PAPER www.rsc.org/jem | Journal of Environmental Monitoring
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A multibiomarker approach in Mytilus galloprovincialis to assessenvironmental quality
Alexandra Cravo, Belisandra Lopes, Angela Serafim, Rui Company, Luısa Barreira, Tania Gomesand Maria Joao Bebianno*
Received 19th May 2009, Accepted 24th July 2009
First published as an Advance Article on the web 7th August 2009
DOI: 10.1039/b909846a
A multibiomarker approach was carried out for the first time in the South Portuguese Coast using
Mytilus galloprovincialis, to assess environmental quality, establish if there are adverse biological
responses associated to different sources of anthropogenic contamination and to determine spatial and
seasonal trends. For this purpose the battery of biomarkers selected was: superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidases (GPx total and Se dependent), Cytochrome P450 component
system, Glutathione-S-transferase (GST) and acetylcholinesterase (AChE), metallothionein (MT) and
lead-d-aminolevulinic acid dehydratase (ALAD), lipid peroxidation (LPO) and Condition Index (CI)
along with the determination of PAHs and metals (Cd, Cu, Ni, Pb and Zn). Results show that despite
the levels of both organic and metallic contaminants in these eight spots in the South Coast of Portugal
not being particularly high compared with other contaminated/polluted sites worldwide, the selected
battery of biomarkers responded efficiently to the environmental changes and allowed an
environmental assessment between seasons and sites. Different spatial and seasonal responses were
evident along the South Coast of Portugal, meaning that the contamination is not homogeneous. This
does not only reflect different competition, origin and intensity of contamination, but also different
environmental conditions (e.g. temperature, salinity). Along the South Portuguese Coast site 8 was the
most contaminated, while site 2 was considered the least contaminated. Despite environmental factors
possibly causing difficulties in the general interpretation of biomarker data, those that better responded
to environmental contamination were CYP450, SOD-mit and T-GPx for theP
PAHs, MT (digestive
gland) for metals (especially Cu), ALAD for Pb and LPO for both organic and metallic contamination.
These biomarkers were also positively correlated with temperature in summer, revealing this as a more
stressful/critical season. In future environmental contamination assessments there is no need to analyse
the components b5, P418, NADH and NADPH of phase I MFO system, and MT in the gills, since their
responses are not evident.
Introduction
Coastal areas including estuaries are usually highly productive.
However, over the years these ecosystems became increasingly
affected by anthropogenic activities, mainly due to urban
development, industrialization and tourism. In those areas,
complex mixtures of contaminants including metallic, organo-
CIMA - Faculty of Marine and Environmental Sciences, University ofAlgarve, Campus de Gambelas, 8005-130 Faro, Portugal. E-mail:[email protected]
Environmental impact
The present paper describes a multibiomarker approach used for the
galloprovincialis to assess the impact of contaminants in this Europe
commonly in ecotoxicology studies, this work integrates a battery
organic) and environmental physico-chemical parameters. This rep
ronment subjected to the impact of mixtures of contaminants and p
that allows pollution effects to be distinguished from those induced
This journal is ª The Royal Society of Chemistry 2009
metallic and persistent organic pollutants (POP; e.g. polycyclic
aromatic hydrocarbons (PAHs), polychlorinated byphenyls
(PCBs), fertilizers and pesticides) from different origins exist.
Due to the ecological relevance of such areas new tools to eval-
uate the environmental quality became necessary.1 Analysing all
the contaminants/pollutants present in the water is virtually
impossible and does not directly reflect the effects upon the biota.
Therefore, measuring the biological effects of pollutants as early
warning signals became increasingly important to assess the
‘‘health status’’ of the environment.2 Bivalve mussels in particular
Mytilus spp. have been widely used in environmental quality
first time in the south coast of Portugal with the mussel Mytilus
an coastal area. Contrary to the single biomarker approach used
of biomarkers to assess the effect of pollutants (metallic and
resents a new tool to evaluate the quality of the coastal envi-
rovides valuable information for environmental risk assessment
by natural factors using the mussels as biological indicators.
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Fig. 1 Sampling sites along the South coast of Portugal.
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assessment.3,4 These studies enable the evaluation of integrated
biological effects in the environment and usually include the
determination of one or several biomarkers in target species. The
advantages of using biomarkers in the assessment of environ-
mental contamination are also highlighted by van der Oost et al.5
The use of a battery of different biomarkers (a multibiomarker
approach) is essential, particularly in environments where
complex mixtures of contaminants are present, for the assess-
ment of responses that reflect the environmental quality and for
the identification of the sources of contaminants in the envi-
ronment.6,7 Nevertheless, the use of biomarkers does not
completely replace the more common approach of chemical
analysis, but their integration provides a qualitative and/or semi-
quantitative approach to determine the effects and the possible
nature of contaminants. In addition it provides a unique
contribution to determine the synergistic effect of a mixture of
pollutants, even in low amounts.8
Biological responses are also influenced by natural environ-
mental factors. Studies using biomarkers pointed to the need to
incorporate the effects of abiotic (temperature, salinity, diet, etc.)
and biotic factors (reproduction cycle, growth, age, sex, etc.)9–11
to a correct interpretation of biomarker responses.
In the South Coast of Portugal, a highly touristic region, the
population increases markedly during the summer as well as the
volume of sewage discharges and maritime activities. Conse-
quently at some places which are already identified as hotspots of
contamination, complex mixtures exist and are magnified during
this season. In this region, the contaminants known to be present
include metals,12–14 PAHs, PCBs15,16 and organotin compounds
(mainly TBT).17–19 Nevertheless in this coastal area the biological
effects of pollutants in mussels are scarce and only include MT
and GST changes in M. galloprovincialis.12,20 Therefore, in order
to assess if and how mussels M. galloprovincialis are affected by
these contaminants, a multibiomarker approach was carried out
to establish if there are adverse biological responses associated
with different sources of anthropogenic contamination and to
determine spatial and seasonal trends. Moreover, it was also
aimed to discriminate between useful biomarkers and those less
helpful for environmental assessement.
The battery of biomarkers selected was: (i) oxidative stress
biomarkers (antioxidant enzymes – superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidases (GPx total and Se
dependent), (ii) biomarkers of exposure to organic compounds,
including pesticides – Cytochrome P450 component system
including the ‘418-peak’, Cytochrome b5, NAD(P)H cytochrome
c reductase), Glutathione-S-transferase (GST) and acetylcho-
linesterase (AChE); (iii) biomarkers of metal exposure – metal-
lothionein (MT) and lead – d-aminolevulinic acid dehydratase
(ALAD) and (iv) biomarkers of effect – lipid peroxidation
(LPO).
Material and methods
Sampling sites and collection
Eight sites were selected at the South Coast of Portugal (Fig. 1),
and mussels Mytilus galloprovincialis collected at low tide, during
summer (2005) and winter (2006) namely at: 1. (37� 00,5880 N; 8�
55,7770 W), 2. (37� 06,4610 N; 8� 40,2810 W), 3. (37� 08,0870 N;
1674 | J. Environ. Monit., 2009, 11, 1673–1686
008� 32,0890 W), 4. (37� 04,4060 N; 008� 07,2950 W), 5. (37�
00,1660 N; 7� 54,9940 W), 6. (37� 01,3670 N; 7� 50,2120 W), 7. (37�
06,9950 N; 7� 37,7320 W) and 8. (37�10,6780 N0; 007� 24,5080 W).
