Anal Bioanal Chem (2006) 384: 1331–1340
DOI 10.1007/s00216-005-0249-5
ORIGINA L PA PER
J. Sanz-Landaluze . L. Bartolome . O. Zuloaga .
L. González . C. Dietz . C. Cámara
Accelerated extraction for determination of polycyclic aromatic
hydrocarbons in marine biota
Received: 19 September 2005 / Revised: 17 November 2005 / Accepted: 18 November 2005 / Published online: 25 February 2006
# Springer-Verlag 2006
Abstract A rapid and simple method is proposed for
determination of polycyclic aromatic hydrocarbons (PAH) in
complex matrices such as marine biota. The method uses
sonication, by means of an ultrasonic probe, as a new tool for
assisted extraction, coupled with reversed-phase liquid
chromatography (RP-LC) with fluorescence detection (FL)
for determination of 16 US EPA priority PAH. Separation
and detection of the 16 PAH were complete in 45 min by RP-
LC with a C18 column and acetonitrile–water gradient
elution. Multivariate optimisation of the variables affecting
extraction (ultrasound radiation amplitude, sonication time,
and temperature of the water-bath in which the extraction
cell was placed) was conducted. The accuracy of the method
was determined by analysis of a certified reference material
and comparison of the results obtained with those from
another method (microwave-assisted extraction and GC–
MS). The new technique avoids the main problems
encountered in the determination of PAH in complex
matrices such as marine biota, and no clean-up step is
necessary. The method was applied to determination of PAH
in estuarine biota samples from the Urdaibai estuary (Biscay,
Spain).
Keywords PAH . Extraction . Ultrasonic probe .
Biota samples
J. Sanz-Landaluze . L. González . C. Dietz . C. Cámara (*)
Department of Analytical Chemistry,
Faculty of Chemistry, University Complutense de Madrid,
Ciudad Universitaria,
28040 Madrid, Spain
e-mail: ccamara@quim.ucm.es
L. Bartolome . O. Zuloaga
Department of Analytical Chemistry,
Faculty of Technology,
Euskal Herriko Unibertsitatea,
644 P.K., 48080 Bilbao, Spain
Introduction
Development of human activity has always been closely
associated with different water resources but, unfortu-
nately, anthropogenic activity has often resulted in chem-
ical contamination of these resources. Lately mankind has
become aware of the potential long-term adverse effects of
these chemicals in general and their potential risks for
aquatic and terrestrial ecosystems in particular. Several
national and international organizations (ONU, WHO, EU,
USEPA …) have classified some PAH as among the most
hazardous and persistent organic pollutants originating
from a wide variety of natural and anthropogenic sources.
PAH are a class of hydrophobic organic contaminant
comprising two or more fused benzene rings [1]. High
molecular weight PAH (e.g. benzo(a)pyrene) are known to
be carcinogenic and di and triaromatic compounds have
been shown to be narcotic to marine organisms [2].
Quantification of these compounds enables better under-
standing of their behavior and, for example, the level of
contamination of a site and its remediation after an oil spill
can be followed quantitatively [3]. Quantification of PAH
in complex matrices, for example environmental samples
and petroleum, can be difficult, because many compounds
can interfere during the analysis.
There is, therefore, a basic need for reliable analytical
procedures for determination of these pollutants to correctly
monitor their distribution in time and space. The best methods
for analysis of PAH are high-performance liquid chromatog-
raphy (HPLC) coupled with fluorescence (FL) or with UV
absorbance detection or gas chromatography (GC) with mass
spectrometric detection (MS). These two separation–detec-
tion steps were chosen as US Environmental Protection
Agency (EPA) methods 8310 and 8100, respectively [4].
To characterize PAH in solid matrices such as biota, an
extraction step is required before the analysis; many well
established extraction techniques are commonly used for
isolation and analysis of PAH and similar organic com-
pounds. Traditionally, Soxhlet extraction has been used for
solid samples and liquid-liquid extraction for aqueous
samples. These techniques have inherent disadvantages,
1332
however, for example the large volumes of organic solvents
required; they are also time-consuming and involve multi-
step processes that always risk loss of some analytes [5–7].
Supercritical-fluid extraction (SFE) [8–10], pressurized
liquid extraction (PLE) [11, 12], microwave-assisted extrac-
tion (MAE) [10, 13], ultrasonic assisted extraction [10, 14],
and subcritical water extraction [10, 15] have been devel-
oped as alternative techniques to replace classical extraction
methods. All these methods reduce time consumption and
the volumes of solvent required, but have the disadvantages
of high investment and maintenance costs of the instruments,
especially for SPE, ASE, and MAE. For evaluation of PAH
