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Validating a multi-biomarker approach with the shanny Lipophrys pholis to
monitor oil spills in European marine ecosystems
M.M. Santos
a,
⇑
,1
, M. Solé
b,1
, D. Lima
a
, B. Hambach
b
, A.M. Ferreira
c
, M.A. Reis-Henriques
a
a
CIMAR/CIIMAR – Interdisciplinary Centre of Marine and Environmental Research, Laboratory of Environmental Toxicology, University of Porto, Rua dos Bragas 177,
4050-123 Porto, Portugal
b
Institut de Ciències del Mar (ICM-CSIC), Pg. Marítim de la Barceloneta, 37-49 08003 Barcelona, Spain
c
IPIMAR – Institute for Fisheries and Sea Research, Avenida Brasilia, 1449-006 Lisbon, Portugal
article info
Article history:
Received 12 May 2010
Received in revised form 21 July 2010
Accepted 28 July 2010
Available online 24 August 2010
Keywords:
Hydrocarbon
Fish
Water Framework Directive
Biomarkers
Oil spill
EROD
abstract
Oil spills are an importance source of polycyclic aromatic hydrocarbons (PAHs) in the aquatic environ-
ment. Intertidal communities are particularly sensitive since most organisms from these ecosystems
are sessile or present reduced mobility. Hence, it is important to validate the use of resident species as
sentinels to characterize the impact of oil spills on the rocky shores and the improvement during the res-
toration process. Recently the advantages of using the shanny Lipophrys pholis in pollution monitoring
within the northwestern Atlantic coast has been pointed out. Therefore, with the aim of further validating
the use of L. pholis in pollution monitoring associated with petrogenic hydrocarbon contamination, a
multi-biomarker approach study was carried out 1 week after a moderate oil spill from the waste treat-
ment plant (WTP) of the major Portuguese refinery in the north of Portugal (Petrogal). Fish collected at
2 km from the accident displayed a significant induction of ethoxyresorufin-O-deethylase activity (EROD)
and fluorescent aromatic compounds (FACs) in bile (up to a 5-fold induction) in comparison with the pre-
spill scenario, and a 15% induction in erythrocytic nuclear abnormalities (ENA), a biomarker of genotox-
icity. In contrast, no significant differences were recorded in the reference site. In order to better charac-
terize the time-course accumulation of FACs in bile after a PAH insult, laboratory exposure of L. pholis to
benzo[a]pyrene (B[a]P) was performed. A clear dose–response accumulation of B[a]P metabolites was
observed that closely reflected nominal exposure concentrations already after 3 d. Overall, the findings
of the present study highlight the potential of L. pholis in pollution monitoring dealing not only with
chronic contamination, but also with oil spill accidents of a moderate scale. Taking into consideration that
EROD and FACs determinations in L. pholis are cost effective, rapid and easy to use, they offer a great
potential to be incorporated into risk assessment of PAHs in the scope of national monitoring programs
and the European Water Policy legislation.
Ó2010 Elsevier Ltd. All rights reserved.
1. Introduction
The presence of petrogenic hydrocarbons in marine ecosystems
is a ubiquitous phenomenon due to its generalized use (Hannam
et al., 2010). In addition to the chronic hydrocarbon contamination
of many aquatic ecosystems, mostly in the vicinity of urban and
industrialized areas due to point and non-point sources, oil spills
are another important source of contamination (Viñas et al.,
2009). Long lasting effects in marine organisms have been reported
after oil spills, which will depend on several factors such as the
habitat sensitivity, the amount and nature of the oil spilled, the
hydrographic and geomorphologic characteristics of the affected
areas. Intertidal rocky shores are usually among the most impacted
ecosystems, since the majority of the marine organisms inhabiting
these areas are sessile or show a reduced mobility, thus making
them a prime target of oil aggregates or diffuse pollution. Since
oil spills are likely to affect different phyla and distinct trophic lev-
els within the intertidal ecosystem, it is important to use and val-
idate species from different groups as sentinels in order to evaluate
the impact on rocky shores and follow up their improvement/ame-
lioration during the restoration process. Furthermore, it is of major
importance from a legal point of view, the development of ade-
quate monitoring tools, which can be used to demonstrate the eco-
logical loss in the process of claims and compensations after spills.
In the past, the use of fish as sentinel organisms in monitoring pro-
grams associated with hydrocarbon contamination has been
shown to be an adequate methodology (Huggett et al., 2006; Lima
et al., 2008). In fact, the utility of the shanny Lipophrys pholis in
0045-6535/$ - see front matter Ó2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2010.07.065
⇑
Corresponding author. Tel.: +351 22 340 1812; fax: +351 22 340 1838.
