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Effects of the fuel oil spilled by the Prestige tanker on reproductionparameters of wild mussel populations

Maren Ortiz-Zarragoitia, Larraitz Garmendia, Mar�ıa Carmen Barbero, Teresa Serrano, Ionan Marig�omezand Miren P. Cajaraville*

Received 23rd April 2010, Accepted 2nd August 2010

DOI: 10.1039/c0em00102c

The aim of this work was to assess possible effects of the Prestige oil spill on reproduction parameters of

mussels along the Galician and Bay of Biscay coast. Studied endpoints included sex ratio, gonad

histology and vitellogenin-like proteins using the alkali-labile phosphate (ALP) method. A high

prevalence of haemocytic infiltration of follicles and severe oocyte atresia was found in most localities

in April 2003. Spawning gonads were observed in most impacted populations in the same sampling. In

April 2004 mature small sized follicles were observed. No histopathological changes were observed in

April 2005 and 2006, except a high prevalence of necrotic gametes in 6 out of 22 localities in April 2006.

Female ALP levels showed high interindividual variability in April 2004, which was reduced in April

2005 and 2006. No xenoestrogenic effects were observed in male mussels. Overall, gamete alterations

were detected during 2003–2004 and a recovery trend was observed afterwards.

Introduction

On November 19th, 2002, the oil tanker Prestige broke in half

and sunk in international waters 133 miles from the north

western coast of the Iberian Peninsula. The Prestige transported

77 000 tones of heavy fuel oil no. 2 according to the French

classification (UK classification type no. 6; Russian classification

M-100). More than 60000 tones of fuel oil were spilled affecting

mainly the coast of Galicia on the north west of the Iberian

Peninsula. During the following months spilled oil also affected

the whole northern coast of the Iberian Peninsula reaching up to

Brittany.1,2 The Prestige oil spill (POS) has been considered the

most important ecological disaster ever to occur in the affected

Biologia Zelularra eta Histologia Lab., Zoologia eta Biologia ZelularraSaila, Zientzia eta Teknologia Fakultatea, University of the BasqueCountry UPV/EHU, Sarriena z/g, 48940 Leioa, Basque Country(Spain). E-mail: mirenp.cajaraville@ehu.es; Fax: +34 946013500; Tel:+34 946012697

Environmental impact

Several works have used mussels as sentinel organism for chemical

addressed the impact of oil spills on reproduction-parameters in wil

term (2003–2006) biomonitoring survey studying reproduction-rela

genin-like proteins, in 22 mussel populations from Galicia and the

alterations in gamete development in mussels from the whole studied

applied reproduction-related biomarkers have been demonstrated to

in ongoing worldwide biomonitoring programmes studying mussel

84 | J. Environ. Monit., 2011, 13, 84–94

area.1,3 Severe loss of intertidal invertebrate communities has

been measured on POS affected beaches.4,5

The area impacted by the POS is rich in bivalve production,

Galicia being the most important European producer of mussels.

Mussel production surpasses 200 000 Tn per year and employs more

than 8000 full-time workers in Galicia.6 Mussels have been used

worldwide as sentinel species of environmental pollution in coastal

areas.7,8 Mussels are excellent indicators of contamination levels in

the marine environment as they are sessile and filter feed from

surrounding water. Furthermore, they show a low ability to metab-

olize xenobiotic compounds like polycyclic aromatic hydrocarbons

(PAHs) and accumulate them at high levels.9,10 Mussels are sensitive

species to accidental oil spills as effects on different biomarkers have

been observed after Exxon Valdez in Alaska,11 Aegean Sea in Gali-

cia,12 Sea Empress in Wales,13 Coral Bulker in North Portugal,14

Erika in Brittany15 and the case of Prestige.16,17 Studied biological

endpoints in mussels affected by an oil spill usually show a rapid

recovery in 1 to 2 years after the oil spill.12,13,18 However, long lasting

effects could not be discounted as shown in the case of wildlife

biomonitoring studies after oil spill events, but no studies have

d mussel populations. Here we present for the first time a long-

ted parameters, such as sex ratio, gonad histology and vitello-

Bay of Biscay after the Prestige oil spill. Our results showed

area in 2003 and 2004, but recovered later on (2005–2006). The

be a useful tool for pollution assessment and could be included

s.

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populations affected by the Exxon Valdez oil spill.11 Bivalve pop-

ulations from the Prince William Sound area were not completely

recovered 17 years after the oil spill.19

Studies dealing with reproduction effects after oil spill events are

scarce and most of them focus only on population abundances. It is

well known that complex oil mixtures can provoke deleterious

effects on wildlife reproduction.20 Fish populations inhabiting oil

and PAH polluted environments show endocrine disruption

effects, such as reduced plasma hormonal levels and reduced

reproduction success.21 After the Exxon Valdez oil spill, reduced

estradiol levels in plasma from marine fish species such as dolly

varden, yellowfin solea and pollock were measured. In dolly varden

low levels of gonadotropin-I, a neurohormone responsible for

steroid biosynthesis and gamete development, were also observed

together with delayed gamete development.22 Reduction in repro-

ductive success was observed in pink salmon and Pacific herring

from the Exxon Valdez oil spill area.23,24 Embryos obtained from

both species showed low survival ratios and severe morphological

deformities in the following 4 years after the oil spill.23,24

Most of the few works dealing with reproduction alterations

after oil spills have been carried out in vertebrates, few of them

focusing on invertebrates such as bivalve mollusks. Therefore, it

is important to study the possible effects of the POS on repro-

duction parameters in local mussel populations. The endocrine

system of bivalve mollusks is not well understood, but effects on

reproduction parameters in mollusks have been described after

exposure to environmental contaminants, both in the laboratory

and in the field (see review by Porte et al.).25 Mussels have been

shown to be sensitive to possible endocrine disruption effects

provoked by PAHs.25–27 Female mussels exposed in the labora-

tory during gametogenesis to North Sea oil showed low levels of

vitellogenin-like proteins in comparison to control females and

delayed gamete development accompanied by high percentage of

atretic oocytes, indicating possible anti-estrogenic effects.27

Similarly, high prevalences of atretic germ cells were observed in

mussels exposed to diesel oil and PAH derivatives.28,29 Anti-

estrogenic effects were also observed in field studies using soft

shell clams. Female clams inhabiting a PAH contaminated

harbor showed low vitellogenin-like protein levels and retarded

gamete development.30,31 Although several works have demon-

strated the anti-estrogenic activity of oil and PAHs,21,32 some

authors have observed estrogenic effects provoked by PAHs.33,34

The water soluble fraction of the Prestige fuel oil reduced clam

embryo survival35 but did not alter reproduction ability using

Daphnia magna bioassays.36 Polychaetes exposed to fuel oil no. 2

during the whole life cycle showed accelerated oocyte maturation

and suppression of fecundity.37 Furthermore, mussel seed

collected from POS affected areas showed reduced growth in

comparison to mussel seed from non-affected populations.38

Affected mussel seed populations showed higher levels of

triglycerides and carbohydrates.39,40

In the present work we aimed to investigate possible reproduction

effects after POS in local mussel populations, which could compro-

mise their reproduction ability and survival. For this purpose we

studied vitellogenin-like protein levels as a biomarker of endocrine

disruption. In fish, vitellogenin is a bulky phospholipoglycoprotein

synthesized by the liver of female fish under the regulation of estra-

diol. After being synthesized it is secreted to the blood and trans-

ported to the ovary, where it is endocytosed by growing oocytes.

