Journal of Hazardous Materials 161 (2009) 1391–1398

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Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Evaluation of an ultrasonic acid digestion procedure for total heavy metals

determination in environmental and biological samples
Tasneem G. Kazi a,∗ , Mohammad K. Jamali a,1 , Mohammad B. Arain a,1 , Hassan I. Afridi a,1 ,
Nusrat Jalbani b,2 , Raja A. Sarfraz a,1 , Rehana Ansari a,1
a
Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080, Pakistan
b
Pakistan Council for Scientific and Industrial Research, University Road, Karachi 75280, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: In this study, a sample preparation method based on ultrasonic assisted acid digestion (UAD) has been eval-
Received 5 April 2008 uated for total heavy metals (Cd, Cr, Ni and Pb) determination in different environmental (soil, sediment
Received in revised form 25 April 2008 and sewage sludge), and biological (fish muscles, vegetables and grains) samples, using electrothermal
Accepted 25 April 2008
atomic absorption spectrometry (ETAAS). The investigated parameters influencing UAD such as preson-
Available online 2 May 2008
ication time, sonication time, temperature of ultrasonic bath, and different acid mixtures were fully
optimized, whereas power was maintained constant at 100% of nominal power of ultrasonic bath. Six
Keywords:
different sets of above parameters were applied on six certified reference materials (CRMs) having differ-
Heavy metal
Ultrasound energy
ent matrices. The accuracy of the method was also tested by comparing the results with those obtained
Environmental sample from conventional hot plate assisted acid digestion method on same CRMs. Analytical results for HMs
Biological sample by both methods showed no significant difference at 95% confidence limit (p < 0.05). Recoveries of HMs
Certified reference material ranging from 96.2% to 102% and 96.3% to 98.6% were obtained from biological and environmental sam-
ples, respectively. The average relative standard deviation of UAD method varied between 3.5% and 8.2%,
depending on the analyte.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
Heavy metals (HMs) pollution is of great concern because their
toxicity threatens the human life and the environment [1]. The high
levels of HMs in sediments, sludges, soils, and through transfer pro-
cesses, also in groundwater and plants, may have a negative effect
on animals and human health [2]. The most common methods
used nowadays for the determination of HMs in environmen-
tal and biological samples involve highly sensitive spectroscopic
techniques, such as atomic absorption spectroscopy, inductively
coupled plasma-optical emission and mass spectrometry (ICP-AES
and ICP-MS). These techniques generally require the destruction of
the sample matrix to render a solution of the analyte ready for anal-
ysis [3,4]. This step is time-limiting, requiring more than 60% of the
∗ Corresponding author. Tel.: +92 22 2771379; fax: +92 22 2771560.
E-mail addresses: tgkazi@yahoo.com (T.G. Kazi), mkhanjamali@yahoo.com
(M.K. Jamali), bilal ku2004@yahoo.com (M.B. Arain), hassanimranafridi@yahoo.com
(H.I. Afridi), nusratjalbani 21@yahoo.com (N. Jalbani), rajaadilsarfraz@gmail.com
(R.A. Sarfraz), rehana ansari 57@yahoo.com (R. Ansari).
1
Tel.: +92 22 2771379; fax: +92 22 2771560.
2
Tel.: +92 21 8142895–8; fax: +92 21 8141847.
total time to perform the complete analysis, and is responsible for
20% to 30% of the total analysis error [5,6]. Sample digestion tech-
niques, such as microwave, and conventional wet acid digestion for
total metals determination have been used widely for the dissolu-
tion of elements [7,8]. Such digestion techniques require the use
of concentrated acids and high temperatures, and often-high pres-
sures, to affect the total dissolution of elements from solid samples
[9].
One of the techniques that have shown promise for speeding
up and simplifying sample treatment, with minimal contamina-
tion, low reagent consumption, and generation of minimal residue
or waste is ultrasonic assisted treatment of samples [10,11]. Ultra-
sound (US) can be considered an alternative for solid sample
pre-treatment because this energy facilitates and accelerates some
steps, such as dissolution, fusion and leaching, among others [12].
Accordingly, the digestion process must be performed with the
assistance of heat, chemical reagents or pressure. Heating can be
substituted or aided by auxiliary energies, such as microwaves or US
in order to accelerate sample dissolution. Because heating occurs
within the digestion medium, microwave digestion is more effi-
cient than conventional heating [13]. Each type of energy assists
digestion differently; thus, microwaves raises the temperature very
rapidly, whereas US increases it slowly to an equilibrium value [14].

