ARTICLE IN PRESS

Experimental and Toxicologic Pathology 61 (2009) 113–121

www.elsevier.de/etp

Sublethal and lethal toxicity in juvenile Senegal sole (Solea senegalensis)

exposed to copper: A preliminary toxicity range-finding test
M. Olivaa,, M.C. Garridob, D. Sales Márquezb, M.L. González de Canalesa
a
Biology Department, Marine and Environmental Sciences Faculty, University of Cadiz, Spain
b
Chemical Engineering, Food Technology and Environmental Technologies Department,
Marine and Environmental Sciences Faculty, University of Cadiz, Spain

Received 5 March 2008; accepted 6 June 2008

Abstract
Static bioassay test for acute toxicity of copper in Senegal sole juveniles (Solea senegalensis) was conducted. The 96 h
LC50 value of metal was found to be 0.32 mg/L Cu. The intensity of histological alterations was increased gradually
with the copper concentration and the exposure time. Numerous aneurysms, hypertrophy, hyperplasia and fusion of
the lamellar epithelium in gills and a disorganization of the hepatic parenchyma and vacuolization in liver were
common lesions in S. senegalensis juveniles exposed to copper. The results of the study indicate the sensitivity of
juvenile S. senegalensis to copper (II), the persistence of sublethal effects and histology as a tool capable of revealing
the sublethal effects of heavy metals on the environment and aquatic biota.
r 2008 Elsevier GmbH. All rights reserved.

Keywords: Toxicity; Histopathology; LC50; Copper; Solea senegalensis

Introduction Copper-containing substances are used to combat

Heavy metals encompass a wide range of substances
that, in general, are non-degradable, highly persistent in
the environment and can be incorporated by organisms
(Ortiz et al., 1999). Toxic metals enter surface waters
from a variety of sources including industries discharges,
domestic sewage, urban storm runoff, and atmospheric
precipitations. The bioavailability and toxicity of metals
depend on the particular physiology of the target
organisms, behavior of metals, and the environment
conditions.
Corresponding author. Tel.: +34 956 016 016;
fax: +34 956 016 019.
E-mail address: milagrosa.oliva@uca.es (M. Oliva).
biological fouling on mariculture nets, boats, and other
marine facilities. Copper sulfate (CuSO4) is one of the
most widely used algaecides for the control of phyto-
plankton in fish ponds, reservoirs, and lakes, as well as
an herbicide used in aquatic weed control (Effler et al.,
1980; Carbonell and Tarazona, 1993). Of particular
interest is the pesticide application among different
sources of copper pollution such as mining, industrial
discharge, sewage sludge disposal, and fertilizers (Nor,
1987). Actually, the extensive use of copper has raised
some concern on the effect in the natural or farmed
organisms in the environment (Karan et al., 1998;
Zyadah and Abde-Baky, 2000; Mazon et al., 2002;
Krishnani et al., 2003; Van Herden et al., 2004).
Acute toxicity studies help in the detection, evalua-
tion, and abatement of pollution by providing reliable

