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Chapter 20

In Vitro Toxicity and Histopathological Effects

Induced in the Mantle Tissue of Males of Mytilus
galloprovincialis During Short-Term Exposure
to a Tar Mixture
Ana Alonso Martinez, P. Suarez, Y. Ruiz, A. Vidal, and F. San Juan

Despite international legislation, environmental pollution has increased, mainly
in coastal and estuarine areas, due to increases in population density, industrialization, ship traffic, port activities, fishing, and tourism in these areas (Srogi
2007). Polycyclic aromatic hydrocarbons (PAHs) are considered to be amongst the
most ubiquitous marine pollutants. Marine organisms acquire these compounds by
ingestion and diffusion across the epithelium, and then accumulate them due to their
lipophilic nature.
The acute and chronic toxicity of PAHs presents a risk for organisms, as some
chemical species and their intermediate biotransformation products are highly toxic,
mutagenic and carcinogenic. Because of this, PAHs are under constant investigation
and review by international organizations (IARC 1983, 1996; ATSDR 1995; WHO
1998; Bostrom et al. 2002; EPA 2010). The toxic effects of these pollutants, as
well as its toxic mechanism are well understood in vertebrates, but fewer studies
have been conducted in invertebrates. However, although much remains to be
discovered about biotransformation and toxic action of PAHs in invertebrates, the
toxic mechanism seems to follow those of vertebrates.
Several neoplastic diseases as well as endocrine and metabolic disruptions have
been described among the main toxic effects of PAHs. These pathologies are related
with mutagenicity and carcinogenicity of hydrocarbons and their capacity to bind
estrogen receptors and to induce P450-dependent enzymes, leading to reproductive

A.A. Martinez ()

Department of Biochemistry, Genetics and Immunology, Faculty of Sciences,
University of Vigo, Lagoas-Marcosende s/n., 36310 Vigo, Spain
P. Suarez Y. Ruiz A. Vidal F. San Juan
Department of Biochemistry, Genetics and Immunology, University of Vigo, Vigo, Spain
G. Sauve (ed.), Molluscan Shellfish Safety, DOI 10.1007/978-94-007-6588-7 20,
Springer ScienceCBusiness Media Dordrecht 2014



A.A. Martinez et al.

and growth alterations (Santodonato 1997; Morton 1988; Bostrom et al. 2002;
Pufulete et al. 2004; Ono et al. 2008; Veeramachaneni 2008; Sanders et al. 2009).
In marine invertebrates, histopathological and endocrine alterations have also been
related to exposure by PAHs and others chemicals (Alonso et al. 2001; Aarab et al.
2011; Au 2004; Lavado et al. 2006; Ortiz-Zarragoitia and Cajaraville 2006; JingJing et al. 2009; Schafer and Kohler 2009; Ruiz et al. 2011). However, few studies
have been undertaken to demonstrate these alterations in the laboratory.

Taking into account the concentrations of accumulated hydrocarbons measured in
mussels cultured in the Ra of Vigo by Ruiz et al. (2011), we conducted a study,
under laboratory controlled conditions, to determine the gonadal histopathologies
induced by a tar mixture, commonly used in cleaning mussel culture rafts.

Analytical Composition of Tar
The chemical analysis of the tar mixture was contracted to the Spanish Institute of Oceanography (IEO, Center of Vigo), where analytical quality control is
supported by the international intercalibration exercises from Quality Assurance
of Information for Marine Environmental Monitoring in Europe (QUASIMEME
2003) or International Atomic Energy Agency (IAEA) (Villeneuve et al. 2004). The
separation and quantification of PAHs was carried out by high-performance liquid
chromatography (HPLC) with fluorimetric detection according to Vinas Dieguez
(2002). A total of 13 PAHs were quantified: phenanthrene (Phe), anthracene
(An), fluoranthene (Flt), pyrene (Py), benzo(a)anthracene (BaA), chrysene (Chry),
benzo(e)pyrene (BeP), benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF),
benzo(a)pyrene (BaP), dibenzo(a,h)anthracene (DBA), benzo(g,h,i)perylene (Bper)
and indeno(1,2,3,-c,d)pyrene (IP). Results were expressed as mg/kg of mussel

Experimental Design
A total of 200 mussels (Mytilus galloprovincialis), between 6 and 8 cm in valve
length were randomly collected from floating rafts in the Ra of Vigo (NW Spain)
in April of 2009. In the laboratory, mussels were placed in five 30 L polyethylene
tanks in an open system of 20 m filtered seawater, with a flow rate of 2 mL/min.

