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Environmental Pollution 151 (2008) 631e640www.elsevier.com/locate/envpol
Field validation of a battery of biomarkers to assess sedimentquality in Spanish ports
M.L. Martın-Dıaz a,b,*, J. Blasco b, D. Sales c, T.A. DelValls a
a Departamento de Quımica Fısica, Facultad de Ciencias del Mar y Ambientales, Polıgono Rıo San Pedro s/n, 11510 Puerto Real, Cadiz, Spainb Consejo Superior de Investigaciones Cientıficas, Instituto de Ciencias Marinas de Andalucıa, Polıgono Rıo San Pedro s/n,
11510 Puerto Real, Cadiz, Spainc Departamento Ciencias Ambientales y Tecnologıa de los Alimentos, Facultad de Ciencias del Mar y Ambientales, Polıgono Rıo San Pedro s/n,
11510 Puerto Real, Cadiz, Spain
Received 10 August 2006; received in revised form 13 March 2007; accepted 16 March 2007
A battery of biomarkers shows exposure to metals and organic compounds.
Abstract
Two marine invertebrates, the crab Carcinus maenas and the clam Ruditapes philippinarum, were used as bioindicator species to assess con-tamination when exposed in situ to sediment from different sites from four Spanish ports Cadiz (SW Spain), Huelva (SW Spain), Bilbao(NE Spain) and Pasajes (NE Spain). In an attempt to determine sediments toxicity, a combination of exposure biomarkers was analyzed inboth species: metallothionein-like-proteins (MTLPs), ethoxyresorufin O-deethylase (EROD), glutathione S-transferase activity (GST), glutathi-one peroxidase (GPX) and glutathione reductase (GR). In parallel, physical and chemical characterization of the different sediments was per-formed and biological responses related to the contaminants. Significant induction of MTLPs was observed when organisms were exposed tometal contaminated sediments (port of Huelva), and EROD and GPX activities after exposure to sediments containing organic compounds (portof Bilbao and Pasajes). No significant interspecies differences were observed in biomarker responses except for the GST and GR.� 2007 Published by Elsevier Ltd.
Keywords: Carcinus maenas; Ruditapes philippinarum; Dredged material; Oxidative stress; Metallothionein
1. Introduction drawbacks and allows field assessment to be integrated with
Ecotoxicological data obtained in laboratory studies isoften difficult to translate into accurate predictions of possibleeffects in the field. Since both overestimation and underesti-mation of effects may occur, laboratory biomarker resultsare best validated by field research. With respect to sediment,and especially dredged material, observations of a range of pa-rameters in individuals sampled from the field do not necessar-ily provide relevant information. The transplantation of cagedorganisms to evaluate dredged material toxicity avoids such
* Corresponding author. Departamento de Quımica Fısica, Facultad de Cien-
cias del Mar y Ambientales, Polıgono Rıo San Pedro s/n, 11510 Puerto Real,
Cadiz, Spain. Tel.: þ34 956 016423; fax: þ34 956 016040.
E-mail address: laura.martin@uca.es (M.L. Martın-Dıaz).
0269-7491/$ - see front matter � 2007 Published by Elsevier Ltd.
doi:10.1016/j.envpol.2007.03.019
laboratory assessment. Prior studies include the analysis ofdistinct biomarkers in caged mussels transplanted to NW Med-iterranean sediment (Munns et al., 2002) as well as in cagedcrabs and clams exposed to Spanish ports’ sediment (Martın-Dıaz et al., 2005). Dredged material has been found to haveadverse environmental effects, resulting in internationaltreaties and protocols for their proper environmental manage-ment. To this end, the OsloeParis Convention (OSPAR) andHelsinki Conventions (North Sea, North-East Atlantic, BalticSea) proposed guidelines to control the disposal of dredgedmaterial. Physical and chemical characteristics of the sedimentare treated in these guidelines, together with biological effects.Nevertheless, biological effects are focused on the measure-ment of acute responses. The use of biomarkers for dredgedmaterial assessment is currently under review because of the
632 M.L. Martın-Dıaz et al. / Environmental Pollution 151 (2008) 631e640
inherent capability of detecting early occurrence of variousstress conditions within organisms and monitoring temporalprogression (or regression) of the disturbance at various levelsof biological organization. When assessing exposure to a varietyof contaminants in the aquatic environment, biotransformationenzymes and metal-binding proteins, metallothionein-like-proteins (MTLPs), play an important role in organic and metalpollutant contamination respectively.
The present study was undertaken to test the potential im-pact of distinct contaminants present in dredged material onthe crab Carcinus maenas and the clam Ruditapes philippina-rum in four Spanish ports (Fig. 1). Few studies have used mul-tiple biomarkers in more than one species, to determinesublethal effects of contaminants. In an attempt to achievethis main objective, the following were employed: (a) Specieswith differing feeding styles and distinct physiological charac-teristics; (b) a battery of exposure biomarkers; and (c) in situassays. The battery of biomarkers was selected in order to
Bilbao
Huelva
Iberian
Peninsula
Bi3
Hu2
Hu3
Bi2
Fig. 1. Localization of sampling p
examine different defense mechanisms of the organism. Thedefense mechanisms studied were: metallothionein-like-pro-teins (MTLPs), proteins for detoxification of metal contamina-tion; mixed function oxidase (EROD), phase I detoxificationenzyme; glutathione S-transferase (GST) phase II detoxifica-tion enzyme; glutathione peroxidase (GPX) and glutathionereductase (GR) antioxidant enzymes. The first of these bio-markers has been reported to be induced by metal exposure,while the remainder are implicated in the detoxification of or-ganic compounds (EROD and GST) as well as the protectionof the organism against oxidative stress (GPX and GR).