This journal is ª The Royal Society of Chemistry 2011

Vitellogenin is the precursor protein of vitellins present in the oocytes

to feed the embryos.41 In fish, vitellogenin levels are used as a marker

of xenoestrogenicity in adult males and juvenile organisms.41–44 In

bivalve mollusks, vitellogenin synthesis occurs in the follicular cells

of the female gonad.45,46 Regulation of vitellogenin synthesis is not

well understood and hormonal control by estrogens is under

discussion.47–49 Nevertheless, bivalve mollusks are known to

synthesize steroid hormones50 and a relationship between estradiol

levels and vitellogenesis has been suggested.48 Due to the lack of

specific antibodies, measurement of vitellogenin or vitellogenin-like

protein levels in bivalves has been based on indirect methods, such as

the alkali-labile phosphate (ALP) method.25,51 Thus, we applied

measurement of ALP levels in combination with gonad histology to

study possible reproduction alterations in 22 mussel populations

differently affected by the POS in Galicia and the Bay of Biscay, for

a period of three years (2003–2006).

Materials and methods

Samplings and sample processing

For the purpose of the present work a biomonitoring study was

performed along the North Atlantic coast of the Iberian Penin-

sula. Mussels (Mytilus galloprovincialis) were collected from 22

different locations (Fig. 1) selected on the basis of the severity of

the impact of the oil spill and on the oil arrival conditions to the

coast (fresh spilled oil versus weathered oil). Based on the first

reports after the oil spill2 the impact was most severe in the coast

of Galicia, where we studied 8 locations comprised of highly

impacted sites (Agui~no, Caldebarcos, Camelle and Sega~no) and

less impacted sites (Estaca de Bares, Ons, C�ıes and Oia), all

affected by fresh spilled oil. We studied a further 13 localities in

the Bay of Biscay affected later on by weathered oil. We also

included one sampling point in the Natural Park of North

Littoral of Esposende (Sao Bartolomeu do Mar, Portugal) as

a possible non-impacted site. Samplings were performed in April,

July and September 2003 (17 localities), February 2004

(19 localities), April, July and October 2004 and 2005, and April

2006 (22 localities). At each sampling time 30–60 adult mussels,

3.5–4.5 cm long, were collected from each locality and directly

processed in the field. For histological studies 10–30 mussels

from each site were carefully opened by sectioning the adductor

muscle and directly fixed in 10% neutral buffered formalin. For

the study of vitellogenin-like protein levels, mantle samples

collected during April 2004, 2005 and 2006 were used since this is

the period when mussels at this latitude show the most developed

gonads.52–55 Mussel gonads (mantle tissue) from 20 animals were

dissected out, chilled on ice and a small piece of tissue was

removed to do a smear on a microscope slide. The sex of each

animal was determined after visualization of the smear under the

microscope. Afterwards the rest of the tissue was frozen indi-

vidually in liquid nitrogen. Once in the laboratory they were

stored at �80 �C until subsequent analysis.

Sex ratio, gamete development and histopathology of the gonad

The gonad (mantle tissue) from each mussel was dissected out

after fixation, placed in histological cassettes in 70� ethanol and

routinely processed for paraffin embedding in a Leica ASP300

Automatic tissue processor (Nussloch, Germany). 7 mm thick

J. Environ. Monit., 2011, 13, 84–94 | 85

Fig. 1 Map showing the study area, the location where the Prestige sunk and selected sampling points. In the adjacent table geographical coordinates

for the location of each sampling point are shown.

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sections were cut in a Leitz 1512 microtome (Vienna, Austria)

and stained with hematoxylin and eosin.56 The sex of each animal

was recorded and the total number of female and male mussels

was calculated for each sampling time and location, including the

sex data collected from mussels used for ALP measurements.

With these data, the sex ratio was calculated for each sampling

time and location.

Gamete developmental stages were distinguished in mussel

gonads following the classification of Seed.57 Then, a gonad

index (GI) value was assigned to each developmental stage

adapted from the description of Kim et al.:58 1 resting stage

(inactive or undifferentiated); 1.5 early gametogenic stage

(gametogenesis has begun but no ripe gametes visible);

3.5 advanced gametogenic stage (gametogenesis still progressing

and ripe gametes and developing gametes have about equal

proportions); 5 mature stage (gonad fully mature, follicles full of

ova or sperm); 3.5 spawning stage (active emission of gametes,

some follicles appear empty); 1.5 post-spawning stage (empty

follicles and only residual gametes remain). Afterwards, a mean

GI value for each location and sampling time was calculated.

In samples corresponding to April 2003, 2004, 2005 and 2006

a detailed histopathological study was carried out. Histopatho-

logical alterations such as haemocytic infiltration of follicles

containing mature gametes, severe gamete atresia, necrosis and

abnormal follicle development were scored for each animal and

reported as a percentage of total animals in each group, for males

and females separately.

Terminal transferase dUTP nick end labeling (TUNEL) assay

With the aim of identifying apoptotic gametes in the gonads of

studied mussel populations, we used a commercial TUNEL assay

system (DeadEndTM Colorimetric TUNEL system, Promega

86 | J. Environ. Monit., 2011, 13, 84–94

Corporation, Madison, WI, USA). Apoptotic cells were detected

in non-stained gonad histology sections. The fragmented DNA

of apoptotic cells was detected by incorporating a biotinylated

nucleotide at the 30–OH ends of the DNA fragments using the

enzyme terminal deoxynucleotidyl transferase and then staining

with diaminobenzidine (DAB) in a reaction catalyzed by the

enzyme streptavidin horseradish peroxidase.

Briefly, paraffin-embedded sections (7 mm thick) were depar-

affinized and rehydrated in graded ethanol steps. Then, slides were

washed in a 0.85% NaCl solution for 5 min and subsequently

immersed in phosphate buffered saline (PBS) solution for 5 min.

Sections were fixed in 10% neutral buffered formalin for 15 min

and then washed in PBS for 2 � 5 min. Following the manu-

facturer’s instructions, sections were covered with a 20 mg ml�1

proteinase K solution and incubated for 10–30 min at room

temperature in a wet chamber. Afterwards, sections were washed

in PBS for 5 min, refixed in 10% neutral buffered formalin for

5 min and rinsed in PBS for 2 � 5 min. The excess of liquid was

removed by tapping the slides and sections were covered for 5 min

with the equilibration buffer supplied in the commercial kit. After

carefully removing the excess of liquid, sections were covered with

recombinant terminal deoxynucleotidyl transferase (rTdT) reac-

tion mix and incubated at 37 �C for 60 min. Then, slides were

immersed in SSC buffer for 15 min. After rinsing the slides with

PBS for 2� 5 min, endogenous peroxidase was blocked with 0.3%

hydrogen peroxide solution for 5 min. Slides were washed in PBS

for 2� 5 min and incubated in a solution (1 : 500) of streptavidin

horseradish peroxidase for 30 min. Before reaction visualization,

slides were rinsed in PBS for 2 � 5 min and subsequently incu-

bated in the DAB solution supplied in the kit for 10 min in the

dark. Finally, slides were rinsed in deionized water for 2 � 5 min

and mounted in Kaiser’s glycerol gelatine. Apoptotic nuclei

appeared stained dark brown. Negative control sections were run

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in parallel, where autoclaved deionized water was used instead of

rTdT in the reaction mix. Positive control slides of human pro-

myelocytic leukemia cells (HL60) previously treated with acti-

nomycin to induce apoptosis, were also included.

