Journal of Scientific Research & Reports

8(7): 1-9, 2015; Article no.JSRR.19717

ISSN: 2320-0227

SCIENCEDOMAIN international
www.sciencedomain.org

Comparison of Acid Mixtures Using Conventional

Wet Digestion Methods for Determination of Heavy
Metals in Fish Tissues
Fabunmi Idera1*, Olumodeji Omotola1, Adeleye Adedayo1
and Uyimadu John Paul1
1
Department of Physical and Chemical Oceanography, Nigerian Institute for Oceanography and
Marine Research, Nigeria.

Authors’ contributions

This work was carried out in collaboration between the authors. Author UJP designed the study,
contributed to the correction of the draft and supervised the work. Authors OO and AA designed the
protocol, author FI wrote the draft of the manuscript, managed the literature searches, designed the
figures and made statistical analysis. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/JSRR/2015/19717
Editor(s):
(1) Surapong Pinitglang, Department of Food Science and Technology, School of Science and Technology, University of the
Thai Chamber of Commerce, Thailand.
Reviewers:
(1) Kpobari W. Nkpaa, University of Port Harcourt, Nigeria.
(2) Anonymous, Rai Technology University, Bangalore, India.
(3) Anonymous, Muğla Sıtkı Koçman University, Turkey.
(4) Anonymous, University of Benin, Nigeria.
Complete Peer review History: http://sciencedomain.org/review-history/11356

Received 24th June 2015

th
Accepted 29 August 2015
Original Research Article th
Published 12 September 2015

ABSTRACT

Sample preparation is an important step of any analytical procedure. Considering low budget and
limited unsophisticated technology available in small laboratories, it is necessary to carefully
evaluate and optimise the efficiency of the conventional wet digestion method. The study focuses
on comparison of different acid mixtures for fish tissue pre-treatment. Four of the most commonly
used acid mixtures: nitric/peroxide (NO), nitric/perchloric (NP), aqua regia (NH) and
sulphuric/perchloric/nitric (SPN) acid mixtures were evaluated for accurate determination of Pb, Zn,
Cd, Cu, Cr and Fe in tuna fish homogenate standard reference material (SRM) and fish tissues by
flame atomic absorption spectroscopy. SPN mixture was found to be the most suitable for
determination Pb, Cd, Cr and Fe in fish tissue. NO and NH gave good extraction for Zn and Cu
while NH showed the least extraction capacity for determination of all the metals in fish tissue.
_____________________________________________________________________________________________________

*Corresponding author: Email: ideragaffar@yahoo.com;

 Idera et al.; JSRR, 8(7): 1-9, 2015; Article no.JSRR.19717

Highest recovery efficiency of the methods were Zn(98%) and Cu(97%) for NP; Cr(101%) and
Fe(102%) for SPN and Cd(101%) for NH. On the basis of this, the two preferred acid mixtures are
NP and SPN. Application of method SPN is recommended for both extraction and recovery
analysis of fish tissue. However, to avoid potential hazards with the use of perchloric acid, it should
be added gradually at the later stage of the digestion process.

Keywords: Heavy metals; fish tissue; standard reference material; conventional wet digestion
methods.