2. Materials and methods
2.1. General approach
The four Spanish ports chosen (Fig. 1) for the assessment of sediment toxi-
city were as follows. The Port of the Bay of Cadiz (SW Spain): the bay of
Cádiz
Pasajes
Pa2
Pa3
Ca2
Ca3
Ca1
oints in the Iberian Peninsula.
633M.L. Martın-Dıaz et al. / Environmental Pollution 151 (2008) 631e640
Cadiz has been studied in some depth (DelValls and Chapman, 1998; Riba
et al., 2004, 2005) and is characterized by an absence of significant contami-
nation. The sampling sites corresponding to this port are Ca1 (negative toxicity
control), Ca2 and Ca3. The Port of Huelva (SW Spain): characterized by well
extended heavy metal contamination owing to historic mining activity. The
sampling sites corresponding to this port are Hu2 and Hu3. The Port of Bilbao
(NNE Spain): this port is characterized by significant shipping activity. It is
predominantly associated with organic contamination, especially hydrocar-
bons. The sampling sites corresponding to this port were Bi2 and Bi3. The
Port of Pasajes (NNE Spain): This port is also characterized by significant
shipping activity, and by contamination associated with organic compounds
similar to the Port of Bilbao. The sampling sites corresponding to this port
were Pa2 and Pa3.
2.2. Selection of organisms
Individuals of two invertebrate species with distinct feeding styles and dif-
ferent physiological characteristics were used: the crab Carcinus maenas (bur-
rowing) and the clam Ruditapes philippinarum (filter feeder). This allowed the
analysis of a variety of exposure routes to the sediment matrix. These species are
widely studied and have been used previously in the biomonitoring of contami-
nated sites (Astley et al., 1999; Hoarau et al., 2004). In the present experiment,
individuals of the intermoult female Carcinus maenas and Ruditapes philippina-
rum, of standardized size (Martın-Dıaz et al., 2004, 2005) were purchased
from an aquaculture farm located at a clean site on the coast. Individuals were
acclimatized for 48 h in the laboratory before being transplanted to the field.
2.3. Sample collection
Surface sediment samples (5e10 cm) were collected at the four Spanish
Ports with a 0.025 m2 Van Veen grab. Samples were transported to the labora-
tory and subsampled prior to physical-chemical characterization. Sediment
samples were sieved through a 0.5 mm mesh into a tank in order to remove
any associated macrofauna and larger sediment granules. They were subse-
quently kept at 4 �C in the dark, up to their use in toxicity tests. Sediments
were stored no more than 2 weeks prior to the toxicity test.
2.4. Field assay
Animals were transported from the laboratory in refrigerated polystyrene
boxes to the boat. Rectangular plastic netting cages (50 � 25 � 15 cm) were
employed for the study. Two cages containing crabs (n ¼ 24) and clams
(n ¼ 50) respectively were utilized per sampling point. Cages were divided
in two so as to provide duplicate assays. They were immersed with the assis-
tance of scuba diving apparatus and anchored in the sediment at each of the
stations (Fig. 1) at all 4 ports. Following a 28-day exposure period, cages
were collected and individuals sampled. The retrieval of the cages was carried
out with the help of scuba-diving.
2.5. Chemical analysis
Sediment characterization was performed according to Spanish guidelines
for dredged material (CEDEX, 1994). Content of metals was determined in the
<0.5 mm grain size fraction of the sediment using atomic absorption spectro-
photometry, according to the methodology used by Riba et al. (2002a). poly-
chlorinated biphenyls (PCBs) (congeners #28, 52. 101, 118, 138, 153 and 180)
and polycyclic aromatic hydrocarbons (PAHs) were analyzed according to
U.S. EPA SW-846 method 8270/8082 (Riba et al., 2002b). All analytical pro-
cedures were verified using reference material (MESS-1 NRC and CRM 277
BCR, for heavy metals and NCR-CNRC HS-1 for organic compounds), result-
ing in a greater than 90% agreement with certified values.
2.6. Biochemical analysis
Intermoult female crabs Carcinus maenas and clams Ruditapes philippina-rum were withdrawn on day 28 for biochemical analysis. Following dissection,
the hepatopancreas and the digestive gland from crabs and clams respectively
were kept at �80 �C prior to homogenization. Samples were homogenized
following the procedure described by de la Lafontaine et al. (2000). Once
homogenized the samples for enzymatic activity and total protein content
determination were centrifuged at 10,000 � g for 30 min, and the supernatant
fraction extracted. Samples obtained for the determination of metallothionein-
like-protein-like-proteins content were centrifuged at 28,000 � g for 40 min.
The supernatant obtained was utilized for total protein determination. The
methodology used to determine total protein content was that described by
Bradford (1976).