Alkali-labile phosphate (ALP) method

Levels of vitellogenin-like proteins in mussel gonads were

measured by the ALP method following the protocol described in

Gagn�e et al..59 Briefly, the gonad from each animal was homog-

enized in 25 mM Hepes-NaOH buffer, pH 7.4, containing 125 mM

NaCl, 1 mM dithiothreitol and 1mM EDTA using a glass-teflon

pestle in an ice containing bath with a Braun Potter S homogenizer

(Melsungen, Germany). The obtained homogenate was centri-

fuged at 12 000 g for 30 min at 2 �C in a Beckman Coulter Optima

L-90 K ultracentrifuge (Palo Alto, CA, USA). The obtained

supernatant was carefully removed and an aliquot of 200 ml was

mixed with acetone (35% final acetone concentration) and

centrifuged at 10 000 g for 5 min for the precipitation of proteins.

Another aliquot (75 ml) of the original supernatant was stored at

�80 �C for protein concentration determination. The obtained

pellet was dissolved in 200 ml of 1 M NaOH solution for 30 min at

60 �C in a water-shaking bath. Levels of inorganic free phosphates

were determined using the phosphomolybdenum assay adapted

from Stanton.60 A subsample of 75 ml was mixed with 125 ml tri-

chloroacetic acid, 630 ml ultrapure water, 170 ml molybdenum

reagent (0.02 M ammonium molybdate tetrahydrated and 5.25 M

H2SO4 solution), and 50 ml Fiske-Subbarow reducer (Sigma, St.

Louis, MO, USA). After incubating for 10 min, the absorbance

was measured at 660 nm using a Multiskan Spektrum spectro-

photometer (Thermo Labsystems, Chantilly, VA, USA). For

inorganic phosphate standard curve, a series of KH2PO4

concentrations was used. ALP levels in gonads are given as mg

phosphates/mg protein. Protein concentration was measured

using the DC Protein Assay of Bio-Rad (Hercules, CA, USA)

based on the Lowry method adapted to microtiter plates.61

Statistics

Sex ratio bias was studied using the G test of association,

comparing total number of female and male mussels and

normalizing for theoretical gender bias (1 : 1). Other statistical

analyses were performed using the SPSS 10.0 software (SPSS,

Inc., Chicago, IL, USA). After testing normality (Kolmogorov–

Smirnov’s test) and homogeneity (Levene’s test) of the data, the

effect of factors sampling time and location, as well as their

interaction on ALP levels was studied by two-way analysis of

variance (ANOVA). Statistically significant differences in ALP

levels among groups were studied using one-way ANOVA fol-

lowed by the Duncan’s post-hoc test. In case of GI values, non-

parametric ANOVA (Kruskall–Wallis) was applied followed by

Mann–Whitney’s U test. The interactive effect of both sampling

time and location on GI values was analyzed by Friedman’s test.

Correlation studies between GI and ALP levels were performed

using the Spearman’s correlation index. Differences in percent-

ages of histopathological alterations among groups were studied

by Chi-squared test. Statistical significance was established at

p < 0.05 for all studied tests. Finally, classification studies based

on hierarchical cluster analysis were performed with GI and ALP

This journal is ª The Royal Society of Chemistry 2011

values and a principal component analysis was run to study the

association between studied endpoints and to explain the vari-

ability among sampled populations.

Results

Sex ratio

Sex ratio values for each sampling location at each sampling time

are shown in Table 1. No statistically significant bias in the sex

ratio of the whole studied population (G value ¼ 0.59, p ¼ 0.44)

was detected in comparison with the theoretical sex ratio in

mussel populations (1 : 1). The observed sex ratio was 1 : 1.02

(female : male) for the total of three years of study. When the sex

ratio was analyzed separately for each sampling time, a statisti-

cally significant bias to more males was observed in September

2003 (G value ¼ 27.98; p < 0.01), July 2004 (G value ¼ 4.14,

p < 0.05) and October 2004 (G value ¼ 6.86, p < 0.05). On the

other hand, in April 2004 significantly more females were

observed (G value ¼ 7.09, p < 0.05). Among studied locations

only C�ıes (G value ¼ 7.77, p < 0.05) showed a significant bias to

females (1.7 : 1). Finally, we found two hermaphrodite animals,

one in Mutriku (April 2005) and the other in Ons (July 2005).

They both showed separate male and female follicles in the

mantle tissue (see Fig. 2c below).

Gamete development and gonad index

Microscopical observation of the gonad showed differences in

gamete development patterns among stations (Fig. 3). Further-

more, Friedman’s test showed that both sampling time and

location had significant effects on gamete development (c2 ¼2495.48, p < 0.05). At the beginning of the present study in April

2003, most mussel populations showed mature gonads, with the

exceptions of mussels from Oia, Estaca de Bares and San Vicente

that showed a high number of animals in advanced gametogen-

esis. It is interesting to note that mussels from Agui~no and Cal-

debarcos, located close to the most impacted area, showed

gonads mainly at spawning stage in April 2003. In July 2003,

mussels from all locations were at more advanced developmental

stages than in April 2003, except for mussels from Llanes, which

remained in the mature stage. In most locations, some mussels

were at the post-spawning stage and in Camelle and Suances the

beginning of a second gametogenesis cycle could be observed. In

September 2003, the gonad of most mussels was at a post-

spawning stage and mussels from most locations had started the

next reproductive cycle.

In February 2004 most mussel populations showed gonads in

advanced gametogenesis or in mature phases, with the exception

of mussels from Suances where all mussels showed early game-

togenic gonads (Fig. 3). In April 2004, mussels from most loca-

tions were spawning and some had reached the post-spawning

stage. However, most mussels from Caldebarcos, Estaca de

Bares, Pedre~na and all in Muskiz showed mature gonads (Fig. 3).

In July 2004, spawning but also mature gonads were predomi-

nant in studied mussel populations, indicating the possibility of

a second gametogenic cycle in some mussels of 16 out of 22

locations. Only mussels from Ons, C�ıes, Caldebarcos, Camelle

and Muskiz did not show this process. In October 2004, most

J. Environ. Monit., 2011, 13, 84–94 | 87

Table 1 Detailed description of sex ratio (female to male; F : M) data in studied mussel populations. — indicates no samples