0304-3894/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jhazmat.2008.04.103
1392 T.G. Kazi et al. / Journal of Hazardous Materials 161 (2009) 1391–1398
US processing applies intense, high frequency US energy to
liquids, producing intimate mixing and powerful chemical and
physical reactions. The process is, in effect, “cold boiling” (“cav-
itation”) and results from the creation and collapse of countless
micro-bubbles in the liquid, producing shock waves [15]. Cavitation
accelerates both chemical and physical reactions, and combined
effects produce high temperatures and pressures at the interface of
the sonicated solution and the solid matrix, along with the oxidative
power of strong acids results in high extractive power [16–18].
In the few studies published on ultrasonic assisted extraction
of metals, some controversial results concerning the extraction
behavior of metals have been reported. Thus, Mierzwa et al. [19]
found a non-quantitative recovery of Se from two biological mate-
rials using an US cleaner preheated at 70 ◦ C for 18 min. In contrast,
Minami et al. [20] reported the quantitative extraction of Cd, Cu,
Pb and Mn from powdered biological samples with the use of a
similar device operated at 40 ◦ C for 5 min. Bermejo-Barrera et al.
[21] studied the effects of several variables on the extraction of As,
Ca, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Pb, Se, and Zn from lyophilized
seafood samples by using an US bath. This technique is extensively
used for extraction of HMs from different matrices [22,23]. Because
of the novelty of ultrasound-assisted sample preparation proce-
dures, quite often they are compared to other more conventional
or microwave-assisted preparation methods.
There is no risk that the extraction solution evaporates to dry-
ness, which is often a problem with hot plate digestion [24]. US
possessions have also exploited for total metals determination in
biological, foods, soft drinks and environmental samples [25,26].
Both US baths and probes are useful for UAD, while use of ultra-
sonic bath in laboratories is preferred over that of a probe, because
baths are cheaper and easily available.
The aim of this work was to improve sample preparation perfor-
mance, in view of the present trend to develop new methodologies
or improving sample preparation, proposing the development of
an ultrasound-assisted acid digestion method (UAD) for fast and
reproducible recovery of some heavy metals (Cd, Cr, Ni and Pb)
in environmental (soil, sediment, sewage sludge) and biological
samples (vegetables, grains, chicken and fish muscles), in order
to establish an alternative to classical and non-classical diges-
tion techniques. Parameters influencing UAD such as presonication
time, temperature of US bath, and sonication time, were fully inves-
tigated. The optimal set of parameters were chosen by comparing
the determined concentration of Cd, Cr, Ni and Pb with certified
values of six certified reference materials (CRMs) having different
matrices as well as by comparison with those obtained by con-
ventional wet acid digestion (CAD) method. Measurements were
carried out by electrothermal atomic absorption spectrometer.
2. Experimental
2.1. Reagents
Ultrapure water obtained from ELGA labwater system (Bucks,
UK), was used throughout the work. Hydrogen peroxide (30%),
nitric acid (65%), hydrofluoric acid (48%), perchloric acid (60%) and
hydrochloric acid (37%) were spectroscopic grades (Merck, Darm-
stadt, Germany). Standard solutions of Cd, Cr, Ni and Pb were
prepared by dilution of certified standard solutions (1000 ␮g mL−1 ;
Fluka Kamica Switzerland) of corresponding metal ions. Chemi-
cal modifiers, Mg(NO3 )2 stock standard solution, 2.00 g L−1 , was
prepared from Mg(NO3 )2 (Merck) and Pd stock standard solution,
3.00 g L−1 , was prepared from Pd 99.999% Sigma–Aldrich (Milwau-
kee, WI, USA).
Table 1
Measurement conditions for electrothermal atomization AAS 700.
Parameters Cd Cr Ni Pb
Lamp current (mA) 4 30 30 10
Wave length (nm) 228.8 357.9 232.0 283.3
Dry temperature 140/15/5 140/15/5 140/15/5 140/15/5
(◦ C)/ramp/hold(s)
Ashing temperature 850/10/20 1650/10/20 1400/10/20 700/10/20
(◦ C)/ramp/hold(s)
Atomization temperature 1650/0/5.0 2500/0/5.0 2500/0/5.0 1800/0/5.0
(◦ C)/ramp/hold(s)
Cleaning temperature 2000/1/3 2600/1/3 2600/1/3 2200/1/3
(◦ C)/ramp/hold(s)
Slit width = 0.7 L, sample volume = 20 (␮L), cuvette = pyrocoated graphite tube, back
ground correction = D2 lamp, carrier gas = 200 mL/min.
Accuracy was evaluated by using six certified reference materi-
als: BCR 144R (sewage sludge), BCR 141R (soil), BCR 601(sediment),
BCR 189 (whole meal flour) obtained form Community Bureau of
Standards (BCR, Brussels, Belgium), NIST SRM 1571(Orchard Leaves)
National Institute of Standards and Technology (NIST, Gaithersburg,
USA) and DORM-2 (Dog fish Muscle certified reference material for
trace metals) was purchased from the National Research Council
Canada. These reference materials were prepared according to the
instructions provided by producer. The all materials were dried in
an oven at 80 ◦ C for 4 h before use. All glass wares and plastic mate-
rials used were previously treated for 24 h in 5 M supra pure nitric
acid and rinsed with double distilled water and then with ultrapure
water.
2.2. Instrumentation
The UAD was carried out with a Sonicor, Model No. SC-
121TH, (Sonicor Instrument Corporation Copiague, N.Y., USA) with
technical specifications; timer 0–30 min, volts 220 V, 50/60 Hz,
intensification frequency 35 kHz, programmable for temperature
ranging from 0 to 90 ◦ C and a total volume of 4 L. For analysis of
metals a double beam PerkinElmer Atomic Absorption Spectrome-
ter (Model 700; Norwalk, CT, USA) equipped with a graphite furnace
HGA-400, pyrocoated graphite tube with integrated platform, an
autosampler AS-800, interfaced to a personal computer for the
execution of analysis and data collection. Hollow cathode lamps
(PerkinElmer) were used as radiation sources. Experimental condi-
tions for the determination of all elements are shown in Table 1. A
MM 2000 Retsch mixer mill (Haan, Germany) was used for grinding
the samples and sieved made of nylon to obtained particle size of
<50 ␮m.
2.3. Materials
Soil samples were collected from an agricultural land at
(0–25 cm in depth) with a stainless steel auger. Sediment sam-
ples were collected from fresh water lake on monthly basis in
2005. The untreated domestic sewage sludge sampling was per-
formed at sites where the wastewater was separated from solid
waste, from Hyderabad city, Pakistan in 2005. The 50 individual
environmental samples (EV) of each soil, sediment and sewage
sludge were collected. Once in laboratory the EVs were spread
on plastic trays in fume cupboards and allowed to dry at ambient
temperature for 4 days. The biological samples (BIOs), vegetables,
potato (Solonum tuberosum), spinach (Spinacia oleracea L.), pepper-
mint (Mentha piperita L.), radish (Raphanus sativus), turnip (Brassica
napus L.) cauliflower (Brassica oleracea), grains, Barley (Hordeum
vulgare), wheat (Triticum aestivum), maize (Zea mays), sorghum
(Sorghum bicolor), rice (Oryza sativa), fish (Oreochromis mossam-
 T.G. Kazi et al. / Journal of Hazardous Materials 161 (2009) 1391–1398 1393