2.7. Metallothionein-like-protein (MTLP) concentration
A volume of 0.1 ml of the supernatant (homogenized at 28,000 � g for
40 min) was added to 0.9 ml of NaCl (0.9%), heated to 95 �C for 4 min,
then centrifuged at 10,000 � g for 15 min at 4 �C. The supernatant was stored
at �80 �C prior to MT concentration determinations by Anodic Stripping Vol-
tammetry (ASV) (Olafson and Olsson, 1987) using purified rabbit metallothio-
nein-like-protein (Sigma-Aldrich). MTLP concentrations were expressed as
mg MTLP mg�1 total protein.
2.8. Ethoxyresorufin O-deethylase (EROD) activity
Mixed function oxidize activity was measured using the adapted EROD
assay (Gagne and Blaise, 1993) used for rainbow trout. 50 ml of supernatant
(homogenized at 10,000� g for 30 min) were added to 10 mM 7-ethoxyresorufin
and 10 mM reduced NADPH in 100 mM KH2PO4 buffer (pH 7.4). The reac-
tion was initiated by the addition of NADPH, allowed to proceed for 60 min at
30 �C, and stopped by the addition of 100 ml of 0.1 M NaOH. 7-Hydroxyresor-
ufin was determined fluorometrically using 520 nm (excitation) and 590 nm
(emission) filters. Determination of 7-hydroxyresorufin in the samples was car-
ried out using a standard calibration curve of 7-hydroxyresorufin concentra-
tion. Results were expressed as (pmol min�1 mg�1 total protein).
2.9. Glutathione S-transferase (GST) activity
The procedure utilized for the determination of GST activity was adapted
from McFarland et al., 1999. The activity was analyzed using 42 mM
1-chloro-2,4-dinitrobenzene (CDNB) and 1 mM GSH as substrates and mea-
sured spectrophotometrically at 340 nm every 30 s for 3 min. Results were
expressed as (nmol min�1 mg�1 total protein).
2.10. Glutathione reductase (GR) activity
Glutathione reductase activity was measured using the GR assay (McFar-
land et al., 1999). The reaction mixture contained 10 mM oxidized glutathione
as the substrate. Activities were determined spectrophotometrically at 340 nm
every 2 min during 10 min. The decrease in NADPH absorbance measured at
340 nm during the oxidation of NADPH to NADP, was indicative of GR activ-
ity. Results were expressed as (nmol min�1 mg�1 total protein).
2.11. Glutathione peroxidase (GPX) activity
The methodology used for the determination of this activity was that used
by McFarland et al. (1999). GPX activities were measured spectrophotometri-
cally at 340 nm every 2 min for 10 min, using 1 mM cumene hydroperoxide as
the substrate. The decrease in NADPH absorbance measured at 340 nm during
the oxidation of NADPH to NADP, was indicative of GPX activity. Results
were expressed as (nmol min�1 mg�1 total protein).
2.12. Statistical analysis
Different biomarker responses were analyzed using the SPSS/PCþ statis-
tical package. Significant differences between individuals exposed to control
sediment and individuals exposed to contaminated ones were determined using
Zn
PC
BPA
H
90
37
8.2
50
.11
0.1
1
61
13
5.5
00
.01
0.0
1
70
18
57
.00
0.0
10
.01
60
11
76
.00
0.0
10
.01
50
77
7.5
00
.23
66
.77
90
12
2.3
50
.01
13
.90
70
76
3.0
00
.74
1.0
6
00
57
6.0
00
.24
0.2
6
634 M.L. Martın-Dıaz et al. / Environmental Pollution 151 (2008) 631e640
a one-way ANOVA followed by a multiple comparison of Dunett’s tests. The
significance level was set at p < 0.05.
Factorial analysis was also performed in order to determine the major re-
sponses with respect to sediment contaminant levels. A MAA (Factor Analysis
using the Principal Component Analysis (PCA) extraction procedure) was ap-
plied to the original set of variables using the STATISTICA/PCþ statistical
package (Frane et al., 1985).
Correlation between chemical concentrations in sediments and interspecies
biomarker responses was undertaken using a Pearson correlation analysis
using the SPSS/PCþ statistical package. The level of significance was set at
p < 0.05.
3. Results
Pb 86
.
17
.
38
4.
21
7.
14
7.
28
5.
29
3.
24
6.
3.1. Chemical concentration in sediment
u%
Fe
Hg
Mn
Ni
02
.80
26
.50
1.9
82
01
.60
20
.14
46
.76
19
.63
0.2
82
94
.40
16
.90
49
.70
57
.13
1.9
93
03
.60
7.1
0
72
.50
41
.25
1.2
03
54
.45
12
8.5
5
04
.10
42
.00
1.4
33
96
.60
32
.00
23
.03
16
.98
0.1
81
91
.35
15
.72
67
.10
31
.80
1.2
91
80
.00
28
.48
62
.50
22
.00
1.3
61
52
.60
19
.61
Summarized results of the chemical analysis are shown inTable 1 and are represented in Fig. 2. The chemical character-ization of the sediment samples indicates that they containmixtures of contaminants (metals, PCBs and PAHs). The sum-marized results for conventional sediment parameters (Table 1)such as organic matter and grain size percentages were foundto vary depending on the sediment and area of concern.Sediment from the port of Huelva was characterized by highconcentrations of metals such as As, Cu, Pb, Zn, Ni and Cdwhile sediment from the ports of Pasajes and Bilbao was pre-dominantly characterized by contamination due to PCBs andPAHs, although contamination by metals such as Cr, Mn,Ni, Pb and Hg was also found. Sediment from the port ofCadiz was not found to be highly contaminated and provedto be a good negative toxicity control for the experiment.
