Microchemical Journal 95 (2010) 169173

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Microchemical Journal
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m i c r o c

Optimization of a centrifugation and ultrasound-assisted procedure for the

determination of trace and major elements in marine invertebrates by ICP OES
Wagna P.C. dos Santos a,, Vanessa Hatje b, Darlan da S. Santil b, Andra P. Fernandes b,
Maria Graas A. Korn b, Manuel M. de Souza b
a
b

Instituto Federal de Educao, Cincia e Tecnologia da Bahia, IFBA, Salvador, BA, Brazil
Laboratrio de Oceanograa Qumica, Departamento de Qumica Analtica, Instituto de Qumica, UFBA, Campus de Ondina, 40170-280, Salvador, BA, Brazil

a r t i c l e

i n f o

Article history:
Received 3 November 2009
Accepted 11 November 2009
Available online 26 November 2009
Keywords:
Centrifugation
Sonication
ICP OES
Trace elements
Marine invertebrate
Todos os Santos Bay

a b s t r a c t
A three factor Doehlert design was applied to optimize the extraction of elements in marine invertebrate
samples (oyster, mussel and clam) using centrifugation with diluted acids. The factors were: concentration
of acid mixture (HCl + HNO3 + H3CCOOH; 13 mol L 1), centrifugation time (1040 min), and rotation
velocity (600010,000 rpm). In order to evaluate the ultrasound effect on the extraction of trace metals, a
portion of the certied oyster tissue sample (NIST 1556b; 0.250 g) was sonicated for 15 min and then the
solution was centrifuged at the optimized conditions. The optimized procedure was then applied to nine
marine invertebrate samples (oysters, clams and mussels) collected at Todos os Santos Bay, Bahia, Brazil. The
optimized condition of the developed method was: 20 min of centrifugation at 9000 rpm using the acid
mixture 1.0 mol L 1. The effect of sonication prior centrifugation was very variable, but it promoted a
signicant increase in the extraction (12 to 44%) of some trace elements. Moreover, the combination of the
centrifugation with sonication offered other advantages, such as improvement of the precision and accuracy,
both demonstrated using the oyster tissue reference sample. The results of major and trace elements in
marine invertebrate samples showed that elements such us Cd and Cu in oyster samples and Zn in mussel
and clam samples were above recommendations, suggesting that its consumption may impose health risks.
2009 Elsevier B.V. All rights reserved.

1. Introduction
Many species of marine invertebrates (e.g. clams and mussels) are
known to accumulate trace metals in its tissues and are widely used in
environmental monitoring programs. The accumulated metal concentrations provide a relative measure of the total metal intake by an
organism, including all routes, integrated over a preceding time period
[13]. At the same time, marine invertebrates represent a major part of
the human diet, especially for coastal populations, which are more
susceptible to the toxic effects that elevated metal concentrations in
oysters, clams and mussels may pose to them. Considering that each
biological species has its own pattern of accumulation that reects a
wide response of the environment contamination, it is important to use
a suite of organisms to evaluate metal contamination [3].
Several analytical techniques are available for trace elements
determinations in invertebrates, such as inductively coupled plasmaoptical emission spectrometry (ICP OES) [26], inductively coupled
plasma-mass spectrometry (ICP-MS) [611], ame atomic absorption
spectrometry (F AAS) and electrothermal-atomic absorption spectrometry (ETAAS) [1219]. For trace metal determination in mussels,
the matrix usually needs to be digested to convert the solid sample in
Corresponding author. Tel.: + 55 71 2102 9516; fax: + 55 71 2102 9506.
E-mail address: wagna@ifba.edu.br (W.P.C. dos Santos).
0026-265X/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.microc.2009.11.004