Sites 1 and 6 are near fishing harbours, and sites 2 and 4 near
recreational marinas, while sites 3, 7 and 8 are close to the mouth
of the major rivers, whereas site 5 is close to a commercial
harbour.
From each sampling site about 100 mussels (5.2 � 0.8 cm shell
length) were collected for both biochemical (biomarker
measurements) and chemical (PAHs and metal) analyses.
Mussels were transported alive (at �4� C) to the laboratory. To
assess the physiological condition of native mussels, 10 animals
per site were randomly collected for condition index determina-
tion. Following dissection, gills (for acetylcholinesterase and
MT) and digestive gland of 40 mussels were immediately frozen
in liquid nitrogen and kept at –80 �C until analyses. The
remaining mussels were maintained at �20 �C for chemical
analyses. Environmental parameters (temperature and salinity)
were also measured at each site.
Condition index (CI)
The condition index (CI) was determined as a percentage of the
ratio between dry weight of the soft tissues and the dry weight of
the shell.
Biochemical analysis
Antioxidant enzymes. Antioxidant enzymatic activities were
determined in the digestive gland, the major site of organic
xenobiotic metabolism. Three pools (5 digestive glands each) of
M. galloprovincialis were used after homogenisation in 20 mM
Tris buffer, pH 7.6, containing 1 mM of EDTA, 0.5 M of sac-
charose, 0.15 M of KCl and 1 mM of DTT. The homogenates
were centrifuged at 500 g for 15 min at 4 �C to precipitate large
particles and recentrifuged at 12 000 g for 45 min at 4 �C to
precipitate the mitochondrial fraction. Gel filtration was used to
eliminate low molecular weight impurities. Hence, all cytosolic
fractions were chromatographed on a Sephadex G-25 column
(PD10, Pharmacia) to remove small weight proteins.
SOD activity (EC 1.15.1.1) was determined in the cytosolic
(SOD-cyt) and mitochondrial (SOD-mit) fractions by measuring
the reduction of cytochrome c by the xanthine oxidase/hypo-
xanthine system at 550 nm.21 One unit of SOD is defined as the
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amount of enzyme that inhibits the reduction of cytochrome c by
50%. SOD activity was expressed in U mg�1 total protein
concentrations. CAT activity (EC 1.11.1.6) was determined
according to Greenwald22 by the decrease in absorbance at
240 nm due to H2O2 consumption. CAT activity was expressed
as mmol min�1 mg�1 of total protein concentrations. GPx activ-
ities were measured following NADPH oxidation at 340 nm in
the presence of excess glutathione reductase, reduced glutathione
and corresponding peroxide.23 The Se-GPx (EC 1.11.1.9) and
Total GPx activities were measured by using H2O2 and cumene
hydroperoxide as substrates, respectively. GPx activities are
expressed as mmol min�1 mg�1 of total protein concentrations.
CYP450 and GST enzymes
Like for the antioxidant enzymes, for the analysis of CYP450,
‘‘418 peak’’, b5, NADPH, NADH and GST, 3 pools (5 digestive
glands each) of mussels were used. Cytosolic and microsomal
fractions were prepared at 4 �C by differential centrifugation as
described by Livingstone.24 Samples were homogenized in 1 : 3.5
(tissue weight : buffer volume) ratio, at 4 �C with 10 mM Tris–
HCl pH 7.6, containing 1 mM dithiothreitol, 0.15 M KCl and 0.5
M sucrose using an ultra-Turrax homogenizer. Following
centrifugations of 500 g for 15 min, 10 000 g for 30 min and
100 000 g for 90 min, the resulting microsomal pellet was
resuspended in 10 mM Tris–HCl, pH 7.6, containing 20% (w/v)
glycerol to give a protein concentration of approximately
10 mg mL�1. Biochemical measurements were carried out on
microsomal samples either immediately (reductase activities), or
after overnight storage at �80 �C (CYP450, putative denatured
CYP450, cytochrome b5). The cytosolic fraction (GST) was also
stored at �80 �C until analyses.
CYP450 and reductase activities were assayed as described in
Livingstone and Farrar.25 Cytochrome P450 concentration and
‘‘418 peak’’ was measured by the carbon monoxide difference
spectrum of sodium dithionite reduced samples using an extinc-
tion coefficient of 91 mM�1 cm�1 (450–490 nm)26 as follows: 1 mL
100 mM Tris–HCl pH 7.6, 900 mL water and 100 mL of the
mussels digestive gland microsomes were mixed and divided into
two semi-micro cells. CO was bubbled (with a flow of 70 bubbles
per minute) in the cell containing the sample and the baseline
recorded. A few grains of sodium dithionite were added to both
cells and the cytochrome P450 content was measured. CYP450
concentration was expressed in pmol mg�1 proteins. Total ‘‘418
peak’’ was calculated in arbitrary units as described in Living-
stone,24 i.e. peak height (lmax – 490 nm) � 1000 mg�1. Total
cytochrome b5 was similarly analysed by difference spectroscopy
using 50 mL of microsomal sample, 30 mM NADH and an
extinction coefficient of 185 mM �1 cm �1 (426–409 nm). For the
difference spectra, each sample was scanned from 3 to 4 times,
and mean values were calculated from replicate spectra.
NADPH-dependent cytochrome c and NADH-dependent cyto-
chrome b5 reductase activities were measured, by the increase in
absorbance at 550 nm (3 ¼ 19.6 mM�1 cm�1). Final assay
conditions in a final volume of 1 mL were: 50 mM Tris–HCl pH
7.6, 1 mM KCN, 0.30 mM NADPH (or NADH), and 0.60 mM
cytochrome c. Sample volumes were: 50 mL of microsomes for
NADPH and 10 mL for NADH dependent reductases.
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Microsomal protein concentration was determined by the
Lowry method using bovine serum albumin as standard.27
Protein yield of the microsomal fraction was measured and
expressed as protein content per gram of weight of fresh digestive
gland tissue.
Glutathione-S-transferase activity was measured spectropho-
tometrically in the cytosolic fraction of the 100 000 g centrifuge,
using 1-chloro 2,4 dinitrobenzene (CDNB) and reduced gluta-
thione (GSH) as co-substrate, according to the method of Habig
et al.28 Specific activity was expressed as nmol min�1 mg�1
protein, using a molar extinction coefficient of 49.6 mM�1 cm�1.
Acetylcolinesterase (AChE)
The activity of AChE was assayed in the gills of the mussels.
Measurements of AChE activity were performed using the col-
orometric method of Ellman.29 The absorbance at 405 nm was
recorded for samples and blanks. AChE activity was expressed in
nmol min�1 mg�1 protein using a molar extinction coefficient of
13.6 mM�1 cm�1.