in biota Soxhlet is, even nowadays, the principal extraction
technique, especially for routine studies [16–18]. Very few
studies have been performed using these new extraction
procedures [14, 19]. Several studies have compared the
efficiency of extraction of PAH using these techniques [5,
10, 11], but only for soil, sediment, and sewage samples. All
these studies concluded that, once optimized, these extrac-
tion techniques are comparably efficient, with similar
standard deviations.
Ultrasonic probe-assisted extraction is a relatively new
technique being used with success for analysis of a variety of
compounds [20–23] including PAH in soil [24]. In this
technique solid–liquid extraction occurs as a consequence of
cavitation phenomena—implosion of cavitational bubbles
leading to extreme temperatures and pressures. Furthermore,
when a cavitating bubble collapses near the surface of a solid
sample particle, micro-jets of solvent propagate toward the
surface at great velocity, causing pitting and mechanical
erosion of the solid surface, and leading to particle rupture.
This is the main characteristic of focussed ultrasound, and it
makes this extraction method unique, because the smaller the
particle size, the higher the total area exposed to the solvent
[24].
For complete development of an analytical method,
optimization of all the variables involved in the analysis is
fundamental [25, 26]. Most optimisation procedures,
however, are conducted one-factor-at-a-time. Generally
speaking, optimisation procedures that alter all variables at
the same time are more advantageous, because interactions
among them are considered. Use of an experimental design
(e.g. full factorial, fractional factorial, Plackett and Burman
designs, central composite, face-centred cube, routable
central composite, Doehlert, etc.) combined with analysis
of variance (ANOVA) is much more reliable, and avoids
the limitations mentioned above, but, surprisingly, is still
not extensively applied in analytical applications. Multi-
variate methods involve simultaneous combination of
several conditions enabling evaluation of the interdepen-
dence of variables and their effect on the response, and
optimization of these influential factors.
The method proposed here is based on use of static
ultrasound-assisted extraction for leaching of PAH from
natural contaminated estuarine biota before separation and
determination by HPLC–FL. This analytical procedure was
then applied to a reference material, a mussel tissue (NIST-
2977), to validate the procedure. The sample-preparation
procedure developed was then used to assess the extent of
contamination of oysters and mussels collected along the
Urdaibai estuary (Biscay, Spain). Urdaibai is a protected
estuary, situated in a rural area, which in 1984 was declared
Reserve of the Biosphere by the United Nations for the
Education, Science, and Culture Organization (UNESCO).
Experimental
Cleaning procedures
All laboratory ware and other equipment that comes into
contact with samples must be rigorously cleaned to avoid
contamination. All glass and plastic ware was washed with a
common detergent, thoroughly rinsed with MilliRo quality
water (Milli-Q Model 185, Millipore; Bedford, MA, USA),
and soaked in clean dilute (15%) HNO3 for 24 h. The
equipment was then rinsed with Milli-Q (Millipore) quality
water and then soaked for another 24 h in acetone (HPLC
grade, Scharlau; Barcelona, Spain). Finally, glassware was
placed overnight in an oven at 150°C and plastic in an oven
at 90°C.
Reagents
Working standard PAH mixtures were prepared by suitable
dilution of stock standard solutions (Supelco; Barcelona,
Spain) with acetonitrile. HPLC-grade n-hexane (Scharlau;
Barcelona, Spain) was used. Other chemical reagents, for
example methanol and acetonitrile (Carlo Erba; Milan,
Italy) were also of HPLC purity. Commercial acetone was
purchased from Dismadel (Madrid,Spain). Water was
purified with a Milli-Q system (Millipore).
Stock solutions and certified reference materials
Stock solutions, Dr Ehrenstorfer PAH mix 64 (17 PAH
each at 2,000 μg mL−1), were purchased from Supelco
(Walton-on-Thames, UK), in a 1:1 mixture of HPLC grade
dichloromethane and benzene. The stock solutions were
stored under refrigeration and protected against light.
Intermediate dilution of this stock solution was performed
for calibration. More dilute solutions (∼1,000 ng mL−1)
were prepared weekly by appropriate dilution of the stock
solution with acetonitrile. These solutions were used to
produce calibration solutions, following the standard
addition method.
A certified reference material was used to check the
accuracy of the proposed method and for validation. NIST-
2977 mussel tissue was provided by the National Institute of
Standards and Technology (Gaithersburg, MD, USA). The
mussels (Perna perna, edible brown mussel) used for the
preparation of NIST 2977 were collected in Guanabara Bay,