solution prior to the measurement. Many procedures have been

developed in order to shorten the analysis time and also to minimize
the problems associated with solid sample pre-treatment, such as
sample contamination and analyte loss [20]. Microwave digestion of
solid samples followed by ICP OES analysis, for instance, is a powerful
method for complete decomposition and accurate determination of
metals in marine species [7,8,1824]. In contrast to intensive sample
preparation methods, involving high temperatures and pressures,
acid extraction, under mild conditions, appears to be a promising
alternative, particularly, when associated with ultrasonication.
Ultrasonic extractions have proven to be successful for achieving
quantitative recoveries for an array of biological matrices. Parameters
associated with the ultrasonic acid extraction for elemental analysis
have already been reviewed [2527]. Acid extraction methods usually
involve the use of diluted acid to remove elements from solid samples,
providing a signicant speeding up in the pre-treatment step. Various
ultrasound-assisted extraction procedures have been reported for
biological tissues [6,1214], but the use of centrifugation and ultrasound-assisted multielemental extraction procedure, has not yet been
sufciently explored.
The aim of the present work was to develop and to apply a
centrifugation and ultrasound-assisted multielemental extraction
procedure, using a Doehlert design, for the analysis of marine
invertebrate (oyster, mussel and clam) samples by ICP OES.

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W.P.C. dos Santos et al. / Microchemical Journal 95 (2010) 169173

2. Materials and methods

2.1. Instrumentation
An ICP OES with axially-viewed conguration (VISTA PRO, Varian,
Mulgrave, Australia) was used for Ca, Cd, Cu, K, Mg, Mn, Na, Sr, and Zn
determination. This equipment includes a Littrow optical system with an
echelle grating and a simultaneous charge coupled device (CCD) detector
that allows readings from 167 to 785 nm. The pre-optical system was
purged with Ar in order to enable readings below 190 nm. The
polychromator was thermostatized at 34 C and purged with argon
end-on-gas interface was used to minimize interferences caused by autoabsorption processes. The operational parameters are described in
Table 1.
An analytical balance (Sartorius Ag Gottiengen, Germany) was
used to weigh the marine invertebrate samples, a freeze dryer
(Modulyo D, Thermo Scientic, USA) was used to dry samples, an
ultrasonic bath VWR, model 75D (Cortland, New York, USA) and a
centrifuge Eppendorf model 5804 (Germany) were employed to
promote the extraction of target elements. A ball mill, model 8000M
(Spex Sample Prep, USA) with a tungsten carbide vial set and tungsten
carbide ball was used for grinding samples.
A closed-vessel microwave oven (ETHOS EZ, Milestone, Italy) was
used for sample decomposition with the aim to evaluate, by comparison, the efciency of extraction methodology.

Santos Bay, Bahia, Brazil. In the laboratory, bivalve shelves were

washed with deionized water, opened and the tissue was removed
with Teon tweezers. The tissue, after rinsing with deionized water,
was freeze dried, and maintained in a dessiccator at room temperature.
Dried samples were grinded in a ball mill with a tungsten carbide vial
set.

2.4. Multivariate optimization of extraction method

A three factor Doehlert design was applied to optimize the
extraction of elements in marine invertebrate samples using centrifugation with diluted acids. The factors were: concentration of acid
mixture (HCl + HNO3 + H3CCOOH; 1.0, 2.0, and 3.0 mol L 1), centrifugation time (10, 15, 20, 25, 30, 35 and 40 min) and rotation (6000,
7000, 8000, 9000, and 10,000 rpm). Mass of sample (250 mg) and the
nal volume (10.0 mL) were the xed variables.
The analytical response was the percentage of recovery of the
studied elements using as reference the certied concentration values
of the oyster tissue sample (NIST, SMR 1566b). To optimize a
procedure that satises an efcient simultaneous extraction of all
studied elements, the overall response (OR) was adopted. The overall
response (OR) is given by the following expression:
OR = RX1 = LSX1 + RX2 = LSX2 + + RXn = LSXn

