Fresenius J Anal Chem (1999) 363 : 5–11
O R I G I N A L PA P E R
F. Lagarde · M. B. Amran · M. J. F. Leroy ·
C. Demesmay · M. Ollé · A. Lamotte · H. Muntau ·
P. Michel · P. Thomas · S. Caroli · E. Larsen ·
P. Bonner · G. Rauret · M. Foulkes · A. Howard ·
B. Griepink · E. A. Maier
Improvement scheme for the determination
of arsenic species in mussel and fish tissues
Received: 31 March 1998 / Revised: 20 July 1998 / Accepted: 25 July 1998
Abstract Six interlaboratory studies were organised by
the Standard, Measurement and Testing Programme of the
European Commission on the determination of arsenic
F. Lagarde · M. B. Amran · M. J. F. Leroy
Laboratoire de Chimie Analytique et Minérale,
URA 405 (CNRS-ECPM-Université Louis Pasteur),
ECPM, 1 rue Blaise Pascal BP 296,
F-67008 Strasbourg Cédex, France
C. Demesmay · M. Ollé · A. Lamotte
Service Central d’Analyse, Echangeur de Solaize BP22,
F-69390 Vernaison Cédex, France
H. Muntau
Joint Research Center,
Commission of the European Communities, I-21020 Ispra, Italy
P. Michel
IFREMER, BP 1049, F-44037 Nantes Cédex, France
P. Thomas
Institut Pasteur de Lille, Départment Eaux Environnement,
BP 245, F-59019 Lille Cédex, France
S. Caroli
Istituto Superiore de la Sanita, Lab. Igiene del Teritorio,
299 viale Regina Elena, I-00161 Roma, Italy
E. Larsen
Institute of food chemistry & nutrition, National Food Agency,
DK 2860 Soeborg, Denmark
P. Bonner
State Laboratory Abbosttown, IRL-15 Dublin, Ireland
G. Rauret
Universidad de Barcelona, Dept. Quimica Analitica,
Av. Diagonal 647, E-08028 Barcelona, Spain
M. Foulkes
University of Plymouth, Dept. of Environmental Sciences,
UK-PL4 8AA Plymouth, Great Britain
A. Howard
University of Southampton, Dept. of Chemistry,
UK-SO9 Highfield, Southampton, Great Britain
B. Griepink · E. A. Maier (쾷)
European Commission, Standards,
Measurement and Testing Programme, rue de la Loi 200,
B-1049 Brussels, Belgium
© Springer-Verlag 1999
species (arsenobetaine, arsenocholine, monomethylarson-
ic acid, dimethylarsinic acid, As(III) and As(V)) in marine
matrices and soil. A step-by-step approach was used and a
meeting was held at the end of each study to help the par-
ticipants to discover errors and to improve their analytical
methods. The successive steps investigated the calibration
procedures on various solutions, the separation and de-
rivatisation techniques on solutions and extracts and the
extraction on mussel and fish tissues. All materials used
for the study were monitored for their stability. Verified
calibration solutions and compounds were distributed to
the participants in each exercise in order to trace calibra-
tion problems. The agreement between the results im-
proved regularly and at the end of the six campaigns al-
lowed the certification of a reference material of tuna-fish
tissue (BCR-CRM 627) for its total arsenic, arsenobetaine
and dimethylarsinic acid contents.
1 Introduction
It is known for a long time that some marine organisms
may contain high amounts of arsenic [1]. Concentrations
generally lie in the range of 10 to 500 µg · g–1 but may
sometimes exceed 1000 µg · g–1. The highest content re-
ported (2739 µg · g–1) was found by Gibbs et al. in a poly-
chaeta Tharyx Marioni living in a contaminated area [2].
The first arsenic compound contained in marine animals
was isolated from the western rock lobster Panulirus
Cygnus in 1977 and found to be arsenobetaine [3]. Since
then, other species such as arsenocholine, the tetramethyl-
arsonium ion, trimethylarsenoxide, dimethylarsinic acid
and arsenosugars have been identified [4–11]. Very per-
forming hyphenated techniques involving separation by
liquid chromatography and detection by Inductively Cou-
pled Plasma – Mass Spectrometry (ICP-MS) [8, 9, 11–14],
Inductively Coupled Plasma – Atomic Emission Spec-
trometry (ICP-AES) [15], Hydride Generation – Quartz
Furnace Atomic Absorption Spectrometry (HG-QFAAS)
[15, 16], or UV degradation – Inductively Coupled Plas-
ma Atomic Emission Spectrometry (UV-HG-ICP-AES)
6

