e),
n r
en
nvi
s is a
concern. Many are the causes of such degradation that go from
tivities
of such cases is the abandoned uranium mine in Cunha Baixa
usually presents a seriousmenace to the local fauna (Jarvis and
physical and chemical parameters of which the pH, alkalinity
Amphibians are, in many cases top predators, playing an
development stage and a highly permeable skin (Duellman
S C I E N C E O F T H E T O T A L E N V I R O N M E N T 4 0 2 ( 2 0 0 8 ) 2 9 – 3 5
ava i l ab l e a t www.sc i enced i r ec t . com
m/Younger, 1997) frequently compromising the survival, growth
and behaviour of organisms (Lefcort et al., 1998; Antunes et al.,
2007a) especially in aquatic systems.
In Cunha Baixa mine besides the presence of metals in the
effluent there are additional concerns resulting from the
and Trueb, 1994). Several studies have shown the toxic effects
of metals in this kind of animals. Among the most common
deleterious effects of metals on amphibians, reduction of
immune functions (Linzey et al., 2003), limb, mouth and tail
malformations and other kind of abnormalities (Calevro et al.,(Mangualde, Central Portugal). Uranium mining activity in this
area has contributed since the beginning of the extraction and
even after the activity has ceased for the production of toxic
metal rich-effluent (Antuneset al., 2007a,b). Thiskindofeffluent
important role in the aquatic community and bioaccumulat-
ing contaminants (Loumbourdis et al., 1999). In addition they
are some of the most sensitive vertebrates to environmental
changes, due, in most cases, to an early aquatic-dependentagriculturalpractices to industrial acpresence of radioactive elements such as
daughter radionuclides (Oliveira and Ávila
et al. (2005) reviewed, uranium may exert
radiological toxicity on terrestrial and
⁎ Corresponding author. Tel.: +351 234 370 20
E-mail address: s.reis.marques@gmail.com
0048-9697/$ – see front matter © 2008 Elsevi
doi:10.1016/j.scitotenv.2008.04.005suchasmining.One and hardness are the most important (Sheppard et al., 2005).Rana perezi Seoane
1. Introduction
Degradation of freshwater resourcethis study aimed to assess the ecotoxicological effects promoted by the mine effluent in the
early-life stages of this species. To attain this objective, eggs (collected in a nearby reference
river) and laboratory hatching larvae were exposed during 96 h to different dilutions of the
effluent. All the effects on the hatch success were recorded. The highest concentration of
the effluent produced a significant decrease in body length of larvae, as well as a decrease in
stimulus reactions and an increase in pigmentation along with tail deformities. A recovery
assay showed an increased bioaccumulation of metals, uranium included, resulting from
increased effluent exposure.
© 2008 Elsevier B.V. All rights reserved.
world-wide growing
Nonetheless the main concern regards the aquatic compart-
ment (Antunes et al., 2007a), where the bioavailability and
toxicity of U for freshwater species depends on severalAvailable online 6 June 2008
Keywords:
Uranium
Amphibians
Embryo and larvae acute toxicity
assays
Heavy metalsAccepted 3 April 2008 (Rana perezi Seoane) are able to survive in the ponds of a uranium mine (Central Portugal)Effects of a uranium mine efflu
Rana perezi Seoane
S.M. Marques⁎, F. Gonçalves, R. Pereira
Departmento de Biologia/Centro de Estudos do Ambiente e doMar (CESAM
A R T I C L E I N F O A B S T R A C T
Article history:
Received 15 January 2008
Received in revised form 3 April 2008
Amphibians have bee
by several environm
inhabiting extreme e
www.e l sev i e r. couranium and their
, 2001). As Sheppard
both chemical and
aquatic organisms.
0x22711, 22712; fax: +351
(S.M. Marques).
er B.V. All rights reservednt in the early-life stages of
Campus de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal
eported as sensitive organisms whose survival has been impaired
tal factors. Nevertheless, sometimes amphibians are found
ronments. Thus, in order to perceive how Iberian green frogs
l oca te / sc i to tenv1998; Rowe and Freda, 2000; Linzey et al., 2003), behaviour
alterations, growth reduction and survival decrease (Lefcort
et al., 1998) were recorded. Even though much information is
available on the effects of metals to amphibians, few studies
234 426 408.
.

nated segment of the Vouga River (VR) located in the north part
V I Rof the city ofViseu, a fewkilometres fromMangualde,where the
presence of the Iberian green frogs has been recorded.
2.2. Water sampling
The water samples for the acute assays were collected in both
VR and M sites with 20 L polystyrene containers, which were
filled to the brim and tightly capped to exclude oxygen
preventing oxidations. These flasks were previously filled
with nitric acid (50%, v/v) left overnight and after this period
thoroughly rinsed with distilled water. Afterwards the sam-
ples were immediately transported to the laboratory, within a
maximum period of 1 h, where they were stored for no longer
than 48 h, in the dark, at 4 °C until further usage in the assays.
Water samples for chemical analyses were also collected in
the same locations in 0.5 L polystyrene bottles, washedhave focused on the effects of radioactive metal rich effluents
in these organisms.