2.2. Reagent and analytical solutions

All solutions were prepared from analytical reagent grade chemicals
and using high purity deionized water obtained by Milli-Q water
purication system (Millipore, Belford, MA, USA; 18.2 m cm 1). High
purity analytical stock solutions of 1000 mg L 1 (Titrisol, Merck,
Germany) of each element were used daily to prepare the multielement
reference analytical solutions. Diluted acid solutions of HNO3, HCl and
CH3COOH (1.0, 2.0 and 3.0 mol L 1) were prepared by dilution with pure
water.
All glassware and polypropylene asks were cleaned with
detergent solution, soaking in 10% (v/v) HNO3 for at least 24 h, rinsed
with deionized water (Milli-Q system) and stored into a cleaned
polypropylene container.

where R(Xn) is the analytical signal of an n element in a particular

experiment and LS(Xn) is the largest signal in the set of experiments
for that n element [26]. The statistical signicance of the effects of the
variables were studied and the interactions among them was
evaluated by applying analysis of variance (ANOVA) using the
software Statistica for Windows [28].
In order to evaluate the ultrasound effect on the extraction of trace
metals, a portion of the certied oyster tissue sample (NIST, 1556b;
0.250 g) was weighed in polypropylene centrifuge tube (50 mL
capacity) and 10.0 mL of the diluted acid mixture was added.
Afterward, the solution was sonicated for 15 min, in an ultrasonic
bath at 2530 C, and then the solution was centrifuged at the
optimized conditions.

2.3. Samples
2.4. Sample digestion in closed system
All experiments were realized with the certied material oyster
tissue (NIST, SRM 1566b National Institute of Standard and
Technology, USA). The certied sample was also employed to verify
the accuracy of the proposed method.
The optimized procedure was applied to the analysis of oyster
tissues (Crassostrea rhizophora), mussels (Mytella guiyanensis) and
clams (Anomalocardia brasiliana) collected at three sites along Todos os

Table 1
Operational parameters used in axial view ICP OES.
Characteristics

Instrument conditions

RF generator
Power
Spray chamber
Nebulizer
Plasma gas ow
Auxiliary gas ow
Nebulizer gas ow
Injector tube diameter
Emission lines (nm)

40 MHz
1.3 kW
Cyclonic chamber
SeaSpray
15.0 L min 1
1.5 L min 1
0.7 L min 1
2.4 mm
Ca 422.673; Cd 214.439; Cu 324.754;
K 766.491; Mg 279.553; Mn 257.611;
Na 588.995; Sr 407.771; Zn 213.857

In order to validate the developed method, the certied oyster

tissue sample (NIST, 1556b) was also digested by a microwave closed
system. For these digestions, a mass of about 250 mg of the certied
oyster tissue sample was directly weighed in a TFM digestion vessel
and 1 mL of H2O2 conc. and 7 mL of HNO3 conc. were added. The
vessels were placed on the turntable of the microwave. The
microwave oven was operated according to the parameters listed in
Table 2, for the simultaneous digestion of eight to ten vessels. The
digests were diluted to 25 mL with deionized water and the analytes
were determined by ICP OES.

Table 2
Heating program for microwave assisted digestion.
Steps

Power (W)

Time (min)

Temperature (C)