1. INTRODUCTION acid procedure was also found to be effective,

The determination of heavy metal in fish tissues
at trace levels has become an important process
in monitoring the aquatic environment. Several
analytical methods have been used in
determination of these metals in fish tissues.
Sample preparation is an important step of
analytical process in the determination of heavy
metals. Methods of sample treatment for the
determination of heavy metals include wet
digestion and dry ashing followed by acid
dissolution [1]. However, wet digestion is more
commonly employed due to reduced loss of
analyte at low temperature and speed [2]. Wet
digestion utilizes various mineral acids such as
HCl, HNO3, H2SO4 for decomposition of the
organic matrice of samples and can be carried
out either in closed or open system by use of
conventional heating or microwave digestion [3].
Microwave digestion procedure is more efficient
than the conventional means of heating as it
reduces time as well as risk of contamination of
the digest [4]. Yet, the conventional wet digestion
procedure remains popular and important in most
laboratories due to its economic aspect
(inexpensive and low technology). The
conventional wet digestion entails a system
equipped with a temperature source operating
either at a fixed temperature or in response to a
temperature program [5]. Wet digestion
determination of heavy metals in fish tissues
utilizes acids such as perchloric acid, nitric acid,
sulphuric acid and hydrochloric acid followed by
analysis using atomic absorption spectroscopy
[6-10]. To optimize organic phase separation with
inorganic acid, combination of these acids has
been used to reduce sample preparation [11-13].
The digestion process is the determining stage
as to the procedural time and the efficiency of the
recovery of the actual metal present in the
sample [14]. Tinggi et al. [15] reported that nitric
acid/perchloric acid procedure was the most
efficient, whereas the nitric acid/hydrogen
peroxide procedure was the least efficient in the
determination of cadmium and lead in biological
tissue. He further noted that nitric acid/sulphuric
but less effective for determination of lead, as the
sulphuric acid suppressed the absorbance
signals. HNO3-HClO3 and HNO3-H2SO4 were
shown by Dhindsa [16] to be a more superior
method in the decomposition of biological tissue
for the determination of Selenium when
compared to the use of aqua-regia and reverse
aqua-regia. Chad [17], revealed in his analysis
that measurement of mercury using aqua-regia
was much lower when compared to nitric-
sulphuric acid. He further explained that aqua-
regia did not completely extract mercury bound in
tissue and rapid heating during digestion using
aqua-regia led to loss of mercury, since mercury
is highly volatile.
In view of the importance of fish to human health
as a source of food and sustenance; it is
necessary to carefully evaluate a digestion
method considering the low budget and
unsophisticated technology available, by
comparing different acid mixtures to determine
levels of heavy metals in fish tissue. Therefore,
the aim of this study is to determine the most
efficient, convenient and inexpensive way for
sample preparation of fish tissue in a low
technology environment by comparing different
acid mixtures in conventional hot plate wet
digestion. For this reason, different acid mixtures
were adapted from other methods for the
determination of cadmium, chromium, lead,
copper and zinc, Nickel and Cobalt in seven
species of Croaker and Sole fish tissues using
the conventional wet digestion method. The
effectiveness of these methods was also
compared with the use of standard reference
material.
2. MATERIALS AND METHODS
Seven fish species were used for this study,
Cynoglossus senegalensis (sole), Pomadasys
jubelini (grunter), Brachydeuterus auritus
(grunter), Galeoides decadactylus (croaker),
Pseudotolithus senegalensis (croaker),
Pseudotolithus elongathus (croaker) and

2

Cynoglossus brownii (sole). The species were
selected on the basis of their abundance and
distribution in the marine system. Fishes were
purchased from fishermen on the shores of
Makoko in Lagos. All samples were obtained
fresh and were packed in ice and transported in
an insulated container back to the laboratory.
Fish species were identified in the laboratory and
allowed to thaw at room temperature. The fish
samples after defrosting were dissected with the
help of a stainless steel knife to obtain the
boneless tissue. Based on literature survey [18-
20], four most commonly used acid combinations
were applied herein; nitric-peroxide, aqua-regia,
nitric-perchloric and nitric-sulphuric-perchloric for
the determination of Cd, Cr, Pb, Cu, Zn and Fe
for decomposition of each fish tissue and results
compared to determine their efficiencies.
2.1 Nitric-peroxide Acid Digestion (NO)
Nitric-peroxide acid digestion was carried out
using 5 ml of (65%) nitric acid and 5 ml of (30%)
hydrogen peroxide added to 5 g each of sample
on a hot plate for about forty five minutes until
the solid dissolved and the volume of contents
was reduced to about 5 ml [21]. Contents were
then filtered through a 0.45-µ Millipore
membrane filter paper transferred quantitatively
Fig. 1. Map of the study area
3
Idera et al.; JSRR, 8(7): 1-9, 2015; Article no.JSRR.19717
to a 50 ml volumetric flask by adding de-ionized
water and analysed for heavy metals in an
atomic absorption spectrophotometer.
2.2 Aqua-regia Acid Digestion (NH)
Five grams each of samples was weighed
accurately and freshly prepared 15 ml HCl and 5
ml HNO3 using ratio 3:1 was added to the
sample. The mixture was heated on a hot plate
till the volume was reduced to about 5 ml. The
mixture was filtered through a 0.45-µ Millipore
membrane filter paper transferred quantitatively
to a 50ml volumetric flask by adding de-ionized
water and analysed for heavy metals in an
atomic absorption spectrophotometer.
2.3 Nitric-perchloric Acid Digestion (NP)
This method of wet digestion method was
described by Ferreira et al. [22]. Five grams each
of sample was accurately weighed into a 250 ml
conical flask followed by the addition of 15 ml
HNO3. The conical flask was heated on a hot
plate at about 100°C until all the solid
disappeared and vigorous reaction stopped. The
conical flask was then removed and allowed to
cool at room temperature. Next, 5 ml HClO4