bicus) and chicken (Broiler) were purchased from local market on The stock sample solutions of certified and real BIOs and EVs
alternate week to collect six replicate (n = 30) of each BIOs. After samples were diluted appropriately according to concentration of
delivery to the laboratory, vegetables, grains, chicken and fish mus- different HMs obtained.
cles samples were washed twice with distilled water then with
deionized water and dried for 72 h in an oven at 60 ◦ C. The dried EVs 2.4.3. ETAAS determinations
and BIOs were homogenized by grinding in an agate ball mixer mill The four HMs understudy were analysed by electrothermal
and sieved through a nylon sieve (<50 ␮m mesh size) and stored atomic absorption spectrometry (ETAAS) under optimum condi-
in prewashed and dried polyethylene vessels separately at room tions (Table 1). Pd(NO3 )2 was used as a chemical modifier for Pb
temperature in desiccators until further treatment. determination, Mg(NO3 )2 for Cr and Ni, while a solution contain-
ing Pd(NO3 )2 + Mg(NO3 )2 was used for Cd determinations. 10 ␮L of
digested solution of certified and real samples and 10 ␮L modifiers
2.4. Methodology
were simultaneously injected into the pyrocoated graphite tube of
furnace, using autosampler AS-800. Aqueous calibration was a real
2.4.1. Ultrasound-assisted acid digestion method
possibility for Cd, Cr, Ni and Pb.
For optimization of the UAD procedure, six replicate of each
CRMs (BIOs and EVs) were prepared by applying different sets of
3. Results and discussion
presonication time [(2, 4, 6, 8, 10 . . . 20 min) and (5, 10, 15, 20, 25 . . .
50 min)], sonication times [(2–20 min) and (5–50 min)], tempera-
The environmental (soil, sediment and sewage sludge), biolog-
ture of US bath (30–80 ◦ C), different solvent systems, a mixture of
ical samples (vegetables, grains, fish and chicken muscles) and
concentrated HNO3 –HClO4 –HF [(2:1:1, v/v/v) [A1], HNO3 –HCl (1:3,
certified reference material having matched matrices to real sam-
v/v) [A2], HNO3 –H2 O2 (2:1) [A3] and HNO3 [A4], while 100 mg sam-
ples were used for optimization purposes. Variables influencing the
ple mass and particle size <50 ␮m were applied for all experiments.
UAD were optimized within the intervals as shown in Table 2. Each
To evaluate the efficiency of the process, the results obtained with
result was the average value of three determinations performed for
the UAD were compared with those obtained from CAD.
real samples and average value of six for CRMs in replicate manner
About 100 mg of all samples were directly weighed into
(Tables 3 and 4).
polypropylene flasks (25 mL capacity) and 1.0 mL acid mixtures of
A1 and A2 were added to soil, sediment and sewage sludge sam-
3.1. Optimization of the UAD
ples separately. While vegetables, grains, chicken and fish muscles
were treated with 1.0 mL of A3 and A4 separately. The all flasks were
The evaluation of results provided by six sets of three vari-
allowed to stand for different time intervals (2, 4, 6, 8, 10 . . . 20 min)
ables (Table 2) was based on the recovery of four HMs (Cd, Cr,
and (5, 10, 15, 20, 25 . . . 50 min), for BIOs and EVs samples, respec-
Ni and Pb) from CRMs of different matrices and real samples
tively at room temperature, marked as presonication time (PST).
under study. All the results were compared with those obtained
After each time intervals of the PST, flasks were immersing into the
by applying the conventional wet acid digestion method (CDM)
ultrasonic water bath and were subjected to US energy at 35 kHz
on the same materials. The use of validated methodology for both
for different time interval, termed as sonication time (ST), 5–50 min
sample digestion and measuring step is mandatory to assure that
were applied to EVs, while for BIOs 2–20 min were tested. The tem-
reliable results and conclusions are obtained. The mean values
perature range of US water bath (TB) was 30–80 ◦ C. After sonication
obtained after the application of the digestion procedures to a given
for different time intervals and temperature, the contents of the
number of sample replicates were compared with the certified
flasks were diluted with 2 mL deionized water and subjected to son-
values.
ication for another 2 min. Then, the solutions were centrifuged at
In present work, 100 mg sample mass and 1.0 mL of acids mix-
3000 rpm for 10 min, and the final volume was made up to 10 mL in
ture volume were investigated, the results achieved are comparable
volumetric flasks with deionized water for EVs, while BIOs diluted
to those work with high amount of sample mass and extracting
up to 5 mL as stock sample solutions, and stored in polyethylene
acids [27,28]. A sample mass of up to 200 mg has been reported
tubes at 4 ◦ C for analyses. Blanks were also treated in the same way
in our previous work with an US assisted metals extraction [29]. In
without samples for each experiment.
proposed method the particle size of EVs and BIOs were >50 ␮m, the
After validation the process, the optimized UAD was applied to
efficiency of lower particle size for accuracy and precision is consis-
determine the four HMs understudy in a wide range of environmen-
tent with other study [30]. It was observed that the concentration
tal (agricultural soils, sewage sludge domestic in origin, sediment
of polluted lake) and biological samples (vegetables, grains, chicken
Table 2
and fish muscles). All experimental procedures were performed on
Operating conditions for ultrasound-assisted pseudo-digtestion of Cd, Cr, Ni and Pb
a clean bench. from environmental and biological samples