C 2 1 7 2 1 1
3.2. Biomarker responses
Cr
14
.94
8.4
3
24
.10
8.1
3
23
.11
3.4
8
23
.42
18
.61
No significant mortality compared with the control site wasobserved.
Cd
1.3
2
1.2
3
2.5
0
1.3
2
2.0
0
0.0
4
0.7
0
0.0
4
3.3. Metallothionein-like-protein (MTLP) concentration
Sp
anis
hp
ort
sse
dim
ent
%S
and
%F
ine
%M
OA
s
40
.42
59
.53
13
.75
30
.77
17
.80
81
.90
20
.30
16
.61
56
.02
90
.21
10
.64
53
2.2
7
16
.13
43
.95
6.3
02
72
.78
14
.48
47
.40
15
.07
10
4.4
9
6.2
29
3.5
91
6.7
32
1.7
1
5.0
89
1.2
41
8.4
72
8.7
6
38
.53
59
.65
19
.81
23
.78
CB
con
cen
trat
ion
isex
pre
ssed
asm
gk
g�
1.
Concentrations of metallothionein-like-protein in Ruditapesphilippinarum and Carcinus maenas deployed at the sites inthe port of Huelva (Hu2 and Hu3) exhibited significantlyhigher levels when compared to the same tissue analyzed inclams and crabs at the reference site (Ca1). Clams showed sig-nificant induction of these proteins when exposed to sedimentfrom Hu2 (242.97% of control, p < 0.05), Hu3 (237.43% ofcontrol, p < 0.05), Bi3 (243.03% of control, p < 0.05) andBi2 (210.80% of control, p < 0.05). In crabs significant induc-tion was observed in those individuals exposed to Hu3 (212%of control, p < 0.05) and Hu2 (203.68% of control, p < 0.05).No significant differences were observed between crabs andclams in metallothionein-like-protein induction.
of
fou
r
se Han
dP
3.4. Glutathione S-transferase (GST) activity
Tab
le1
Ch
arac
teri
zati
on
Sit
e%
Co
ar
Ca2
0.0
5
Ca3
0.3
0
Hu
20
.19
Hu
30
.03
Bi2
38
.12
Bi3
0.1
9
Pa2
3.6
7
Pa3
1.8
2
Hea
vy
met
al,
PA
As found with MTLPs induction, the GST activity observedin individuals exposed to sediments from the port of Huelva(Hu3) showed the highest induction when compared to the ref-erence site’s. In clams, significant differences were observed
EROD CLAM
0
5
10
15
20
25
30
35 EROD CRAB
pmol
min
-1 m
g-1
pmol
min
-1 m
g-1
0
5
10
15
20
25
30
**
**
**
**
GST CLAM
nmol
min
-1 m
g-1
nmol
min
-1 m
g-1nm
ol m
in-1
mg-1
nmol
min
-1 m
g-1nm
ol m
in-1
mg-1
nmol
min
-1 m
g-1
0
20
40
60
80
100
120 GST CRAB
0
10
20
30
40
50
60
70
80
* **
*
* *
mg
mg-1
mg
mg-1
0
10
20
30
40
MET CLAM
CA2 HU2 BI3 BI2 PA3 PA20
10
20
30
40
50
60
MET CRAB*
*
*
**
*
GR CLAM
0
20
40
60
80
100 GR CRAB
0
20
40
60
80
100
120
140*
*
*
*
GPX CLAM
0
200
400
600
800 GPX CRAB
0
200
400
600
800**
**
*
HU3CA3CA2 HU2 BI3 BI2 PA3 PA2HU3CA3
CA2 HU2 BI3 BI2 PA3 PA2HU3CA3 CA2 HU2 BI3 BI2 PA3 PA2HU3CA3
CA2 HU2 BI3 BI2 PA3 PA2HU3CA3CA2 HU2 BI3 BI2 PA3 PA2HU3CA3
CA2 HU2 BI3 BI2 PA3 PA2HU3CA3 CA2 HU2 BI3 BI2 PA3 PA2HU3CA3
CA2 HU2 BI3 BI2 PA3 PA2HU3CA3CA2 HU2 BI3 BI2 PA3 PA2HU3CA3
50 70
Fig. 2. Representation of metallothioneins (MTs) concentration, ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST), glutathione reductase
(GR) and glutathione peroxidase (GPX) activities determined in digestive gland of Ruditapes philippinarum and hepatopancreas of Carcinus maenas after
a 28-day exposure period to sediments from the port of Cadiz (Ca2, Ca3), the port of Huelva (Hu2, Hu3), the port of Pasajes (Pa2, Pa3) and the port of Bilbao
(Bi2, Bi3). The line represents the average value for each biomarkers and species that correspond to the negative toxicity control (Ca1). Asterisk (*) represents
significant induction ( p < 0.05) compared with control treatment.
636 M.L. Martın-Dıaz et al. / Environmental Pollution 151 (2008) 631e640
in those exposed to Hu3 (255.37% of control, p < 0.05), Ca3(255.02% of control, p < 0.05), Ca2 (251.56% of control,p < 0.05) and Pa2 (239.16% of control, p < 0.05). GST activ-ities determined in the crabs were significantly different fromthe control in those deployed in Hu3 (179.91% of control,p < 0.05) and Bi2 (67.75% of control, p < 0.05). No signifi-cant differences were observed between GST induction incrabs and clams.