was added and heated until HClO4 fumes were
visible. The temperature was regulated until
HClO4 fumes completely disappeared. The flask
was cooled at room temperature. The mixture
was filtered through a 0.45-µ Millipore membrane
filter paper transferred quantitatively to a 50 ml
volumetric flask by adding de-ionized water and
analysed for heavy metals in an atomic
absorption spectrophotometer.
2.4 Nitric-sulphuric-perchloric Acid Dige-
stion (SPN)
This approach was partly modified from that of
Tinggi et al. [23]. Five grams of each sample was
weighed into a conical flask and 10 ml of
concentrated HNO3 was added. The conical flask
was placed on a hot plate and heated until white
fumes evolved. The conical flask was removed
from the hot plate and allowed to cool at room
temperature. After cooling, 3 ml of concentrated
H2SO4 was added and further heated to release
orange fumes. The conical flask was removed
from the hot plate to cool at room temperature. 2
ml of HClO4 was added and heated until the
solution was reduced to about 5 ml. Each
digested sample was filtered through a 0.45-µ
Millipore membrane filter paper and made up
quantitatively to 50 ml with deionised water in a
volumetric flask and analysed for heavy metals in
an atomic absorption spectrophotometer.
2.5 Quality Control
In order to avoid contamination, all glasswares
were acid soaked in 10% HCl for 24 hrs and later
rinsed with double distilled water. Standard
Reference Material (SRM) IAEA-436 Tuna fish
flesh homogenate from the International Atomic
Energy Agency, analytical quality control
services, Austria was digested using the four
methods to check the quality of the methods. The
analytical results for reference material and its
certified values showed satisfactory agreement
with the recoveries (Table1). A quality control
sample was analysed in every seven samples
during AAS metal analysis.
% Recovery = Found concentration x 100
True concentration
Agilent 240AA air-acetylene Flame Atomic
Absorption Spectrometer was used for the
determination of all the elements, simultaneous
background corrections were made using a
deuterium lamp.
4
Idera et al.; JSRR, 8(7): 1-9, 2015; Article no.JSRR.19717
2.6 Statistical Analysis
A one way factor analysis of variance (ANOVA)
and Pearson correlation was used to interpret the
relationships between the different digestion
methods and heavy metals in fish samples.
Significantly different means were separated
using the methods of Duncan. Statistical
hypothesis testing was used to determine the
significance of the result obtained using 95%
confidence interval where p<.05 are considered
to be statistically significant [24].
3. RESULTS
The effectiveness of the digestion method in
recovery of heavy metals Cd, Cr, Cu, Zn and Fe
in standard reference material (SRM) IAEA-436
is shown in Table 1, expressed in percentage
recovery of each metal. Results obtained were in
good agreement with the certified values of all
the metals analysed. Recoveries of Zn ranged
from 92-98%, Cd 94-101%. Cu 91-97%, Cr 90-
101%, and Fe 92-102%. The effectiveness of the
digestion methods for determination of Cd, Cr,
Pb, Cu, Zn and Fe in fish samples were given in
Table 2.
SPN was shown to have the highest efficiency in
the determination of Pb, Cr, Cd and Fe in all the
fish species with the exception of Cd in
C. brownii which had the highest value of 0.870
mg/kg for NO digest and Fe in P. jubelini which
gave the highest value of 65.910 mg/kg for NP
digest. Values obtained using the different acid
mixture on the fish species were found to be
relatively close for Cu and Zn with efficiency
ranging from SPN≈NP>NO and SPN>NO>NP
respectively. Data obtained indicates that there
was no significant difference (p>.05) in the acid
mixture used. This was demonstrated by a one
way factor analysis of variance (ANOVA),
however, opposite results were obtained for the
relation between metals in the fish samples
using Pearson correlation analysis. Fe was found
with the highest concentration which ranged from
10.56 mg/kg – 141.04 mg/kg while Cu was found
to have the least concentration found in the fish
tissues with values ranging from 0-1.96 mg/kg.
Concentration of Pb, Zn, Cd and Cr were in
ranges of 0-13.622 mg/kg, 2.111-5.069 mg/kg,
0.76-1.63 mg/kg and 6.49-12.66 mg/kg
respectively.
4. DISCUSSION
Statistical analysis (ANOVA) indicates that there
was no significant difference (p>.05) in the