0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved.
doi:10.1016/j.etp.2008.06.001
 ARTICLE IN PRESS
114 M. Oliva et al. / Experimental and Toxicologic Pathology 61 (2009) 113–121
estimates of safe concentration, from which water
quality criteria can be derived (Ahsunullah et al.,
1981). Utilization of acute toxicity studies for assessing
water quality can be employed by examining different
life stages of important species (NAS/NAE, 1973).
Fish, in comparison to invertebrates, are more
sensitive to many pollutants and are the convenient
test-objects for water quality assessment (Forstner and
Wittmann, 1983; Spry and Wiener, 1991). Sublethal
effects in fish allow us to define toxicity of the
environment and understand the potential danger of
pollutant inputs.
Last years, the culture of the teleostean fish Solea
senegalensis has achieved a great economic and com-
mercial interest. It is also one of the most abundant and
representative species of Atlantic and Mediterranean
coasts. S. senegalensis is a well-adapted species to warm
climates and is commonly exploited in extensive aqua-
culture production in Spain and Portugal (Drake et al.,
1984; Dinis, 1992; Sarasquete et al., 1998).
In the present article, the acute toxicity of copper in
juvenile S. senegalensis and the associated histopatho-
logical changes in two organs, gills and liver, were
studied.
Materials and methods
Experimental animals
One hundred and eighty-day-old juvenile S. senega-
lensis (mean: 0.07 g; 2.08 cm; n ¼ 15) were obtained
from the aquaculture facilities of the Faculty of Marine
and Environmental Sciences (University of Cadiz,
Spain). During the acclimatization (24 h), no mortalities
were detected. Juveniles selected for tests were checked
to avoid the use of organisms with obvious disease or
apparent stress (due to handling or culture conditions)
that could fake the results of the test.
Toxicity screening test
When preparing to perform a static test with a sample
of completely unknown quality, or before initiating a
flow-through test, it is advisable to conduct a prelimin-
ary toxicity range-finding test.
A toxicity range-finding test ordinarily consists of a
down-scaled, abbreviated static acute test in which
groups of organisms are exposed to several widely
spaced sample dilutions in a logarithmic series, and a
control, for periods between 8 and 96 h.
It should be noted that the toxicity (LC50) of a sample
observed in a range-finding test may be different from
the toxicity observed in the follow-up definitive test
because: (1) the definitive test is usually longer and (2)
the test may be performed with a sample collected at a
different time (USEPA, 2002).
In this study, static acute (96 h) toxicity tests using
different copper concentrations were performed at the
Marine Species Pathology and Histophysiology Labora-
tory (University of Cadiz, Spain). The experimental
design was adapted to meet the procedure proposed by
USEPA (2002).
Copper solutions (CuSO4  5H2O PRS Pure, Panreac,
SA) and controls of the tests were prepared with filtered
natural and unpolluted seawater (45 mm fiber-glass filter)
from Sancti Petri Beach (South Atlantic Spanish Coast)
(copper (II) concentration lower than 0.1 mg/L). Con-
centrations of the test are expressed in mg/L Cu (II).
A 1.5 L of each test solution (and controls) was added
to 2 L glass vessels at nominal concentrations of 0.01,
0.1, and 1 mg/L. Three replicates for each concentration
or treatment including control group were realized. Two
replicates were used to obtain the mortality data and the
third one was used for histopathological analysis.
Fifteen animals were placed in each vessel with
continuous slight aeration. Juveniles were not fed during
the bioassay. Temperature was maintained at
2070.5 1C, salinity of 39.670.66 ppt, pH 8.270.03,
photoperiod 12 h light/12 h darkness, and dissolved
oxygen 95% saturation. Physical and chemical para-
meters were monitored daily in each vessel. Dead and
living juveniles were counted daily throughout the 96 h
period. The absence of response of juveniles to gentle
mechanical stimulus was taken as the criterion for death.
Dead juveniles were removed immediately to avoid
fungal and bacterial infection of the other organisms.
The survival, mortality, and corrected mortality
percent were calculated for each concentration:
No:of living juvenile
% Survival ¼  100,
No:of total of juvenile exposed
% Mortality ¼ 100  % Survival;
% Mortality  % Control mortality
% Corrected mortality ¼  100.
100  % Control mortality
Histopathology
On 24, 48, 72 and 96 h, samples of tissues (gill and
liver) from two specimens of each copper concentration
(third replicate) were collected and immediately pro-
cessed for the histopathological studies. In the case of
the treatments of 1 mg/L of copper, the last histopatho-
logical study was realized at 72 h exposure because at
96 h all specimens were died.
The samples for the histological examination were
fixed in Bouin’s solution during 24 h. After dehydration
in graded concentrations of ethanol, the samples were
 ARTICLE IN PRESS
M. Oliva et al. / Experimental and Toxicologic Pathology 61 (2009) 113–121 115

embedded in paraffin wax. Sagittal sections of 6–8 mm Table 1. Histopathological alterations observed in gills of
thickness were stained with hematoxylin/eosin and juvenile S. senegalensis exposed to 0.01, 0.1, and 1 mg/L of
hematoxylin/VOF from Gutiérrez (1967). copper at 24 and 96 h exposure

Lesion Exposure Copper (mg/L)

Statistical analysis period (h)
0.01 0.1 1a

Accumulative mortalities of juveniles were used to Aneurysms 24 + + ++

estimate the LC50. Data analysis scheme published from 96 + + ++
USEPA (2002) was used to select the most appropriate
Hypertrophy of 24  + ++
analysis method. As insufficient numbers of partial the lamellar 96  ++ ++
kills were present in the bioassay, the data were epithelia
analyzed by the trimmed Spearman–Karber (TSK)
method (Trimmed Spearman–Karber Program Version Hyperplasia of 24  ++ ++
the lamellar 96 + ++ +++
1.5, Environmental Monitoring Systems Laboratory,
epithelia
Cincinnati, OH). Mortality data were corrected for
control responses using Abbott’s procedure. Results are Hypertrophy of 24 + + +
reported as means7S.D. cartilage 96 + + ++
Fusion of 24 + + ++
lamellae 96 + + +++