3.5. Ethoxyresorufin O-deethylase (EROD) activity
A distinct pattern of EROD activity induction, comparedwith MTLPs and GST activities was detected in the clamRuditapes philippinarum and the crab Carcinus maenas, themost significant induction being observed for exposure tosediments from the port of Bilbao (Bi2 and Bi3) and theport of Pasajes (Pa2 and Pa3). Significant induction was foundin clams compared to the control for those individuals de-ployed at Pa2 (132.54% of control p < 0.05), Pa3 (120.23%of control, p < 0.05), Bi2 (113.31% of control, p < 0.05)and Bi3 (97.76% of control, p < 0.05). Significant differencescompared to the reference site were observed in crabs exposedto sediments from Pa3 (149.43% of control, p < 0.05), Bi2(138.37% of control, p < 0.05), Pa2 (132.21% of control,p < 0.05) and Ca2 (127.63% of control p < 0.05). Significant( p < 0.05) differences were observed between EROD activityinduction in clams and crabs.
3.6. Glutathione peroxidase (GPX) activity
Glutathione peroxidase activities determined in the crabCarcinus maenas and the clam Ruditapes philippinarum, fol-lowing exposure to sediments from the various ports showeda significant induction related to sediments from the ports ofBilbao and Pasajes, when compared with those individuals ex-posed to sediments from the reference site. More importantly,these activities followed the same pattern as EROD induction.In clams, significant induction was seen for Pa3 (224.21% ofcontrol, p < 0.05), Bi3 (217.75% of control, p < 0.05), Bi2(114.75% of control, p < 0.05) and Pa2 (84.65% of control,p < 0.05). In crabs, significant induction was observed inPa3 (150.43% of control, p < 0.05), Bi2 (146.4% of control,p < 0.05), Pa2 (142.21% of control, p < 0.05) and Ca2(137.64% of control p < 0.05). No significant differenceswere found between GPX activity induction in clams andcrabs.
3.7. Glutathione reductase (GR) activity
Significant differences were found between glutathione re-ductase activities determined for crabs and clams exposed tosediments from the ports of Pasajes and Huelva compared tothe control. For the crab Carcinus maenas significant induc-tion was observed at Hu3 (179.90% of control, p < 0.05).Clams showed enzymatic activity induction when exposed toPa3 (100.43% of control, p < 0.05), Hu3 (91.90% of control,p < 0.05) and Hu2 (91.74% of control, p < 0.05). Significant
( p < 0.05) differences were observed between GR inductionin clams and crabs.
3.8. Interspecies biomarker response correlation
The correlation between species for each biomarker deter-mination, following the 28-day exposure period to the Spanishports’ sediment, was analyzed. Biomarker responses of each ofthe species followed a similar trend, nevertheless the only sig-nificant correlation between biomarker responses in clams andcrabs was found for EROD ( p < 0.01), MTLPs ( p < 0.01)and GPX ( p < 0.05).
Possible correlations among chemical concentration values,MTLP concentration and enzymatic activity (GST, GR,EROD, and GPX) were investigated analyzing responses forboth species in situ. Two multivariate analysis approaches(MAA) were undertaken in order to analyze chemical concen-trations in the sediment from the different ports and their rela-tion to biological adverse effect variables, the concentration ofMTLPs and the enzymatic activities registered on day 28 infemale crabs and clams exposed to sediment from the 4 ports(8 sampling points in situ). The MAA was performed using theset of data obtained for the 8 cases defined by the separatesampling sites Ca3, Ca2, Hu2, Hu3, Bi3, Bi2, Pa3 and Pa2.In total we applied the MAA to 21 variables for the 8 cases.The application of MAA indicates that the original set of vari-ables can be narrowed down to three variables or factors(Tables 2 and 3). These factors provide a more concise descrip-tion than the original data set. The criteria for consideration ofa variable as being associated with a particular factor was de-fined as its having a loading of 0.4 or higher; which approxi-mates Comreys’ cut-off (Comreys, 1973) of 0.6 or higher fora reasonable association between an original variable and afactor, and also takes into account discontinuities in the magni-tudes of the loadings of the original variables. Each componentis described according to the dominant group of variables. Thecomponents for each species exposed in situ are described asfollows.
With regard to the clam Ruditapes philippinarum exposedto different sediments:
e The first principal factor, #1, accounts for 28.60% of thevariance. Factor 1 (the positive value) accounts for the re-lationship between MTLPs induction, acting as a defensebiomarker related to exposure to the metals As, Cd, Cr,Fe, Hg, Pb, and Zn - and the induction of GR activities.
e The second factor, #2, accounts for 19.00% of the vari-ance. This factor (the positive value) is related to the in-duction of EROD and GPX activity, as defensebiomarkers of PCBs and the metals Cr, and Pb. However,the negative value of Factor 2 is associated with the induc-tion of GST enzymatic activity due to the presence of Cu,Mn, and Ni.
e The third factor, #3, accounts for 18.67% of the variance. Itrepresents the relationship between the induction of ERODactivity and contamination by PAHs, Cd, Cr and Mn.