E-mail address: santos@ciimar.up.pt (M.M. Santos).
1
These authors contributed equally to the study.
Chemosphere 81 (2010) 685–691
Contents lists available at ScienceDirect
Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
Author's personal copy
pollution monitoring within the northwestern Atlantic coast rocky
shores has been recently evidenced (Lima et al., 2008; Solé et al.,
2008a; Ferreira et al., 2009). The major advantage of this species,
in relation to other intertidal rocky shore fish species, lies on the
fact that they are abundant and easy to catch, ubiquitous, its ecol-
ogy and behavior has been intensively studied, (Faria et al., 1996;
Monteiro et al., 2005), and it has a restricted home range. Hence,
this species is representative of local environmental conditions
and pollutants exposure (Faria et al., 1996; Monteiro et al.,
2005). In a previous study (Lima et al., 2008) with adult L. pholis,
a clear correlation between the distance to polycyclic aromatic
hydrocarbons (PAHs) sources and the response of several biomark-
ers such as ethoxyresorufin-O-deethylase activity (EROD) and bili-
ary fluorescent aromatic compounds (FACs) was found. Hence, this
data supports the use of L. pholis in monitoring PAH contamination.
In February 2007, a moderate scale spill from the waste treat-
ment plant (WTP) of the major Portuguese refinery, located in
the north of Portugal (Petrogal), occurred. While no information
on the amount of hydrocarbons spilled to the vicinity coast was
available, it was possible to detect traces of spilled oil in the sand
in approximately 3–5 km of coastline nearby. Therefore, with the
aim of further validating the use of L. pholis in pollution monitoring
regarding PAH contamination, fish were collected 1 week after the
accident at approximately 2 km north of the refinery and also in a
control reference site. In order to get more information into the
time-course responses of selected biomarkers after a PAHs insult,
controlled laboratory experiments were undertaken with L. pholis,
and the findings compared with the field observations.
2. Material and methods
2.1. Field studies
2.1.1. Study area
Animals were collected at the site of Cabo do Mundo, located at
approximately 2 km north from the Petrogal WTP, where the spill
took place. This site was selected because we had former baseline
data on biomarker responses in L. pholis, and PAHs levels in mus-
sels, for over 1 year before the accident. Hence, direct comparison
with the pre-spill situation during the winter period could be
made. This is an important aspect to consider since some biomark-
ers, such as ethoxyresorufin-O-deethylase activity (EROD), are sea-
son dependent (Santos et al., unpublished data). In parallel, a
control location (Vila Praia de Âncora) where baseline data was
also available was selected for comparison purposes. A global posi-
tion system (GPS) was used to determine the coordinates of the
sampling sites, which are briefly described below:
Cabo do Mundo (C. Mundo), N 41, 22401; W 008, 71667: this
site is located 2 km north of an important oil refinery and 4.6 km
north of Leixões harbour, the biggest commercial harbour in the
North of Portugal. It is also in the vicinity of a small water house
(Joane), which has been reported to be contaminated both with
industrial and urban effluents (Lima et al., 2008).
Vila Praia de Âncora (V. P. Ânc.), N 41. 79726; W 008. 87306: lo-
cated 3 km south of a small urbanized area under the same name.
No human settlement exists near this sampling site.
2.1.2. Sampling and tissue handling
In February 2007, 1 week after the accident, specimens of L.
pholis were collected with hand-nets in rocky pools that emerged
during ebb tides. Approximately 15 individuals were sampled at
each site. Typically, juveniles of this species suffer a shift in their
pattern of microhabitat occupation and feeding behavior when
they are about 7 cm, and they migrate to bigger rocky pools, lo-
cated in a lower part of the intertidal zone. By that time, the onset
of reproduction takes place and animals of about 8 cm start to
present mature ovaries and testis (Monteiro et al., 2005). Thus, in
order to avoid using animals at different maturation stages, only
adults were selected for the present study (between 8 and
15 cm). After collection, animals were transported alive to the lab-
oratory in a refrigerated and aerated container. This approach was
validated in previous studies with L. pholis (Lima et al., 2008).
Once in the laboratory, fish were anaesthetized in saline water
and ice and immediately killed by decapitation, body length and
weight were determined. After dissection, liver was weighted
and immediately frozen in liquid nitrogen and stored at 80 °C.
Enzymatic assay was performed no later than 2 months after col-
lection. The gallbladder was removed, frozen in liquid nitrogen
and stored at 80 °C until analyses. Gonads were weighted, sexed
and the maturation condition was determined and the presence of
food in the stomach annotated. Mussels from the same locations
were also collected in February 2007 and used to determine tissue
levels of PAHs.