Sampling month

2003 2004 2005 2006

TOTALApril July September February April July October April July October April

Sao Bartolomeu do Mar — — — — 1 : 1.1 1 : 2.3 1 : 9 1 : 1.1 1 : 2.3 1 : 2.3 1.5 : 1 1 : 1.4Ons — — — — 1 : 2.5 9 : 1 1 : 1.5 1 : 1.8 1 : 3.5 1 : 1 2.7 : 1 1 : 1C�ıes — — — — 1.3 : 1 1 : 1 9 : 1 1.1 : 1 1 : 1 4 : 1 2.6 : 1 1.7 : 1Oia 1 : 1 1 : 2.3 1 : 9 2 : 1 1.7 : 1 1 : 1 1.5 : 1 2 : 1 1 : 2.3 3 : 1 1 : 1.1 1.3 : 1Agui~no 1 : 4 1 : 1.5 1 : 2.3 1 : 1 1 : 1.8 1 : 1 1 : 2.3 1 : 1 1 : 1.5 2.3 : 1 1.5 : 1 1 : 1.1Caldebarcos 1 : 2.3 1 : 1.5 1 : 4 1.5 : 1 1.1 : 1 1 : 2.3 2.3 : 1 1 : 1 1 : 1.5 1 : 1 1.7 : 1 1.1 : 1Camelle 1 : 2.3 1.5 : 1 1 : 1.5 1.5 : 1 1.6 : 1 1.5 : 1 1 : 1.5 1.1 : 1 1 : 1.5 2.3 : 1 1 : 1.1 1.2 : 1Sega~no 1 : 9 1 : 1.5 1 : 1 1 : 1 1.1 : 1 1 : 1.5 1 : 1.5 1.9 : 1 1 : 1.5 1.5 : 1 1.9 : 1 1.3 : 1Estaca de Bares 2.3 : 1 1.5 : 1 1 : 9 4 : 1 1.4 : 1 1 : 1 1 : 1.5 1 : 1.3 1.5 : 1 1 : 1.5 1.1 : 1 1 : 1Llanes 2.3 : 1 2.3 : 1 1 : 1 1.5 : 1 3.3 : 1 1 : 1 1 : 2.3 1 : 1.5 1 : 1 1.5 : 1 1 : 1.3 1.1 : 1San Vicente 1 : 1 4 : 1 1 : 4 1 : 1 1.7 : 1 1 : 4 1 : 1 1.3 : 1 1 : 1 1.5 : 1 1 : 1.3 1.1 : 1Suances 1.5 : 1 1 : 1.5 1 : 9 1 : 1.5 1 : 1.3 1 : 1 1 : 2.3 1 : 1.1 1 : 1 1 : 1 1.3 : 1 1 : 1.1Pedre~na 1 : 2.3 1 : 1 2.3 : 1 1.5 : 1 1 : 1.3 1 : 4 1 : 4 1 : 1.1 2.3 : 1 1 : 9 1.7 : 1 1 : 1.2Laredo 1.5 : 1 1 : 2.3 1 : 9 1 : 1.5 1.1 : 1 1 : 1 1 : 2.3 1 : 1.6 1.5 : 1 1 : 1 1.1 : 1 1 : 1.1Muskiz 1 : 4 1 : 1.5 1 : 9 1 : 1 1.1 : 1 1 : 1 2.3 : 1 1.1 : 1 1 : 1 1 : 1.5 1 : 1.1 1 : 1Arrigunaga 1.5 : 1 1 : 9 1 : 4 1.5 : 1 1 : 1.2 1 : 1.5 1 : 4 1 : 1.1 1 : 1 1 : 1.5 1 : 1.3 1 : 1.3Gorliz 1.5 : 1 1 : 1 1 : 1 1 : 1.5 1.5 : 1 1 : 1.5 1 : 1.5 1.5 : 1 1 : 2.3 1 : 4 1.1 : 1 1 : 1Bakio — — — 1 : 1 1.1 : 1 2.3 : 1 1 : 1.5 1.3 : 1 1 : 2.3 1 : 1.5 1 : 1.1 1 : 1Mundaka 2.3 : 1 1 : 1 1 : 1.5 1.2 : 1 1.1 : 1 1 : 2.3 1.5 : 1 1.7 : 1 1 : 1 1.5 : 1 1 : 1.3 1.2 : 1Mutriku — — — 2.3 : 1 1.7 : 1 1 : 1 1 : 1 1.2 : 1 1.5 : 1 1 : 1 1 : 1.3 1.3 : 1Orio 1 : 4 1.5 : 1 1 : 1.5 1 : 1 1.5 : 1 1 : 9 1 : 4 1 : 1.3 1 : 2.3 1 : 1.5 1 : 1.4 1 : 1.2Hondarribia 1 : 1.5 1 : 1 1 : 1.5 2.3 : 1 1.7 : 1 1 : 1 1 : 2.3 1 : 1.1 1 : 1 1.5 : 1 1.1 : 1 1.1 : 1

TOTAL 1 : 1.3 1 : 1.1 1 : 2.3 1.3 : 1 1.2 : 1 1 : 1.3 1 : 1.5 1.1 : 1 1 : 1.1 1 : 1 1.1 : 1 1 : 1.02

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mussel populations showed gonads in resting and post-spawning

stages.

In April 2005, the predominant stage was the mature phase

and in some stations mussels at spawning stage were also

observed, mainly in Llanes and San Vicente (Fig. 3). In July 2005

most mussels showed spawning and post-spawning gonad stages,

except mussels from Estaca de Bares and Llanes where all indi-

viduals were still at mature stage. Some localities, such as

Camelle, Sega~no and Gorliz showed the start of a second

gametogenic cycle. In October 2005 all studied mussel pop-

ulations showed gonads mainly at post-spawning and resting

stages. Some locations, such as Ons, Caldebarcos, Sega~no,

Pedre~na, Muskiz, Arrigunaga, Mundaka and Hondarribia had

already started the new reproductive cycle.

In April 2006, most mussel populations showed gonads in

mature and spawning stages, except those from Suances and

Mutriku where all mussels were spawning (Fig. 3). Some few

mussels from Agui~no, Arrigunaga and Gorliz were at post-

spawning stage. Overall, most studied mussel populations

showed one reproductive cycle in 2003 and 2005, and seemingly

two reproductive cycles in 2004.

A hierarchical cluster analysis of GI values showed two main

branches separating samplings of February, April and July (mainly

gonads in mature and spawning stages) from those of September

and October (gonads mainly at post-spawning or resting and early

gametogenesis stages) (Fig. 4a). Furthermore, the dendrogram

shows that data from samplings performed in April 2003, 2005 and

2006 are closely related but April 2004 was more related with

February 2004 and July 2003, 2004 and 2005. On the other hand, no

significant discrimination between most oil impacted and less

impacted localities was observed (data not shown).

88 | J. Environ. Monit., 2011, 13, 84–94

Gonad histopathology

The histopathological study of the gonad (Table 2) showed

a high prevalence of oocyte atresia in female mussels in

April 2003 (Fig. 2d), together with a high prevalence of haemo-

cytic infiltration of gonad follicles (Fig. 2e). In April of the

following year 2004 the atresia disappeared but widespread small

sized mature follicles were observed in most mussel populations

(Table 2). Both male and female mussels showed small follicles

containing developing and full developed gametes, together with

follicles of larger size with no gametes and surrounded by a high

amount of reserve material (Fig. 2f). Overall, relative volume of

gonad follicles was small compared with the rest of the speci-

mens. Haemocytic infiltration of follicles was present in several

stations. In April 2005, few histopathological alterations were

observed. In females, atresia was scarce and low levels of infil-

tration of follicles by haemocytes were observed (Table 2).

However, in April 2006 a high prevalence of abnormal gametes

was observed in female mussels from six out of 22 studied pop-

ulations (Table 2). Some abnormal gametes showed dispersed

cytoplasm in the form of eosinophilic spheres (Fig. 2g). Other

abnormal gametes were characterized by higher staining inten-

sity than normal gametes. They progressively loose structure

(nuclei and cytoplasm became indistinguishable) and finally they

were released to the follicle lumen. Both types of abnormal

gametes were studied by TUNEL assay to establish whether they

appear as a result of apoptotic events. Whereas positive nuclei

were evidenced in the positive control slides containing apoptotic

HL60 cells, no positive nuclei were detected in studied mussel

gonad sections indicating that abnormal gametes could be orig-

inated by degenerative processes such as necrosis and not by

This journal is ª The Royal Society of Chemistry 2011

Fig. 2 Micrographs showing gonad sections stained with hematoxylin/

eosin from studied mussels. (a) Male gonad section from Caldebarcos

(April 2004) showing advanced gametogenic stage. (b) Female gonad

section from Pedre~na (April 2004) showing mature stage. (c) Hermaph-

roditic gonad section from Mutriku (April 2005). Separate male and

female follicles are observed. (d) Female gonad section from Mundaka

(April 2003) showing widespread gamete atresia. (e) Female gonad

section from Muskiz (April 2003) showing severe haemocytic infiltration

(asterisks) of mature follicles. (f) Female gonad section from Suances

(April 2004) showing small sized follicles with mature gametes at

spawning stage. (g) Female gonad section from C�ıes (April 2006) showing

necrotic oocytes (arrows) with dispersed cytoplasm in the form of

eosinophilic spheres. (h) Gonad section showing trematode parasite

infestation of gonad follicles causing castration of a mussel from

S. Bartolomeu do Mar (April 2005). Scale bars in (a), (b), (d), (e), (f), (g)

and (h) represent 100 mm and in (c) represents 200 mm.