Metallothionein and ALAD
Metallothionein concentrations were determined in the gills and
digestive gland of M. galloprovincialis. Three replicates of tissues
were weighed and homogenized in 3 volumes of 20 mM Tris-HCl
(pH 8.6) in an ice bath (4 �C). Subsamples of the total homog-
enate were used for wet/dry weight ratio determination and metal
analysis. A further aliquot of the homogenate (3 mL) was
centrifuged (30 000g for 1 h at 4 �C) to separate the soluble and
insoluble compounds. Two aliquots of the supernatant were
further collected to be used in LPO and protein determinations.
The supernatant was then heat treated at 80 �C and centrifuged
(30 000g for 1 h at 4 �C) to precipitate the denatured proteins.
Aliquots (50–250 mL) of the heat treated cytosol were used to
quantify MT concentrations by differential pulse polarography,
according to the method described by Bebianno and Langston.30
The standard addition method used for calibration of MT
concentrations was rabbit liver MT (MT-I from Sigma) (working
standard 10 mg L�1 in distilled water). MT concentrations in the
tissues of mussels were expressed as mg g�1 protein.
d-Aminolevulinic acid dehydratase activity (ALAD -
E.C.4.2.1.24) was determined according to the European stan-
dardized method for determination of d-ALAD activity in the
blood.31 The whole soft tissues of mussels were homogenized
with 0.1 M phosphate buffer (pH 6.6). The homogenates were
centrifuged at 10 000 g for 15 minutes at 4 �C. The resulting
supernatants were then separated in 5 aliquots of 50 ml each and
200 mL of phosphate buffer was added to 2 aliquots and 200 mL
of ALA-reagent (d-aminolevulinic acid) was added to the others.
The mixture was incubated for 2 hours at room temperature and
afterwards 750 mL of the precipitation reagent (containing tri-
chloroacetic acid) was added, mixed for 30 minutes and then
centrifuged at 2500 g for 5 minutes. 500 mL of the resulting
supernatant was transferred to a plastic cell (1 mL) mixed with
500 mL of the Ehrlich chromogenic reagent (dimethylamino-
benzaldehyde) and incubated for 15 minutes at 25 �C. The UV
absorbance of the amount of porphobilinogen (PBG) was
determined in the bivalve samples and blanks at 550 nm. The
J. Environ. Monit., 2009, 11, 1673–1686 | 1675
Table 1 Certified metal concentrations and analysed concentrationsexpressed by mean � 95% confidence limits and mean � standard devi-
�1
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activity of ALAD was expressed in ng PBG min�1 mg�1 total
protein.
ation, respectively in TORT-2 reference materials (RM) (mg g dw)Metal Certified Analysed
Cadmium 26.7 � 0.6 25.3 � 1.1Copper 106 � 10 106.5 � 1.8Chromium 0.77 � 0.15 0.80 � 0.21Lead 0.35 � 0.13 0.30 � 0.02Nickel 2.50 � 0.19 2.32 � 0.12Zinc 180 � 6 186 � 3.1
Lipid peroxidation
Lipid peroxidation was determined in the same homogenate as
MT in the digestive gland. The method, described by Erdelmeier
et al.,32 measures the amount of malondialdehyde (MDA) and 4-
hydroxyalkenals (4-HNE) produced in the peroxidation of
membrane lipids. LPO was expressed as nmol MDA + 4-HNE
g�1 protein.
Total protein concentrations
Total protein concentrations were determined by the method of
Lowry.27 The quantification was performed using bovine serum
albumin as standard. Protein concentrations are expressed as mg
mL�1.
PAHs analysis
PAHs were determined in the whole soft tissues of M. gallopro-
vincialis. Pools of mussels (n ¼ 5) were homogenised with
Na2SO4 and Soxhlet extracted with n-hexane/dichloromethane
(4 : 1) for 24 h, which was further separated by liquid chroma-
tography (silica–alumina). The PAH containing fraction was
eluted with n-hexane : dichloromethane, as described else-
where.33 Individual PAHs (ng g�1 dw) were identified and
quantified by HPLC-UV, by comparison of retention times and
library spectra of reference compounds. A standard mixture
containing 16 individual PAHs, (EPA 610 PAH Mix, Sigma)
namely naphthalene, acenaphthylene, acenaphthene, fluorene,
phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)an-
thracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene,
benzo(a)pyrene, dibenzo(a,h)anthracene, benzo(g,h,i)perylene
and indeno-(1,2,3-cd) pyrene was used. Detection limit ranged
from 0.01 to 0.24 ng g�1 dw, for individual PAHs. PAH
measurements were validated using a standard reference material
of mussel tissue (SRM 2977; NIST, USA), that was extracted and
analyzed as the samples. PAH recoveries from the analyses of
certified material ranged between 73% and 112%. Blanks were
performed extracting an amount of sodium sulfate equivalent to
that used with the samples. Hydrocarbons were not detected in
the blanks.
Table 2 Temperature (�C) and salinity (PSU) from the sites in the SouthCoast of Portugal
Sites
Temperature (�C) Salinity (PSU)
Summer Winter Summer Winter
1 18.8 14.4 36.0 35.32 19.0 14.3 36.9 33.73 19.5 13.7 36.0 26.34 19.3 15.3 34.0 335 19.6 14.4 36.5 35.36 21.5 15.5 36.3 35.77 20.0 14.8 36.0 30.68 20.0 13.7 32.9 30.1
Metals
Metals (Cd, Cu, Ni, Pb and Zn) were analysed in the whole soft
tissues of M. galloprovincialis on dried subsamples by atomic
absorption spectrophotometry (SpectrA-A10/20; Varian,
Oxford, UK) after wet digestion with nitric acid. The accuracy of
the analytical procedure was checked using certified reference
material (TORT-2) from the National Research Council (Can-
ada). The results were in good agreement with the certified values
(Table 1). Metal levels were expressed as mg g�1 dry weight of soft
tissue.
1676 | J. Environ. Monit., 2009, 11, 1673–1686
Statistical analysis
Results are expressed as means � standard deviation (SD). The
data were tested for normality and homogeneity and analyzed by
analysis of variance (ANOVA). Duncan test was used to deter-
mine significant differences between variables. Pearson’s corre-
lation analysis was also applied between all the biomarkers and
between biomarkers and contaminants. Principal component
analysis (PCA – ordination method) was used to discriminate the
main variables responsible for the variance of biomarkers and
environmental factors. Canonical Correspondence Analysis
(CCA) was used to assess the influence of biomarkers associated
with the contaminants on the different sites. Statistical analysis
was carried out with Statistica 5.1 and CANOCO 4.5 package. A
minimum significance level of 0.05 was used for all statistical
analysis, i.e. a probability of p # 0.05 was considered significant.
Results
Environmental factors
Temperature and salinity data from each site are shown in Table 2.
Temperature in summer increased eastward, i.e., 18.8 �C (site 1) to
21.5 �C (site 6). In winter, the range was smaller, from 13.7 �C
(sites 3 and 8) to 15.5 �C (site 6) and lower than in summer (p <
0.05). Salinity ranged from 32.9 (site 8) to 36.9 (site 2) in summer
and was significantly higher than in winter (ranged from 26.3 (site
3) to 35.7 (site 6); p < 0.05). The lowest salinity was measured at the
sites close to the mouth of the rivers (sites 3, 7 and 8).