2.1.3. Biochemical measurement
Each liver was homogenized independently in a 1:3
(weight:volume) ratio in ice-cold buffer (50 mM Tris–HCl, pH 7.4,
containing 0.15 M KCl). Microsomes were prepared in 50 mM
Tris–HCl buffer containing 1 mM NaEDTA, pH 7.4, 1 mM dithio-
threitol, 20% (v/v) glycerol, and were obtained by centrifugation
of the 9000gsupernatant at 36 000gfor 90 min in a SIGMA 3K30
centrifuge. The obtained pellet was resuspended, washed in the
above mentioned buffer, and spun down at 36 000gfor 120 min
(Fent and Bucheli, 1994). Microsomes resuspended in EDTA free
buffer were stored at 80°C until use. CYP1A1-dependent EROD
activity was determined essentially as described in (Burke and
Mayer, 1974), with some modifications. A detailed description
can be found in Lima et al. (2008). Fluorescence was determined
using a BIOTEK SFM25 fluorimeter at excitation/emission wave-
lengths of 530 and 585 nm. Microsomal proteins were measured
by the method of Lowry et al. (1951), using bovine serum albumin
as standard. Liver EROD activity is reported in pmol min
1
mg
protein
1
.
2.1.4. Fluorescent aromatic compounds (FACs) analysis
Fluorescent aromatic compounds (FACs) in the bile were deter-
mined by Fixed Wavelength Fluorescence (FF). Five microliters of
bile were diluted in 5 mL of ethanol 48% and were centrifuged
for 5 min at 1800g,4°C. The supernatant was then used for FF
determination at the excitation/emission wavelength pairs 260/
380, 290/335, 341/383 and 380/430 nm, denoted FF260/380,
FF290/335, FF341/383 and FF380/430, respectively. Phenanthrene
type metabolites are detected at FF260/380. At FF290/335, mainly
naphthalene type of metabolites, typically associated with petro-
leum products are detected, and benzo[a]pyrene type of metabo-
lites are more efficiently detected at FF380/430. At FF341/383,
mainly pyrene-derived metabolites are detected (Lima et al.,
2008). Measurements were performed on a BIOTEK SFM25 fluo-
rimeter. To allow samples comparisons, a calibration curve was
made for each metabolite, using the following standards: 1-
hydroxypyrene (10, 5, 2.5, 0.625 and 0.156
l
gL
1
), Phenanthrene
(400, 100, 25, and 2.5
l
gL
1
), naphthalene (1000, 750, 375 and
75
l
gL
1
) and B(a)P (5, 1, 0.3 and 0.1
l
gL
1
)(Lima et al., 2008).
Biliverdin content was also determined to normalize FACs concen-
tration, but since no differences between normalized and non-nor-
malized data was seen, only non-normalized data is shown.
2.1.5. Erythrocytic nuclear abnormalities
Genotoxic damage was evaluated using the erythrocytic nuclear
abnormalities (ENA) assay. Briefly, the ENA test was performed
according to Carrasco et al. (1990) and Pacheco and Santos
686 M.M. Santos et al. / Chemosphere 81 (2010) 685–691
Author's personal copy
(1996) with slight modifications (Micael et al., 2007). Approxi-
mately 5
l
L of blood per specimen was collected, smeared in clean
slides and allowed to air-dry. After fixation in absolute methanol
for 10 min, slides were once more allowed to air-dry and were fur-
ther stained with 5% Giemsa for 45 min. Four thousand erythro-
cytes per fish were scored for the presence of ENA under a
1000magnification lens. Slides were coded and scored blindly
by the same observer, with two replicate slides per fish. ENA were
classified into one of the following categories: (1) micronuclei,
small (<1/3 of the main nucleus) non-refractive, circular or ovoid
chromatin bodies, showing the same pattern as the main nucleus
(AI-Sabti and Metcalfe, 1995); (2) cells with two nuclei were con-
sidered as binuclei; (3) nuclei with two lobes were classified as
segmented nuclei; (4) nuclei with a central and unilateral constric-
tion were classified as kidney shaped nuclei. The final results were
expressed as the sum for all individual lesions per 1000
erythrocytes.