Variable studied Interval Best conditions

2.4.2. Conventional wet acid digestion method (CAD)
Environmental samples (soil, sediment and sewage sludge samples)
About 100 mg replicate six samples of CRMs and triplicate sam- Presonication time (min) 5–30 15
ples of each EVs and BIOs were weighed in PTFE digesting flasks, Sonication time (min) 5–50 25
and added 5 mL mixture of acids A1 and A2 to EVs, while 5 mL Solvent systems HNO3 –HCl, (1:3) HNO3 –HCl (1:3)
A3 and A4 were added to BIOs separately. The flasks were covered HNO3 –HClO4 –HF
(2:1:1)
with watch glasses and then digested at 60–80 ◦ C for 3–4 h, added ◦
Temperature ( C) 40–80 80
more acid mixture and heating was continued until the color of the
Biological samples (vegetables, grains and fish muscles samples)
digestion solution became transparent. Evaporated the extra acid
Presonication time (min) 2–12 8
to semidried mass, after cooling, added 2 mL 0.5 M HNO3 and cen- Sonication time (min) 2–20 10
trifuged at 3000 rpm for 10 min, and the final volume was made Solvent systems HNO3
up to 5.0 and 10.0 mL with 0.5 M HNO3 as stock sample solutions HNO3 –H2 O2 (2:1) HNO3 –H2 O2
for BIOs and EVs, respectively. Blanks were treated in the same Temperature (◦ C) 40–80 70
way. Sample mass 100 mg, particle size <50 ␮m.
1394 T.G. Kazi et al. / Journal of Hazardous Materials 161 (2009) 1391–1398