20 In Vitro Toxicity and Histopathological Effects Induced in the Mantle. . .


During each experiment, the mussels were maintained under constant aeration and
temperature (20 C), with natural photoperiod. They were fed a daily microalgae
diet (0.0224 g microalgae/mussel) comprised of Isochrysis galbana, Tetraselmis
suecica and Chatoceros gracilis in a 50:30:20 proportions.
After 15 days acclimatization to the described conditions, the mussels from each
tank were exposed to different concentrations of the tar mixture. Previous studies
by our research team regarding total hydrocarbon accumulation in mussels from the
Ra of Vigo, had shown mean PAH concentrations of 400 g/kg (Ruiz et al. 2011).
This value was the lowest concentration measured in mussel tissues after laboratory
exposure to 10 ppm of the tar mixture, applied for 17 days. We also employed three
higher concentrations: 40, 60 and 80 ppm. Control mussels from the fifth tank were
not treated with tar. The tar mixture was heated to 200 C and added in 5 L of
seawater, together with the microalgal diet, at a 2 mL/min flow rate. Treatment was
continued for 17 days and samplings made at days 0, 10 and 17.

Tissue Preparation for Histology

In species of the genus Mytilus, the proximity of the genital papilla to the mantle
tissue is used for the expansion of gonad and the gametogenic development taking
place mainly in this tissue. For each sampling, the mantles from 10 individuals from
each assay tank were dissected, fixed in 10 % neutral buffered formalin, dehydrated
and processed in paraffin. Sections of 5 m thickness were mounted on microscope
slides and stained with Carazzi haematoxilin and eosin. Slides were examined by
light microscopy (Nikon 90i Eclipse). The images were captured by an 8Mpx high
resolution Nikon camera using Nis- Elements BR 2.30 version software.
The gametogenic stage of each mussel was determined according to Lubet (1959)
and Suarez et al. (2005). Histological preparations were examined comparatively
with those of the control mussels as a reference.

Statistical Analysis
A statistical survival analysis with a PROBIT regression test was performed
(statistical package SPSS 17.0, Microsoft Co) in order to calculate the median lethal
concentration (LC50 ) of the tar mixture.

The concentration of total hydrocarbons analyzed in the tar mixture (13 parent
PAH species) was 40,364 mg/kg. Therefore, the dilutions of 10, 40, 60 and 80 ppm
employed in this experiment represent exposure concentrations of 400, 1,600, 2,400

Table 20.1 Tar composition
and percentage of each PAH

A.A. Martinez et al.