Due to the unexpected presence of the Iberian green frog
(Rana perezi Seoane) in the effluent ponds of Cunha Baixa
uraniumminearea, our study intended toassess lethal andsub-
lethal effects, yieldedby short-termexposures to themetal rich-
effluent in the hatching and early-life stages of this species. In
order to do so, egg masses collected from a contaminant-free
nearby freshwater resource were used. In addition we also
aimed to assess the recovery ability of these animals, regarding
parameters impaired and bioaccumulatedmetals by comparing
larvae hatched in the 100% treatment (96 h embryo assay) that
were further exposed to the 100% treatment for more 96 h, with
those that were immediately transferred to FETAX medium, at
the end of the 96 h embryo assay.
2. Materials and methods
2.1. Study site
The study site was an abandoned uraniummine located in the
small village of Cunha Baixa (Mangualde, Central Portugal).
Themining area is included in the designated uraniferous belt
of the Iberian Peninsula and the ore extraction occurred
between 1967 and 1993 (Santos Oliveira and Ávila, 1998). After
the operation period themine pit was filledwith low-grade ore
and it was flooded with sulphuric acid to extract uranium
through an in situ leaching process (Santo and Freire, 1998).
Presently, there are 3 small ponds in this area which have a
variable water volume depending on the uprising of the
underground acidic effluent which, in turn, is determined by
fluctuations in the underground water level. This effluent,
because of its origin, has a complex mixture of metals and an
extremely low pH (Pedrosa and Martins, 1999). Despite the
proximity of the ponds they showed very different chemical
characteristics, being the largest (the M pond) the one that
provided extremer conditions on account of the direct
connection with the underground tunnels of the mine pit
and consequently with the mine effluent.
The reference site chosen for this study was an uncontami-
30 S C I E N C E O F T H E T O T A L E Nfollowing the procedure described above and, acidified with
pro analysis nitric acid (65%), MERCK® to a pH below 2, toprevent metal adsorption. These samples were then stored at
4 °C until chemical analyses was possible.
2.3. Test organisms
Egg masses from the Iberian green frog (R. perezi Seoane) less
than 36 h in age were collected in the Vouga River (VR) for the
96 h embryo test. For the larvae acute toxicity test (96 h) the
organisms were obtained from the remaining egg masses,
maintained inVRwaterwithaeration,ata constant temperature
(24 °C±2) and photoperiod (14 hL:10 hD), until hatching occurred.
2.4. Acute assays
The bioassays were performed according to ASTM recommen-
dations for conducting acute toxicity assays with effluents
(ASTM, 1997). The VR water was used to obtain the different
effluent dilutions (6.25%, 12.5%, 25%, 50%, 75%, and 100%) for
both assays. Both effluent and dilution water were filtered by
cellulose nitrate ALBET® filters with a 47 mm diameter and a
0.20 µm pore to reduce possible bacteria and parasite
contamination. A FETAX (Dawson and Bantle, 1987) control
and aVR controlwere considered to ascertain thequality of the
water from the reference site. For each concentration the pH
was adjusted to 8, with 5MNaOH, to eliminate the effect of the
effluent acidity. Animals were not fed during the larvae assay.
For the 96 h embryo toxicity test the jelly coat of the egg
masses was removed by gently stirring for 2–3 min in a 2%
cistein (Sigma®) solutiondilutedwith FETAXsolution andwith
a 8.1 adjusted pH (Mann and Bidwell, 2000). Afterwards, for
eachof the 2 replicas of effluent concentrations and eachof the
4 replicas of the controls, 20 eggs from the same R. perezi egg
mass were placed, with plastic pipettes, in plastic Petri dishes
with 20 ml of the respective effluent dilution and of the
controls. This assay was carried out at constant temperature
(24±2 °C) and photoperiod (14 hL:10 hD) conditions. Embryo
mortality was recorded and dissolved oxygen and pH were
checked every 24 h during the test, using a WTW 315i/SET Oxi
meter and aWTW330/SET-2pHmeter, respectively. At the end
of the assay larvae were checked for abnormalities and the
body length measured using an Olympus SZX9 stereoscope.
For the acute larvae toxicity test the organisms were
obtained from the laboratory hatching eggs. In this assay
larvae from the same egg mass less than 6 days in age were
placed in 500 ml plastic vessels in groups of 5 for each of the 5
replicas for every effluent concentration and controls (FETAX
and VR water). The test was performed in the conditions
already described. The body sizewas recorded at the beginning
and at the end of the assay, and the pH, the dissolved oxygen
and the mortality were registered every 24 h. The dead larvae
found were removed from the vessels in each observation.
Larvae movement was checked daily for any alterations by
gentleproddingusing aplastic pipette andat the endof the test
the existence of abnormalities in the organisms was verified.
An extra 96 h assay (recovery assay) was carried out using
the larvae obtained from the embryo test. In this assay the
larvae obtained from the FETAX control were kept in FETAX
and were also used as a control. The 20 larvae were divided in
O N M E N T 4 0 2 ( 2 0 0 8 ) 2 9 – 3 54 replicas of 5 organisms placed into 500ml plastic vessels and
maintained under the above described laboratory conditions.