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apoptotic events. In addition, in April 2006 haemocytic infiltra-

tion of gonad follicles was widespread among most studied

populations, being again female mussels more affected than male

mussels.

Parasite prevalence during the whole study period was very

low in the gonads. Only 4 individuals showed trematode infes-

tation of gonad follicles, which provoked castration of infected

mussels (Fig. 2h).

Statistical analysis of histopathology data showed that sex

dependent differences occurred, female mussels showing higher

prevalence of histopathologies than male mussels. Females

showed statistically significant higher prevalence of haemocytic

This journal is ª The Royal Society of Chemistry 2011

infiltration of follicles than male mussels in April 2003 (c2 ¼20.73; p < 0.05) and April 2006 (c2 ¼ 6.13; p < 0.05). In April

2004, small sized follicle prevalence was higher in female mussels

than in male mussels (c2 ¼ 12.48; p < 0.05). Regarding station

dependent effects, we did not observe any significant differences

among studied stations. Only mussels from Arrigunaga, both

female and male, showed significantly higher prevalence levels

for all studied pathologies. As to possible statistically significant

differences between sampling years, in the case of female mussels

prevalence of oocyte atresia was significantly higher in 2003 than

in other years (c2 ¼ 38.85, p < 0.05). Similarly, prevalence of

oocyte atresia was higher in 2005 (c2 ¼ 8.33, p < 0.05) and 2006

(c2 ¼ 9.42, p < 0.05) than in 2004. Finally, abnormal gamete

prevalence was higher in 2006 (c2 ¼ 5.67; p < 0.05) than in other

studied years.

ALP levels

Levels of vitellogenin-like proteins in mussel gonads measured

as ALP levels are shown in Fig. 5. Female mussels showed

significantly higher ALP values than male mussels in the three

samplings (Fig. 5), in agreement with the female-specific

synthesis of vitellogenin-like proteins. In female mussels, ALP

levels in April 2004 were highly variable among stations and

inside each population (Fig. 5). Accordingly, ALP levels and

GI values were not significantly correlated during April 2004

sampling (Spearman’s r2 ¼ 0.339). In April 2005, differences

among stations in ALP levels of female mussels were reduced in

comparison with April 2004 (Fig. 5), which was related with

a more homogeneous gamete development observed during

April 2005, most females being at mature gonad stage (Fig. 5).

ALP levels and GI values were significantly correlated (Spear-

man’s r2 ¼ 0.557) in female mussels in the April 2005 sampling.

In April 2006, ALP values in female mussels were overall lower

than in 2005 but more homogeneous within each location

(Fig. 5). These lower values were related with gonads mainly at

spawning stage observed in April 2006 (Fig. 3). The association

between ALP and GI values was confirmed by a significant

correlation (Spearman’s r2 ¼ 0.773), which was stronger than in

April 2005. A hierarchical cluster analysis of ALP levels in

female mussels showed that April 2005 and 2006 were closely

associated between them and separated from April 2004

(Fig. 4b). As in the case of GI values, no discrimination

between most oil impacted locations and less impacted loca-

tions was obtained (data not shown).

In the case of male mussels, no differences in gonad ALP levels

between sampling localities or sampling years were observed

(Fig. 5). No correlation between ALP levels and GI values was

obtained at any sampling time for male mussels.

Principal component analysis

Principal component analysis was performed with data on GI,

ALP and histopathology obtained in April 2004, 2005 and

2006. More than 70% of the variability was explained by two

components (Fig. 4c). The first component was highly asso-

ciated with GI (association coefficient: �0.904) and ALP

levels (association coefficient: �0.754) and separated samples

of 2005 and 2006. The second component showed high

J. Environ. Monit., 2011, 13, 84–94 | 89

Fig. 3 Graphs showing gamete development and gonad index from each station at each sampling time. Stacking-bar graphs show the percentage of

individuals at each gametogenic stage at each sampling time. Superimposed continuous lines represent gonad index values for each sampling point at

each sampling month. F: February; A: April; J: July; S: September; O: October.

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association with small sized follicle prevalence (association

coefficient: �0.696) and haemocytic infiltration of gonad

follicles (association coefficient: 0.654) and separated samples

of 2004 from those of 2005 and 2006. Therefore, variability in

90 | J. Environ. Monit., 2011, 13, 84–94

the spatial distribution of studied samples was based on

gamete development (GI and ALP levels) and observed

histopathologies such as small sized follicles and haemocytic

infiltration.

This journal is ª The Royal Society of Chemistry 2011

Fig. 4 Statistical classification analysis of studied endpoints. Dendro-

grams showing results of the hierarchical cluster analysis for GI values

(a) and ALP levels (b) at each sampling time following the squared

Euclidean distance between groups. Principal component analysis (PCA)

of all studied endpoints for each sampling point at each sampling time is

shown in (c). Circle groups mussel populations belonging to April 2005

and 2006 samplings. PC1: principal component 1. PC2: principal

component 2.

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Discussion

In the present work we investigated the effects caused by the POS

on reproduction of wild mussel populations from Galicia and the

Bay of Biscay, which were differently impacted by the oil spill.62

Most alterations occurred during 2003 and 2004 and disappeared

later on.

There were differences in gamete development from year to year

but not between sexes. The present results demonstrated that in

studied mussel populations gametogenesis proceeded during winter

months and the active reproductive period occurred in spring

months. These observations agree with reproductive cycles

described before by other authors for the north western Iberian

Peninsula and in the Bay of Biscay for mussel populations.52–55,63

During 2003 and 2005 there was mainly one reproductive cycle but

in 2004 two reproductive peaks appeared to occur. These differences

This journal is ª The Royal Society of Chemistry 2011

in reproductive output from year to year are common to mussel

populations worldwide. Gamete development and reproduction are

controlled by biotic and abiotic factors such as food availability,

temperature and salinity, which are highly variable.53,63–65

Alterations in sex ratios have been observed in freshwater

mussels inhabiting rivers polluted with endocrine disruptors.66

No significant bias on sex ratio was measured in the present

study, and the resulting ratio close to 1 : 1 was as previously

reported53,67 except in C�ıes. Only two hermaphrodite animals

were detected during the whole study. This is in accordance with

previous data reported by Villalba53 on the north western coast

of Spain. An increase in hermaphroditism has been reported in

bivalves inhabiting highly polluted environments67 and in

mussels from the locality of Txatxarramendi in the Biosphere’s

Reserve of Urdaibai.69 The histopathological study of mussel

gonads in April showed a high prevalence of atresia in female

mussels in 2003. This could lead to a possible decrease in gamete

quality in affected female mussels. A high prevalence of atretic

gametes has been observed in mussels exposed to diesel oil and

PAH derivatives,28,29,70 to North Sea oil and to a mixture of oil,

alkylphenols and PAHs similar to produced water released by

offshore oil-extraction platforms.27 However, in the following

year 2004, no atresia was observed but a striking observation was

the reduced size of follicles both in male and female mussels.

These small follicles with mature gametes have been described

before by Villalba53 in mussels from Galicia. This author used the

term ‘‘imperfect ripeness’’ to describe this process, but the reason

and possible effects on mussel reproductive ability are

unknown.53 It is unclear whether the presence of small sized

follicles in 2004 could be related to the two reproductive peaks

found in the same year.