Condition index
No significant differences between the two seasons were observed
in the condition index of the mussels (8.8%� 2.5 and 9.0%� 2.1,
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respectively; p > 0.05), with the exception of sites 2, 7 and 8. The
lowest values, that indicate poor physiological condition were at
sites 8 (4.8%) and 1 (6.5%) in summer and 2 (7.2% in winter). The
highest CI was at sites 2 (14.6% in the summer) and 7 (12.1% in
winter).
Biomarker levels
The biomarkers analysed in the digestive gland of M. gallopro-
vincialis are shown in Figs. 2–7. The antioxidant enzymes activity
(SOD, CAT, GPx) are shown in Fig. 2. Each of the antioxidant
Fig. 2 Cytosolic (A) and mitochondrial SOD (B), CAT (C), total (D) and Se-
M. galloprovincialis collected in South coast of Portugal. Different letters me
CAT in mmol/min/mg prot, Total and Se- dependent GPx in nmol/min/mg p
This journal is ª The Royal Society of Chemistry 2009
enzymes responded distinctively. The variation pattern of SOD-
cyt was more similar to that of CAT and Se-GPx than to the
SOD-mit.
SOD activity in the cytosolic fraction (SOD-cyt; Fig. 2A) was
higher than in the mitochondrial fraction (SOD-mit; Fig. 2B);
(pv< 0.05). The variation of SOD-cyt activity (Fig. 2A) was
similar between surveys except at sites 1 and 2. SOD-cyt activities
were significantly higher and similar at sites 1 and 6 (summer)
and 3 (winter) while a minimum was observed at site 4 for both
seasons. SOD-mit activity (Fig. 2B), showed a different pattern
with almost 2-fold higher activity in the summer (p < 0.05), than
dependent GPx (E) activities (mean� sd) in the digestive gland of mussels
an significant differences. Activities of SOD are expressed in U/mg prot,
rot.
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Fig. 3 CYP450 concentration (A), ‘‘peak 418’’ (B), b5 (C), NADPH (D) and NADH (E) activities (mean � sd) in the digestive gland of mussels M.
galloprovincialis collected in South coast of Portugal. Different letters mean significant differences. CYP450 is expressed in pmol/mg prot, ‘‘peak 418’’ in
arbitrary units, b5 in pmol/mg prot and NADP(H) in nmol/min/mg prot.
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in winter (<3.5 U mg�1 prot). The maximum SOD-mit activity
was at site 6 (summer) and minimum at sites 4 and 8 in winter.
The highest CAT activity (Fig. 2C) was at sites 1 and 3 (62 and
58 mmol min�1 mg�1 prot in summer and winter, respectively).
Like for SOD-cyt, CAT activity was similar between surveys at
sites 2, 4, 6 and 7, (p > 0.05).
With respect to T-GPx and Se-dependent GPx activities
(Fig. 2D and E), both were of the same order of magnitude
despite slightly higher for T-GPx but showed a distinct pattern.
The maximum activity of T-GPx was at site 3 (9 nmol min�1 mg�1
prot in winter) but lower and similar at the other sites in both
seasons (# 5.5 nmol min�1 mg�1 prot) (Fig. 3D). Se-GPx the
1678 | J. Environ. Monit., 2009, 11, 1673–1686
highest activities (5–6 nmol min�1 mg�1 prot) were measured at
sites 1, 7 (summer) and 3 (winter), and minimum at site 4 (p <
0.05).
The activity of the phase I enzymes (CYP450 and the related
components ‘‘418 peak’’, cytochrome b5, NADPH cytochrome c
reductase and NADH-cyt b5) and of phase II GST in the
digestive gland of mussels M. galloprovincialis are shown in Fig. 3
and 4, respectively. In general, all biomarkers associated with
phase I (Fig. 3) showed a similar trend, higher in summer than in
winter. Levels generally increased from sites 1 to 6, decreasing
progressively at sites 7 and 8. The CYP450 activity (Fig. 3A) was
highest at site 6 (62 pmol mg�1 prot in summer), followed by 3, 4
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Fig. 4 GST activity (mean � sd) in the digestive gland of mussels M.
galloprovincialis collected in South coast of Portugal. Different letters
mean significant differences. GST is expressed in nmol/min/mg prot.
Fig. 5 AChE activity (mean � sd) in the gills of mussels M. gallopro-
vincialis collected in South coast of Portugal. Different letters mean
significant differences. AChE is expressed in nmol/min/mg prot.
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and 2 (p < 0.05). The ‘418- peak’ (Fig. 3B) as representative of
putative denatured cytochrome P450, followed a similar trend
(Fig. 3A), with the highest value at site 6 (9.5 a.u; p < 0.05).
Cytochrome b5 activity (Fig. 3C), as CYP450 (Fig. 3A) was also
highest at site 6, although in summer the values were similar at all
sites (p > 0.05). In winter cytochrome b5 activity decreased
drastically, with the maximum at sites 3 and 8 (�14 pmol mg�1
prot; p < 0.05). NADPH cytochrome c reductase activity
(Fig. 3D) like CYP450, ‘‘418 peak’’ and cytochrome b5 (Fig. 3A,
B, C) was also significantly higher at site 6 (14 nmol min�1 mg�1
prot, p < 0.05) in summer. Only at site 8 could no significant
difference between sites be detected. NADH-cyt b5 reductase
activity (Fig. 3E), was also significantly higher at site 6 (85 nmol
min�1 mg�1 prot in summer; p < 0.05), 4- to 6- fold higher than
NADPH-cyt c reductase (Fig. 3D) and like for NADPH cyt c
reductase activity levels were similar at site 8 between surveys.
GST (Fig. 4), however, showed a different spatial and seasonal
pattern of phase I enzymes, but followed that of SOD-cyt
(Fig. 2A), with a maximum at sites 2 (13.5 nmol min�1 mg�1 prot
in summer) and 3 (10.8 nmol min�1 mg�1 prot in winter). In
winter, the GST activity decreased reaching a minimum at site 4
(6.5 nmol min�1 mg�1 prot, p < 0.05).
The AChE activity in the gills of the M. galloprovincialis is
shown in Fig. 5. For this biomarker, although levels were in
general lower in summer than in winter, no seasonal variation
This journal is ª The Royal Society of Chemistry 2009
was observed between sites (except for site 3 and 7). Interestingly,
at site 5, AChE activity was lower than anywhere else and similar
to that of site 7 in the winter (5.5–6.0 nmol min�1 mg�1 prot),
suggesting an inhibition of this biomarker at these two sites.