suitability of either of the acid mixtures for fish
tissue decomposition for determination of Pb, Zn,
Cd, Cu, Cr and Fe. Although no single acid
mixture was ideal for all the metals, particular
methods were better for the extraction of some
metals. For example, SPN acid mixture gave
good extraction of metals sampled in the different
fish species with the highest concentration found
for Pb, Cr and Fe (with the exception of
P. jubelini, which gave the highest extraction with
NP acid mixture) in all the fish species.
Correlation analysis showed that Zn, Cd, Cu, Cr
and Fe were not dependent on the acid mixture
for decomposition of fish tissue as p>.05,
however, Pb was significantly correlated to the
type acid mixture with p=.017. In terms of the
standard reference material IAEA-436, the
criteria used for determining the suitability of acid
mixture were based on the completeness of
decomposition as determined by the percentage
recovery. The order of preference for the
different acid mixture (Table 1) shows SPN was
best for Cr and Fe; NP for Zn and Cu; and NH for
Cd. On the basis of this, the two preferred acid
mixtures are NP and SPN.
Of all the acid mixture methods used, the SPN
method proved to be the most suitable for
accurate determination of Pb, Zn, Cd, Cu, Cr and
Fe in fish tissue sampled. Owing to the oxidizing
power of nitric acid, this is aided by the addition
of H2SO4 which helps to complete digestion [25].
In addition, HClO4 acid prevents the excessive
frothing which occurs frequently when HNO3 or
H2SO4 is used [26]. Also with the AAS technique,
the use of SPN for digestion of fish tissue was
suitable for analysis of heavy metals as it
eliminates the problem of clogging of the
aspirator tube of the atomic absorption unit [12].
NP acid mixture was next to SPN in extraction
performance and this has been shown in past
studies [15,27]. NP gave the highest extraction
for G. decadactylus, P. senegalensis and
C. brownie for Cu and P. jubelini for Zn and Fe.
This is in agreement with Ferrieira et al. [28] who
developed an acid digestion procedure of HNO3:
HClO4 (3:1) for the determination of Cu in various
food samples of animals and plants determined
by flame atomic absorption spectrophotometry.
Although the use of NP and SPN would be a
Table 1. Percentage recovery of heavy metals in standard reference
Acid mixture Zn
SNO 92
SNH 95
SNP 98
SSPN 97
5
Idera et al.; JSRR, 8(7): 1-9, 2015; Article no.JSRR.19717
preferred approach, however, some
consideration had been given to the explosive
nature of perchloric acid when in contact with
organic matrix [5], as it was added at the later
stage of digestion of the fish tissue to prevent
explosion.
Though SPN and NP gave better results
compared to NH and NO, high decomposition of
organic matter was also recorded for NO. H2O2 is
a popular oxidizing and addition to HNO3 has
been said to increase the oxidizing power when
used to decompose biological materials [5]. NO
gave the highest extraction of Cu in P. jubelini;
Zn in P. senegalensis; Zn and Cd in C. brownii.
The relatively low recovery and incomplete
digestion showed by NO was also shown by
Matusiewic et al. [29] and Yaru et al. [30] when
NO was used for digesting biological materials.
However, this acid mixture has been shown to be
relatively quick and easy to use for routine
analysis of environmental samples [29] and has
been used extensively for determination of heavy
metals in fish tissues [31-34].
The lowest concentration generally obtained by
acid mixture NH mixture was due to incomplete
decomposition of some of the organic matrix.
Similar observations have been made in past
studies [16,17] where NH has been found to be
less effective in the digestion of heavy metal in
biological tissues when compared with acid
mixtures such as nitric-sulphuric acid and nitric-
perchloric.
Correlation analysis showed statistical
differences between the metals concentrations
analysed (p<.05), this could be due to the
uptake, function and elimination rate of the
metals in the fish tissue. Also, some metals are
essential and play important roles in the fish
biological system and thereby have affinity for
specific organs where they are needed [35]. The
accumulation of metals in fish species was found
to be in the order Fe> Pb > Cr > Zn > Cd > Cu,
this was similar to the trend reported by Authman
[36] in order of Al > Mn > Fe > Pb > Cu > Zn >
Cd. Accumulation of heavy metals in fish tissues
has been reported to be regulated by physical
factors of the water body in which they live in
such as salinity, temperature and pH [37].
Cd Cu Cr Fe
98 93 97 96
101 91 90 92
96 97 93 98
94 95 101 102
 Idera et al.; JSRR, 8(7): 1-9, 2015; Article no.JSRR.19717