[17] have been recently developed and applied to the spe-

ciation of arsenic in fish and mussel tissues. Nevertheless,
the quality of analytical results can only be guaranteed if
methods are properly validated. In order to facilitate such
validations and in parallel to allow the emergence of new
techniques, the Standard, Measurement and Testing Pro-
gramme of the European Commission (formerly BCR)
decided to support a project on arsenic speciation and pos-
sibly to produce (a) certified reference material(s).

2 Background of the project and method used

At the beginning of the project, it had been envisaged to study the
content of 6 arsenic species: arsenobetaine (Asbet), arsenocholine
(Aschol), monomethylarsonic acid (MMA), dimethylarsinic acid
(DMA), As2O3 (As(III)) and As2O5 (As (V)) in four materials (fish,
mussel, soil and sediment). Nevertheless, the workload and the an-
alytical investments necessary for the investigation of the four ma-
trices in parallel was too high for several laboratories and it was fi-
nally decided, in the course of the project, to restrict the study to
marine animal tissues, mussels and fish. As the analytical methods
required for speciation studies are very complex, a stepwise ap-
proach was adopted in order to study each step of the procedures
individually. Six interlaboratory studies were performed with the
appropriate samples as shown in Fig. 1.
1. Solutions of six pure arsenic species: arsenate (As(V)), mono-
methylarsonic and dimethylarsinic acids (MMA and DMA), ar-
senobetaine and arsenocholine, arsenite (As(III)) were prepared
extemporaneously by the participants or were distributed in a
NaOH solution.
2. Solutions containing a mixture of the six same analytes (arsen-
ite was added extemporaneously by the participants).
3. Solutions containing the six arsenic compounds together with
interfering cations and anions to mimic a fish and a sediment
extract. Fig. 1 Overall scheme for the stepwise method validation studies
These three steps on solutions helped the participants to of forms of As in matrix materials. To verify the calibration step,
evaluate the performance of their final detection including standard solutions were distributed in each interlaboratory study;
quantification and the separation power of the HPLC system. in the third study the solutions were mimicking soils, sediments
4. Solutions mimicking fish and mussel extracts allowed to fi- and fish tissue (see Table 3). At the end of the third exercise it was
nalise the separation and final quantification. decided to focus efforts on fish and mussel matrices. For the fourth
5. Fish and mussel clean extracts were used to validate the separa- exercise only solutions mimicking fish and mussel tissues were
tion and quantification on real samples. analysed. At the end of the project the group of laboratories
6. A mussels raw extract, shark and mussel tissue powders to- achieved to certify a fish tissue for Asbet and DMA [19]. Mussel
gether with a common extract allowed to verify the clean-up tissue could not be certified
and the total analytical procedure including extraction.
At each step several sources of errors were detected and were fur-
ther corrected. calibrate or verify the calibration of his own system. All solutions
were prepared on a mass basis using calibrated balances. In total
25 laboratories expressed their interest and participated at one or
Samples and substances more exercises. A total of 10 laboratories from 6 European coun-
tries participated in all six interlaboratory studies.
Shark and mussel powders were provided by the Joint Research
Center (Ispra, I). Arsenobetaine and arsenocholine were synthe-
sised at the Laboratoire des Matériaux Organiques à Propriétés Stability studies of the materials
Spécifiques (Vernaison, France). The preparation of these two
compounds is described in this issue [18]. The preparation of the All distributed materials were studied for their stability over the
various solutions as well as the study of their homogeneity and sta- entire period of the interlaboratory study. Samples of the solid ma-
bility was performed in collaboration between the Service Central terials were studied for their homogeneity and stability. The ap-
d’Analyse and the Laboratoire de Chimie Analytique et Minérale plied methods used for homogeneity and stability testing were de-
(Strasbourg, France). veloped during the interlaboratory studies and are described in the
Commercially available As compounds were purchased by the respective publications: certification of CRM 626 – arsenobetaine
latter two laboratories from suppliers at the highest stated purity solution [18], and certification of CRM 627 – tuna fish tissue [19].
possible. Before use and distribution, each set of pure calibrant The stability studies were performed at three temperatures: +4 °C
was verified for purity and stoichiometry; results are given in and +20 °C and +40 °C. Samples stored at –20 °C served as refer-
Table 1. These “pure” compounds were used to prepare the un- ences as explained in ref. [19].
known solutions distributed to the participants. Each laboratory
also received a concentrated solution of each substance in order to
 7

