This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright

Author’s personal copy
Aquatic Toxicology 91 (2009) 187–195
Contents lists available at ScienceDirect
Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox
Histopathological changes and erythrocytic nuclear abnormalities in Iberian
green frogs (Rana perezi Seoane) from a uranium mine pond
S.M. Marques
a,∗
, S.C. Antunes
a
, H. Pissarra
b
, M.L. Pereira
c
, F. Gonc¸alves
a
, R. Pereira
a
a
Departamento de Biologia/Centro de Estudos do Ambiente e do Mar (CESAM), Campus de Santiago, Universidade de Aveiro,
3810-193 Aveiro, Portugal
b
Laborato´rio de Anatomia Patolo´gica, Centro de Investigac¸a˜o Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterina´ria,
U.T.L., Lisboa, Portugal
c
Departamento de Biologia/Centro de Investigac¸a˜o em Materiais Ceraˆmicos e Compo´sitos (CICECO), Campus de Santiago, Universidade de Aveiro,
3810-193 Aveiro, Portugal
article info
Article history:
Received 16 April 2008
Accepted 19 April 2008
Keywords:
Amphibians
Metals
Rana perezi
Histopathology
Genotoxicity
abstract
In spite of their sensitivity to anthropogenic stressors, adults of Rana perezi Seoane were found inhabit-
ing effluent ponds from a uranium mine. Due to the presence of such organisms in this environment, it
becomesof paramount importance to assess thedamages inducedby local contaminationon these aquatic
vertebrates, in order to integrate this information on a site-specific risk assessment that is being carried
out in the area. To attain this purpose an ethically and statistically acceptable number of green frogs were
captured in the mine pond (M) and in a pristine river (VR), a few kilometres from the mine. Bioaccumu-
lation of metals and histopathological alterations were evaluated in the liver, kidneys, spleen, lungs and
testes of the animals. Simultaneously, blood samples were collected for the evaluation of genotoxic dam-
age on erythrocytes. Animals captured in the M pond showed significantly increased levels of Be, Al, Mn,
Fe and U in the liver, as well as Pb and U in the kidney. The liver was the main target organ for the bioac-
cumulation of Be, Al, Fe and U. However, renal histopathologies were more severe than those of liver. The
main tissue alterations recorded in animals from the mine were: a slight increase in melanomacrophagic
centers (MMC) in liver, lung and kidneys; dilatation of the renal tubules lumen associated with tubular
necrosis. A significantly higher number of erythrocytic abnormalities (lobed, notched and kidney shaped
nuclei and micronuclei) were recorded in frogs from M than in frogs from VR, along with a significantly
lower frequency of immature erythrocytes. Both observations suggested that the removal of abnormal
blood cells might be compromised.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
The mining activity is a source of physical, chemical, biological
and landscape alterations. Usually large amounts of solid residues
are produced and accumulated in the vicinity of themines (Oliveira
and
´
Avila, 2001; Arau´jo et al., 2002). These solid wastes exposed
to atmospheric conditions as well as the processes carried out to
treat poreoreusually give rise to theproductionofmetal-rich acidic
effluents (e.g. Lopes et al., 1999; Antunes et al., 2007a). As a con-
sequence of low pH values, and of the high persistency of metals
in the environment, fauna and flora may suffer alterations, which
are associated with a decrease in the population’s density or even
the total disappearance from the site (Linzey et al., 2003). Further-
∗
Corresponding author. Tel.: +351 234 370 200x22711/22712;
fax: +351 234 426 408.
E-mail address: s.reis.marques@gmail.com (S.M. Marques).
more the situation is worsened when radioactive elements, like
uranium, are extracted as they exert both chemical and radiologi-
cal toxicity (Domingo, 2001). Despite the hazard posed by this kind
of contamination few studies assessing its impact on indigenous
species are available (e.g. Antunes et al., 2007a,b), and frequently
they do not cover all the trophic levels, or main functional groups,
of food chains. Regarding vertebrates, several studies have already
shown that chronic exposures to toxic metals both in laboratory
(e.g. Donnadieu-Claraz et al., 2007) and in field conditions (e.g.
Pereira et al., 2006) might damage internal organs. Mainly owing
to their role in detoxification and homeostasis (Kerr, 1999), liver
and kidneys are usually target organs and may accumulate met-
als and display damages (Cooley and Klaverkamp, 2000; Cooley et
al., 2000; Loumbourdis, 2005; Pereira et al., 2006). These organs
may undergo alterations in their enzyme activities (e.g. Fenoglio
et al., 2005) and, either mild (e.g. tissue swelling) (Linzey et al.,
2003; Pereira et al., 2006) or severe (e.g. necrosis) (Cooley and
Klaverkamp, 2000) histopathological damage. Besides these two
0166-445X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2008.04.010