In female mussels, ALP levels in April 2004 were highly vari-

able among stations, while in April 2005 and 2006 the variability

among groups was reduced and values were more homogeneous.

In male mussels ALP levels were always low, suggesting that the

fuel oil spilled by the Prestige did not cause xenoestrogenic

effects. In female mussels, ALP levels and gamete development

(gonad index) were positively correlated in April 2005 and 2006,

as it could be expected based on the seasonal variation of vitel-

logenin-like proteins and oocyte growth.47,69,71 This correlation

was not found in April 2004, indicating that natural environ-

mental factors were not the only factors controlling observed

differences in reproductive cycle and that a possible alteration in

mussel reproduction occurred during 2004. Similarly, mussels

collected in Galicia during the winter of 2003–2004 lost seasonal

variability of nitric oxide synthesis in haemocytes.72 This could

indicate altered endocrine regulation and reproduction as nitric

oxide is a signaling molecule controlled by estradiol in mussels

and participates in the immunoresponse and reproduction.73,74

Effects on gamete development and reduction in ALP levels in

female mussel gonads have been described after exposure to

0.5 ppm North Sea oil for 3 weeks.27 Intermoult female crabs

Carcinus maenas exposed for 28 days to sediments obtained from

POS affected areas showed a trend to reduced vitellogenin

levels.75 Low ALP levels have been measured in bivalve pop-

ulations inhabiting PAH contaminated sites30 together with

delayed gametogenesis.31 Delayed gametogenesis was also

observed in clams feed on PAH contaminated algae and exposed

throught sediments76 and in mussels exposed for three months to

J. Environ. Monit., 2011, 13, 84–94 | 91

Table 2 Histopathology observed in the gonad of studied mussel populations. Data are indicated as percentage of individuals showing each pathology.— indicates no data

Histopathology

Haemocytic infiltrations Small sized follicles Oocyte atresia Abnormal gametes

Females 2003 2004 2005 2006 2003 2004 2005 2006 2003 2004 2005 2006 2003 2004 2005 2006

Sao Bartolomeu do Mar — 60 0 0 — 70 0 0 — 0 0 12 — 0 0 0Ons — 50 0 40 — 25 0 0 — 0 0 40 — 0 0 20C�ıes — 20 0 30 — 0 40 0 — 0 0 0 — 40 0 28Oia 60 0 0 0 0 100 0 0 100 0 0 0 0 0 0 0Agui~no 0 0 0 20 0 100 0 0 0 0 0 40 0 0 0 0Caldebarcos 0 0 0 20 0 0 0 0 0 0 0 25 0 0 0 0Camelle 0 0 0 100 0 0 0 0 0 0 20 30 0 0 0 0Sega~no 100 0 20 30 0 16 0 0 100 0 20 14 0 0 20 0Estaca de Bares 30 20 0 20 0 0 0 0 60 0 50 0 0 0 0 0Llanes 15 10 25 16 0 44 25 0 30 0 0 0 0 0 0 0San Vicente 0 12 25 0 0 37 0 0 0 0 50 0 0 12 0 0Suances 50 0 33 50 0 80 33 0 0 0 33 0 0 0 0 66Pedre~na 70 0 25 0 0 0 0 0 70 0 25 0 0 0 0 0Laredo 50 100 25 28 0 75 0 0 100 0 0 0 0 0 0 0Muskiz 100 50 0 20 0 0 0 0 100 0 25 0 0 0 0 60Arrigunaga 0 100 65 100 0 100 0 0 100 0 0 0 0 0 0 25Gorliz 0 0 0 20 0 50 0 0 100 0 0 0 0 0 0 25Bakio — 20 0 0 — 100 0 0 — 0 0 0 — 0 16 0Mundaka 30 0 14 0 0 80 0 0 100 0 14 0 0 0 0 0Mutriku — 0 0 33 — 40 0 0 — 0 0 0 — 0 0 0Orio 0 0 0 66 0 0 0 0 0 0 0 33 0 0 0 0Hondarribia 25 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0

Histopathology

Haemocytic infiltrations Small sized follicles

Males 2003 2004 2005 2006 2003 2004 2005 2006

Sao Bartolomeu do Mar — 0 0 0 — 33 37 0Ons — 16 0 20 — 50 0 0C�ıes — 0 0 0 — 20 0 0Oia 0 25 0 0 0 50 0 0Agui~no 0 0 0 0 0 50 0 0Caldebarcos 0 0 0 12 0 0 0 0Camelle 0 0 0 0 0 0 0 0Sega~no 0 0 0 0 0 0 0 0Estaca de Bares 0 0 0 0 0 0 0 0Llanes 0 0 0 0 0 0 0 0San Vicente 0 0 0 0 0 50 0 0Suances 50 60 14 25 0 40 0 0Pedre~na 15 16 0 0 0 0 0 0Laredo 0 0 0 33 0 0 0 0Muskiz 12 20 0 0 0 0 0 0Arrigunaga 25 60 50 67 0 80 0 0Gorliz 0 0 0 0 0 50 0 0Bakio — 0 0 0 — 0 0 0Mundaka 0 0 0 0 0 0 0 0Mutriku — 0 20 25 — 0 0 0Orio 0 0 50 66 0 0 0 0Hondarribia 0 60 70 40 0 0 0 0

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low doses of the water accommodated fraction of three crude

oils.77 However, accelerated spawning occurred at high oil

exposure doses,77 similar to that observed in stations severely

impacted by the POS (Agui~no and Caldebarcos) in April 2003.

According to biomarker analysis in mussel digestive gland,

mussel health started to recover after the POS at the end of

2004.18 The results of the present work also indicate no

significant gamete alterations in 2005 and 2006, but long-lasting

effects on mussel reproduction ability, embryo development

92 | J. Environ. Monit., 2011, 13, 84–94

and growth can not be discarded. Blue mussels exposed to

North Sea oil during the whole gametogenic cycle showed early

gamete atresia and reduced fertility. Obtained larvae showed

delayed development and severe abnormalities.78 Similarly,

scallops (Mizuhopecten yessoensis) from polluted areas in the

Sea of Japan showed reduced fertilization, delayed larval

development and increased larval abnormalities,68 as shown in

oysters (Crassostrea virginica) and Dwarf surfclams (Mulinia

lateralis) exposed to water soluble fractions of three crude oils79

This journal is ª The Royal Society of Chemistry 2011

Fig. 5 Graphs showing ALP levels in the gonads of mussels in studied

April months (2004, 2005 and 2006). Bars represent ALP levels (mean �standard deviation) in gonads of female mussels. Matrices at the top of

each graph represent results after ANOVA analysis of data from female

mussels. Localities belonging to the same statistical group are marked

with a black dot. As no significant differences were observed between

male mussels, the mean value at each sampling is represented with a black

line for comparison purposes.

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and in mussel embryos exposed to different PAHs.80 Seawater

samples collected in December 2002 from Galicia after POS

were embryotoxic to oyster (Crassostrea gigas) and clam

(Venerupis pullastra).35 A reduction in survival ability has been

This journal is ª The Royal Society of Chemistry 2011

described in mussel seeds collected in February 2003 in areas

highly impacted by the POS.39 Mussel seeds affected by POS

showed lower growth than non-impacted or less impacted

mussels.38 Therefore, long-lasting effects on affected pop-

ulations can not be ruled out.

