Journal of Trace and Microprobe Techniques

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MICROANALYTICAL DETERMINATION OF METALS

IN SELECTED ORGANS OF ZEBRA MUSSELS
BY TOTAL REFLECTION X-RAY FLUORESCENCE
SPECTROMETRY

I. Bohuss , A. Varga , K. Barkács , N. Oertel & Gy. Záray

To cite this article: I. Bohuss , A. Varga , K. Barkács , N. Oertel & Gy. Záray (2001)
MICROANALYTICAL DETERMINATION OF METALS IN SELECTED ORGANS OF ZEBRA
MUSSELS BY TOTAL REFLECTION X-RAY FLUORESCENCE SPECTROMETRY, Journal of
Trace and Microprobe Techniques, 19:1, 177-182, DOI: 10.1081/TMA-100001472

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Published online: 16 Feb 2007.

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J. TRACE AND MICROPROBE TECHNIQUES, 19(1), 177–182 (2001)

MAJOR AND TRACE BIOELEMENTS

MICROANALYTICAL DETERMINATION OF
METALS IN SELECTED ORGANS OF ZEBRA
MUSSELS BY TOTAL REFLECTION X-RAY
FLUORESCENCE SPECTROMETRY

I. Bohuss,1 A. Varga,2 K. Barkács,1,∗ N. Oertel,3 and Gy. Záray2

1
Department of Chemical Technology and Environmental Chemistry,
L. Eötvös University, P.O. Box 32, H-1518 Budapest 112, Hungary
2
Research Group of Environmental and Macromolecular Chemistry,
Hungarian Academy of Sciences, L. Eötvös University, P.O. Box 32,
H-1518 Budapest 112, Hungary
3
Hungarian Danube Research Station, Hungarian Academy of Sciences,
Jávorka S. 14, H-2131 Göd, Hungary

ABSTRACT

Selected organs (gill, mantle, muscle, gonad) and the total soft tissue of
zebra mussels (Dreissena polymorpha) collected from a 54-km long Danube-
branch near Budapest were analysed by total reflection X-ray fluorescence
spectrometry. For sample preparation a microwave assisted vapour-phase di-
gestion technique was used. Nine elements (Ca, Cu, Fe, K, Mn, Ni, Pb, Sr,
Zn) were detectable in the samples. The K distribution was nearly homo-
geneous among the organs investigated. A considerable enrichment for Ca
and Sr in the mantle and a moderate enrichment of Fe, Zn and Mn also in
the mantle and/or the gill, of Ni in the muscle, and of Cu in the gill were
observed.

Key Words: Zebra mussel; Metal accumulation; Biomonitoring; TXRF

∗
Corresponding author. E-mail: [email protected]

177

Copyright 
C 2001 by Marcel Dekker, Inc. www.dekker.com
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178 BOHUSS ET AL.

INTRODUCTION

Bioaccumulation monitoring is a valuable tool for checking the quality of

environment and mapping the sources of pollution. Bivalve molluscs are often
applied as sentinel organisms for monitoring heavy metal pollution. The freshwa-
ter zebra mussel, which is spread in North-America and in Europe, especially in
Hungarian surface waters, has been widely used in biomonitoring tests (1,4). Con-
centration levels and kinetics of uptake and depuration of heavy metals by zebra
mussels have been published earlier (5,8). In these tests in most cases the total
soft tissues were removed from the shells, and 5–10 g samples were digested and
analysed by various methods such as flame-, graphite furnace- or cold flameless
AAS, differential pulse anodic stripping voltametry or γ -radiation spectrometry
(5,9). Although a significant metal uptake by various organs of mussel species
has been observed (6), no such study has still been carried out with the zebra
mussel. To do that, due to the relatively low mass of total dry tissue (20–30 mg)
and even more of the selected organs (1–10 mg) of the zebra mussels, we had
to use powerful microanalytical techniques. Total reflection X-ray fluorescence
spectrometry (TXRF) was chosen because of its multielement capability, low de-
tection limits (ng–pg range) and simple calibration by internal standardisation
(3,7).

MATERIALS AND METHODS

Sampling

1–1.5 years old (size 1.3–1.8 cm) zebra mussels were collected during a week-
long period (June 20–28, 1998) along the 54-km long Danube branch between
Budapest and Tass. The sampling sites were located at kms 54, 44, 38, 32, 19, 15,
and 1 south of Budapest (referred to as sites Nos 1–7). The water level is regulated in
this Danube branch which receives unknown industrial and communal pollutants.
The mussels were carried to the laboratory in polyethylene bags and placed into
cold tap water for 24 h to remove the surface contamination.