1
2
3
4
Ventilation

1000
1000
1000
1000
10 min

4
2
8
15

90
90
180
180

W.P.C. dos Santos et al. / Microchemical Journal 95 (2010) 169173

171

3. Results and discussion

3.1. Optimization
The percentage of recovery obtained by Doehlert varied from 45 to
95, 8 to 52, 35 to 71, 67 to 102, 38 to 78, 61 to 92, 58 to 100, 40 to 85
and 51 to 91%, respectively, for Ca, Cd, Cu, K, Mn, Na, Mg, Sr and Zn
(Table 3). ANOVA was used to assess the signicance of each studied
variable. The quadratic model employed did not present lack of t for
the overall response and for individual responses of the studied
elements. The quadratic mean of the pure error was 0.3, which
represents less than 3% for the overall response.
In general terms, the variance analysis showed that centrifugation
time (quadratic and linear term), concentration (linear term) and the
interaction time and rotation were the signicant (P b 0.05) factors. It
is worth mentioning that the interaction presented the highest
signicance for the overall response and also the individual responses
of the studied elements. Results showed that high rotation and
extraction time favor high recoveries (Fig. 1), whereas the acid
concentrations presented the opposite effect, i.e. extractions are
favored when more diluted acids are employed (Table 3, experiments
11 and 23). When individual responses are compared to the overall
response it can be seen that, for Cu, while centrifugation time and the
interaction time and rotation were signicant (P b 0.05), the concentration of the acid mixture did not show a signicant effect.
Nevertheless, maximum regions were not observed and response
surface was characterized as saddle point. It was considered that the
optimized condition was 20 min of centrifugation at 9000 rpm using
the acid mixture 1.0 mol L 1.
In order to evaluate the effect of sonication in the extraction of
trace and major elements in the CRM oyster sample, the matrix
with the acid mixture 1.0 mol L 1 was sonicated for 15 min prior
to centrifugation at the optimized condition. The use of the
sonication prior to the centrifugation promoted an increase in the
percentage of recovery for most studied elements. The efciency of

Fig. 1. Contour graph obtained for the optimization procedure using Doehlert design.

sonication upon the analyte extractions nevertheless was very

variable. For major elements the enhancement promoted by
sonication was less than 5%, and the improvement was not
signicant. Nevertheles, sonication had a signicant effect in the
extractions of Cu, Mn and Sr. The increases in trace element
recovery suggest that the use of sonication associated with
centrifugation is recommendable (Fig. 2).
3.2. Merit gures
Figures of merit can be used to characterize an analytical method
by ICP OES, including the number of elements, repeatability, accuracy
and limit of quantication. Analytical characteristics of the developed

Table 3
Doehlert matrix for the optimization procedure.
Exp

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27

t
(min)

Rotation
(rpm)

C
(mol L 1)

25
25
25
25
40
25
10
10
40
20
20
30
35
15
30
25
40
25
10
10
40
20
20
30
35
15
30

8000
8000
8000
10,000
9000
6000
7000
9000
7000
7000
9000
7000
8000
8000
9000
10,000
9000
6000
7000
9000
7000
7000
9000
7000
8000
8000
9000

2
2
2
2
2
2
2
2
2
1
1
3
1
3
3
2
2
2
2
2
2
1
1
3
1
3
3

OR = overall response.

Extraction (%)

OR

Ca

Cd

Cu

Mn

Na

Mg

Sr

Zn

83.5
73.7
85.5
88.9
72.3
83.9
59.7
71.5
84.9
86.6
94.3
80.2
85.1
73.3
83.2
86.3
57.8
81.1
45.2
87.7
83.8
91.2
87.6
78.8
94.6
73.3
80.4

45.0
34.0
44.0
46.9
29.6
44.0
20.9
33.0
44.1
44.3
52.0
39.8
44.6
36.5
45.5
44.6
17.2
40.7
8.1
46.4
45.7
50.4
49.6
43.7
48.4
34.8
39.9

67.1
56.6
68.1
70.0
53.9
68.0
45.0
56.9
69.9
63.2
71.0
66.3
61.2
62.4
70.8
69.2
40.9
65.0
34.6
71.2
69.5
69.1
66.8
63.7
69.9
59.9
65.5

93.8
87.5
94.3
96.1
89.1
93.1
77.6
85.1
95.2
97.8
101
91.3
98.1
87.4
92.8
95.2
81.3
91.5
66.5
94.7
95.0
98.6
98.0
88.5
102
86.1
91.9

71.0
61.3
71.1
73.5
59.1
70.9
49.5
60.5
73.0
71.2
78.1
67.7
70.4
63.8
71.6
73.3
45.7
68.0
37.8
74.6
72.2
75.2
74.0
66.2
76.5
61.6
67.0

85.9
77.9
85.1
87
80
84.1
69.2
76.5
87.3
88.2
92.2
83.0
88.9
80.2
85.0
86.7
71.5
83.2
60.8
87.3
86.0
89.0
88.8
78.4
91.1
79.2
83.6

