified BCR three-step sequential extraction pro-
cedure for the fractionation of Cd, Cr, Cu, Ni, Pb and Zn in
soil reference materials of different origins. Talanta, 70,
973–978.