MT concentrations in the gills and digestive gland of M. gal-
loprovincialis are shown in Fig. 6A, B, respectively. As expected,
MT levels were higher in the digestive gland than in the gills and
both tissues showed a distinct spatial and seasonal variation. MT
concentrations in the gills (Fig. 6A) were significantly higher in
summer than in winter, at sites 3, 4 and 8 (p < 0.05), while the
opposite occurs at sites 5 and 7. The highest concentration was in
winter at site 7 (�12 mg g�1 prot). Like for the gills, MT levels in
the digestive gland were 2-fold higher (10–18 mg g�1 prot; p <
0.05) in summer than in winter (Fig. 6B), particularly at site 4.
Only at sites 7 and 8 no significant differences of MT exist
between seasons (p > 0.05), with the minimum at site 8 (5 mg g�1
prot in winter). In winter the highest concentration was at site 7
(11 mg g�1 prot).
The ALAD activity in the whole soft tissues of M. gallopro-
vincialis is present in Fig. 6C. For this biomarker a marked
seasonal pattern exists. ALAD activity was significantly lower in
summer than in winter (p < 0.05) at all sites and varied in summer
within a narrow range (�0.1–0.3 ng PBG min�1mg�1 prot; p >
0.05). The minimum was at sites 3 and 4, which reveals that this
biomarker is particularly inhibited at these sites. In winter, ALAD
significantly increased particularly at sites 1 and 2 (�1.6 ng PBG
min�1mg�1 prot; p < 0.05). In general, ALAD activity showed an
opposite variation of that of MT in the digestive gland (Fig. 6A).
LPO concentrations in the digestive gland of M. gallopro-
vincialis are shown in Fig. 7. This biomarker also showed
a significant seasonal variation with levels significantly higher in
summer than in winter for all sites, with a maximum at site 8
(�13.5 mmol g�1 prot; p < 0.05). In winter, LPO levels were
similar at all the sites (�1 mmol g�1 prot; p > 0.05).
Influence of biotic and abiotic factors on biomarkers
The condition index varies with the reproductive cycle and the
food availability, but also with environmental factors. Despite
being similar between sites and seasons, a significant negative
relationship exists between CI and LPO (r ¼ �0.566; p < 0.05).
In order to minimize the influence of physiological conditions
upon biomarker levels, when the data matrix is normalised by the
condition index, GST was positively correlated with phase I
biomarkers (except the ‘‘418 peak’’), SOD (cyt and mit), CAT
and LPO (r > 0.580; p < 0.05).
The influence of abiotic parameters temperature and salinity
on biomarkers was also studied. Phase I (CYP450 and related
components) and Phase II (GST) enzymes, SOD-mit, MT in the
digestive gland and LPO increased when temperature increases (r
> 0.680; p < 0.05) while ALAD activity decreases (r ¼ �0.720; p
< 0.05). SOD-mit activity was also positively related to salinity
while T-GPx activity was inversely related (r¼�0.640; p < 0.05).
A PCA was applied to all biomarker data along with the
environmental variables (temperature and salinity) and CI of
mussels (Fig. 8). Biomarkers show a clear seasonal behaviour in
most of the sites (except sites 3 and 5) reflecting the seasonal
environmental changes associated with the bioavailability of
contaminants and abiotic factors.
J. Environ. Monit., 2009, 11, 1673–1686 | 1679
Fig. 6 MT concentrations (mean � sd) in the gills (A and digestive gland (B) and ALAD activity in the whole soft tissues (C) of M. galloprovincialis
collected in South coast of Portugal. Different letters mean significant differences. MT is expressed in mg/g prot and ALAD in ng/PBG/min/mg prot.
Fig. 7 LPO activity (mean � sd) in the digestive gland of mussels M.
galloprovincialis collected in South coast of Portugal. Different letters
mean significant differences. LPO is expressed in MDA + 4-HNE mmol/g
prot.
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In Fig. 8, PC1 (explaining 55% of the variance) shows that the
most evident biomarker response was in summer, for CYP450,
SOD-mit, MT in the digestive gland and LPO associated with
1680 | J. Environ. Monit., 2009, 11, 1673–1686
highest temperature and salinity values (and condition index)
against ALAD. During winter, ALAD activity shows greater
response than in the summer, indicating that this biomarker
during summer was more inhibited. Moreover, sites in PCA are
closer in the winter period than in summer. PC2 (that explains
26% of the variance), shows an increase of antioxidant enzymes
activities SOD-cyt, CAT, T- and Se-GPx together with GST,
AChE and MT in the gills against MT in the digestive gland.
Contaminant levels
Since in summer there was an evident seasonal influence on the
biomarker levels with a general increase of SOD-mit, CYP450,
MT in the digestive gland and LPO along with an evident
decrease of ALAD, analyses of contaminants were carried out on
the same samples to help to interpret the biomarker results.
PAHs and metals (Cu, Cd, Cr, Ni, Pb and Zn) were analysed in
the whole soft tissues of M. galloprovincialis (Tables 3 and 4,
respectively).
Total PAHs concentrations (Table 3), showed an increasing
gradient from site 1 (<60 ng g�1) to 6 (�900 ng g�1; p > 0.05), as
occurred for the MFO system and SOD-mit. For the 16
This journal is ª The Royal Society of Chemistry 2009
Fig. 8 PCA of the biomarkers battery in mussels Mytilus gallopro-
vincialis for both summer and winter surveys showing the data scores
labeled as sites, temperature, salinity and condition index.
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individual PAH concentrations, 2 + 3 rings were predominant
(>79%) at sites 4, 5, 6 and 8, while 4 ring PAHs (64–80%) were at
the other sites. Five ring PAHs, which are usually more toxic,
were only detected at site 8 (2%). The Phenanthrene/Anthracene
Table 3 PAHs concentrations (ng g�1 dw) in the soft tissues of mussels Myt
PAH compound
Sites
1 2 3 4
Naphthalene nd nd nd 323Acenaphthylene nd nd nd ndAcenaphthene nd nd nd 106Fluorene 11.3 � 2.0 6.1 � 1.5 6.3 � 1.9 6.8Phenanthrene 10.1 � 0.8 14.4 � 3.9 6.7 � 0.6 20.3Anthracene 0.63 � 0.05 0.94 � 0.36 2.6 � 0.03 0.65Fluoranthene 7.2 � 1.4 1.4 � 0.27 28.7 � 4.0 9.0Pyrene 2.5 � 0.03 7.7 � 0.17 15.1 � 2.9 10.1Benzo(a)anthracene 37.6 � 7.1 26.5 � 6.7 15.8 � 3.3 29.3Chrysene 5.3 � 1.2 2.9 � 0.01 3.6 � 0.08 5.5Benzo(b)fluoranthene nd nd nd ndBenzo(k)fluoranthene nd nd nd ndBenzo(a)pyrene nd nd nd ndDibenzo(a,h)anthracene nd nd nd ndBenzo(g,h,i)perylene nd nd nd ndIndeno-(1,2,3-cd)pyrene nd nd nd ndSPAH 74.7 � 1.8 (b) 59.9 � 2.1 (b) 78.8 � 2.1 (b) 511P/A 15.9 15.4 2.6 31.3F/P 2.9 0.2 1.9 0.9BaA/Chrys 7.1 9.2 4.3 5.32 + 3 rings (%) 29.5 35.8 19.8 89.44 rings (%) 70.5 64.2 80.2 10.65 + 6 rings (%)
a nd – not detected; () – Different letters mean significant differences.