2.1.6. Polycyclic aromatic hydrocarbons determination in mussels
Approximately 100 mussels of the species Mytilus galloprovin-
cialis (2–3 cm size) per site were homogenized, and the pool used
for PAHs determinations. In a former study (Lima et al., 2008)we
determined that a pool of 100 mussels was representative of PAHs
tissue accumulation in the field. Mussel tissue was Soxhlet ex-
tracted with acetone/hexane (1:1) for 24 h and after addition of
pre-deuterated surrogate PAH standards from Supelco. Isolation
and purification clean-up was completed by silica gel/alumina col-
umns (1:1). Extracted PAH were separated with dichlorometane/
hexane (9:1 and 4:1) and measured by gas chromatography mass
spectrometry (GC–MS), using a Trace GC Ultra connected to a
DSQ (Thermo) in the selected ion mode according to the method-
ology described in detail in Lima et al. (2008). Quantifications of
16 PAHs (acenaphethylene, fluorene, phenanthrene, anthracene,
fluoranthene, pyrene, chrysene, benz[a]anthracene, benzo[b]fluo-
ranthene, benzo[k]fluoranthene, benzo[a]pyrene, benzo[e]pyrene,
perylene, indeno[1,2,3-cd] pyrene, dibenz[a,h] anthracene and
benzo[g,h,i] perylene) were done using a 9-point calibration curve
for each compound. Per sampling site, a single determination was
performed. Quality control was done analyzing reference material
NIST, SRM 2977 and blanks. Recoveries, relatively to certified val-
ues, ranged from 75% to 125% for all PAHs analyzed. Detection lim-
its ranged from 0.2 to 1.0 ngg
1
wet weight.
2.2. Laboratory exposures
In order to get more insights into the time-course responses of
selected biomarkers after PAHs insult, controlled laboratory expo-
sures with the model PAH benzo[a]pyrene (B[a]P) were performed
using adult L. pholis. Fish for the exposure studies could not be col-
lected in Vila Praia de Âncora (the control location in the field
study), due to the reduced extent of this rocky shore area. Alterna-
tively, we selected Mindelo/Vila Chã which had also been reported
to display low levels of contaminants. In two consecutive days, 120
adult animals were collected and let to acclimatize for 1 month in
two 200 L aquaria filled with artificial sea water, provided with
aeration and biological filtration. During the acclimatization peri-
od, animals were fed once a day with frozen hake. After this period,
six animals per replicate were transferred to 30 L aquaria (four rep-
licates per treatment) filled with artificial sea water (salinity 35‰),
and maintained at 15 °C ± 1 in a room under natural photoperiod.
‘‘Sera premium” salt and carbon activated filtrated tap water were
used to prepared artificial sea water (pH = 8.3, conductiv-
ity = 48 ms cm
1
, redox potential = 76 mv). Water was changed
daily (80%), and fish were fed hake three times during the course
of the experiment (8 d). Fish were not fed the day before sacrific-
ing. Because of the low solubility of B[a]P in water, a carrier had
to be used (acetone) at a concentration of 0.002%. Five treatments
were established, with four replicates per treatment: control, sol-
vent control, B[a]P at 0.1
l
gL
1
;B[a]P at 1
l
gL
1
;B[a]P at
10
l
gL
1
. Actual B[a]P concentration of the stock solution used
to prepare working solutions was confirmed. Animals from two
replicates per treatment were sacrificed at day 3 and the additional
two replicates were sacrificed at the end of exposure (day 8). No
mortality was recorded during the course of the exposure and fish
fed regularly during the exposure period.
At the end of 3 and 8 d exposure, a similar methodology to that
described for the field study was used. Liver was collected for
EROD activity measurement and B[a]P determination, the gallblad-
der for FACs and blood for ENA determinations. Additionally, mus-
cle was also collected in order to investigate any effects due to
oxidative stress damage, measured as lipid peroxidation (LP) levels
and potential neurotoxicity as cholinesterases (ChEs) activity (Solé
et al., 2008b). Unfortunately, due to a failure in the 80 °C freezer,
the material collected for EROD determination was damaged and
the assay could not be performed in laboratory exposed fish.
A portion of the muscle of about 0.4 g was homogenised in a
50 mM buffer phosphate pH 7.4 in a 1:5 (w:v) ratio using a poly-
tron
Ò
blender. The homogenate was centrifuged at
10 000g20 min and the supernatant used for biochemical deter-
minations. Assay conditions were 1 mM for each substrate: acetyl-
thiocholine iodide (ASCh), butyrylthiocholine iodide (BSCh),
propionylthiocholine iodide (PrSCh). In each microplate well,
150
l
L of 5,5
0
-dithio-bis-2-nitrobenzoat (DTNB; 270
l
M) were
mixed with 25
l
L of sample (diluted or undiluted) and after
2 min pre-incubation, the reaction was started adding 50
l
Lof
the substrate. A 5-fold dilution of the sample was necessary for
AChE determination but not for any of the other esterases. Deter-
mination of esterase activities was done using the principle of Ell-
man et al. (1961) with appropriate modifications for microplate.