Table 1 Purity of the pur-

chased As compounds Compound Origin Claimed purity Verified purity
(mass fractions) (mass fractions ± SD)
a) based on the As measure-

ments by ICP-AES As2O3 (As(III)) Aldrich 99.999% > 99.9%

b) Laboratoire des Matériaux Na2HAsO4,7H2O (As(V)) Aldrich – 98.8 ± 0.3% a)
Organiques à Propriétés Spéci- AsCH3O3Na2,6H2O (MMA) Carlo Erba 99.4% 98 ± 3% a)
fiques (Vernaison, France). AsC2H6O2Na,3H2O (DMA) Sigma 98% 98 ± 6% a)
c) Service Central d’Analyse
AsC5H11O2,H2O (Asbet) b) – > 99% c)
(Vernaison, France) (see ref. b)
AsC5H14OBr (Aschol) – > 99% c)
[18])

to check the quality of the separation and not to test the

3 Results and discussions
of the interlaboratory studies
First interlaboratory study
The first interlaboratory study consisted in the preparation,
distribution and determination of the 5 single unknown
solutions (between 5 and 20 mg · L–1) and individual calibra-
tion solutions (each of 1.0 g · L–1, except As2O5 : 0.5 g · L–1)
expressed as mass fractions of the As compound. As(III)
was distributed as As2O3 in pure crystallised form. The
participants were advised to dissolve the substance in a
0.4% NaOH solution. An unknown As2O3 powder (pure
As(III) diluted with NaCl) was also provided. Each par-
ticipant had to prepare the As(III) calibration solution and
unknown solution extemporaneously, following a detailed
protocol. One solution contained three As species (As2O5:
2.5 mg · L–1, DMA: 7.5 mg · L–1, Aschol: 6 mg · L–1). No
instability of the solutions was detected. It was left free to
each participant to analyse or not the latter solution. The
outcome of this exercise demonstrated that several im-
provements were necessary and that an additional trial on
solutions had to be performed to allow participants to set
up their analytical systems. Only ten laboratories deliv-
ered results in the first study.
Second interlaboratory study
For this study, five mixtures containing different propor-
tions of arsenocholine, arsenobetaine, DMA, MMA, As(III)
and As(V) were prepared in freshly boiled de-ionised wa-
ter and distributed to the participants together with fresh
individual standard solutions. The aim of that study was
performances of the detector. It was therefore decided to
take relatively high concentrations of each arsenic com-
pound (see Table 2). The five solutions were found to be
stable (except for As(III)) for four months if kept in the
dark at +4 °C. Storage in the dark at +40 °C leads to the
formation of As(III) in some solutions. A significant for-
mation of arsenobetaine resulting from the degradation of
arsenocholine was observed when solutions are stored at
+20 °C in day light but no trace of degradation was de-
tected at +4 °C in the dark. A total of 13 laboratories de-
livered results. Only few of them were able to quantify all
species in the five solutions. Separation of arsenic species
was mainly performed by HPLC (11 laboratories). Gas
Chromatography (GC) with cold trapping (CT) and Capil-
lary Zone Electrophoresis (CZE) with UV detection (ab-
sorption at 190 nm) were also applied by one laboratory
each. Some participants determined As(III), As(V), DMA
and MMA concentrations by atomic absorption (AAS) or
atomic emission spectrometry with inductively coupled
plasma, following a hydride generation. The hydride gen-
eration of arsenobetaine and arsenocholine was not quan-
titative and no results could be delivered for these com-
pounds unless strong UV irradiation could be applied.
The adequate UV irradiation systems were not available
in this second study. Some laboratories using off-line meth-
ods, e.g., Instrumental Neutron Activation Analysis (INAA),
Energy Dispersive X-Ray Fluorescence (EDXRF), Elec-
troThermal (ET)- or Zeeman Effect (ZE) -AAS, encoun-
tered difficulties in collecting the entire right fractions af-
ter separation. Others claimed that they had to work close
to the detection limits (UV-CZE, ICP-AES) of their meth-
ods for some compounds. Users of an ICP-MS detector
had to remove chloride ions which interfere with the As
signal (m/z = 75 for As as well as for Ar+Cl–). The use of