This journal is ª The Royal Society of Chemistry 2009
(Phen/Ant) and Fluoranthene/Pyrene (F/P) ratios, used to iden-
tify the origin of PAHs, showed that PAH sources are different
along the South Coast of Portugal (Table 3). PAHs have a pet-
rogenic origin (fuel), (Phen/Ant ratio > 10 and F/P ratio # 1) at
sites 2, 4 and 8. At site 3, the origin is pyrogenic (pyrolysis) (Phen/
Ant and F/P ratios < 10 e > 1, respectively) while at sites 1, 6 and
7 the origin is both petrogenic and pyrolitic.
Concerning metal levels accumulated in the mussel tissues
(Table 4), different gradients were found. Cd, Ni and Pb levels
increased from site 4 to 8, as for the antioxidant enzymes SOD-
cyt, CAT and Se-GPx (Figs. 2A, C and E, respectively). Copper
showed a different pattern, increasing from site 1 to 3, where the
maximum occurred, and then decreased progressively from site 4
to 8. Cr and Zn were similar between sites (p > 0.05). Zn
concentrations were, like for Cd, maximum at site 8 (�380 mg
g�1) and similar between sites 7, 5, 3 and 1 (p > 0.05). Further-
more, a direct significant relationship exists between Cd and Zn
(r¼ 0.88, p < 0.01), Ni and Pb (r¼ 0.80, p < 0.05) and Zn and Ni
(r ¼ 0.74, p < 0.05) while Pb was only positively related with $ 5
ring PAHs (p < 0.01).
Considering the effect of abiotic parameters (temperature,
salinity) and CI with contaminants, it is observed that total PAH
concentrations were directly related with temperature, Cd was
inversely related with CI while LPO was negatively associated
with salinity. Total PAH concentrations were maximum at the
sites where temperature was highest (i.e. at site 6) and Cd was
maximum where CI was minimum (i.e. at site 8).
Relation between biomarkers and contaminant levels
CCA was applied to biomarkers, contaminants, condition index
and environmental factors, to provide insight into the factors
that explain the variance of biomarkers in the summer (Fig. 9). In
ilus galloprovincialis at the eight sites in summer 2005a
5 6 7 8
� 92 nd nd nd ndnd nd nd nd
� 8 128 � 44 735 � 129 nd 157 � 11� 1.3 6.6 � 0.7 31.5 � 3.8 3.4 � 0.2 12.8 � 0.2� 5.4 8.6 � 1.8 19.4 � 2.4 11.0 � 1.0 18.1 � 5.2� 0.17 0.38 � 0.07 0.33 � 0.07 0.90 � 0.16 1.5 � 0.33� 1.5 nd 58.5 � 5.1 3.5 � 0.5 2.6 � 0.03� 0.02 7.3 � 0.32 22.1 � 5.6 1.5 � 0.26 2.6 � 0.21� 7.8 8.6 � 3.0 25.8 � 7.0 21.9 � 2.2 24.6 � 0.13� 0.73 2.8 � 0.59 18.5 � 3.0 7.2 � 0.29 16.8 � 3.6
nd nd nd ndnd nd nd 4.8 � 0.50nd nd nd ndnd nd nd ndnd nd nd ndnd nd nd nd
� 13.9 (a) 162 � 7.2 (b) 911 � 19.5 (a) 49.3 � 0.66 (b) 241 � 2.4 (b)22.6 65.3 12.2 12.50 2.7 2.4 1.03.1 1.4 3.0 1.588.5 86.3 30.9 78.711.5 13.7 69.1 19.4
2.0
J. Environ. Monit., 2009, 11, 1673–1686 | 1681
Table 4 Metal concentrations of Cd, Cu, Cr, Ni, Pb and Zn (mg g�1 dw) in the soft tissues of mussels Mytilus galloprovincialis at the eight sites insummer 2005a
Metal
Sites
1 2 3 4 5 6 7 8
Cd 1.56 � 0.77 (b) 0.37 � 0.15 (c) 0.58 � 0.14 (c) 0.49 � 0.10 (c) 1.22 � 0.54 (bc) 0.46 � 0.05 (c) 1.19 � 0.51 (bc) 2.35 � 0.57 (a)Cu 10.2 � 1.3 (b) 16.8 � 3.9 (ab) 24.9 � 13.0 (a) 16.3 � 4.2 (ab) 12.5 � 7.3 (b) 10.0 � 2.8 (b) 10.9 � 1.7 (b) 11.4 � 0.4 (b)Cr 0.86 � 0.41 (a) 0.80 � 0.58 (a) 0.67 � 0.13 (a) 0.64 � 0.21 (a) 0.50 � 0.12 (a) 0.43 � 0.06 (a) 0.66 � 0.30 (a) 0.85 � 0.26 (a)Ni 0.03 � 0.02 (c) 0.05 � 0.02 (c) 0.26 � 0.07 (a) 0.05 � 0.02 (c) 0.21 � 0.12 (ab) 0.08 � 0.01 (bc) 0.16 � 0.04 (abc) 0.29 � 0.11 (a)Pb 0.66 � 0.17 (bc) 0.40 � 0.27 (c) 6.91 � 4.61 (b) 2.69 � 1.33 (bc) 3.51 � 2.05 (bc) 5.62 � 3.23 (bc) 4.14 � 2.55 (bc) 14.45 � 4.75 (a)Zn 238 � 73 (ab) 143 � 60 (b) 243 � 141 (ab) 181 � 47 (b) 268 � 99 (ab) 111 � 16 (b) 287 � 91 (ab) 380 � 171 (a)
a () – Different letters mean significant differences.
Fig. 9 CCA of the biomarkers battery in the mussels Mytilus galloprovincialis for the summer survey showing the data scores labeled as: (A)
contaminants, temperature (T), salinity (S) and condition Index (CI) and (B) sites.
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Fig. 9A is represented the association of biomarkers responding to
contaminants while at Fig. 9B, the projection of the sites is asso-
ciated with the same contaminant data. The two main axes explain
68% of the total variance, where only PC1 represents 50%.
Axis 1 confirmed that the highest damage expressed by the
increase of LPO was at site 8, associated with the presence of 5–6
PAHs rings (2%), high levels ofP
PAHs (�240 ng g�1 dw) and
the maximum metal concentrations (Table 3 and 4). These metal
concentrations also induce an increase of MT in the gills. At site
6, the increase of CYP450, SOD-mit and T-GPx may be asso-
ciated withP
PAHs whose concentration was the maximum
(�900 ng g�1 dw; Table 3) of the whole South Portuguese Coast
while MT in the digestive gland with Cu.
In axis 2, the increase of SOD-cit, CAT and Se-GPx at sites 1, 2
and 3 responded to the moderate levels of PAHs (50–80 ng g�1
dw) mainly of 4 rings (65–80%) (Table 3). In opposition, the
general inhibition of antioxidant enzymes at site 4 and 5 is mainly
associated to 2 + 3 ring PAHs (�90%) within the range�160–500
ng g�1 dw (Table 3).