Reading was performed in triplicate at 405 nm in a microplate
reader (TECAN Infinite200) during 5 min at 25 °C. Activity was ex-
pressed in nmol min
1
mg prot
1
.
LP was determined in muscle using 200
l
L of the same superna-
tant homogenate, as for esterases, and mixed with 650
l
Lof1-
methyl-2-phenylindole in acetonitrile:methanol (1:3) and 150
l
L
of 37% HCl. This mixture was incubated at 65 °C for 60 min, the
reaction was stopped in ice and further centrifuged at
13 000 rpm 10 min to precipitate proteins, following a modifica-
tion of Shaw et al. (2004) protocol. Absorbance was read at 586 nm
versus a standard solution of 1,1,3,3-tetramethoxypropane treated
similarly. LP content was expressed as nmol MDA (malondialde-
hyde) g
1
wet weight.
2.3. Statistical analyses
In order to evaluate differences among groups, a one-way anal-
ysis of variance (ANOVA) was performed, followed by the multiple
comparison test fisher LSD. When data did not fit ANOVA assump-
tions of normality and homogeneity of variance, a non-parametric
ANOVA Kruskal Wallis followed by multiple comparisons of means
ranks was applied. The Man-Witney U test was used for a direct
comparison between two groups. All tests were performed using
the software Statistica 7.0.
3. Results
3.1. Field studies
Fig. 1 reports on EROD activity in L. pholis from the impacted
site (Cabo do Mundo) and the reference site (Vila Praia de Âncora),
during the pre-spill period (January 2006) and 1 week after the
M.M. Santos et al. / Chemosphere 81 (2010) 685–691 687
Author's personal copy
spill (February 2007). A 5-fold induction in EROD activity was ob-
served in Cabo do Mundo after the spill, if compared with the pre-
spill period (p< 0.001, Kruskal Wallis non-parametric ANOVA fol-
lowed by multiple comparisons of means ranks). No significant dif-
ferences were recorded in the reference site at both sampling
periods.
Similarly, the comparison of FACs concentrations in L. pholis bile
between the pre-spill situation and 1 week after the spill shows an
approximately 2-fold increase for naphthalene and B[a]P type
metabolites and a 3-fold increase for phenanthrene and 1HO-pyr-
ene type metabolites in Cabo Mundo (Fig. 2). This increase reached
statistical significance (p< 0.05, Kruskal Wallis non-parametric
ANOVA followed by multiple comparisons of means ranks) for
phenanthrene, naphthalene and 1HO-pyrene type metabolites.
Fig. 1. Liver ethoxyresorufin-O-deethylase (EROD) activity in L. pholis collected in
January 2006 and February 2007 in Cabo do Mundo (spilled area) and Vila Praia de
Âncora (reference site). Values are mean ± SE (n= 14). Significant differences
(p< 0.05) are indicated by different letters.
Fig. 2. Levels of fluorescent aromatic compounds (FACs) in L. pholis bile in Cabo do Mundo and Vila Praia de Âncora in January 2006 and February 2007 (A–D). Values are
mean ± SE (n= 14). Significant differences (p< 0.05) are indicated by different letters.
Fig. 3. Sum of PAHs (ng g
1
wet weight) in mussel tissues in Cabo do Mundo and
Vila Praia de Âncora in January 2006 and February 2007.
688 M.M. Santos et al. / Chemosphere 81 (2010) 685–691
Author's personal copy
On the contrary, the reference site showed similar levels for all
FACs tested between 2006 and 2007. These results are in agree-
ment with an increase in the total PAHs detected in mussels col-
lected at Cabo do Mundo in 2007, if compared with 2006 (an
approximately 3-fold increase), whereas the same range of PAHs
levels were recorded in the reference site, Vila Praia de Âncora, be-
tween 2006 and 2007 (Fig. 3). In 2006, the levels of FACs in fish bile
(i.e., phenanthrene and 1HO-pyrene type metabolites) and total
PAHs in mussel tissues were already elevated in Cabo do Mundo
if compared with the control site.
The percentage of ENA in L. pholis from Cabo do Mundo in Jan-
uary 2006 (49‰) were already higher (although it did not reach
significance) than the levels recorded in the control site Vila Praia
de Âncora (30‰)(Fig. 4). After the spill, a further 15% increase in
the percentage of ENA was observed in Cabo do Mundo although
differences were not significant in comparison with the 2006 sam-
pling period due to high sample variability (p> 0.05, Kruskal Wallis
non-parametric ANOVA followed by multiple comparison of means
ranks). In 2007, a significant elevation of ENA was observed in Cabo
do Mundo in comparison with the control site.