Table 2 Composition of the solutions distributed for the second and third interlaboratory studies (all concentrations in mg · kg–1)
Compound 2nd interlaboratory study 3rd interlaboratory study
Solution 1 Solution 2 Solution 3 Solution 4 Solution 5 Solution 1 Solution 2 Solution 3 Solution 4 Solution 5
Aschol 5 1 1 3 1.5 0.25 – – 0.5 –
Asbet 5 – 10 – 1.5 1 0.5 – 1.25 –
As2O3 5 – 1 2 – 0.3 0.3 0.4 – 1
DMA 5 10 10 0.75 – 0.15 0.3 0.2 0.5 0.75
MMA 5 – 1 – 1 0.15 0.2 0.1 0.5 0.5
As2O5 5 10 – – 2 0.3 0.75 1 0.75 2
8

Table 3 Concentrations of interfering ions (mg · kg–1) added in

solutions 4 and 5 in the third interlaboratory study
Interfering ion Solution 4 Solution 5
Mn2+ – 6.75
Na+ 1100 3.45
Al3+ – 48.5
K+ 240 5.35
Fe2+ 1.1 36.5
Cu2+ 0.95 0.18
Fig. 2 Second interlaboratory study: bar-graph of results of DMA Zn2+ 16.3 0.52
expressed in µg/g. The analysed solution contained As(III), As(V),
MMA, DMA, Aschol and Asbet. The results correspond to 4 or 5 Ca2+ 25 46.3
replicate determinations. 왎 shows the mean value and the vertical Pb2+ 1.1 –
bar the standard deviation of laboratory x; m represents the mean Cl– 1680 473
of means and the between laboratory standard deviation. (target Br– 3.3 –
value = 5 µg/g represented by the horizontal dotted line). Methods NO3– 22 –
used: LC-ICP-MS: lab 1 and 2; LC-HAAS: lab 4, 5, 6 and 9, LC-
PO43– 257 2360
ETAAS: lab 12; GC-CT-HAAS: lab 3; LC-ICP-EAS: lab 7 and 8;
LC-EDXRF: lab 10; CZE-UV: lab 11. (Lab numbers on the x axis
do not correspond to the lab numbers in Figs. 3 and 4)

a properly selected pre-column eliminates the chlorides.