92.8
82.9
92.9
95.5
81.9
91.8
69.9
81.1
94.1
93.5
99.5
91.3
91.8
86.9
93.8
94.7
69.1
89.0
58.0
96.4
93.7
96.2
94.7
86.4
98.2
84.3
90.6

78
62.2
72.2
77.1
61.4
75.1
55.0
63.8
72.8
75.5
84.7
71.9
80.7
64.4
68.6
77.0
50.7
69.3
39.5
74.4
72.2
82.3
83.7
65.8
85.1
60.7
70.3

82.6
73.8
83.0
85.1
72.7
82.1
64.1
72.6
84.3
85.0
90.7
80.2
83.6
77.3
83.0
84.7
60.8
79.6
51.0
85.7
84.4
88.1
86.7
76.3
89.5
74.2
80.0

8.23
7.11
8.18
8.48
6.95
8.15
5.88
7.01
8.29
8.26
9.00
7.87
8.26
7.40
8.18
8.36
5.65
7.83
4.54
8.46
8.27
8.72
8.60
7.63
8.87
7.17
7.84

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W.P.C. dos Santos et al. / Microchemical Journal 95 (2010) 169173

also using the background equivalent concentration (BEC) according

to the following equations [29]:
LOD = 3 BEC RSD = 100

2
3

LOQ = 3:3 LOD:

1

The obtained LOQs (g g ) for the centrifugation and centrifugation associated with ultrasound procedures are presented in
Table 4. The paired t-test at 95% condence level showed that there
was no signicant difference between the LOQs (P N 0.05) obtained for
the tested procedures. However, it seems that there is a slight improvement in this parameter for Mg and Zn.
The repeatability and accuracy of the proposed procedure were
evaluated by the determination of trace and major elements in 10
replicates of the oyster tissue certied material. The relative standard
deviations (% RSD) for the centrifugation procedure varied between
2.4 and 8.8% (Table 4). For all elements, an increase in the precision
when sonication was applied prior to the centrifugation (i.e. RSD from
1.9 to 3.8%) was observed.
The recovery of the tested elements for the certied oyster tissue
(SRM 1566b) varied from 72 to 100%. The only elements that presented
recovery below 85% were Cd and Sr. Elements such as Ca, K, Mg and Na
did not present signicant difference in the recoveries obtained by
centrifugation associated with sonication and the microwave digestion.
3.3. Application
Fig. 2. Concentration of trace metals and major elements in oyster sample (NIST 1566b)
after treatment with centrifugation and sonication following centrifugation.

After the optimization of the procedure employing centrifugation

associated with ultrasonication, and the verication of the precision and
accuracy (Table 4), the developed procedure was then applied to three
species, thoroughly consumed by local population, of marine invertebrates (i.e. C. rhizophora oyster; M. guiyanensis mussel; A. brasiliana
clam) collected at Todos os Santos Bay, northeast Brazil. The results are
presented in Table 5. It was observed a large variability in metal

method were evaluated after centrifuge optimization and also for the
procedure combining centrifugation and sonication.
The limit of detection (LOD) and limit of quantication (LOQ) were
estimated using the RSD obtained for ten experimental blanks, and

Table 4
The relative standard deviations (n = 10) and limit of quantication (LQ) for the centrifugation procedures and CRM NIST 1566b certied and obtained values for microwave
digestion and centrifugation associated with ultrasonication.
Element

Oyster tissue (CRM 1566b)

Centrifugation

Ca
Cd
Cu
K
Mg
Mn
Na
Sr
Zn
a

Centrifugation + US

RSD (%)

LQ (g g

5.9
6.2
8.8
2.4
5.8
6.1
2.6
6.0
4.7

8.59
0.30
0.48
3.86
1.55
0.10
3.05
0.05
1.44

RSD (%)

LQ (g g

3.1
5.7
3.8
1.7
2.4
3.0
1.9
3.5
2.3

8.66
0.32
0.33
3.01
0.39
0.08
4.30
0.04
0.89

Certied value

Centrifugation + US

Microwave digestion

(g g 1)