Conclusions

Mussel populations appeared to be affected in the whole studied

area and most alterations in studied endpoints were observed

after 1–2 years of POS. Thus, high prevalences of oocyte atresia

and haemocytic infiltration were found in most localities in 2003

whereas small sized follicles and very variable ALP levels were

observed in 2004. Gamete alterations disappeared in 2005, but in

2006 a high prevalence of necrotic gametes was found at six

localities, which deserves further research. In agreement, cluster

analysis and PCA clearly distinguished mussels sampled in April

2004 from those sampled in April 2005 and April 2006. PAH

accumulation levels in the same mussel populations were high in

Galicia and Bay of Biscay during 2003 and 2004, and returned to

pre-spill levels in 2005 and 2006.62,81,82 At present, the meaning

and relevance of gamete alterations found in 2003–2004 are not

known. Gamete atresia accompanied by haemocytic infiltration

observed in most localities in 2003 could be associated with an

increased energy consumption and demand to cope with detox-

ification and defense metabolism.

Acknowledgements

This work was supported by the Spanish Ministry of Science and

Technology through ‘‘Urgent Research Actions on Coastal

areas’’ and project PRESTEPSE (VEM2003-20082-CO6-01) and

by the Basque Government through ETORTEK actions

(IMPRES) and through a grant to Consolidated Research

Groups (Ref: GIC07/26-IT-393-07).

Notes and references

1 J. Albaig�es, B. Morales-Nin and F. Vilas, Mar. Pollut. Bull., 2006, 53,205.

2 M. Gonz�alez, A. Uriarte, R. Pozo and M. Collins, Mar. Pollut. Bull.,2006, 53, 369.

3 A. Del Valls, Ciencias Marinas, 2003, 29, i.4 R. De la Huz, M. Lastra, J. Junio, C. Castellanos and J. M. Vi�etez,

Estuarine, Coastal Shelf Sci., 2005, 65, 19.5 J. Junoy, C. Castellanos, J. M. Vi�eitez, M. R. de la Huz and

M. Lastra, Mar. Pollut. Bull., 2005, 50, 526.6 G. Caballero-Miguez, M. D. Garza-Gil and M. M. Varela-Lafuente,

Mar. Policy, 2009, 33, 288.7 E. D. Goldberg, Mar. Pollut. Bull., 1975, 6, 111.8 M. P. Cajaraville, M. J. Bebianno, J. Blasco, C. Porte, C. Sarasquete

and A. Viarengo, Sci. Total Environ., 2000, 247, 295.9 J. Oehlmann, U. Schulte-Oehlmann, 2002. Molluscs as bioindicators.

In: Markert, B. A., Breure, A. M., Zechmeister, H. G. (ed.),Bioindicators and biomonitorings. Elsevier, New York, USA, pp. 577.

10 R. Smolders, L. Bervoets, V. Wepener and R. Blust, Hum. Ecol. RiskAssess., 2003, 9, 741.

11 C. H. Peterson, S. D. Rice, J. W. Short, D. Esler, J. Bodkin,B. E. Ballachey and D. B. Irons, Science, 2003, 302, 2082.

12 C. Porte, X. Biosca, M. Sol�e and J. Albaig�es, Biomarkers, 2000, 5, 436.13 R. J. Law, J. E. Thain, M. F. Kirby, Y. T. Allen, B. P. Lyons,

C. A. Kelly, S. Haworth, E. A. Dyrynda, P. E. J. Dyrynda,J. S. Harvey, S. Page, M. D. Nicholson, D. R. P. Leonard, 1998.The impact of the Sea Empress oil spill on fish and shellfish. In:Edwards, R., Sime, H. (ed.), The Sea Empress Oil Spill: Proceedings

J. Environ. Monit., 2011, 13, 84–94 | 93

Publ

ishe

d on

29

Oct

ober

201

0. D

ownl

oade

d on

13/

01/2

014

15:2

5:06

.

View Article Online

of the International Conference held in Cardiff, 11–13 February 1998,Chartered Institute of Water and Environmental Management,London, UK, pp. 109–136.

14 S. M. Moreira, M. Moreira-Santos, R. Ribeiro and L. Guilhermino,Ecotoxicology, 2004, 13, 619.

15 G. Boquen�e, S. Chantereau, C. Cl�erendeau, E. Beausir, D. M�enard,B. Raffin, C. Minier, T. Burgeot, A. P. Leszkowicz andJ. F. Narbonne, Aquat. Living Resour., 2004, 17, 309.

16 I. Marig�omez, M. Soto, C. Cancio, A. Orbea, L. Garmendia andM. P. Cajaraville, Mar. Pollut. Bull., 2006, 53, 287.

17 A. Orbea, L. Garmendia, I. Marig�omez and M. P. Cajaraville, Mar.Ecol.: Prog. Ser., 2006, 306, 177.

18 M. P. Cajaraville, L. Garmendia, A. Orbea, R. Werding, A. G�omez-Mendikute, U. Izagirre, M. Soto and I. Marig�omez, Mar. Environ.Res., 2006, 62, S337.

19 Exxon Valdez Oil Spill Trustee Council, 2006. Exxon Valdez Oil SpillRestoration Plan. Update on injured resources and services, 2006,Anchorage, AK, USA. pp. 42.

20 WHO/IPCS (World Health Organization/International Progamme onChemical Safety), Global assessment of the state-of-the-science ofendocrine disruptors, 2002, WHO/IPCS/EDC/02.2, Geneva,Switzerland.

21 P. Matthiessen, Pure Appl. Chem., 2003, 75, 2197.22 S. Y. Sol, L. L. Johnson, B. H. Horness and T. K. Collier, Mar. Pollut.

Bull., 2000, 40, 1139.23 S. D. Rice, R. E. Thomas, R. A. Heintz, A. C. Wertheimer,

M. L. Murphy, M. G. Carls, J. W. Short, A. Moles, Synthesis oflong-term impacts to pink salmon following the Exxon Valdez oilspill: persistence, toxicity, sensitivity, and controversy. Final Reportproject 99329, 2001,pp. 77.

24 M. G. Carls, G. D. Marty and J. E. Hose, Can. J. Fish. Aquat. Sci.,2002, 59, 153.

25 C. Porte, G. Janer, L. C. Lorusso, M. Ortiz-Zarragoitia,M. P. Cajaraville, C. Fossi and L. Canesi, Comp. Biochem. Physiol.Part C, 2006, 143, 303.

26 J. Lintelmann, A. Katayama, N. Kurihara, L. Shore and A. Wenzel,Pure Appl. Chem., 2003, 75, 631.

27 M. Ortiz-Zarragoitia and M. P. Cajaraville, Arch. Environ. Contam.Toxicol., 2006, 50, 361.

28 D. M. Lowe, Mar. Ecol. Res., 1988, 22, 243.29 D. Lowe and R. K. Pipe, Mar. Environ. Res., 1987, 22, 243.30 F. Gagn�e, C. Blaise, J. Pellerin and S. Gauthier-Clerc, Mar. Environ.

Res., 2002, 53, 295.31 S. Gauthier-Clerc, J. Pellerin, C. Blaise and F. Gagn�e, Comp.