From this study, it became clear that washing and precon-
ditioning of the liquid chromatographic column is a criti-
cal step. After a short period, the separation efficiency of
the anion-exchange columns can be seriously reduced,
causing As(III) and arsenobetaine to co-elute. It was sug-
gested to thoroughly wash the columns at the beginning of
each analytical cycle and after three consecutive runs.
As an example, Fig. 2 shows the bar-graph obtained for
the DMA content in mixture 1 after identification and cor-
rection of the calculation errors (e.g. dilution errors, con-
fusion of units: mole instead of g etc.). The mean of the
mean values is very close to the target value and the coef-
ficients of variation between laboratories is only 7.6%.
For Asbet and Aschol only three sets of data could be de-
livered. This demonstrated that, at that stage of the study,
no method was able to deliver reliable results for all com-
pounds if compared to the target values calculated from
the preparation procedure. Therefore, it was decided that
an additional intercomparison on five synthetic solutions
would be useful before starting the exercises on real ex-
tracts.
Third interlaboratory study
For the third interlaboratory study, participants were pro-
vided with:
– 3 different solutions containing the arsenic species of
interest. The concentrations were relatively low com-
pared to the second trial, in order to be close to the con-
centrations of the different forms of arsenic typically
found in fishes, soils and sediments;
– 1 solution to mimic a fish extract containing ions, e.g.
Fe2+, Cl-, PO43–, Pb2+, NO3– etc. that might interfere
with the applied techniques;
– 1 solution to mimic a soil extract using the same ap-
proach
Fig. 3 Third interlaboratory study: bar-graph of results of DMA
expressed in µg/kg. The analysed solution contained Asbet,
Aschol, DMA, MMA, As(V), As(III). (As(III) was added extem-
poraneously by the participants). The results correspond to 3 to 5
replicate determinations. 왎 shows the mean value and the vertical
bar the standard deviation of laboratory x; m represents the mean
of means and the between laboratory standard deviation. (target
value = 150 µg/kg represented by the horizontal dotted line). One
laboratory submitted values of 1303 ± 76 µg/kg (calculation error
due to a forgotten dilution factor), they are not included in the
graph. Methods used: LC-HAAS: lab 2 and 5; GC-HAAS: lab 1;
LC-HICP-AES: lab 3; LC-ICP-MS: lab 6; LC-EDXRF: lab 5 (Lab
numbers on the x axis do not correspond to the lab numbers in
Figs. 2 and 4)
The composition of these solutions are presented in Ta-
bles 2 and 3. As(V), DMA, MMA, arsenobetaine and ar-
senocholine were found stable in these solutions if they
were kept at +4 °C in the dark. In the solution mimicking
the sediment extract, about 50% of As(III) was oxidised
into As(V) after 45 days of storage at +4 °C. In compari-
son with the previous studies, results appeared to be more
comparable between laboratories and closer to the target
values for As(III), As(V), MMA and DMA in solutions
1 to 3. Figure 3 shows the bar-graph obtained for DMA in
solution 1 (relative BLSD of 23%). Nevertheless, some
laboratories encountered difficulties in the separation of
some compounds. The collection of the fractions after
separation in off-line techniques was not satisfactory (lack
of reproducibility). As in the previous study, most of the
laboratories used HG with AAS or ICP-AES detection.
 9
Table 4 Mean of means, coefficient of variation between labora- given because they have not been determined at all stages by a suf-
tories and range of values, obtained for various materials in the in- ficient number of participants)
terlaboratory studies. (Arsenocholine, As(III) and As(V) are not

Study & Matrices Arsenobetaine Monomethylarsonic acid Dimethylarsinic acid

m CV% range target m CV% range target m CV% range target
3rd sol. 4 1.05 7.6 1.0–1.1 1.25 0.4 41 0.4–0.5 0.5 0.46 9 0.4–0.5 0.5
3rd sol. 5 – – – – 0.4 43 0.4–0.6 0.5 0.8 19 0.6–1.0 0.75
4th sol. 1 – – – – 0.3 39 0.2–0.4 0.35 0.15 40 0.1–0.3 0.12
4th sol. 2 1.0 26 0.8–1.5 0.87 0.25 24 0.2–0.3 0.25 0.25 12 0.2–0.3 0.24
5th fish extract 2.5 17 2.0–3.0 – 0.3 27 0.1–0.3 – 0.3 45 0.2–0.6 –
5th mussel extract 3.3 33 2.5–5 – – – – – 0.4 31 0.3–0.7 –
5th spiked mussel 3.5 29 2.5–4.5 – 0.25 28 0.15–0.3 – 0.35 24 0.25–0.5 –
extract
6th raw mussel 6.3 22 5.1–8.0 – – – – – 0.74 2.7 0.6–0.9 –
extract
6th fish 5.7 45 0.4–9.8 – – – – – 0.3 15 0.2–0.6 –
6th mussels 6.8 54 0.4–13.1 – 0.32 50 0.06–0.6 – 1.0 27 0.7–1.5 –
sol.: aqueous solution (composition are given in the text and Table 2), m: mean of means, range: highest and lowest mean values, CV%:
coefficient of variation between laboratories, target: target value of the weighing preparation procedure