Discussion
Mussels are suitable organisms for environmental quality
assessment since they are able to provide cellular and
1682 | J. Environ. Monit., 2009, 11, 1673–1686
physiological responses to contamination.34 M. galloprovincialis
is a very common species along the South Coast of Portugal,
where it is subjected to different contamination loads. In envi-
ronmental risk assessment, the use of a battery of biomarkers is
strongly recommended7,8 because a multibiomarker approach
gives a more comprehensive and integrated view of the biological
responses, even where the levels of contaminants are not
particularly high, like in the South Coast of Portugal.
PAH levels are of the same order of magnitude of those found
previously in the South Coast of Portugal,15,35 in the NW
Portuguese coast,36 but lower than in mussels from areas affected
by oil spills or tanker accidents, such as the Prestige37 and the
Aegean Sea, at La Coru~na38 and the Erika, along the Bay of
Biscay.39 Nevertheless, the highest PAH concentrations are
similar to those in some polluted areas of the NW Mediterra-
nean.40–43 Metal concentrations are similar to those early repor-
ted, for the South Coast of Portugal12,44 and in reference sites in
the Adriatic Sea and Greece coast45,46 but lower when compared
to contaminated coastal areas in the Atlantic and the Mediter-
ranean (such as the Spanish,47 Italian,48 Moroccan49,50 and in the
NW Mediterranean coasts).43
The overall biomarker data along with contaminants and
environmental variables revealed that in the South Coast of
Portugal, biomarkers in M. galloprovincialis varied spatially and
This journal is ª The Royal Society of Chemistry 2009
Ta
ble
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77
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seasonally depending upon both the origin and the magnitude of
the contaminants present. Considering both summer and winter
periods, PCA analyses demonstrated that the biomarkers
response differs markedly between seasons and was more
important in the summer (Fig. 8). In the winter, the major
biomarker changes were in mussels from site 3, with an increase
of all antioxidant enzymes (except SOD-mit), GST and AChE
(Figs. 2, 4 and 5), which suggests that contamination at this site
was more important in winter. Considering only the summer
data, the response of the biomarkers (Fig. 9A) reflects a rise in
the contamination levels during this period, leading to a clear
separation of sites (Fig. 9B). There was an increase of most of the
biomarkers responses, except for ALAD, which had an opposite
trend. Moreover, the selected battery of biomarkers reflects
different sources of contamination. The CCA analysis (Fig. 9)
discriminate that the principal factor responsible for the primary
biomarkers response, considered as the ‘‘contamination
component’’, depends on the nature and intensity of contami-
nation. At site 6, CYP450, SOD-mit and T-GPx, are responding
to maximumP
PAH concentrations, mainly of 2 + 3 rings and
MT in the digestive gland responds particularly to Cu. At site 8,
LPO responded primarily to high levels of PAH, including those
of high molecular weight, and the highest metal concentrations.
The secondary factor explaining the variability of the biomarkers
data, elected as the ‘‘oxidative stress component’’, was mainly
associated with the antioxidant enzymes Se-GPx and CAT, along
with SOD-cyt, responding to 4-ring PAHs, that caused oxidative
stress at sites 1 to 5. A very important feature related to the use of
a multibiomarker approach in risk assessment is the need for
having a detailed knowledge of basal levels of the biomarkers,
and of its seasonal variation, in order to distinguish pollution
induced effects from those induced by the natural biological cycle
of mussels. From these results, most of the biomarkers at site 2
can be used as baseline levels.
Despite the fact that the activity of antioxidant enzymes in the
mussels from the South Coast of Portugal (Fig. 2) is similar to
those of the same species of mussels from the Mediterranean sea,
farmed in the Ebro Delta,51 Italian,52 French coast53 and in the
Adriatic Sea46,54 (Table 5), spatially, their behaviour was inter-
esting, varying in an inconsistent way. Mussels from site 1
revealed an increase of SOD-mit, SOD-cyt, CAT and Se-GPx,
(Fig. 2 A, C, E) indicating a cascade of antioxidant enzymes
working to counteract the presence of ROS. The levels of anti-
oxidant enzymes at this site are associated with moderate levels
of PAHs (mainly of 4-rings) (Table 3) or other organic
compounds such as lindane, identified in the sediments as one of
the highest concentrations in the South Coast of Portugal.55 At
this site, the presence of PAHs and possibly other organochlorine
compounds were also reflected by the increase of GST activity
(Fig. 4). The levels of Cd (and Zn) are also important (Table 4)
and can be responsible for stimulating the lipid peroxidation56
(Fig. 7). Similarly, the increase of MT levels in the gills (Fig. 6A)
indicates the presence of bioavailable metals at this site, partic-
ularly Cd. This protein can, due to its high cysteine content, also
participate in defence against oxidative stress, not only acting as
oxyradical scavenger but also through metal binding/release
dynamics.57 The increase in the activity of antioxidant enzymes in
sites without significant differences in lipid peroxidation reflect
an adaptation to a chronic exposure to contaminants at this
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site.36,58 Opposed to what occurred at site 1, the inhibition of
most antioxidant enzyme systems (SOD-cyt, CAT and Se-GPx)
at site 4 (Fig. 9) points out the difficulty that mussels have at this
site in defending themselves against oxidative stress. Moreover,
CYP450 and MT were induced in the digestive gland suggesting
the presence of organic compounds and metals that can compete
to inhibit the antioxidant defence systems.
CYP450 followed the trend of variation of the concentration
of SPAH, expressed by a direct relationship between CYP450
and SPAHs, as previously reported and attained values similar
to those previously found in the South Coast of Portugal,15,35 in
the Spanish coast40,59 and in Venice lagoon.60 The highest
CYP450 values were found, like for SOD-myt and SOD-cyt
activities, in mussels from site 6. This suggests an increase of
oxidative stress and subsequent activation of the biotransfor-
mation enzyme GST (Fig. 4) indicating the formation of super-
oxide anion due to the presence of organic compounds. At this
site,P
PAHs were highest (Table 3), pesticides such as feni-
trothion and parathion-methyl were also identified,55 as well as
Cu, Ni and Pb (Table 4) that also induced MT in the digestive
gland. Moreover, at sites 3 (and 7) Se-GPx increases along with
CYP450, which indicates the presence of organic peroxides,
possibly due to the presence of low PAHs levels (Table 3). At site
2, CYP450 was induced along with of GST indicating the pres-
ence of organic pollutants (PAHs and possibly other organic
contaminants). However, the highest SPAHs concentrations
(sites 4 to 6 and 8) were not followed by the highest GST activity
(at sites 2, 3 and 1). The apparent lack of response of GST
activity in M. galloprovincialis to PAHs concentrations was also
found either from natural field conditions61,62 or under experi-
mental exposure.63 The activity of this biomarker in M. gallo-
provincialis was slightly lower than found previously for the same
area15 and other Portuguese regions36 or at the Mediterranean
and Adriatic Sea.46,52,54 Earlier results in the same area also
showed that when PAH concentrations are highest (sites 4 and
5), GST activity was inhibited, particularly at site 4.15 At site 5,
GST activity may be related to the high concentrations of total
PCBs and PAHs in mussels from this site.20
MT responses in mussels were metal specific (see the review of
Amiard et al.64). MT concentrations in the digestive gland of
mussels are �1 order of magnitude lower than those in mussels
from a heavily metal contaminated site in the NW Mediterra-
nean.43 MTs (gills and digestive gland) (Fig. 6A, B) followed the
trend of metals (Table 4). MTs in the gills were particularly related
with Cr concentrations (Fig. 9A), in agreement with Porte et al.65
At site 3, MT levels increased in both tissues (gills and digestive
gland) indicating the presence of metals, particularly Cu and Ni
that were maximum at this site (Table 4). Bivalve gills are the
primary site of metal uptake from the aqueous phase, suggesting
that bioavailable metals are accumulated in the gills via water. In
the digestive gland, MTs were mainly associated with Cu, which
reflects the transport of this metal by the circulatory system from
gills to the internal organs66 associated with food/diet (particulate
phase). MT concentrations in the digestive gland, in summer, were
relatively similar between sites, despite maximum at site 4, where
none of the analysed metals reached the maximum concentration.