3.2. Laboratory exposure to B[a]P
3.2.1. B[a]P equivalents accumulation
Fig. 5 displays the accumulation of B[a]P equivalents in L. pholis
bile 3 and 8 d after laboratory exposure to B[a]P. Fish exposed to
the lowest concentration (0.1
l
gL
1
) showed already a 10-fold
B[a]P increase in comparison to controls after 3 d exposure. A 10-
fold increase in B[a]P levels in bile was also observed between
the 0.1 and the 1
l
gL
1
exposed groups, which reflects the differ-
ence in the nominal concentrations between both treatment
groups. An approximately 7-fold increase in B[a]P equivalents
was observed between the 1 and the 10
l
gL
1
exposed groups.
Overall, a correlation coefficient of r= 0.99, p< 0.01, was observed
between nominal concentrations of the parent compounds and
B[a]P metabolites in fish bile. These differences were significant
for the two highest B[a]P exposure levels in comparison with con-
trol (p< 0.05, Kruskal Wallis non-parametric ANOVA followed by
multiple comparison of means ranks). However, the non-paramet-
ric ANOVA could not detect significant differences between control
treatments and B[a]P at 0.1
l
gL
1
, despite a 10-fold induction in
the B[a]P exposed animals. The use of the Man-Witney U test for
a direct comparison between solvent control and B[a]P at
0.1
l
gL
1
clearly indicate a significant accumulation (p< 0.001)
at the lowest B[a]P exposure if compared with controls (Fig. 5B).
No significant differences between 3 and 8 d were observed in
any of the treatments, which indicate that the FACs accumulation
in bile is quick, and that after 3 d the equilibrium had been
reached. Despite a clear accumulation of B[a]P metabolites in fish
bile, the determination of B[a]P levels in L. pholis liver showed a
lack of bioaccumulation of the parent compound (data not shown)
which suggests its rapid elimination in L. pholis.
3.2.2. Erythrocytic nuclear abnormalities
Fig. 6 displays the levels of ENA at the end of the exposure per-
iod (8 d) in fish from the controls and B[a]P treatment at the high-
est concentration tested (10
l
gL
1
). Despite a slight trend towards
an increase in ENA values between control and the other condi-
tions, differences did not reach significance (p> 0.05, Kruskal Wal-
lis non-parametric ANOVA followed by multiple comparisons of
means ranks).
Fig. 4. Average erythrocytic nuclear abnormalities frequency (ENA 1000 erythro-
cytes
1
)inL. pholis erythrocytes in Cabo do Mundo and Vila Praia de Âncora in
January 2006 and February 2007. Values are mean ± SE (n= 9).
Fig. 5. Concentration of B[a]P equivalents in L. pholis bile after 3 and 8 d of B[a]P
exposure under laboratory conditions (A – all treatment groups; B – solvent control
versus B[a]P at 0.1
l
gL
1
). Values are mean ± SE (n= 12).
Fig. 6. Average erythrocytic nuclear abnormalities frequency (ENA 1000 erythro-
cytes
1
)inL. pholis erythrocytes in solvent control and B[a]P at 10
l
gL
1
at the end
of exposure. Values are mean ± SE (n= 12).
M.M. Santos et al. / Chemosphere 81 (2010) 685–691 689
Author's personal copy
3.2.3. Cholinesterase activities and lipid peroxidation levels
Table 1 displays the effects of B[a]P exposure in L. pholis muscle
cholinesterases activity and lipid peroxidation levels at both sam-
pling periods. No significant differences were observed due to
treatment and/or length of exposure (p> 0.05, one-way ANOVA).
4. Discussion
Oil spills have an important impact from an ecological and eco-
nomical point of view (Goodlad, 1996; Jewett et al., 2002; Garza-
Gil et al., 2006a,b). It is therefore of great importance to establish
well validated sentinel species that can provide information on
the toxicological consequences over the fauna from the impacted
area soon after the incident and during the recovery process. In
the present study, despite the moderate scale spill of the Petrogal
refinery WTP accident, if compared to other maritime spills re-
ported in the literature, the use of selected biomarkers responses
in L. pholis, 1 week after the accident, clearly identified an exposure
if contrasted with the pre-spill period. A significant 5-fold EROD
induction observed in Cabo do Mundo between both sampling
periods, was contrasted with no significant changes in the refer-
ence site. The degree of EROD induction recorded in the present
study at the vicinity of the WTP and after the spill, is comparable
to the observed differences in L. pholis EROD activity determined
in an earlier study along the Portuguese coast, between reference
sites and chronically polluted areas (Lima et al., 2008). Similarly,
in the Galician coast of Spain, up to a 4-fold EROD induction was
observed in the flatfish Callionymus lyra collected in the continental
shelf in areas severely affected by the Prestige oil spill, in compar-
ison to reference sites (Martinez-Gomez et al., 2006). Supporting
EROD data, FAC levels in L. pholis bile showed a similar trend with
an increase in bile from fish collected at the impacted area,
whereas in the reference site bile levels remained unaffected. The
absolute levels of FACs in L. pholis collected in Cabo do Mundo in
2007 after the spill were elevated by approximately 2-fold if com-
pared with the most PAHs contaminated locations along the Portu-
guese coast reported in a previous field study (Lima et al., 2008).