Brazil. Certified values are given for 10 of the 16 EPA-PAHs
and indicative concentrations for another 16 PAH.
Instrumentation
A Cryodos-50 laboratory freeze dryer from Telstar
Instrumat (Sant Cugat del Valles, Catalonia, Spain) and a
Pulverisette-6 agate mortar (Fritsh, Idar-Oberstein, Ger-
many), respectively, were used to lyophilise and grind the
samples. For PAH extraction, a Bandelin sonifier ultrasonic
cell disruptor/homogeniser (20 kHz, Bandelin Electronic,
Berlin) equipped with a 3 mm titanium microtip was used.
The energy of ultrasonic irradiation was fixed at the desired
level by using power settings up to 40%.
PAH analysis was performed with a Spectra System P
4000 (Thermo Electron Corporation, San Jose, USA)
HPLC chromatograph equipped with an FL 3000 pro-
grammable fluorescence detector (Thermo Electron Cor-
poration). The 16 PAH to be quantified were separated on a
Hypersil Green PAH (200 mm×4.6 mm; particle size,
5 μm) column by gradient elution. Acetonitrile–water
mixtures were used as mobile phase at a flow-rate of
1.2 mL min−1. The composition of the gradient started with
40% water and 60% acetonitrile; the acetonitrile content
was then increased to 65% (0–10 min) and later to 100%
(10–40 min). This level was held constant for 4 min and
finally reduced to 40% at the end of the analysis. Analysis
was performed at room temperature and excitation and
emission wavelength pairs were programmed to change
during the analytical run to optimise the detection of each
component. Data collection and integration were con-
ducted using HPLC ChromQuest software version 4.1-
Surveyor LC systems (Thermo Electron Corporation).
The area studied
The Urdaibai estuary is in the centre of the Reserve and is
formed by waters mainly from the hydrographic basin of
the Oka river, but also from the mouths of the rivers
Golako, Mape, Artike and Laga. The estuary is 13 km long,
has an average width of 500 m, and approximately 80% of
the surface belongs to the inter-tidal zone. A variety of
industrial activity can be found (metallurgic, shipyards,
treatment of surfaces, dies, manufacture of cutlery), mainly
in the surroundings of the town of Gernika. Two
monitoring programmes (spring 2003 and spring 2004)
were conducted at three sampling points (A≡Kanala,
B≡Murueta, C≡Gernika) in the Urdaibai estuary (Fig. 1).
These programmes were designed to evaluate the impact of
the breaking up, in heavy seas off Galicia on the Northwest
coast of Spain, of the large oil tanker Prestige, in November
2002, spilling more than 77,000 tons of crude oil. Its
composition, according to analysis by the Superior Centre
of Scientific Research of Spain (CSIC), was 50% aromatic
hydrocarbons including PAH, 20% saturated hydrocar-
bons, and 35% resins and asphaltene [27].
1333
Sample collection, storage, and preservation
Oyster samples were collected manually at the coast, stored
in sealed plastic bags, transported to the laboratory in cold
boxes and frozen until lyophilisation. Lyophilisation of the
oysters was performed at −45°C and 0.05 mbar for 24 h.
The lyophilised samples were ground and sieved through a
63-μm metal sieve and stored in hermetically closed Pyrex
vials at 4°C until analysis.
Analytical procedures
Extraction
For ultrasound-assisted extraction, 0.1 g lyophilised oyster
tissue was weighed and placed in a 10-mL vial with 5 mL
n-hexane. The vial was immersed in an ice–water bath
(0°C) and the sample was exposed to ultrasonic irradiation
for 2 min, with the titanium tip of the probe 1 cm from the
upper surface in the slurry (output amplitude ∼15% of the
nominal amplitude of the converter, applied power 200 W).
Extracts were filtered through 0.22 μm Millipore Millex-
HV filters (Scharlab) into amber flasks, the solvent was
evaporated with a stream of argon, and the residues were
reconstituted in 0.5 mL acetonitrile.
HPLC analysis
Approximately 20 μL of the reconstituted samples were
injected into the high-performance liquid chromatograph.
As already mentioned, acetonitrile–water mixtures were
used as mobile phase at a flow-rate of 1.2 mL min−1. The
column was maintained at room temperature. The fluores-
cence excitation and emission wavelengths were changed
during chromatographic separation, a powerful technique
for enhancing sensitivity and selectivity (Table 1).
Results and discussion
Chromatographic separation
A Spherisorb ODS-2 column from Waters and a Green
PAH column from Hypersil were evaluated for separation
of the 16 PAH under different gradient elution conditions
with acetonitrile–water mixtures. This study was con-
ducted using a high-concentration PAH standard solution
(1 μg mL−1) and with a fluorescence program using non-
specific excitation and emission wavelengths (λexc=280
and λem=390) [4]. Only the second column (Green PAH
from Hypersil) enabled complete separation of all 16 PAH
at room temperature, as shown by the chromatogram in
Fig. 2. The chromatographic cycle for the complete
separation of the 16 PAH was 45 min, including 5 min
for the equilibration of the column. This time is suitable for
economic and routine analysis of the 16 PAH.
1334