Indene (1,2,3-c,d)pyrene



PAHs species with high mutagenic and

carcinogenic potency

and 3,200 mg/kg, respectively. The concentrations of each PAH specie present in
the mixture are summarized in Table 20.1. Among these, Chry, BaA, BbF, BkF,
BaP, DBA and IP are considered to be highly mutagenic and carcinogenic (EPA
2010) and constitute 29.5 % of the total mixture.
The experiment was carried out in April, which is the main reproductive period
of Mytilus in the Ra of Vigo. At the beginning of the experiment, mussels were
in an advanced IIIC stage, or gonadal restoration stage of Suarez et al. (2005).
Histologically, this stage is characterized by the presence of large follicles filled with
gametes, and scarce reserve tissue, mainly constituted by vesicular cells (Fig. 20.1a).
At the end of the experiment, the reference mussels were at an incipient spawning
stage (IIIB), with larger follicles and scarce reserve tissue (Fig. 20.1b).
An increase in mortality and cellular disorders was observed in the mantle tissue
from mussels, which was dependant on dose and exposure time to the tar mixture.
Survival of control mussels at the end of experiment was 100 %. However, after
12 days of tar exposure, accumulated mortality of mussels treated with 10, 40 and
60 ppm of mixture was 25.2, 26.8 and 48.0 %, respectively. Acute exposure to
80 ppm of tar mixture caused death of 40 % of mussels after 5 days and of 95 %
after 10 days of treatment (Fig. 20.2a). These results suggest a toxic accumulative
effect of tar. The median lethal concentration (LC50 ) calculated was 67.90 ppm
(Fig. 20.2b).
After 10 days exposure to 10 and 40 ppm of tar, an intense desquamation
of germinal cells was observed histologically (Fig. 20.1c). This phenomenon has
also been described in testicular vertebrate pathologies where it is considered as
an untimely spawning induction (Aarab et al. 2011). Moreover, mussels exposed
to 60 and 80 ppm of tar showed an interstitial tissue increase and an abnormal
development of follicles leading to a clear follicular hypoplasia and a loss of
their radial architecture. The basal membrane of these follicles was thickened
and hyalinised and inside them a low mitotic activity of germinal cells was seen.

20 In Vitro Toxicity and Histopathological Effects Induced in the Mantle. . .


Fig. 20.1 Microphotographs of histopathological changes induced in male mantle tissue of

Mytilus galloprovincialis exposed to a tar mixture: Control organisms: (a) Normal histology of
mantle showing gonadal follicles, VCs (arrow) and ADGs (star) (10). (b) Follicles showing
evidence of spawning (IIIB stage) (10). Treated mussels: (c) Spawn induction (arrow) (20).
(d) Hypoplasia follicular (arrow) (40). (e) Auxiliary cells with hyperchromatic nucleus (arrow),
eosinophilic deposits (stars) and thickened basal membrane showing different degrees of hyalinization (asterisk) (60). (f) Aggregation of immature germinal cells (arrow) and hyalinization
of membrane follicular (asterisk) (20). (g) Basal membrane rupture (star), infiltration of
the abnormal cells in the connective tissue (arrow) and fibrosis (discontinue arrow) (60).
(h) Aggregation of spermatocytes with picnotic and karyorhesic nucleus (discontinue arrow) and
disintegrated basal membrane (star) (60). (i) Atypical hypercromatic and hypertrophic germinal
cells (arrow) (60)

Many large auxiliary cells, with hyperchromatic nucleus and clear cytoplasm, with
eosinophilic deposits could be observed (Fig. 20.1d, e). Similar effects have been
related with seminomas in vertebrates (Boekelheide 2005).
After 17 days of exposure to 40 and 60 ppm of tar, similar effects to those
caused by 10 days exposure to 80 ppm of tar could be observed. Moreover, the
following was also observed (Fig. 20.1fi): aggregation of immature spermatocytes;
the occurrence of large and multinucleated germinal cells with picnotic nucleus,
karyorrhexis and vacuolated cytoplasm; the breaking of follicular basal membrane;
infiltration of abnormal cells in the connective tissue and fibrosis. All of these
alterations could be considered as pre-neoplastic lesions of a carcinoma in situ
affecting germinal cells. Along with these neoplastic disorders both hyperplasia and
hypertrophy of vesicular cells with clear signs of edema occurred (Fig. 20.1gi).


A.A. Martinez et al.


Accumulated mortality (%)

80 ppm
60 ppm
40 ppm
10 ppm







Treatment days



- 0.5

- 1.0





Fig. 20.2 (a) Cumulative lethality (percentage) as effect of short term tar exposure at different tar
concentrations. (b) Median lethal concentration of tar. Probit test

Toxic and mutagenic effects of several PAHs species, as well as their relation
with the development of neoplastic diseases are reported in the literature (EPA
2010). However, few studies have been conducted to demonstrate induction of
these pathologies in the laboratory. Mytilus spp. is considered suitable as a sentinel
organism in marine environmental monitoring (Goldberg et al. 1978; Farrington