A consequence of this third interlaboratory study was

Fig. 4 Sixth interlaboratory study: bar-graph of results of arseno-
betaine in a shark tissue powder expressed in µmol/kg. The results
correspond to 2 to 5 replicate determinations. 왎 shows the mean
value and the vertical bar the standard deviation of laboratory x; m
represents the mean of means and the between laboratory standard
deviation. (One laboratory reported a value of 2.4 ± 0.2 µmol/kg
due to an incomplete hydride generation of Asbet, this result is not
shown in the figure; laboratory 1 reported values for all analysed
compounds twice as high as the results of the majority of the par-
ticipants, suggesting a systematic error in the calibration proce-
dure). Methods used: LC-HICP-EAS: lab 1; LC-HAAS: lab 2; LC-
ICP-MS: lab 3, 4, 5 and 6. (lab numbers of the x axis do not corre-
spond to the lab numbers in Figs. 2 and 3)
Consequently, again very few data were provided for ar-
senobetaine and arsenocholine. Participants using hydride
generation were invited to develop an analytical proce-
dure for the determination of arsenocholine and arsenobe-
taine in a separate experiment or to improve their hydride
generation yield by applying strong UV irradiation or
NaOH pre-treatment.
The between laboratory standard deviations (BLSD),
range of results, as well as the agreement with the target
values for solutions 4 and 5 (simulating respectively a fish
and a sediment extract) remained poor. Table 4 lists the
achieved between laboratory results. This lack of compa-
rability between laboratory results was explained by the
presence of high amounts of chlorides (solution 4) and
phosphates (solution 5) which affects the separation power
of the analytical column. A purification procedure appeared
necessary to remove chlorides before any chromatograph-
ic separation.
that many participants could not afford to develop and
validate in parallel methods for four different matrices
(mussels, fish, soil and sediment) and six compounds. The
analytical and manpower investments necessary appeared
to be too heavy. It was therefore decided to focus the pro-
ject on fish and mussel tissues. Therefore, a fourth inter-
comparison was organised to improve methods on these
matrices. Two synthetic solutions simulating a fish and a
mussel tissue extract were prepared.
Fourth intercomparison study
The synthetic solutions distributed did not contain As(III)
and the concentrations of interfering ions did better ap-
proximate real contents of a fish or mussel tissue extract
(e.g. solutions contained less chlorides). Table 4 shows
that the trueness (agreement between the target values and
the between laboratory means) of the results for MMA,
DMA and arsenobetaine was very satisfactory.
Fifth intercomparison study
The fifth interlaboratory study focused on arsenic specia-
tion in cleaned extracts of fish and mussel tissues. One
fish and one mussels extract were prepared from fresh
flesh using a water/methanol [1 : 1; v/v] extraction fol-
lowed by a clean-up with diethylether [20]. No As(III)
could be detected. Arsenobetaine, arsenocholine (very low
contents), DMA, MMA and As(V) were found in both ex-
tracts and were stable at –20 °C and +4 °C for at least
2 months. This period covered the duration of the analysis
in the laboratories engaged in the study. The mussels ex-
tract was divided into two portions, one being enriched
with known amounts of the various As compounds. Par-
ticipants noticed that saturation of the analytical column
(ion exchange or ion-pairing) due to the high amounts of
salts present in the extracts (especially in mussels) could
10
be avoided by dilution of the extract or by using a C18 pre-
column. On chloride cartridges, As(V) and DMA were
partially or totally lost. As difficulties with calibration still
persisted, it was decided that a common unknown standard
solution to check the calibration step would further be dis-
tributed. The main observation reported for the mussel ex-
tract concerned the presence of two unknown compounds.
It was suggested that these peaks might be due to arseno-
sugars as already reported in the literature [4–11]. Only
3 laboratories detected a peak at the retention time of ar-
senocholine but at a level close to the detection limits. As
the results between the laboratories showed a relative
BLSD comparable to the fourth study, it was decided to
pass to the next step on a raw mussel extract and dried
shark and mussel tissue.
Sixth intercomparison study
Arsenobetaine, DMA and traces of arsenocholine found in
the samples, were stable at +4 °C and +20 °C in the dark
for the entire duration of the interlaboratory study. The
different purification methods tested by the participants
on the raw mussel extract were based on solvent extrac-
tion using chloroform, diethylether or petroleum ether,
column elution on a C18 cartridge, dilution or filtration.
Some of the lower values obtained in the extract for ar-
senobetaine could be attributed to losses during the clean-
up process. Some higher values were attributed to a co-
elution of an unknown compound with arsenobetaine and