Table 3
Validation of the ultrasound-assisted digestion (UAD) and conventional wet acid digestion (CAD) against six certified reference materials (␮g g −1 , n = 6)
√
BCR 141R (soil) Certified values CAD x̄ ± ts/ n UAD %Recovery tExperimental

Cd 14.6 ± 0.5 14.5 ± 0.83 14.4 ± 0.47 98.6 0.314

Cr 195 ± 7.0 194 ± 7.57 190 ± 4.5 97.4 0.062
Ni 103 ± 3.0 102 ± 4.9 101 ± 5.0 98.1 0.012
Pb 57.2 ± 1.2 57.0 ± 2.9 55.3 ± 2.52 96.7 0.032

144 R (sewage sludge)

Cd 1.82 ± 0.1 1.81 ± 0.08 1.79 ± 0.081 98.4 0.035
Cr 104 ± 3.0 103 ± 1.87 101 ± 2.03 97.1 0.071
Ni 47.7 ± 1.1 47.6 ± 2.4 46.7 ± 1.67 97.9 0.027
Pb 106 ± 4.0 105 ± 2.1 102.4 ± 2.21 96.6 0.013

BCR 601 sediment

Cd 11.5 ± 1.9 11.4 ± 0.48 11.3 ± 0.43 98.3 0.106
Cr 112 ± 10.0 112 ± 4.99 110 ± 4.01 98.2 0.002
Ni 282 ± 52.0 281 ± 12.7 274 ± 12.1 97.2 0.045
Pb 78.0 ± 6.7 77.3 ± 3.61 75.1 ± 3.47 96.3 0.021

NIST 1571 (Orchard Leaves)

Cd 0.110 ± 0.09 0.113 ± 0.009 0.112 ± 0.007 101.8 0.280
Cr 2.60 ± 0.146 2.59 ± 0.113 2.52 ± 0.087 96.9 0.017
Ni 1.30 ± 0.2 1.29 ± 0.074 1.25 ± 0.075 96.2 0.047
Pb 44.0 ± 3.0 44.0 ± 2.57 42.7 ± 1.44 97.0 0.070

DORM-2 (Dog fish muscles)

Cd 0.043 ± 0.008 0.044 ± 0.004 0.0435 ± 0.002 101.2 0.401
Cr 34.7 ± 5.5 35.7 ± 2.5 34.8 ± 1.49 100.3 0.231
Ni 4.64 ± 0.260 4.72 ± 0.26 4.58 ± 0.14 98.7 0.213
Pb 0.065 ± 0.007 0.065 ± 0.004 0.063 ± 0.004 96.9 0.250

BCR189 (whole meal flour)

Cd 0.0713 ± 0.003 0.073 ± 0.003 0.0725 ± 0.002 101.7 0.152
Cr 0.065 ± 0.001 0.066 ± 0.001 0.0641 ± 0.001 98.6 0.027
Ni 0.348 ± 0.040 0.349 ± 0.0037 0.338 ± 0.002 97.1 0.017
Pb 0.379 ± 0.012 0.38 ± 0.01 0.371 ± 0.002 97.9 0.068

Recovery (%) = [HMS found with UAD/certified values of CRMs] × 100, tCritical = 2.57