Fig. 1 Location of the sampling points in the Urdaibai estuary

When the chromatographic separation had been opti- 10 ng mL−1 PAH mixture was used; the optimized program
mized, a study of the most appropriate excitation and is summarized in Table 1. Problems arose with the last
emission wavelengths for each PAH was conducted, using eluted PAH, indenopyrene, which could be determined at
data from the literature [4, 10, 11]. For this study a very high levels only.

Table 1 Fluorescence detector wavelength program used in this study

Retention time (min) λexc (nm) λemi(nm) PAH determined

0–7.0 226 330 Naphthalene, acenaphthylene

7.0–9.5 276 324 Acenaphthalene, fluorene
9.5–13.5 250 390 Phenanthrene, anthracene
13.5–15.8 280 466 Fluoranthene
15.8–20.0 270 388 Pyrene
20.0–28.0 260 420 Benzo(a)anthracene, chrysene
28.0–40.0 290 430 Benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene
40.0–44.0 290 418 Dibenzo(a,h)anthracene, benzo(g,h,i)perylene
44.0–45.0 300 464 Indenopyrene
 1335
Fig. 2 Typical chromatogram
obtained from the 16 PAH in
the mixed stock solution
(100 ng mL−1 each) using a
Green PAH column and aceto-
nitrile–water mixtures at
1.2 mL min−1

Optimization of the extraction analyzed using a microwave-assisted extraction technique

It is well-known that recoveries obtained with spiked
compounds may not be representative of those obtained
with native compounds. Analytes spiked in real-world
samples are neither situated on the same binding sites as
those of the native analytes nor adsorbed in the same manner.
Spiked analytes are usually lightly coated on the surface of
the matrix whereas native analytes can be strongly adsorbed
inside a porous matrix [28]. Thus use of spiked analytes in
recovery studies may overestimate the efficiency of extrac-
tion methods with real samples. It is, therefore, clearly
necessary to validate the extraction procedures with certified
reference matrices, when available, or at least with unspiked
matrices. Because of the high price of the certified reference
materials, however, optimization of the extraction tech-
niques was performed with biota material collected as
described in the experimental section and previously
which in turn had previously been optimized and validated
[29].
Using the ultrasonic probe and taking into account
previous experiments, it was assumed the conditions
affecting extraction that must be optimized were: the
extractant, the ultrasonic energy (for the ultrasonic device
used, this is defined by the percentage of ultrasonic
amplitude), sonication time, and the external temperature
of the sample. For further optimization it was important to
keep in mind that a total of 16 analytical responses must be
evaluated and the areas of each peak corresponding to each
PAH determined. Occasionally, because of the methodology
used, these responses were evaluated one by one and
compromise conditions had to be finally chosen.
As the first extraction optimization step, a solvent study
was conducted to select the most appropriate extractant. As
is apparent from Fig. 3, different solvents used in previous

Fig. 3 Recovery obtained for Recovery

three of the 16 PAH using 1,6 (refered to
Napht
different extractants. (Ultrasonic Hexane)
probe amplitude 15%, external Anthr
1,4
temperature 0°C, and extraction Benzop
time 2 min)
1,2

0,8

0,6

0,4

0,2

0
Hexan Aceton Hexane- Methan Toluen Dichlorom Dichlorome
(1:1) Hexane
1336
Table 2 Factors and levels considered in the full-factorial design to Table 4 Variables and levels for the central composite design
check the effect of ultrasonic probe amplitude (A), external
temperature (T a) and extraction time (t) on analyte response Variables Levels
−α − 0 + +α
Variable Low (−) Central (0) High (+)