Table 2. Heavy metal content (mg/kg) in fish samples by four digestion methods

Fish species Acid mixture Pb Zn Cd Cu Cr Fe

C. senegalensis NO 0.709a±0.0001 2.479 a±0.0001 0.770 a±0.0002 ND 6.490 a±0.0013 19.640 a±0.0054
NH 0.466 a±0.0021 3.519 a±0.0019 0.900 a±0.0005 ND 8.530 a±0.0022 34.110 a±0.0044
NP 2.418 ab±0.0043 3.673 a±0.0025 0.860 a±0.0001 0.100 a±0.0001 7.140 a±0.0024 49.770 a±0.0015
b a a a a
SPN 13.109 ±0.0165 4.480 ±0.0012 1.330 ±0.0003 0.340 ±0.0001 10.270 ±0.0017 57.840 a±0.1072
P. jubelini NO 1.196 a±0.0071 2.848 a±0.0023 0.810 a±.0001 0.270 a±0.0001 7.610 a±0.0011 23.990 a±0.0005
NH 0.543 a±0.0001 3.340 a±0.0002 0.810 a±0.0002 ND 7.790 a±0.0008 29.370 a±0.0008
NP 3.054 ab±0.0024 3.849 a±0.0043 1.040 a±0.0002 0.130 a±0.0001 8.720 a±0.0019 65.910 a±0.1014
b a a a a a
SPN 13.622 ±0.0204 3.822 ±0.0011 1.630 ±0.0012 0.240 ±0.0002 10.380 ±0.0007 58.480 ±0.0011
a a a a a
B. auritus NO 1.499 ±0.0040 2.677 ±0.0001 1.220 ±0.0004 ND 8.880 ±0.0003 27.310 ±0.0209
a a a a a
NH 0.610 ±0.0001 3.092 ±0.0004 1.000 ±0.0001 ND 8.570 ±0.0004 31.690 ±0.2022
ab a a a a a
NP 1.654 ±0.0022 2.111 ±0.0011 0.870 ±0.0001 0.010 ±0.0001 8.260 ±0.0011 10.560 ±0.0004
b a a a a a
SPN 12.841 ±0.0052 3.856 ±0.0009 1.620 ±0.0014 0.250 ±0.0002 10.220 ±0.0003 56.220 ±0.0319
a a a a a
G. decadactylus NO 0.715 ±0.0011 2.315 ±0.0004 1.030 ±0.0002 ND 9.010 ±0.0002 23.270 ±0.0031
a a a a a
NH 0.863 ±0.0030 2.885 ±0.0003 0.930 ±0.0001 ND 8.680 ±0.0002 30.980 ±0.0098
ab a a a a a
NP 2.991 ±0.0141 3.045 ±0.0001 1.000 ±0.0001 0.260 ±0.0002 10.140 ±0.0019 33.450 ±0.0040
b a a a a a
SPN 12.908 ±0.0306 3.685 ±0.0005 1.550 ±0.0033 0.250 ±0.0001 10.470 ±0.0005 67.790 ±0.0099
a a a a a a
P. senegalensis NO 1.894 ±0.0072 5.069 ±0.0041 1.060 ±0.0002 0.400 ±0.0002 9.990 ±0.0002 36.380 ±0.0017
a a a a a a
NH 0.798 ±0.0027 3.187 ±0.0027 1.120 ±0.0002 0.090 ±0.0001 9.630 ±0.0051 34.630 ±0.0008
ab a a a a a
NP 2.750 ±0.0098 4.107 ±0.0077 0.960 ±0.0001 1.960 ±0.0004 10.210 ±0.0012 34.550 ±0.0301