of HMs obtained by UAD were dependent on the analyte–matrix
interaction and the three variables, presonication time and other
two variables of US bath (temperature and sonication time). Time
and temperature were modified experiment by experiment based
on the achieved results and the target values of analytes understudy
were established by the CAD. The HMs recoveries obtained by UAD
and CAD were calculated on the basis of certified values of CRMs
(Table 3), while in real EVs and BIOs the HMs recoveries obtained
by proposed method was calculated on achieved results using CAD
(Table 4).
The paired t-test was also applied to compare the results of both
methods. A texperimental values was calculated at degrees of freedom
n − 1 = 5, the experimental values are lower than the tcritical (2.57)
at a confidence interval of 95% (p = 0.05), indicate non-significant
difference in obtained values of HMs by CAD and UAD (Table 3).
3.2. Effect of presonication and sonication time
After the treatment with acid–oxidant mixtures, BIOs and EVs
were kept at room temperature for different time intervals, 2–20
and 5–50 min, respectively, (Table 2), before being subjected to the
US bath, denoted as presonication (PST). Optimum effects of PST
were observed in BIOs at 8 min while EVs required 15 min. Longer
PST has no effect on the recoveries of HMs understudy, except in the
case of Cu required 12 and 30 min to achieved optimum recoveries
from BIOs and EVs, respectively. The 10–15% recoveries of all HMs
in EVs and BIOs were enhanced with PST as compared to without
it.
The evaluation of results provided by the six sets of sonication
time (Table 2), was based on the recoveries of Cd, Cr, Ni and Pb
from certified reference materials. The UAD efficiency increased
with increasing sonication time from 5 to 50 and 2 to 20 min for
EVs and BIOs, respectively. The UAD rate curves with different son-
ication time intervals for recovery of four HMs were compared in
Figs. 1 and 2. Each value in line graph represents the mean of gen-
erally six replicates and the estimation of uncertainty for HMs. For
some metals and samples the results were multiplied by a factor
(indicated on the bar) in order to be plotted in the same graph.
The Fig. 1a–c shows effect of the sonication time on the recover-
ies of HMs. It was observed that optimum recoveries of Ni and Cd
required 20 min, while up to 25 min sonication was necessary for
Cr and Pb from CRMs of EVs (BCR 601, 141R and 144R) to reach the
same concentration as of the certified values for all the considered
HMs. There was no significant difference between 25 and 50 min
sonication periods for all HMs at 0.05 probabilities, but were 20
and 25 min for HMs understudy in EVs. Fig. 2 shows that the opti-
mum recoveries of Cd, Cr, Ni and Pb in biological CRMs (NIST 1571,
DORM-2 and BCR 189) and real samples were reached at 6 min for
Cd, 8 min for Ni while Pb and Cr required 10 min sonication to reach
the optimum values, increase of ST up to 20 min did not enhance
the recovery values of all HMs as shows in (Fig. 2a–c). This fact
offers an important practical advantage because the time for the
acid digestion can be shortened, and our results are consistent with
other study [31]. The optimized conditions were applied for further
study on real samples.
3.3. Influence of solvent systems
The influence of solvent systems, such as mixtures of concen-
trated acids HNO3 –HClO4 –HF and HNO3 –HCl (aqua regia) were
used for EVs (soil, sediment and sewage sludge), while for BIOs,
HNO3 and mixture of (HNO3 –H2 O2 ) were studied by fixing the
other variables at their optimal values. Considering the matrices
of EVs (soils, sediments, sewage sludge), a total digestion scheme
 T.G. Kazi et al. / Journal of Hazardous Materials 161 (2009) 1391–1398 1395

Table 4
Determination of heavy metals in environmental and biological samples using ultrasound-assisted digestion (UAD) and conventional wet acid digestion (CAD) (dried basis,
␮g g −1 )

Methods Solonum tuberosum Spinacia oleracea L. Mentha piperita L. Raphanus sativus Brassica napus L. Brassica oleracea
a
Vegetables
Cd
UAD 1.85 ± 0.12 0.078 ± 0.01 0.108 ± 0.01 0.288 ± 0.016 0.448 ± 0.03 0.393 ± .0.02
CAD 1.86 ± 0.13 0.079 ± 0.01 0.109 ± 0.01 0.29 ± 0.012 0.455 ± 0.032 0.395 ± 0.03
%Recovery 99.5 98.7 98.8 99.3 98.5 99.6

Cr
UAD 0.90 ± 0.06 1.21 ± 0.12 1.08 ± 0.12 0.210 ± 0.012 0.393 ± 0.02 0.296 ± 0.02
CAD 0.93 ± 0.07 1.24 ± 0.13 1.1 ± 0.13 0.216 ± 0.014 0.403 ± 0.03 0.302 ± 0.02
%Recovery 96.8 97.6 98.2 97.2 97.5 98.0

Ni
UAD 7.57 ± 0.6 1.08 ± 0.13 1.77 ± 0.12 1.79 ± 0.15 1.28 ± 0.12 0.505 ± 0.04
CAD 7.72 ± 0.5 1.11 ± 0.012 1.80 ± 0.12 1.82 ± 0.17 1.31 ± 0.13 0.515 ± 0.03
%Recovery 98.1 97.3 98.3 98.4 97.7 98.1

Pb
UAD 0.322 ± 0.02 0.794 ± 0.06 0.776 ± 0.05 1.200 ± 0.12 0.737 ± 0.05 0.808 ± 0.05
CAD 0.332 ± 0.02 0.818 ± 0.05 0.801 ± 0.04 1.238 ± 0.11 0.758 ± 0.06 0.832 ± 0.06
%Recovery 97.0 97.1 96.9 96.9 97.2 97.1

Methods Hordeum vulgare Triticum aestivum Zea mays Sorghum bicolor Oryza sativa
a
Grains
Cd
UAD 0.638 ± 0.04 0.645 ± 0.05 0.743 ± 0.05 0.615 ± 0.04 0.292 ± 0.023
CAD 0.640 ± 0.03 0.647 ± 0.06 0.745 ± 0.06 0.620 ± 0.05 0.294 ± 0.024
%Recovery 99.7 99.7 99.7 99.1 99.3