Table 2
Sorted rotated factor loadings (pattern) of 21 variables for the two principal
factors resulting from the multivariate analysis of results obtained from
Ruditapes philippinarum
Ruditapes philippinarum Factor 1 Factor 2 Factor 3
%Variance 28.60 19.00 18.67
GST28 e �0.666 e
GPX28 e �0.4 e
GR28 0.716 e e
MT28 0.514 e eEROD28 e 0.473 0.4
%Coarse e e e
%Sand e e e%Fine e e e
%MO e e e
As 0.940 e e
Cd 0.573 e 0.620
Cr 0.475 e 0.549
Cu e �0.784 e
Fe 0.862 e e
Hg 0.648 e eMn e �0.509 0.682
Ni e �0.806 e
Pb 0.712 0.407 e
Zn 0.960 �0. 530 ePCBs e 0.574 e
PAHs e e 0.819
The variables utilized were the biomarkers of exposure (metallothioneins
(MT), mixed function oxidase (EROD), glutathione peroxidase (GPX), gluta-
thione reductase (GR) and glutathione S-transferase (GST)), determined after
28-day exposure in situ, metal content in the sediment; organic compounds
content and sediment characteristics (grain size, organic matter (MO). Only
loadings greater than 0.4 are shown in the table. Factors (#) are numbered con-
secutively from left to right in order of decreasing variance.
Table 3
Sorted rotated factor loadings (pattern) of 21variables for the two principal
factors resulting from the multivariate analysis of results obtained from
Carcinus maenas
Carcinus maenas Factor 1 Factor 2 Factor 3
%Variance 30.04 24.06 17.70
GST28 0.4 e e
GPX28 e �0.4 e
GR28 e 0.919 e
MT28 0.803 0.513 eEROD28 e �0.5 0.725
%Coarse e e e
%Sand e e e%Fine e e e
%MO e e e
As 0.898 e e
Cd 0.762 e eCr 0.635 �0.4 0.660
Cu e 0.899 e
Fe 0.919 0.4 e
Hg 0.796 e eMn e 0.695 e
Ni e 0.927 e
Pb 0.513 e e
Zn 0.938 e ePCBs e �0.4 0.534
PAHs e e 0.742
The variables utilized were the biomarkers of exposure (metallothioneins
(MT), mixed function oxidase (EROD), glutathione peroxidase (GPX), gluta-
thione reductase (GR) and glutathione S-transferase (GST)), determined after
28-day exposure in situ; metal content in the sediment; organic compounds
content and sediment characteristics (grain size, organic matter (OM). Only
loadings greater than 0.4 are shown in the table. Factors (#) are numbered con-
secutively from left to right in order of decreasing variance.
637M.L. Martın-Dıaz et al. / Environmental Pollution 151 (2008) 631e640
In relation to the female crab Carcinus maenas exposed todistinct sediments:
e The first principal factor, #1, accounts for 30.04% of thevariance. The positive Factor 1 combines MTLPs induc-tion and GST enzymatic activities as defense biomarkersof exposure to As, Cd, Cr, Fe, Hg, Pb and Zn.
e The second factor, #2, accounts for 24.06% of the vari-ance. The positive value of this factor relates MTLP induc-tion with exposure to Cu, Fe, Mn and Ni and the inductionof GR enzymatic activity against oxidative stress. Thenegative value of Factor 2 associates the EROD andGPX activity induction with Cr and PCBs content in thesediment.
e The third factor, #3, accounts for 17.70% of the variance.The positive value of this factor relates the induction ofEROD activities to the presence of Cr, PCBs and PAHs.
The studied areas are characterized by a heterogeneousmixture of contaminants and conventional parameters suchas organic matter and grain size percentages. A graphical rep-resentation of the estimated factor values corresponding toeach case (sampling site), is presented in order to confirmthe descriptions of these new factors for Ruditapes philippi-narum and Carcinus maenas (Figs. 3 and 4). Values corre-sponding to the clam Ruditapes philippinarum following the
28 day exposure period are described in Fig. 3. Factor 1values indicated the induction of Metallothionein-like-proteinconcentrations and glutathione reductase activities in Hu2and Hu3 in exposed individuals due to the presence of metals(As, Cd, Cr, Fe, Hg, Pb, and Zn). Indeed, these stations arecharacterized by high concentrations of metals, in particularAs, Hg and Zn. Meanwhile, Factor 2 negative values are re-lated to the induction of glutathione S-transferase activity dueto the presence in sediments of a distinct group of metals(Cu, Mn and Ni). Highest values for this factor were foundfor Hu3, Bi3, Ca2 and Ca3. Positive values for Factor 2 relateto the induction of EROD and GPX activities in the portof Pasajes (Pa2 and Pa3), characterized by high concentra-tions of PCBs, Cr and Pb. Finally, Factor 3, represents theinduction of EROD activity in Bi3 due to the presence ofPAHs.