identified on similar separation phases by other partici-
pants.
Two major forms of As, DMA and arsenobetaine, were
detected in both fish and mussel tissue powders. Inorganic
forms of As and in particular As(III) could not be identi-
fied; traces of As(V) were reported by only few partici-
pants but could not be accurately determined (close to de-
tection limit). Extraction efficiencies (sum of As in vari-
ous chemical forms compared to total As determination)
were very different from one laboratory to another (50 to
90%). This could be explained either by the poor determi-
nation of the total arsenic content (partial hydride genera-
tion) or by the extraction method itself. Therefore, it was
recommended to check that the quantification of total ar-
senic is properly validated and to verify its accuracy by
using a certified reference material of similar composition
(BCR-CRM 278, NRCC-DORM-1). Correction for water
content was also requested.
Recovery experiments were performed in order to ver-
ify for potential losses or transformation of the species
during clean-up/pre-concentration steps. Figure 4 shows
that good agreement between laboratories could be ob-
tained in particular when using ICP-MS. With strong UV
irradiation, hydride generation techniques (Fig. 4: ICP-AES
of laboratory 1) achieved results close to those of ICP-
MS. Two participants did still not achieve to set-up an UV
irradiation system which delivered a sufficient irradiation
power (Fig. 4: laboratories 1 and 2). At this stage of the
project, several sources of errors were detected and the
quality control points necessary to trace and eliminate
them were known and under control for the fish matrix.
As a consequence, certification of at least major As spe-
cies in a separate fish tissue was considered as possible.
This study is described in this issue [19].
The methods and the results for dried mussel tissue
were not considered as sufficiently reliable to envisage
certification. The total As content in mussels was often
much higher than the calculated total As resulting from
the speciation measurements. The extraction efficiency in
the mussel tissue did never exceed 80% and was often
lower than 50%. This was attributed to incomplete extrac-
tion of the studied compounds or to the presence of some
unidentified forms of As which remained in the solid
residue.
5 Conclusion
All the analytical steps (extraction, purification, separa-
tion, detection) needed for arsenic speciation in fish and
mussel have been studied, optimised and validated in suc-
cessive interlaboratory studies. Each step benefited from
the preparation of adequate samples systematically checked
for homogeneity and stability. The overall progress made
by the group of laboratories for arsenobetaine, DMA and
MMA in the various samples is shown in Table 4. As(III)
could never be detected in natural fish or mussel tissue
samples, therefore participants decided to optimise their
methods towards the determination of the other forms of
As. Arsenocholine was only detected by some methods
and at very low concentrations. The results also confirm
the validity of such interlaboratory studies to achieve a
quality of determinations which allows certification of
reference materials. The methods which demonstrated the
necessary reliability to certify the fish tissue material are
described in detail in this issue [19].
The findings made over the six exercises have been
used to elaborate a detailed analytical protocol. This pro-
tocol included various quality control points to allow to
trace potential sources of errors in the final certification
study. In particular, the group recommended to make
available in the certification study a solution of all studied
substances in order to verify the calibration [18]. It was
recommended to certify an arsenobetaine solution as this
substance is not yet available on the market. Arsenocho-
line could not be determined by all participants in the real
samples studied from the fifth exercise up to the certifica-
tion of the fish tissue. Therefore, no arsenocholine solu-
tion has been certified. Over the study it became clear that
off-line techniques with sampling of fractions eluting from
a liquid chromatography system are inadequate for the de-
termination of several forms of As. On-line methods al-
low much more flexible procedures and achieve better ac-
curacy. Capillary zone electrophoresis was rapidly aban-
doned because of a lack of sensitivity of the UV detection.
Hydride generation with strong UV irradiation [19] al-
lowed to achieve very satisfactory performances for ar-
senobetaine and was finally applied in the certification of

the fish tissue. In general, it became clear over the dura-
tion of these studies that ICP-MS was the most adequate
detection for As speciation work provided the chloride
ions are fully removed. The overall performance of the
group is demonstrated by the certification of a reference
material for some of the studied substances. This certifi-
cation is described in details in this issue [19].
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