Ni
UAD 1.11 ± 0.13 0.947 ± 0.07 1.84 ± 0.13 1.99 ± 0.13 0.741 ± 0.05
CAD 1.14 ± 0.12 0.965 ± 0.06 1.88 ± 0.15 2.03 ± 0.21 0.761 ± 0.06
%Recovery 97.5 98.1 98.0 98.0 97.4

Cr
UAD 0.973 ± 0.07 0.270 ± 0.02 0.212 ± 0.02 0.247 ± 0.015 1.08 ± 0.1
CAD 0.99 ± 0.08 0.274 ± 0.022 0.218 ± 0.03 0.252 ± 0.016 1.10 ± 0.11
%Recovery 98.3 985 97.3 98.0 98.2

Pb
UAD 1.14 ± 0.13 0.788 ± 0.06 2.721 ± 0.22 2.89 ± 0.23 0.944 ± 0.06
CAD 1.17 ± 0.011 0.813 ± 0.05 2.80 ± 0.23 2.98 ± 0.25 0.97 ± 0.08
%Recovery 97.3 96.9 97.2 97.0 97.6

Methods Fish musclesa Chicken musclesa Lakeb sediment Soilb Sewage sludgeb

Cd
UAD 1.38 ± 0.41 2.14 ± 0.18 6.54 ± 0.34 2.38 ± 0.18 18.9
CAD 1.39 ± 0.121 2.16 ± 0.21 6.6 ± 0.66 2.4 ± 0.266 19.1 ± 1.6
%Recovery 99.2 99.0 99.0 99.2 0.99.1

Cr
UAD 0.44 ± 0.036 0.845 ± 0.071 24.7 ± 1.43 12.2 ± 0.58 55.9 ± 3.8
CAD 0.46 ± 0.046 0.862 ± 0.082 25.2 ± 1.76 12.5 ± 0.83 57.3 ± 4.3
%Recovery 98.2 98.0 97.8 97.9 97.5

Ni
UAD 2.19 ± 0.16 3.25 ± 0.14 17.5 ± 1.24 13.34 ± 0.49 38.5 ± 1.8
CAD 2.23 ± 0.2 3.32 ± 0.17 18.0 ± 1.4 13.6 ± 1.11 39.3 ± 2.1
%Recovery 98.2 98.5 97.2 98.1 98.0

Pb
UAD 2.34 ± 0.14 3.83 ± 0.23 19.0 ± 1.35 15.3 ± 1.11 104 ± 7.5
CAD 2.4 ± 0.2 3.92 ± 0.32 19.7 ± 1.63 15.8 ± 1.32 107 ± 9.7
%Recovery 97.5 97.7 96.4 96.8 97.2
a
n = 30.
b
n = 50.

must include the use of hydrofluoric acid (HF) to completely release
the HMs included in the aluminosilicate phase [32]. However, the
use of HF leads to long, dangerous, and cumbersome schemes and
its use is not recommended for routine analyses [33]. The effec-
tiveness of mixtures of concentrated HF and other acids, such as
HNO3 –HClO4 –HF for the US leaching of target metals, followed by
atomic spectrometric analysis, has been demonstrated previously
for bulk certified reference materials, such as sediments, soils and
fly ash. Concentrated HNO3 –HCl (1:1, v/v) reported to be used as
solvents for leaching of metals from EVs [31].
The UAD results obtained with the use of different acid mix-
tures are shown in bar graphs (Fig. 3a–c), it can be seen that high
recoveries of some HMs (Pb and Cr) are obtained with A1 mixture
from EVs as compared to A2 while difference was not significant at
1396 T.G. Kazi et al. / Journal of Hazardous Materials 161 (2009) 1391–1398

Fig. 1. Effects of sonication time on HMs recovery from certified environmental

samples.

Fig. 3. The effect of solvent system on the ultrasonic assisted acid digestion of HMs
from certified environmental samples.