With respect to female crab Carcinus maenas, Factor 1 isrelated to the induction of Metallothionein-like-proteins andglutathione-S-transferase activities in the port of Huelva(Hu2) as well as the port of Bilbao (Bi2). The induction ofthese biomarkers is associated with the presence in sedimentsof metals (As, Cd, Cr, Fe, Hg, Pb and Zn). On the other hand,positive values of Factor 2, demonstrated the induction of Met-allothionein-like-proteins together with glutathione reductaseactivities in Hu3, associated with the metals Cu, Fe, Mn andNi. In relation to the sites Ca2, Bi3, Pa3 and Pa2, Factor 2
Ca2
-2
-1
0
1
2Ca3 Hu2 Hu3
Bi2
FAC
TOR
SC
OR
E
-2
-1
0
1
2Bi3 Pa2 Pa3
F1
F1
F1
F1
F1
F1
F1 F1
F2 F2 F2F2
F2 F2F2 F2
F3 F3 F3 F3
F3
F3
F3 F3
Fig. 3. Representation of Factor scores estimation for each of the 8 cases (Port of Cadiz: Ca2, Ca3; Port of Huelva: Hu2, Hu3; Port of Bilbao: Bi2, Bi3; Port of
Pasajes: Pa2, Pa3) evaluated using the clam Ruditapes philippinarum and after a multivariate analysis approach (MAA) that was applied to the chemical concen-
tration and characteristics of the sediments from the different ports to link with the biological adverse effect determined via biomarker responses.
638 M.L. Martın-Dıaz et al. / Environmental Pollution 151 (2008) 631e640
negative values account for the induction of EROD and GPXactivities due to PCBs and Cr content in sediments. Finally,Factor 3 was associated with the induction of EROD due toPAHs and PCBs presence in Bi2, Bi3, Pa2 and Pa3.
4. Discussion
The use of biomarkers in assessing the toxicity of dredgedmaterial is continuously developing and improvements areneeded. There is a necessity to validate biomarker responsestaking into account: (a) Determination of biomarker responsesin the laboratory and their extrapolation in situ; (b) The spe-cies for testing dredged material; and (c) the link between bio-marker responses and analytical chemistry.
Among the exposure biomarkers tested, the metallothio-nein-like-protein concentrations determined in the hepatopan-creas and digestive gland of crabs and clams respectivelyexhibited significant induction ( p < 0.05) when exposed tosediments from the port of Huelva (Hu2 and Hu3) in the
Ca2
-2
-1
0
1
2 Ca3
Bi2
FAC
TOR
SC
OR
E
-2
-1
0
1
2Bi3
F1
F1
F1
F1
F2 F2
F2F2
F3 F3
F3
F3
Fig. 4. Representation of Factor scores estimation for each of the 8 cases (Port of
Pasajes: Pa2, Pa3) evaluated using the crab Carcinus maenas and after a multivariat
characteristics of the sediments from the different ports to link with the biologica
case of clams and to sediments from the port of Huelva(Hu2 and Hu3) and the port of Bilbao (Bi2 and Bi3) for crabs,in comparison to concentrations shown at the reference site(Ca1). The role of MTLPs in sequestering metals is well estab-lished, while their induction upon exposure to a wide varietyof metals (e.g. Cd, Cu, Zn, Hg, Co, Ni, Bi and Ag) is associ-ated with an exposure protection function (Stegeman et al.,1992). The expression of this protein gene provides evidencethat induction by metals is a direct response to increases inthe intracellular metal concentration, which is mediatedthrough the action of metal-binding regulatory mechanisms(Thiele, 1992). The shore crab Carcinus maenas and theclam Ruditapes philippinarum have been demonstrated tobe useful in metallothionein-like-protein concentration bio-marker studies, having been employed to detect the potentialeffects of metal contamination performed in situ (Bebiannoand Serafim, 2003). In this study, positive values of Factors1 and 2 corresponding to crabs and positive values of Factor1 corresponding to clams relate MTLP induction to metal
Hu2 Hu3
Pa2 Pa3
F1
F1
F1 F1
F2
F2
F2 F2
F3 F3
F3 F3
Cadiz: Ca2, Ca3; Port of Huelva: Hu2, Hu3; Port of Bilbao: Bi2, Bi3; Port of
e analysis approach (MAA) that was applied to the chemical concentration and
l adverse effect determined via biomarker responses.
639M.L. Martın-Dıaz et al. / Environmental Pollution 151 (2008) 631e640
contamination in the sediment for the ports of Huelva andBilbao.
Significant induction of EROD activity ( p < 0.05) was ob-served in both species exposed to sediment containing highconcentrations of PCBs and PAHs. This relationship hasbeen widely reported (e.g. Van der Oost et al., 2003). Theseresults are confirmed by Factors 2 and 3 in clams and crabs,which relate, for each species, the induction of EROD, to ex-posure to sediment from the ports of Pasajes and Bilbao(highly contaminated by organic chemicals). EROD activityis involved in the first phase of metabolism, unmasking or add-ing reactive functional groups, which involve oxidation, reduc-tion or hydrolysis (Goeptar et al., 1995). Its induction is a clearsign of CYP1A1 enzyme activity, which is among the manyknown cytochrome isoforms. Increases in EROD activitieshave been reported in many species of invertebrates, includingclam and crab species, following exposure to organic tracepollutants (Lafontaine et al., 2000). It is suggested thatEROD activity may not only indicate chemical exposure, butmay also precede effects at various levels of biological organi-zation (Whyte et al., 2000).