A (%) 10 15 20 Ultrasonic probe amplitude, A (%) 7,9 10 15 20 22,1

External temperature, T a (°C) 0 5 15 25 30
T a (°C) 0 15 30
t (min) 1 2 3 Extraction time, t (min) 1 2 3 4 5

studies [10, 12, 30] were tested, and it was concluded that
hexane provided the best results for all PAH. For this
particular study the other variables were maintained constant
at standard values (Ultrasonic probe amplitude 15%, exter-
nal temperature 0°C, and extraction time 2 min).
When the best solvent had been chosen, three other
analytical conditions—ultrasonic probe amplitude (A), ex-
ternal temperature (T a), and extraction time (t) were
optimized using a chemometric approach. First, a full-
factorial design with three factors at two levels and four
replicates of the central point (2n+1) [25], involving 12
experiments in random order, was used to establish the effect
or lack of effect of each of these variables on the response to
the PAH. In each experiment, an oyster sample was analyzed
as described in the experimental section, but using the A, T a,
and t defined by the experimental design using the levels for
each factor shown in Table 2. Results obtained in each
experiment of the design and ANOVA of the results
calculated also by means of the YATES algorithm, [31] are
summarised in Table 3. These results confirmed that the
three variables considered have a statistically significant
effect on analyte response for most of the PAH (F>10.13 at a
95% confidence level) [32] and, consequently, all were
considered in the optimization process.
Further optimisation was attempted by means of central
composite design (CCD). The central composite design
included the points of a factorial design (2n) augmented
with six points located at -α and +α from the centre in a star
design (Table 4) [25]. When the experiments defined by the
CCD are conducted to find the optimum values of the
variables, it is necessary to define the response surface of
each PAH. Results obtained are fitted to a general
polynomial equation (Eq. 1) where β are the adjustable
parameters.
Y ¼ β 0 þ β 1 A þ β2 t þ β3 T a þ β11 A2 þ β 22 t2 þ β33 ðT a Þ2
þ β 12 At þ β 13 AT a þ β23 tT a þ β 123 AtT a
(1)
For each PAH response, fitting of data to Eq. (1) was
performed using the multivariate non-linear regression
analysis program NLREG [33], and the β parameters for
which the statistical probability of being zero was greater
than 10% were sequentially eliminated from the model. For
each PAH, a different model was finally selected which
was able to explain between 95 and 98.65% of the total

Table 3 Design matrix, response values, main and F factors obtained from the factorial design for one of the 16 PAH (anthracene),
developed to investigate the conditions of ultrasonic assisted extraction
Combinations Levels Anthracene peak area (a.u.) Main factor F

1 − − − 1.38
A + − − 0.93 0.046375 3.76
Ta − + − 0.669 −0.113875 22.6
AT a + + − 0.7 −0.148625 38.6
t − − + 0.8 0.256375 114.9
At + − + 0.76 0.191125 63.8
T at − + + 0.71 0.351375 215.8
AT at + + + 1.18 −0.073875 9.5
Zero1 0 0 0 0.81
Zero2 0 0 0 0.85
Zero3 0 0 0 0.87
Zero4 0 0 0 0.9
Factors in bold were found to be statically significant after ANOVA of the results (F >10.13 at a 95% confidence level)
 1337
Peak area Table 5 Recoveries and RSD (n=6) obtained from a standard
4,5
solution evaporated to dryness using the developed extraction and
4
determination method
3,5
PAH % Recovery %RSDs
3
2,5 Naphthalene 97.55 24.38
2 Acenaphthalene 6.2 5.05
1,5 Acenaphthylene 41.55 15.6
1 Fluorene 43.74 12.7
0,5 Phenanthrene 73.93 9.54
0 5 0 Anthracene 74.02 7.79
15 10
0,2 0,5 25 20 Fluoranthene 96.68 18.98
1,5 2
3 35 30 Temperature (ºC) Pyrene 73.16 21.9
Time (min) 440
Benzo(a)anthracene 92.22 9.93
Fig. 4 Response surface estimated from the central composite Chrysene 95.85 16.8
design for optimization of extraction conditions. Results for
anthracene maintaining the amplitude of ultrasonic probe (A=15%) Benzo(b)fluoranthene 98.61 14.86
Benzo(k)fluoranthene 94.96 9.54
Benzo(a)pyrene 102.84 8.86
variance of data. As an example, the equation obtained for Dibenzo(a,h)anthracene 90.22 11.12
anthracene, an PAH of intermediate molecular weight, was: Benzo(g,h,i)perylene 86.58 18.2
Indenopyrene 93.85 11.63
Y ¼ 4:9
0:26A þ 5:2  10
2 t
0
2 t
3:2  10
2 T a
þ 3:9  10
2 A2
0:11t2 þ 3:52  10
6 ðT a Þ2 þ 1:1 Sample treatment recovery
 10
2 At þ 6:9  10
3 AT a þ 8:0  10
3 tT a
The whole procedure includes sonication, filtration, and