Fig. 2. Effects of sonication time on HMs recovery from certified biological samples.
0.05 probability, although the sediment and soil have silicate matrix
structure or silicate structure. Therefore, it can be said that, instead
of highly corrosive and explosive acids (HClO4 and HF), aqua regia
can be used for the rest of the EVs. In this work, sample mass/solvent
volume ratio was chosen as 100 mg/1 mL, very low as compared to
other reported work [34].
In the case of BIOs (vegetables, grains, chicken and fish tissues),
the significantly higher recoveries of all HMs were obtained from
acid–oxidant mixture (A3) than those values released with alone
(A4) as shows in Fig. 4a–c. The obtained results indicated that
organic matters had important role in controlling the release of
metals. Because on heating, H2 O2 dissociate to hydroxyl radicals
(OH− ) that could attack proteins, carbohydrates and polyunsat-
urated fatty acids present in the biological samples. Hence, it
improved the efficiency of the extraction of metals from BIOs. It
was reported that oxidation of BIOs with high organic matter is
usually incomplete with only HNO3 and, traditionally, the use of
a strongly oxidizing acid such as HClO4 is necessary; presently
we found that H2 O2 combined with HNO3 yielded clear solu-
tions and improved recovery, is also consistent with other study
[35].
 T.G. Kazi et al. / Journal of Hazardous Materials 161 (2009) 1391–1398
Fig. 4. The effect of solvent system on the ultrasonic assisted acid digestion of HMs
from certified biological samples.
3.4. Effect of temperature
In this work, the 30–80 ◦ C temperature interval of US bath was
investigated. Temperature of the extraction medium increased with
increasing sonication time. The high temperature and pressure
within a collapsing cavitation bubble produced by US irradiation
causes the formation of free radicals, to accelerate the reactions
involved in sample digestion [36]. The optimum amount of Cd was
released from CRMs and real samples of BIOs at 60 ◦ C, while maxi-
mum recoveries of Cr, Ni and Pb from same samples were obtained
at 70 ◦ C. The maximum recoveries of HMs obtained from EVs at
70–80 ◦ C. Therefore, the high temperature was necessary for opti-
mum recoveries of HMs from all samples as compared to the work
reported at room temperature [37].
3.5. Analytical figure of merit
The linear range of the calibration curve reached from the detec-
tion limit up to 10, 50, 100 and 100 ␮g L−1 , for Cd, Pb, Cr and Ni,
respectively. Characteristic masses were 1.0, 3.8, 25 and 32 pg for Cd,
Pb, Cr and Ni. The detection limit (LOD) was defined as 3s/m, where
1397
s is the standard deviation corresponding to 10 blank injections and
m is the slope of the calibration graph. The LODs of 0.05, 0.55, 0.41,
0.15 ␮g L−1 were calculated for Cd, Pd, Cr and Ni, respectively.
The method detection limits were 2.5, 27.5, 20.5 and 7.5 ng g−1
for Cd, Cr, Ni and Pb, respectively. The calculated LOD and MDL of
Cd, Cr, Ni and Pb for Cd, Cr, Ni and Pb were sufficiently low to allow
the determination of these elements in certified and real samples
especially biological samples.
The analytical performance of the novel UAD was evaluated
by the analyses of six certified reference materials with different
matrices. The precision of the methods, expressed as the relative
standard deviation (RSD) of a minimum of eight independent anal-
yses of the same sample, provided values ranging from 1.2% to 6.24%
as a function of the element considered and its concentration level.
Non-significant differences were observed (t-test; p = 0.05) when
comparing the average HMs values obtained from UAD and CAD
(Table 3).
3.6. Analytical application
The optimized values for the different variables (Table 2) were
applied to the analysis of samples with different matrices. The
EVs (soil, sediment, sewage sludge) and BIOs (grains, vegeta-
bles, chicken and fish tissues) were digested with UAD and CAD,
and the results were compared (Table 4). With these real sam-
ples, recovery values obtained with UAD ranged 95.6–99.7% as
compared to those values of HMs obtained from CAD. The con-
centrations of all HMs were found to be high in domestic sewage
sludge, which is due to small local industries situated in under
study domestic areas. The levels of all HMs were observed in soil
and lake sediment, indicating anthropogenic contamination due
to environmental pollution. The HMs transfer from soil and lake
ecosystem to different food commodities, create health hazards
to human beings. The food samples (vegetable, grains, fish and
chicken) contain all HMs, the leafy vegetable contain high level of
all HMs.
4. Conclusion
The method described offers a rapid, easy and efficient sam-
ple preparation; using a low cost and easily available routine US
bath, for determination of HMs in environmental (soil, sediment
and sewage sludge) and biological samples (vegetables, grains and
fish muscles) by ETAAS. All parameters studied (sonication time,
temperature and solvent system) influence the UAD efficiency. The
use of the UAD allowed the digesting of BIOs and EVs for HMs
determination in a shorter time and low volume of acids mixture
than required by the CAD, while providing similar results. The main
goals achieved with the proposed method include, a higher sam-
ple throughput (12–60 samples/h), longer graphite tube lifetime,
and lower analytical costs, when compared with other decompo-
sition procedures prior to analysis. This method reduces the time
required for all treatments (hot plate assisted digestion with high
volume of acids, heating to dryness, cooling) with CAD from approx-
imately 3–4 h to 20–30 min. We can apply the proposed digestion
protocols to samples with a wide range of organic matter such
as sediments, soils, sludges, vegetables, grains, fish and chicken
muscles.
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