One interesting feature is that, except at site 8 where metal levels
were highest, MT was not induced and the general response of this
biomarker reflects the low metal levels.
1684 | J. Environ. Monit., 2009, 11, 1673–1686
ALAD activity, which is a specific biomarker for Pb expo-
sure,67,68 showed a marked seasonal signature. During summer,
ALAD was inhibited at all sites suggesting an increase of Pb
concentration during this season, particularly at sites 3 and 8
(Table 4) probably associated to boat traffic increase, since lea-
ded petrol could still be used in some boat engines. In bivalves,
ALAD data is scarce. Levels were only reported for the clam
Chamelea gallina from the Southern Spanish coast69 and the
freshwater clam Corbicula fluminea along the Guadiana estuary,
within the range found in this study.70 Other ALAD levels in
mussel M. galloprovincialis from site 8 71 are of the same order of
magnitude as the present work and also negatively related to Pb,
which confirms the enzymatic inhibition of ALAD by this metal.
AChE has a fundamental role in the nervous system of both
vertebrates and invertebrates, and is a suitable biomarker to
detect environmental contamination caused by neurotoxic
compounds either organic (organophosphates, carbamates),
metals (Cd, Cu, and Pb)72 or surfactants.73,74 Compounds in
complex mixtures may also inhibit the AChE activity of bivalve
molluscs.75 The activity of this biomarker was similar to that
reported in mussels collected from the French, Italian and
Spanish coasts in the Mediterranean area (Table 5).53,76,77
Nevertheless, it is much higher than those found in species of
mussels at the Adriatic Sea (Table 5).46 AChE levels do not
change noticeably in the South Coast of Portugal between sites,
however lowest levels were found at sites 5 and 7 (Fig. 5), due to
the presence of neurotoxic organic compounds or even metals
(Table 4). Pesticides such as lindane were identified in the sedi-
ments from the Ria Formosa lagoon.55 At site 5 the impact of the
discharges from a small stream in the vicinity of a fruit crop area
can possibly transport neurotoxic compounds including pesti-
cides. Cu, Cd and Pb (Table 4) may in part contribute to the
inhibition of AChE activity. Site 7 is located in the vicinity of
intense agricultural activity where pesticides are used.
Metals are known to produce peroxidation of membrane
lipids.78,79 Lipid peroxidation in mussels from the South Portu-
guese Coast were similar to those found for the same species from
the NW Portuguese coast36 and Balearic Islands80 and relatively
higher than in some Mediterranean areas such as in the French,
Italian and Spanish coasts.76 However, levels were lower than at
the Venice lagoon, considered a highly polluted system81 (Table
5). LPO damage was maximum at site 8, the major estuarine area
in the South Coast of Portugal, where Cd, Ni, Pb and Zn
concentrations were the highest (Table 4). LPO may also be
associated to the exposure of high molecular weight PAHs.82,83
and site 8 was also the only site where 5 + 6 rings PAHs were
detected.
Nevertheless it is important to identify where synergistic,
addition or antagonistic effects exist (including environmental
factors) that might complicate the general interpretation. Abiotic
factors (temperature and salinity) may affect the response of
several biomarkers. Antioxidant enzymes,84 MT85 ALAD, LPO86
and AChE87 are directly affected by temperature variability. The
water temperature in summer (> 18.5 �C) contributes to increase
the environmental stress in M. galloprovincialis, reflected not
only by the variation of biomarker levels but also by a slight
decrease in CI in mussels at the eastern sites (4 to 8). In summer,
the variation of AChE in particular is directly related to
temperature (higher at site 6, while minimum at site 5). In fact,
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temperature is claimed to be the most important natural factor
affecting AChE activity in mussels.75,88 Moreover, temperature
also increases the toxicity of some metals, namely Cd, leading to
elevated oxidative stress.66,89 This may represent a synergistic
effect between temperature and contaminants, particularly in the
summer at site 8 (one of highest temperature (Table 2) and
maximal metal concentrations (Table 4, including Cd). More-
over, changes in salinity are directly related to the bioavailability
of contaminants, either metals or POPs,90 due to modification of
speciation. This may lead to an increase of bioaccumulation with
decreasing salinity,91 changing the response of some biomarkers.
At site 8 (and 3 to a certain extent), the relatively lower salinity
together with a wider range of salinity variation between surveys
(Table 2), increases the environmental stress of mussels and
subsequently LPO levels (Fig. 7), that are negatively related to
salinity. Some other organic or other emerging contaminants not
analysed here may also affect the biomarker responses.
Conclusions
This is the first multibiomarker approach in the South Coast of
Portugal using M. galloprovincialis, while previous studies based
on few biomarkers provided limited information. Despite the
levels of contaminants in these eight hotspots of contamination
in the South coast of Portugal were not particularly high, the
selected battery of biomarkers responded efficiently to the envi-
ronmental changes and allowed an environmental assessment
between seasons and sites. Different responses in space and time
were evident along the South Coast of Portugal, meaning that the
contamination is not homogeneous, reflecting not only different
competition, origin and intensity of contamination, but also
different environmental conditions. Summer represents the most
stressful/critical situation, due to higher environmental contam-
ination and temperature. Despite environmental factors that
may complicate the general interpretation of biomarkers
response, the most suitable battery of biomarkers are: CYP450,
SOD-mit and T-GPx forP
PAHs concentrations (mainly of low
molecular weight), MT in the digestive gland particularly for Cu,
ALAD for Pb and LPO as a damage biomarker for both metals
and high molecular weight PAHs. Based on the interpretation of
PCA and CCA analysis, for further environmental contamina-
tion assessment studies it is possible to reduce the analytical
effort of some biomarkers, by eliminating the components of
phase I MFO system (b5, P418, NADH and NADPH), and MT
in the gills, since their responses are not so evident.
Acknowledgements
This study was financially supported by the FCT project ref:
POCTI/CTA/48027/2002.
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