1:2  10
4 AtT a evaporation of the biota extract before its analysis. Losses
(2) of PAH during these steps are evaluated in this section. A
solution of the 16 PAH at a concentration of 10 ng mL−1 in
5 mL n-hexane was sonicated, filtered, and evaporated to
The response surface predicted by this model can be dryness under a stream of argon. The recoveries are
visualized (Fig. 4) if one of the three variables is reported on Table 5. For the least volatile PAH (from benzo
maintained constant. Variable values that maximize the (a)anthracene to indenopyrene), losses are below 14%. But
response obtained can be calculated mathematically. The losses are more important for the more volatile PAH
optimum conditions were: 15% of maximum amplitude, species (from naphthalene to pyrene), reaching values up to
2 min extraction time, and external temperature 0°C. A 51% or even, exceptionally, 93% for acenaphthalene. This
typical chromatogram obtained from analysis of the sixteen almost complete loss of acenaphthalene is because of
PAH in an oyster sample under these experimental decomposition by the ultrasonic radiation [30]. Taking into
conditions is shown in Fig. 5. account these losses, calibrating solutions were exposed to

Fig. 5 Chromatogram obtained from an oyster sample after optimized extraction with hexane and using the ultrasonic probe (15%, 2 min, 0°C)
1338
Table 6 Results obtained from analysis of the 10 PAH for which has been estimated for the proposed method for PAH
amounts in NIST-2977 mussel tissue are certified (number of determination. When 0.2 g dried mussel sample is processed,
analyses, n=5)
the detection limit is equivalent to approximately 0.5 ng g−1.
PAH Measured value Certified value (ng g−1) This detection limit enables the analysis of most contami-
(ng g−1) nated oyster and marine biota samples and is well above the
reference values established by the Agence Française de
Fluorene 10.2±3.2 10.2±0.4 Sécurité Sanitaire [36] and the Agencia Española de
Phenanthrene 33.6±8.1 35.1±1 Seguridad Alimentaria [37] after the oil spills of the tankers
Fluoranthene 33.1±8.0 38.7±0.8 Erika and Prestige respectively (0.5 mg kg−1 dry mass for
Pyrene 42.1±7.1 78.9±3.5 bivalve molluscs).
Benzo(a)anthracene 21.9±4.3 20.3±0.8
Benzo(b)fluoranthene 12.0±4.3 11±0.3
Benzo(a)pyrene 6.8±2.1 8.35±0.7 Analysis of Urdaibai samples
Dibenzo(a,h)anthracene 1.7±0.5 1.41±0.2
Benzo(g,h,i)perylene 7.0±1.5 9.5±0.4 Oyster samples were collected in different points (Fig. 1) of
Indenopyrene <d.L. 4.8±0.8 the Urdaibai estuary from May 2003 to March 2004 and then
analysed for PAH content using the method proposed here.
The results, summarised in Table 8, are all averages from
the same procedure (sonication, filtration, evaporation, and three independent measurements. The results were also
reconstitution) before analysis by HPLC–fluorescence. compared with those obtained by use of another analytical
method (microwave assisted extraction (MAE) and GC–MS
determination) [29]; these data also are included in Table 8.
Validation of the method Despite of the large repercussions on the scientific and
civil community of the Prestige oil spill [38], very few
The accuracy of the method was checked by using a reports on the PAH content in oysters and other bivalves in
certified reference material suitable for marine biota, NIST- the Cantabrian zone have been published [14, 18, 27, 39,
2977. The results (Table 6) prove recovery is satisfactory– 40]. One of these studies deals with the PAH content of
between 80 and 105% for all the PAH except pyrene and bivalves (mussels and oysters) in the Urdaibai region, but
indenopyrene, for which recovery was only 53% and 0% before the Prestige disaster (sampling from July 1996 to
(the value found was below the quantification limit), February 1997) [39]. The other studies evaluated the
respectively. Consequently, it can be stated that the method impact of the oil spill on mussels, but centred on other
is valid for the other eight PAH only. These results are regions in the north of Spain (Galiza) [14, 27, 39].
similar to those from other studies using much more Because of the few sampling points considered in this
complicated extraction procedures [3, 10, 11, 34, 35]. study the results obtained should be interpreted with
The precision of the analysis was evaluated by repeating extreme care. Looking at the results, however, and taking
the analysis of the certified reference material. Three into account other results obtained from similar samples
measurements per day were repeated on three consecutive before and after oil spills, and also previous values
days (Table 7). Analysis of variance (ANOVA) of the obtained for oyster tissue in the Urdaibai estuary
results indicated that both within-day and the between-day (Table 9), it can be concluded that the preventive acts
precision were statistically comparable (Fcalculated<Fcritical) taken just after the spill and before the first black oil-laden
[32]. The relative standard deviation (RSD) for all the tide reached the estuary, were efficient—the values do not
determinations was 14.44%. reach the high concentrations found in regions where the
A detection limit of below 0.2 ng mL−1 in the extract, spill directly affected the biota. A slight decrease of PAH
calculated as the concentration corresponding to the blank content occurred between the two sampling programmes.
signal plus three times the standard deviation of the blank,