Likewise, the observed increase in PAH accumulation in mussel tis-
sues of approximately 3-fold in Cabo do Mundo, after the spill, cor-
relates well with the induction of biomarkers of exposure in L.
pholis. The total PAH levels determined in Cabo do Mundo mussels
in 2007 are well below those recorded in intertidal mussels from
areas severely impacted by the prestige oil spill (Soriano et al.,
2006), thus confirming that the amount of oil reaching the coast
was not of the same order of magnitude. Nevertheless, total PAHs
levels recorded in mussel tissues 1 week after the accident were
slightly above those determined in some ‘‘hot spots” for PAH con-
tamination such as the Galician Rias (Soriano et al., 2006). Collec-
tively, and from a local perspective, the data obtained in this
study indicates that the spill led to the contamination of several
kilometers of the coastline in the proximity of the oil refinery, with
a clear impact in a local representative species. Records from local
newspapers indicate that problems arising from the refinery WTP
are recurrent, and thus our data highlights the need for improving
their operational procedures.
Laboratory studies with B[a]P further supported the adequacy
of L. pholis as sentinel and the selected exposure biomarkers to fol-
low up petrogenic exposure. After 3-d, a clear dose–response accu-
mulation of B[a]P metabolites was already evident in bile. Since no
differences were observed between both sampling periods (3 and
8 d), we can conclude that for the range of B[a]P concentrations
used in the laboratory studies, B[a]P metabolites in bile quickly
reach an equilibrium. Even at the lowest exposure, a 10-fold in-
crease in B[a]P metabolites was observed, if compared to control
fish. Furthermore, at environmental relevant levels (i.e., B[a]P at
0.1–1
l
gL
1
) a 10-fold increase in bile B[a]P metabolites was ob-
served between the 0.1 and the 1
l
gL
1
treatments, thus reflecting
the nominal water concentrations. Hence, not only B[a]P metabo-
lites in L. pholis bile increased quickly after exposure, they also re-
flected very closely the actual B[a]P exposure concentrations.
Taking into account that L. pholis has a wide geographical distribu-
tion (from Mauritania to Norway including the Azores Islands and
into the Mediterranean), their strong homing behavior, their easi-
ness to catch, and the fact that FACs levels in bile are quickly, easily
determined and cost effective, it becomes clear that FACs levels in
L. pholis bile have a great potential to be included in monitoring
programs dealing with PAH contamination.
Several polycyclic aromatic hydrocarbons, in particular B[a]P,
have been shown to be genotoxic. In fact, a previous study using
L. pholis in environmental monitoring reported higher levels of
DNA adducts in specimens collected in a site affected by the Sea
Empress oil spill, in comparison with the reference sites (Lyons
et al., 1997; Harvey et al., 1999). Therefore, during the present oil
spill accident, genotoxicity occurrence in fish was evaluated using
the presence of ENA. This technique is one of the most well ac-
cepted approaches to evaluate exposure to genotoxic agents (Çavas
and Ergene-Gozukara, 2005; Micael et al., 2007). For most marine
species from relatively clean areas, the normal background ENA
levels are in the range of 3–20‰(Oliveira et al., 2007; Van Ngan
et al., 2007), which is similar to the observed levels in L. pholis
erythrocytes along the Portuguese coast. The background level of
ENA in Cabo do Mundo in 2006 was already twice the recorded
in our reference site, thus indicating that in the pre-spill period fish
were already under chronic levels of genotoxic agents. After the
spill, although a further increase in ENA was observed in fish col-
lected in Cabo do Mundo, this increase did not reach significance
in comparison with the pre-sill period. This could be due to the fact
that ENA was already induced in these animals, and in order to
have a significant induction above the observed levels, exposure
to higher concentrations of genotoxic agents are required. Alterna-
tively, it could be associated with the mechanisms of ENA forma-
tion in erythrocytes. ENA are formed in cells undergoing mitosis.