b a a a a a
SPN 11.591 ±0.0078 4.039 ±0.0008 1.480 ±0.0011 0.650 ±0.0003 10.270 ±0.0034 57.950 ±0.0066
a a a a a a
P. elongathus NO 0.667 ±0.0088 2.247 ±0.0018 0.900 ±0.0001 0.250 ±0.0002 8.660 ±0.0001 23.810 ±0.0034
a a a a a
NH ND 3.080 ±0.0009 0.830 ±0.0001 0.260 ±0.0001 10.780 ±0.0002 30.070 ±0.0122
ab a a a a a
NP 2.893 ±0.0201 2.973 ±0.0056 0.950 ±0.0001 0.400 ±0.0002 9.270 ±0.0022 27.500 ±0.0033
b a a a a a
SPN 12.523 ±0.0650 3.942 ±0.0001 1.190 ±0.0002 0.410 ±0.0002 12.040 ±0.0031 56.280 ±0.0111
a a a a a a
C. brownii NO 0.403 ±0.0003 4.023 ±0.0019 0.870 ±0.0001 0.050 ±0.0001 10.600 ±0.0060 33.170 ±0.0021
a a a a a a
NH 0.613 ±0.0004 3.438 ±0.0008 0.860 ±0.0001 0.110 ±0.0001 11.700 ±0.0009 38.340 ±0.0004
ab a a a a a
NP 2.295 ±0.0015 3.215 ±0.0016 0.760 ±0.0001 0.330 ±0.0001 10.880 ±0.0027 38.470 ±0.0033
b a a a a a
SPN 10.677 ±0.0089 3.367 ±0.0002 0.780 ±0.0001 0.230 ±0.0001 12.660 ±0.0009 141.040 ±0.2031
*Mean values with the same superscript for each fish species in the same row are not significant (p>.05). ± indicates the standard deviation values

6

5. CONCLUSION
The use of wet decomposition methods for
accurate determination of heavy metals in fish
tissue by atomic absorption spectroscopy
requires careful consideration of the digestion
matrice and the heavy metal involved. The wet
decomposition of fish tissue and SRM with SPN
method was found to be generally effective in
both recovery and extraction of the heavy metals
studied while NH gave the least extraction for
heavy metal determination in fish tissues. SPN
method is therefore recommended for
determination of heavy metals Pb, Zn, Cd, Cu, Cr
and Fe in fish tissue. However, to avoid potential
hazards with the use of perchloric acid, it should
be added gradually at the later stage of the
digestion process. Alternatively, NP and NO can
be used when considering cost and safety
precaution.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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