Conclusions
Table 7 Precision of the proposed method for PAH determination:
analysis of variance (ANOVA) of the results obtained for fluorene in
NIST-2977 mussel tissue certified reference material The proposed ultrasound-assisted solvent extraction of
dried, homogenized samples by use of an ultrasound probe
Sample Day 1 (ng g−1) Day 2 (ng g−1) Day 3 (ng g−1)
enables efficient extraction of PAH from biological marine
1 11.48 9.17 9.46 samples. The optimized ultrasonic extraction procedure was
2 12.42 8.43 10.52 found to extract these aromatic hydrocarbons from NIST-
3 8.52 10.46 12.03 2977 with recoveries higher than 80% for most analytes.
Although some losses occur in the evaporation step,
X 10.8±2.0 9.3±1.0 10.6±1.3
application of the same analytical procedure to the calibra-
Results of analysis of variance
tion solutions enables correction for these losses in
Variance between days Fcalculated=0.1 Fcritical=6.94
quantification of PAH in oyster samples. The good results
Variance within a day Fcalculated=2.38 Fcritical=6.94
obtained for the NIST certified material and the comparison
Table 8 PAH content (ng g−1) of oyster samples from the Urdaibai estuary sampled in the two different programmes and analysed using two different methods (method 1: US–HPLC–FLa;
method 2: MAE–GC–MS)
March 2003 March 2004
A B C A B C
Method 1 Method 2 Method 1 Method 2 Method 1 Method 2 Method 1 Method 2 Method 1 Method 2 Method 1 Method 2