Biochem. Physiol., Part C, 2002, 131, 457.32 J. M. Navas and H. Segner, Aquat. Toxicol., 2000, 51, 79.33 J. M. Nicolas, Aquat. Toxicol., 1999, 45, 77.34 M. M. H. Van Lipzig, N. P. E. Vermeulen, R. Gusinu, J. Legler,

H. Frank, A. Seidel and J. H. N. Meerman, Environ. Toxicol.Pharmacol., 2005, 19, 41.

35 R. Beiras and L. Saco-Alvarez, Water, Air, Soil Pollut., 2006, 177,457.

36 J. M. Navas, M. Bab�ın, S. Casado, C. Fern�andez and J. V. Tarazona,Mar. Environ. Res., 2006, 62, S352.

37 S. S. Rossi and J. W. Anderson, Water, Air, Soil Pollut., 1978, 9, 155.38 L. G. Peteiro, J. M. F. Babarro, U. Labarta and M. J. Fern�andez-

Reiriz, ICES J. Mar. Sci., 2006, 63, 1005.39 U. Labarta, M. J. Fern�andez-Reiriz, J. L. Garrido,

J. M. F. Babarro, J. M. Bayona and J. Albaig�es, Mar. Ecol.:Prog. Ser., 2005, 302, 135.

40 L. G. Peteiro, U. Labarta and M. J. Fern�andez-Reiriz, Comp.Biochem. Physiol. Part C, 2007, 145, 588.

41 A. Arukwe and A. Goksøyr, Comp. Hepatol., 2003, 2, 4.42 M. G. Marin and V. Matozzo, Mar. Pollut. Bull., 2004, 48, 835.43 A. Goksøyr, J. Toxicol. Environ. Health, Part A, 2006, 69, 175.44 T. H. Hutchinson, G. T. Ankley, H. Segner and C. R. Tyler, Environ.

Health Persp., 2006, 114(Suppl. 1), 106.45 T. Matsumoto, A. M. Nakamura, K. Mori and T. Kayano, Zool. Sci.,

2003, 20, 37.46 M. Osada, M. Harata, M. Kishida and A. Kijama, Mol. Reprod. Dev.,

2004, 67, 273.47 C. Blaise, F. Gagn�e, J. Pellerin and P. D. Hansen, Environ. Toxicol.,

1999, 14, 455.48 M. Osada, T. Takamura, H. Sato and K. Mori, J. Exp. Zool., 2003,

299a, 172.

94 | J. Environ. Monit., 2011, 13, 84–94

49 A. M. Puinean, P. Labadie, E. M. Hill, M. Osada, M. Kishida,R. Nakao, A. Novillo, I. P. Callard and J. M. Rotchell, Aquat.Toxicol., 2006, 79, 376.

50 G. Janer and C. Porte, Ecotoxicology, 2007, 16, 145.51 V. Matozzo, F. Gagn�e, M. G. Marin, F. Ricciardi and C. Blaise,

Environ. Int., 2008, 34, 531.52 S. Ogueta, B. J. G�omez, T. Serrano, M. Soto, Proceedings of the 5th

National Congress on Aquaculture (Sant Carles de la R�apita, Spain).Barcelona University Press, Barcelona, Spain, 1995, 169.

53 A. Villalba, Aquaculture, 1995, 130, 269.54 A. T. Mikhailov, M. Torrado, J. M�endez and M. J. L�opez, Mar. Biol.,

1996, 126, 77.55 M. P. Su�arez, C. Alvarez, P. Molist and F. San Juan, J. Shellfish Res.,

2005, 24, 531.56 M. Gamble, I. Wilson, The hematoxylin and eosin. In: Bancroft,

J. D., Gamble, M. (ed.), Theory and practice of histologicaltechniques.Churchill Livingstone-Elsevier Science, London, UK,2002, pp. 125–138.

57 R. Seed, Oecologia, 1969, 3, 277.58 Y. Kim, K. A. Ashton-Alcox, E. N. Powell, Gonadal analysis. NOAA

Histological techniques for marine bivalve mollusks: Update. NOAATechnical Memories NOS NCCOS 27,Silver Spring, USA, 2006, pp.1–18.

59 G. Gagn�e, C. Blaise, J. Pellerin, E. Pelletier, M. Douville, S. Gauthier-Clerc and L. Viglino, Comp. Biochem. Physiol., Part C, 2003, 134, 189.

60 M. G. Stanton, Anal. Biochem., 1968, 22, 27.61 H. J. Fryer, G. E. Davis, M. Manthorpe and S. Varon, Anal.

Biochem., 1986, 153, 262.62 L. Bartolom�e, M. Deusto, N. Etxebarria, P. Navarro, A. Usobiaga

and O. Zuloaga, J. Chromatogr., A, 2007, 1157, 369.63 J. C�aceres-Mart�ınez and A. Figueras, Aquaculture, 1998, 162, 141.64 R. I. E. Newell, Species profiles: life histories and environmental

requirements of coastal fishes and invertebrates (North and Mid-Atlantic) –blue mussel. US Fish and Wildlife Service of Biology andReproduction 82 (11.102). US Army Corps of Engineers, TREL-82-4, 1989, 25.

65 F. G. Figueiras, U. Labarta and M. J. Fern�andez-Reiriz,Hydrobiologia, 2002, 484, 121.

66 C. Blaise, F. Gagn�e, M. Salazar, S. Salazar, S. Trottier andP. D. Hansen, Fresenius Environ. Bull., 2003, 12, 865.

67 E. Kenchington, B. MacDonald, L. Cao, D. Tsagkarakis andE. Zouros, Genetics, 2002, 161, 1579.

68 M. A. Vaschenko, I. G. Syasina, P. M. Zhadan and L. A. Medvedeva,Hydrobiologia, 1997, 352, 231.

69 M. Ortiz-Zarragoitia and M. P. Cajaraville, Ecotoxicol. Environ. Saf.,2010, 73, 693.

70 A. Goksøyr, A. Arukwe, J. Larsson, M. P. Cajaraville, L. Hauser,B. D. Nilsen, D. Lowe, P. Matthiessen, Molecular/cellular processesand the impact on reproduction. In: Lawrence, A., Hemingway, K.(ed.), Effects of pollution on fish. Blackwell Publishing, Oxford, UK,2003, pp. 179–220.

71 C. Blaise, F. Gagn�e, J. Pellerin, P. D. Hansen and S. Trottier, Environ.Toxicol., 2002, 17, 170.

72 A. Novas, R. Barcia and J. I. Ramos-Mart�ınez, Aquat. Toxicol., 2007,85, 285.

73 L. Barbin, I. Boarini, P. G. Borasio, P. Barion, S. Fiorini, R. Rossiand C. Bioindi, Gen. Comp. Endocrinol., 2003, 130, 215.

74 G. B. Stefano, P. Cadet, K. Mantione, J. J. Cho, D. Jones andW. Zhu, Endocrinology, 2003, 144, 1234.

75 C. Morales-Caselles, M. L. Mart�ın D�ıaz, I. Riba and T. A. DelValls,Fresenius Environ. Bull., 2009, 18, 140.

76 H. Frouin, J. Pellerin, M. Fournier, E. Pelletier, P. Richard,N. Pichaud, C. Rouleau and F. Garnerot, Aquat. Toxicol., 2007, 82,120.

77 M. P. Cajaraville, I. Marig�omez and E. Angulo, Comp. Biochem.Physiol., Part C: Pharmacol., Toxicol. Endocrinol., 1992, 102, 103.

78 T. Baussant, M. Ortiz-Zarragoitia, M. P. Cajaraville,R. K. Bechmann, I. C. Taban, S. Sanni. Mar. Pollut. Bull., submitted.

79 A. Renzoni, Mar. Pollut. Bull., 1975, 6, 125.80 J. Bellas, L. Saco- �Alvarez, O. Nieto and R. Beiras, Mar. Pollut. Bull.,

2008, 57, 493.81 J. A. Soriano, L. Vi~nas, M. A. Franco, J. J. Gonz�alez, M. H. Nguyen,

J. M. Bayona and J. Albaig�es, J. Environ. Monit., 2007, 9, 1018.82 B. Fern�andez, M. Albentosa, L. Vi~nas, A. Franco, J. J. Gonz�alez and

J. A. Campillo, Ecotoxicology, 2010, 19, 735.

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