Sample Preparation

The organs (gill, muscle, mantle, gonad) of three mussels collected at the
same sampling site were excised using a ruby knife under the microscope and
the selected organs were dried in an oven at 105◦ C till they reached a constant
weight. The dried organs were powdered and homogenised by hand using a plastic
mortar. The dry mass of the total soft tissues varied in the range of 22–35 mg.
The mass of the selected gill, mantle, muscle, and gonad samples amounted to
9.9 ± 1.2, 17.5 ± 1.4, 12.2 ± 0.9, and 34.5 ± 3.1% of the total mass, respectively.
These mass ratios were determined on the basis of 21 mussels collected. 1.5–2.5 mg
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BIOELEMENTS IN ZEBRA MUSSELS 179

of the dried and pulverised tissues were transferred into quartz microtubes and de-
composed using a microwave assisted vapor-phase digestion technique (2), applying
a CEM-2100 type pressure controlled microwave digestion system
(448 kPa, 35 min). After decomposition the samples were filled up by deionized
water to 0.1–0.2 cm3 , thus adjusting an average matrix concentration of 1.4–1.5 mg
dry tissue in 100 mg solution. The samples were mixed with a Ga standard solution
(as internal standard); after homogenisation, an aliquot of 25 µl was dropped onto
the quartz-glass carrier and dried in a clean box at 80◦ C for 30 min on a ceramic-
coated hot plate. Each sample droplet contained 100 ng Ga. The applicability of the
internal standardisation in the case of TXRF spectrometry is strongly dependent
on the thickness of the dried sample layer on the quartz carrier plate (7), therefore,
the decomposition of the organic compounds was checked using a Total Organic
Carbon Analyzer (Beckman 915-B) and micro-COD test (Merck, Germany). The
efficiency of the digestion ranged from 95.1 to 97.8%, therefore, the residual dry
organic material content of a 25 µL droplet did not exceed 20 µg in our sam-
ples. After solvent evaporation the calculated thickness of the thin layer on the
quartz-carrier plate thus remained below the critical value of 5 µm.

TXRF Measurements

Metal concentrations were determined using an EXTRA IIA total reflection

X-ray fluorescence spectrometer (ATOMIKA Instruments GmbH, Oberschleis-
sheim, Germany). The operating parameters were as follows: Mo micro focus
X-ray tube (50 kV, 38 mA) with a high-energy cut-off filter (quartz glass mirror),
an attenuation filter (200 µm Mo, 240 µm Al) and a Si (Li) detector 80 mm2 in area
and 300 s integration time. The energies (keV) of the analytical lines were K Kα
3.312, Ca Kα 3.690, Mn Kα 5.894, Fe Kα 6.398, Ni Kα 7.472, Cu Kα 8.040, Zn Kα
8.630, Pb Lα 10.550 and Sr Kα 14.140. The absolute detection limits expressed in
ng were 11.48 K, 7.78 Ca, 2.28 Mn, 1.64 Fe, 1.08 Ni, 0.94 Cu, 0.89 Zn, 1.57 Pb
and 0.52 Sr.

RESULTS AND DISCUSSION

In the total soft tissues and in the selected organs of the zebra mussels 9 metals
(Ca, K, Sr, Cu, Fe, Zn, Ni, Mn, Pb) were detectable by TXRF spectrometry. Some
of the measured element concentrations are represented in Table 1 calcium and
potassium were present at relatively high concentrations (0.5–15 mg/g dry tissue)
compared to the other metals detected (10–350 µg/g dry tissue). The reproducibility
of the concentration measurements amounted to 0.6–4.2% (Ca, K) and 3–13% (Cu,
Fe, Mn, Ni). The K concentration was less dependent on the sampling site and on
the organ selected than on the other elements. The concentration of calcium varied
significantly according to the sampling site, and it was 2–3 times higher in the mantle
 180

Table 1. Mean Concentration ± Standard Deviation of Various Elements Calculated from Three Simultaneous Determinations in Dry Organ Tissues of Zebra
Mussels at Seven Sampling Sites Along the 54-km Long Danube Branch Between Budapest and Tass

Site Number

Metal Organ No 1 No 2 No 3 No 4 No 5 No 6 No 7

Ca (mg/g) Mantle 2.71 ± 0.09 9.51 ± 0.36 1.41 ± 0.04 2.59 ± 0.08 14.8 ± 0.52 8.08 ± 0.26 5.39 ± 0.21
ORDER

Total Tissue 1.18 ± 0.03 3.70 ± 0.12 0.48 ± 0.04 1.01 ± 0.07 6.48 ± 0.21 2.95 ± 0.11 2.09 ± 0.09
K (mg/g) Mantle 0.85 ± 0.04 1.61 ± 0.08 0.83 ± 0.04 1.67 ± 0.08 1.28 ± 0.06 1.47 ± 0.07 1.12 ± 0.05
Gonad 0.55 ± 0.01 1.59 ± 0.08 0.77 ± 0.02 1.33 ± 0.07 0.99 ± 0.04 1.31 ± 0.06 0.96 ± 0.06
Total Tissue 0.64 ± 0.01 1.57 ± 0.06 0.77 ± 0.03 1.23 ± 0.06 1.07± 0.05 1.28 ± 0.09 0.98 ± 0.04
Sr (µg/g) Mantle 54.0 ± 0.4 16.5 ± 0.5 21.8 ± 0.4 85.5 ± 0.4 43.2 ± 3.8 26.4 ± 0.7 58.0 ± 2.3
REPRINTS