That is, when fish from Cabo do Mundo were sampled 1 week after
the accident, a fair part of the erythrocytes could have already been
formed during the pre-spill period. The laboratory exposures with
B[a]P seems to support this second hypothesis. Although B[a]P has
been described as a model carcinogen (Tsuji and Walle, 2007),
exposure of L. pholis for 8 d did not increase ENA above the back-
ground levels of control animals. In fact, the absolute ENA levels
observed for the highest B[a]P concentration was approximately
half of that recorded in field animals from the impacted area in
2007. Nevertheless, laboratory data from other fish species indi-
cates that ENA induction after a genotoxic insult usually takes
place between 3 and 7 d (Çavas and Ergene-Gozukara, 2005).
Hence, future studies should investigate in detail the kinetics of
Table 1
Acetilcholinesterase (AChE), propionylcholinesterase (PrChE), butyrylcholinesterase
(BChE) activities (in nmol min
1
mg prot
1
) and lipid peroxidation (LP) levels (in nmol
MDA g
1
w.w) in muscle of L. pholis after 3 and 8 d of B[a]P exposure under laboratory
conditions. Values are mean ± SE (n= 8).
Day AChE BChE PrChE LP
Control 3 41.91 ± 2.48 2.34 ± 0.23 19.76 ± 1.44 86.00 ± 8.04
8 44.62 ± 3.77 2.52 ± 0.30 23.24 ± 3.08 55.89 ± 4.16
Sol.
control
3 36.94 ± 3.30 2.11 ± 0.29 16.96 ± 1.59 83.19 ± 10.74
8 45.04 ± 2.48 2.69 ± 0.21 23.49 ± 2.11 54.65 ± 6.83
B[a]P 0.1 3 41.35 ± 3.30 2.17 ± 0.25 19.15 ± 1.59 77.66 ± 8.02
8 38.39 ± 1.93 2.63 ± 0.29 21.35 ± 3.19 61.14 ± 9.44
B[a]P 1 3 38.95 ± 1.70 2.20 ± 0.22 17.82 ± 1.33 81.26 ± 3.71
8 49.63 ± 3.06 2.28 ± 0.26 23.53 ± 2.96 47.32 ± 6.13
B[a]P 10 3 43.25 ± 2.45 2.12 ± 0.23 18.43 ± 1.02 74.35 ± 6.89
8 42.57 ± 3.41 2.37 ± 0.18 21.44 ± 1.89 51.13 ± 6.21
690 M.M. Santos et al. / Chemosphere 81 (2010) 685–691
Author's personal copy
ENA formation in L. pholis in relation to exposure to model carcin-
ogens, in order to further validate the application of this effect bio-
marker in routine monitoring programs.
During the laboratory exposures, in addition to the determina-
tion of FACs and ENA, we also evaluated the effects of B[a]P in L.
pholis muscle cholinesterases activity and LP levels. While AChE
is clearly a neurotoxic marker, pseudocholinesterases (PrChE and
BuChE) physiological role is less clearly understood. The lack of en-
hanced LP levels in muscle tissue of exposed animals and of AChE
inhibition suggest that B[a]P toxic action is through other mecha-
nisms. In fact, ChE activities and LP levels are similar to those ob-
served in a field study using L. pholis as sentinel (Solé et al.,
2008a). Similarly, lack of a neurotoxic response to B[a]P (measured
as brain AChE) was seen in Sparus aurata (Cunha et al., 2007), or in
LP and AChE in juveniles of Solea senegalensis exposed to Prestige
fuel oil for a short period (Solé et al., 2008b).
The European commission aims at reaching a good water qual-
ity status of all European water bodies by 2015. In order to achieve
this, the Water Framework Directive and the European Marine
Strategy are key legal instruments that should be adopted by all
member states. Both legal instruments set the need to consider
both chemical and ecological status. To date, the use of biomarker
responses in key sentinel species as early warning signals in the
context of these legal instruments has played a limited role. Never-
theless, the need for instruments which can anticipate an impact at
higher levels of biological organization (i.e., ecological level) has
been identified (Hagger et al., 2008; Sanchez and Porcher, 2009).
Hence, the validation of biological responses in key sentinel species
within European waters is an important step in the implementa-
tions of these directives. The findings of the present study highlight
the potential of L. pholis in pollution monitoring dealing not only
with chronic PAH contamination, but also with oil spill accidents
even those of a moderate scale. Taking in consideration that EROD
and FACs determination in L. pholis are cost effective, rapid and
easy to use, they offer a great potential and deserve to be incorpo-
rated into risk assessment of PAHs within the European Water Pol-
icy legislation.
Acknowledgements
The present study was funded by and Interreg III B ‘‘Atlantic
Area” project EROCIPS and a GRICES-CSIC agreement (Ref.
2005PT0020). We would like to acknowledge the comments of
two anonymous reviewers that help us improve the manuscript.
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