Naphthalene 8.1±2.2 19.2±2.8 19.0±3.5 234.4±34.5 16.8±3.3 68.9±10.1 4.5±0.9 4.04±0.6 67.3±10.5 57.7±8.5 18.2±3.5 30.1±4.4
Acenaphthalene n.db <d.L.c n.d <d.L. n.d 3.7±0.5 n.d 6.2±0.8 n.d 1.9±0.3 n.d 6.3±0.9
Acenaphthylene 70.4±5.9 0.7±0.1 22.8±2.5 6.9±1.1 86.1±6.9 16.1±2.7 14.3±1.3 13.8±2.3 22.1±4.7 12.0±2.0 17.5±4.0 8.3±1.4
Fluorene 17.2±4.5 1.6±0.3 54.2±6.7 38.7±6.0 19.6±1.2 16.6±2.6 28.5±7.8 30.8±4.8 13.1±0.8 10.0±1.5 51.2±5.8 28.6±4.4
Phenanthrene 40.8±2.0 24.8±1.3 61.9±8.5 61.5±3.3 58.1±5.8 78.8±4.2 44.1±5.0 46.0±2.5 29.5±5.0 41.1±2.2 61.7±7.8 75.1±4.0
Anthracene 7.2±1.9 3.3±0.2 9.0±0.8 <d.L. 8.2±0.8 7.8±0.4 <d.L. 5.3±0.3 2.1±0.8 2.8±0.2 6.9±3.0 7.0±0.4
Fluoranthene 48.4±3.4 56.3±3.7 69.8±3.5 148.8±8.2 127.6±9.8 139.1±9.2 25.8±6.0 52.0±3.5 78.2±3.5 32.7±2.1 46.3±6.7 23.5±1.5
Pyrene 22.6±2.3 46.8±3.2 144.1±12 125.7±8.4 58.6±5.3 178.7±12.2 17.5±3.5 35.6±2.4 50.9±4.9 25.7±1.7 57.1±2.5 73.8±5.0
Benzo(a)anthracene 7.9±0.9 8.1±0.7 19.8±3.9 17.3±1.2 30.5±2.2 31.8±2.6 12.3±2.5 14.9±1.2 10.7±3.1 4.4±0.4 15.7±1.5 41,2±3.4
Chrysene 17.6±2.6 34.1±2.7 28.1±3.5 42.9±3.5 14.5±2.2 79.4±6.4 22.7±5.0 29.7±2.4 30.7±3.5 16.5±1.3 26.0±2.5 54.5±4.4
Benzo(b)fluoranthene 29.8±3.0 25.3±2.2 26.8±3.5 56.6±4.5 27.5±5.5 45.9±3.9 16.6±1.3 31.7±2.7 42.2±3.5 9.2±0.8 33.8±3.1 64.6±5.6
Benzo(k)fluoranthene 7.9±2.1 13.4±1.5 11.8±0.9 13.9±1.2 8.9±1.5 24.6±2.6 3.5±1.1 14.4±1.5 12.9±1.0 2.6±0.3 10.5±2.5 21.1±2.3
Benzo(a)pyrene 0.96±0.1 14.2±1.0 5.1±0.5 17.6±1.9 1.7±0.7 31.9±2.2 6.1±2.7 9.7±0.5 10.5±3.0 1.0±0.1 3.3±0.8 19.9±1.3
Dibenzo(a,h)anthracene 19.7±3.1 < d.L. 27.1±5.6 3.86±0.3 29.0±6.7 22.6±3.6 2.3±0.9 2.8±0.4 7.4±1.5 1.5±0.2 11.7±2.3 0.60±0.1
Benzo(g,h,i)perylene 7.8±1.5 < d.L. 3.5±0.3 < d.L. 8.6±2.3 < d.L. 7.8±2.0 2.1±0.2 13.4±2.0 2.1 ±0.2 7.5±1.0 1.4±0.15
Indenopyrene < d.L. < d.L. < d.L. 3.1±0.6 < d.L. 6.4±1.2 < d.L. 3.5±0.7 < d.L. 2.3±0.4 < d.L. 6.7±1.3
ΣPAH 306.6 247.7 503.3 771.6 495.9 752.4 220.4 302.8 391.6 223.7 367.2 462.4
a
US=ultrasound;
b
n.d=not detected;
c
<d.L.=below detection limit
1339
1340
Table 9 Total PAH content (mg kg−1 dry weight) of some bivalve samples from other locations affected by oil spills
Location Sample Date Oil Spill Σ PAH Refs.

Ría de Arousa (Galiza-Spain) Mussel July 2002 Before Prestige 0.82–3.27 [14]
Ría de Arousa (Galiza-Spain) Mussel July 2003 After Prestige 2.45–9.8 [14]
Betanzos estuary (Galiza-Spain) Mussel October 1995 After Aegean Sea 1.6–45.7 [39]
Betanzos estuary (Galiza-Spain) Mussel July 2003 After Prestige 16.9–17.0 [27]
Prince Willian Sound (Alaska-EEUU) Mussel 1990–1993 After Exxon Valdéz 9.9–15.5 [40]
Urdaibai (Basque Country-Spain) Oyster July 1996 Before Prestige 0.15–1.09 [18]
Urdaibai (Basque Country-Spain) Oyster March 2003 After Prestige 0.2–0.5 This work

with another analytical method validate the method. The
major advantages of ultrasonication are the reduced extrac-
tion time and the elimination of an additional clean-up step,
involving additional glassware and apparatus. Traditional
extraction methods require longer times (Soxhlet >8 h) and
although recently developed methods may reduce this time
substantially (SFE ∼30 min, PLE ∼20–30 min, MAE ∼10–
15 min, ultrasonic bath ∼30 min), they still suffer from the
complexity and high instrument costs. The ultrasonic probe
described here is an attractive alternative which also avoids
additional clean-up steps, which makes this method suitable
for laboratories engaged daily in routine analysis of a large
number of samples.
It can be concluded that levels of PAH in natural oysters
from the Urdaibai estuary did not increase substantially after
Prestige oil spill, and that the PAH levels are far lower than
those reported after other oil spillage episodes. Fortunately,
therefore, the effect of the Prestige oil spill on the ecosystem of
the Urdaibai estuary can be regarded as only moderate to low.
Acknowledgements The authors want to thank the Spanish
Government for financial support through project BQU2002-
01348. J. Sanz is grateful to the European Union for his post-
doctoral fellowship.
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