20 In Vitro Toxicity and Histopathological Effects Induced in the Mantle. . .


et al. 1987; Nelson 1990; OConnor 2002; Monirith et al. 2003). Due to the
commercial importance of mussel culture, the toxic effects of PAHs identified here
could constitute a significant risk for mussel production, as well as a risk to human
health following mussel consumption.
The chemical analysis of tar mixture used for cleaning culture mussel rafts
suggests its potential toxicity. The lethal and histopathological effects induced in
mussels, during this laboratory study, confirm its harmful effects at concentrations
higher than 10 ppm.
Mortality and histopathological effects caused by tar mixtures in the laboratory
depend on dose and time exposure, suggesting a cumulative toxic effect as described
in vertebrates (Richburg et al. 2002; Strmac and Braunbeck 2002). However,
lethality caused by high concentrations of tar (60, 80 ppm) could be enhanced by
asphyxia of different organs as described by Iniesta y Blanco (2005).
Histopathological effects follow a progressive sequence of malignancy until
the development of carcinoma in situ, affecting germinal cells. This sequence
starts with the desquamation of germinal cells by untimely spawning induction,
as described in testicular pathologies from vertebrates, or related to pollution in
invertebrates (Cajaraville 1991; Cajaraville et al. 1992; Tay et al. 2003; Jing-Jing
et al. 2009; Aarab et al. 2011). Next, the atrophy and hypoplasia of follicles
occurs, accompanied by scarce proliferation and differentiation of germinal cells,
as well as by many, large and vacuolated auxiliary cells within them. Similar
histopathologies with abnormal follicles, consisting of abnormal auxiliary cells
with a clear cytoplasm and deposits of eosinophilic material, have been reported
in seminomas from vertebrates, caused by toxic damage (Nistal 1973; Nistal et al.
1998; Chapin et al. 1984; Boekelheide 2005). Membrane hyalinization of gonadal
follicles and fibrosis have also been described in gonadal neoplasias of vertebrates
in individuals exposed to chemicals, where it may become an irreversible process
(Nistal 1973; Nistal et al. 1998). The sea urchin is the only invertebrate exposed
to phenanthrene, where an increase of collagen fibers around the gonadal follicles,
has been described (Schafer and Kohler 2009). Other effects observed in the latter
work, like aggregation of spermatocytes and spermatids, or existence of large
and multinucleated germinal cells, are similar to the spermatogenic alterations
reported after prolonged exposure to pollutants (Ono et al. 2008). Hyperplasia and
hypertrophy of vesicular cells observed in this study are similar to those described in
interfollicular connective tissue cells of gonadal carcinomas in situ from vertebrates
(Dieckmann and Skakkebaek 1999; Ulbright 2005) and of gonadoblastomas from
marine invertebrates (Elston et al. 1992; Peters et al. 1994; Ford et al. 1997). All
of these chemically mediated effects suggest a possible endocrine disruption in
mussels as described in vertebrates (Irvine 2000; Swan et al. 2003), resulting from
the xenoestrogenic activity of several PAHs species from the tar mixture used.
Our results show that the tar mixture tested is lethal for mussels at high
concentrations, but at sublethal doses can cause serious pathologies, sufficient to
affect their reproduction. Moreover, the mussels capacity to accumulate lipophilic
compounds such as hydrocarbons could constitute a risk to human health. So, use of


A.A. Martinez et al.

this tar mixture for cleaning the culture mussels rafts, should be replaced by a less
toxic chemical in order to ensure continued production and sustainability of mussel
farms, as well as to produce a cultured mussel which is safe for human consumption.

The exposure of mussels to PAHs in a tar mixture used for cleaning purposes, causes
histopathological effects, possibly due to endocrine disruption, which lead to the
maturation arrest of germinal cells and to the development of germinal carcinoma,
which could decrease fertility and reproductive capacity of cultured mussels.
Acknowledgements The authors wish to thank Antonio Antepazos and the fishermen of the
Antepazos I, who kindly provided the mussels used in this work. We also thank the Spanish Institute of Oceanography who completed the chemical analysis of the tar used in this project. This work
was supported by a grant from Autonomous Galician Government (PGIDT10MDS312030PR).

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