Total Tissue 10.9 ± 1.0 10.3 ± 0.5 5.0 ± 0.3 8.5 ± 1.0 29.7 ± 0.4 13.9 ± 1.9 14.3 ± 1.6
Fe (µg/g) Mantle 224.4 ± 7.9 130.2 ± 9.9 166.5 ± 7.2 265.1 ± 13.1 117.8 ± 3.6 252.4 ± 9.2 228.0 ± 2.8
Gill 137.7 ± 1.0 138.4 ± 5.1 247.1 ± 2.2 120.6 ± 2.9 137.7 ± 6.6 160.5 ± 3.5 334.1 ± 4.8
Total Tissue 135.6 ± 1.4 143.0 ± 0.7 204.2 ± 5.8 154.6 ± 3.0 85.5 ± 2.5 147.3 ± 4.2 190.4 ± 4.0
Mn (µg/g) Mantle 79.1 ± 4.3 14.3 ± 1.4 29.8 ± 0.9 47.9 ± 0.3 32.5 ± 2.4 18.3 ± 1.1 29.7 ± 0.7
Total Tissue 78.7 ± 4.7 17.2 ± 1.7 22.7 ± 2.1 23.6 ± 1.8 26.1 ± 1.4 18.4 ± 0.3 22.8 ± 1.2
Cu (µg/g) Gill 17.1 ± 1.5 27.0 ± 2.6 16.8 ± 0.8 27.2 ± 0.5 13.4 ± 1.0 24.4 ± 1.5 89.0 ± 1.9
Total Tissue 21.6 ± 1.2 35.2 ± 1.7 23.1 ± 0.8 38.8 ± 0.1 8.6 ± 0.4 29.7 ± 1.2 52.3 ± 1.4
Ni (µg/g) Muscle 62.1 ± 0.4 86.6 ± 4.0 62.8 ± 0.9 21.1 ± 0.5 52.5 ± 2.2 67.3 ± 4.7 121.5 ± 5.3
Total Tissue 15.5 ± 0.7 87.9 ± 0.7 64.3 ± 1.6 99.0 ± 2.9 6.9 ± 0.9 66.2 ± 2.3 200.5 ± 5.0
BOHUSS ET AL.
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BIOELEMENTS IN ZEBRA MUSSELS 181

than in the other tissues at all sites. Strontium was detectable only in the mantle and
in the total soft tissue. The concentration of iron and zinc in the mantle and/or in
the gill exceeded their mean concentration in the total soft tissue in most samples
by a moderate enrichment factor of 1.1–2.0. The fluctuation of Zn concentration
was in the range of 90–320 µg/g in the total soft tissue and in the mantle, and of
70–600 µg/g in the gill. Lead was undetectable except in the total soft tissue, where
its concentration was in the range of 5–40 µg/g. The copper concentration was
similar in all of the separated organs. Mn and Ni were detectable only in the total
soft tissue and in the mantle. According to the critical concentration values of heavy
metal pollutants published in the literature (1,4), only the Cu and Fe concentrations
of the soft tissue samples fluctuated around the values characteristic for unpolluted
areas, whereas in most sampling sites, Zn, Ni, and Pb exceeded the concentration
values characteristic for highly polluted freshwater.
On the basis of these data, the only significant difference in the accumulation
levels was found in the mantle for calcium and strontium, due to the shell formation
process. For the other elements no significant enrichment was observed in any
particular mussel organ compared to the other organs. Therefore, to follow the
accumulation of heavy metals in zebra mussels, there is no particular advantage in
analyzing the separated organs compared with whole body analysis.

CONCLUSION

TXRF spectrometry turned to be a suitable multielement, microanalytical

method for determining the distribution of major and trace elements in zebra mus-
sel’s organs, having detection limits of 1.5–33 µg metal/gram dry tissue and relative
standard deviations of 0.6–13% for the nine elements determined. Taken the or-
gans mass as well as metal concentration, and comparing them to the data of the
soft tissue, we concluded, that the time consuming separation and analysis of the
selected organs in the case of zebra mussel do not bring advantages in freshwater
biomonitoring tests.

ABBREVIATIONS

AAS atomic absorption spectrometry

TXRF Total reflection X-ray fluorescence

ACKNOWLEDGMENTS

The authors gratefully acknowledge the financial support of the Hungarian

National Science Foundation (OTKA) T 030845 and of the Hungarian Academy
of Sciences AKP 98-91 2.4.
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182 BOHUSS ET AL.

REFERENCES

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Submitted August 13, 1999

Accepted April 04, 2000
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