Glutathione S-transferase activity for each species wasfound to be significantly induced by the presence of variousmetals (As, Cd, Cr, Cu, Fe, Hg, Mn and Pb), in Carcinusmaenas and by Cd, Cu, Mn and Ni in the clam Ruditapes phil-ippinarum. Glutathione transferases are a family of enzymesthat utilize glutathione (GSH) as a substrate in reactions whichpermit the biotransformation and disposal of a wide range ofexogenous compounds (Contreras-Vergara et al., 2004). Thesecompounds may be xenobiotics, drugs or products of oxidativestress, but are mainly polar organic compounds. In comparisonto mammals and insects, marine invertebrate GSTs have beenless well studied, although reports about their induction fol-lowing exposure to toxic chemicals are becoming available.These enzymes are phase II type enzymes and catalyze thesynthetic conjugation reactions of the xenobiotic parent com-pounds and their metabolites, in order to facilitate the excre-tion of chemicals. An increase in hepatic GST activity hasbeen reported in several studies following exposure to PAHsand PCBs. Several studies reported GST activities to be signif-icantly increased because of PAH and PCB exposure, but inmost cases no significant differences were observed betweenthe control and polluted sites (Van der Oost et al., 2003). Itis important to note that Cu and Cr belong to a group of metalsthat are redox-active and are capable of directly generatingfree radicals (Ercal et al., 2001) as opposed to the generationof free radicals following biotransformation reactions leadingto excretion.
Many pollutants (or their metabolites) may elicit toxicityreactions related to oxidative stress. Oxygen toxicity may actas a strong oxidant, capable of reacting with critical cellularmacromolecules, possibly leading to DNA damage and celldeath. Defense systems that tend to inhibit oxyradical forma-tion include the antioxidant enzymes such as glutathione re-ductase (GR) and glutathione peroxidase (GPX).
Significant induction of GPX activity ( p < 0.05) in com-parison to the control was registered in both species exposed
to sediment containing high PCB concentrations. The factoranalysis revealed a significant relationship between inductionof GPX activity on day 28 and the contaminants PCBs, Crand Pb, thereby providing an explanation for the results ob-served in Pasajes (Pa2 and Pa3). GPX induction has been as-sociated with EROD activity in crabs ( p < 0.05).
Glutathione peroxidase is involved in the inhibition of oxy-radical formation in the presence of redox-active compoundssuch as PCBs and PAHs. Increased GPX activity was observedin experiments with fish exposed to PCBs and PAHs (Van derOost et al., 2003); nevertheless more research is required oninvertebrate species.
Glutathione reductase is not always recognized as an anti-oxidant enzyme. It can nevertheless be included in this cate-gory given that it is responsible for oxidized glutathionereduction (GSS G) via a NADPH-dependent process. There-fore, it is essential to the regeneration of reduced GSH, neces-sary for the operation of GPXs and many other cell enzymes(Manduzio et al., 2003). Glutathione reductase enzyme isalso involved in antioxidant defense in the same way as gluta-thione peroxidase. Significant induction was only observed inexposed clams. Significant ( p < 0.05) induction was observedin the clams exposed to sediment from the port of Huelva (Hu2and Hu3) and the port of Pasajes (Pa3). In crabs, significantinduction was observed only in those exposed to sedimentfrom the port of Huelva (Hu3). Glutathione reductase induc-tion has been associated with metal contamination in sedi-ments for both species. Results from studies of this enzymein different species are contradicting. Increased and decreasedactivity have been reported in individuals exposed to PCBsand PAHs (Van der Oost et al., 2003), while little change inactivity has been reported in relation to GR induction andmetal contamination. Although GR responses to pollutantshave seemingly received little attention, this enzyme playsa fundamental role against oxidative stress by maintainingthe redox status of glutathione, this being important in itsdual role as cofactor of several antioxidant enzymes and asan indirect scavenger of oxyradicals (Regoli et al., 2002).
All biomarker responses were significantly correlated( p < 0.001) between species, with the exception of GST andGR activities. These differences may be due to the distinct bio-availability of contaminants in the sediment to species withdifferent feeding habits. When selecting a single species forthe evaluation of sediment or dredged material toxicity, factorssuch as the sensitivity of the species, the life stage tested, itsdegree of phylogenetic and ecological relatedness to receptorsat the disposal site, its preferences and tolerance to the particlesize makeup of the test sediment should be taken into consid-eration among other characteristics. Consideration of thesefactors allows each test species to be established as a surrogatefor organisms living at the disposal site (Munns et al., 2002).
These results demonstrate the induction in situ of a range ofbiomarkers associated with chemicals present in the sediment.Consistent responses were observed for the following bio-markers: metallothionein-like-proteins concentration, ERODand GPX activities. Enzymatic activities however, such asGST and GR, showed contradictory trends.
640 M.L. Martın-Dıaz et al. / Environmental Pollution 151 (2008) 631e640
5. Conclusions
In the present study, the use of a battery of exposure bio-markers has been validated for in situ determination ofdredged material toxicity. The battery of biomarkers proposedhas addressed a potential link between metal and organic sed-iment contamination and bioavailability of contaminants. Theclam Ruditapes philippinarum and the crab Carcinus maenashave been found to be potential bioindicators of sediment con-taminant exposure and no significant differences were ob-served between interspecies biomarker responses with theexception of the biomarkers GST and GR.
The use of in situ toxicity testing procedures coupled withanalytical characteristics of sediment and biomarker responsesin species with distinct feeding habits provides an estimate ofthe potential risk associated with contamination.
Acknowledgments
This research has been funded by the Project of the SpanishGovernment (CTM2005-07282-C03-C01/TECNO). Authorswould like to thank the Port Authorities of Cadiz, Huelva, Pa-sajes and Bilbao. We would also like to thank the members ofAZTI for their support and help during the performance of thisstudy. English in the manuscript has been edited by TomRansome.
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