(g g 1)

(g g 1)

0.0838 0.0020a
2.48 0.08
71.6 1.6
0.652 0.009a
0.1085 0.0023a
18.5 0.2
0.3297 0.0053a
6.8 0.2
1424 46

0.0838 0.0025a
1.78 0.10
63.9 2.44
0.637 0.0113a
0.0973 0.0024a
15.7 0.47
0.2834 0.0053a
4.99 0.18
1264 29

0.0852 0.0009a
2.32 0.06
76.3 0.31
0.614 0.0117
0.1028 0.033a
17.7 0.3
0.2914 0.0080a
6.69 0.12
1590 45

Concentration in % (m/m).

Table 5
Results of application of the optimized procedure in clams, oysters and mussels (n = 3) from Todos os Santos Bay, Brazil.
Sample

Clam 1
Clam 2
Clam 3
Mussel 1
Mussel 2
Mussel 3
Oyster 1
Oyster 2
Oyster 3

Concentration, g g 1
Ca

Cd

Cu

Mg

Mn

Na

Sr

Zn

8030 196
7774 248
9774 371
2994 263
3712 58
3804 24
4835 166
6101 966
6033 459

1.04 0.03
bd
bd
bd
bd
bd
12.6 0.71
bd
bd

8.69 0.13
5.14 0.06
5.33 0.08
7.69 0.64
13.8 0.29
14.1 0.74
137 8.87
26.9 0.30
28.9 0.17

14,372 509
13,028 461
12,453 91.9
7630 564
10,567 435
10,711 336
10,633 583
14,541 172
15,370 165

5741 39.3
6458 97.0
6507 44.9
3325 83.1
4158 50.5
4824 81.7
3829 143
5203 62.4
5742 68.0

22.3 0.33
19.0 0.89
25.6 0.26
36.8 3.04
36.8 0.56
40.1 0.8
24.9 1.43
16.5 0.32
20.4 0.24

6034 136
8416 342
9527 330
4668 150
5722 58.6
5870 135
3911 114
6209 212
7179 674

97.9 3.90
111 3.20
135 6.67
52.1 4.71
64.2 1.95
65.3 0.68
49.1 2.86
65.9 4.46
67.8 2.48

58.9 2.04
62.8 0.88
59.8 1.35
57.2 6.65
64.8 0.92
60.0 1.15
2334 88.1
1230 14.4
1015 7.78

bd = below detection; Clam 1, 2, and 3; Mussel 1, 2 and 3; Oyster 1, 2 and 3 are independent samples collected at different sites at Todos os Santos Bay.

W.P.C. dos Santos et al. / Microchemical Journal 95 (2010) 169173

concentrations between the analised species, which is expected seen the

differences in the feed behaviour and ecology of each tested sample. The
highest concentration of Zn, Cu, Cd and K was observed in oyster samples,
which are well known to accumulate trace metals. Concentrations of Cd
and Cu in oyster samples and Zn in mussel and clam samples were above
recommendation values of the Brazilian Legislation for Food Contamination [30,31]. The obtained results are in good agreement with the review
on contamination of Todos os Santos Bay [32].
4. Conclusions
The centrifugation associated with ultrasonication is a versatile,
cheap and simple procedure for quantitative determinations of Ca, K,
Cu, Zn and Na in marine invertebrates. In general, studied elements
presented a better precision and accuracy with the procedure
employing centrifugation associated with sonication, compared with
the use of centrifugation only. The application of the proposed
procedure in marine invertebrate samples of Todos os Santos Bay,
Brazil, showed that concentrations of Cu, Zn and Cd in some especies
consumed by local population are well above recommended values.
Acknowledgements
This research was funded by a grant from CNPq (Proc. 505924/
2004-8), and FAPESB. V. Hatje and M.G. Korn express their gratitude to
CNPq-Brazil by sponsorships provided.
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