Results Epithelial 24   +
peeling of the 96  + +
Toxicity screening test lamellae
Epithelial 24   +
No fish in the control group and in the two lower lifting of the 96   ++
concentrations of copper (0.01 and 0.1 mg/L copper) lamellae
died during the experiment. Percent of corrected Empty mucous 24 + ++ ++
mortality for sole juvenile exposed to 1 mg/L copper at cells 96 + ++ ++
different exposure time was 26.6670.00% (24 h),
83.3374.70% (48 h), 96.6674.71% (72 h) and The number of plus symbols is proportional to the assessed damage.
Lesion grade: () absent, (+) light, (++) moderate, and (+++)
100.0070.00% (96 h) (mean7S.D., n ¼ 2). The LC50
high.
values for the different exposure times were a
In the case of 1 mg/L of copper, the last histopathological study was
0.4070.02 mg/L (48 h), 0.3370.01 mg/L (72 h) and realized at 72 h exposure because at 96 h all specimens were died.
0.3270.00 mg/L of copper (96 h) (mean7S.D., n ¼ 2).
At 24 h, the LC50 value was not calculable by STK
program because the minimum required trim was too affected and at the maximum concentration (Fig. 1E)
large (73.3). The LC50 values showed gradual decrease completely altered.
with an increase in time. In general, the increase in Generalized hypertrophy and hyperplasia of second-
percent mortality was related to both time and copper ary lamellae as well as hypertrophy of the cartilage were
concentrations. The higher increase of mortality was observed. Lamellae tended to shrink and disappear in
observed at 48 h in highest concentration of metal. some cases. Additional observations included numerous
aneurysms in gill filaments, lifting of gill epithelia as well
as a decrease of the mucus secretion and empty mucous
Histopathology cells.
Table 1 shows the histopathological manifestations in
Gills gills of S. senegalensis juveniles exposed to copper at 24
Exposure to copper (96 h) caused severe histopatho- and 96 h exposure.
logical changes in gill epithelia (Table 1). Gills of
juvenile S. senegalensis specimens (Fig. 1A and B) Liver
exposed to 0.01, 0.1, and 1 mg/L of copper showed how Exposure to copper (96 h) caused severe histopatho-
the histopathological alterations increased with the logical change in liver (Table 2). Liver of juvenile
copper concentration (Fig. 1C–E). Considerable changes S. senegalensis specimens (Fig. 2A) exposed to 0.01, 0.1,
are observed only after extended exposure times at 1 mg/L and 1 mg/L of copper (Fig. 2B–D) showed an increase of
copper. At 0.01 mg/L copper (Fig. 1C), the gill alterations the histopathological alterations with the copper con-
were mild or non-existent. At 0.1 mg/L copper concentra- centrations (Fig. 2D). Considerable changes were
tion (Fig. 1D), the secondary lamellae became seriously observed only after extended exposure times at 0.1 mg/L
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116 M. Oliva et al. / Experimental and Toxicologic Pathology 61 (2009) 113–121

Fig. 1. Histological sections of gills in Solea senegalensis specimen. (A) Normal aspect of secondary lamellae and gill filament.
Secondary lamellae (sl), pillar cell (pc), gill filament (gf), capillary (c), cartilage (ct) (hematoxylin/eosin  25). (B) Detail of chloride
(cc) and mucous cells (mc) in branchial epithelium (Alcian Blue  40). (C) Histological sections of gills in specimen exposed to
0.01 mg/L copper at 96 h exposure (hematoxylin/eosin  10). Note the aneurysms (*). (D) Gill sections of a specimen exposed to
0.1 mg/L copper at 96 h exposure (hematoxylin/eosin  10). Note the hyperplasia of the lamellar epithelia and fusion of lamellae (*).
(E) Gill sections of specimen exposed to1 mg/L copper at 72 h exposure (hematoxylin/eosin  10). Note the hyperplasia of the
lamellar epithelium, the fusion and the epithelial lifting of the lamellae (*).

copper (Fig. 2C). At 0.01 mg/L copper, the liver
alterations were mild or non-existent (Fig. 2B). At 0.1
and 1 mg/L copper concentration, a general disorgani-
zation of hepatic parenchyma was observed (Fig. 2C
and D).
A disorganization of hepatic parenchyma was related
to the increase of exposure time to copper. Necrotic
zones, dilatation of the blood capillaries, and high
concentration of sanguineous cells in the blood vessels
were also observed. The hydropic vacuolization was
observed in the totality of tissue, this vacuolization
increased with the copper concentration. The hepato-
cytes presented a diffuse degeneration of the cytoplasm
and several zones with lipid degeneration were observed.
Moderate steatosis was observed in some zones of tissue.
Hepatocytes showed diffuse degeneration of cytoplasm.
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M. Oliva et al. / Experimental and Toxicologic Pathology 61 (2009) 113–121 117

Table 2. Histopathological alterations observed in liver of

juvenile S. senegalensis exposed to 0.01, 0.1, and 1 mg/L of
copper at 24 and 96 h exposure

Lesion Exposure Copper (mg/L)

period (h)
0.01 0.1 1a

Blood stagnation 24 + + ++
96 + ++ ++
Steatosis 24  + + 2A
96  + +
Necrosis 24   +
96  + ++
Parenchyma 24 + ++ +++
disorganization 96 + +++ +++
Vacuolization 24 + ++ ++
96 + ++ ++
Capillary diameter 24  ++ ++
increase 96  ++ ++

The number of plus symbols is proportional to the assessed damage.

Lesion grade: () absent, (+) light, (+ +) moderate, (+++) high.
a
In the case of 1 mg/L of copper, the last histopathological study was
realized at 72 h exposure because at 96 h all specimens were died.

The grade of these lesions in S. senegalensis juveniles at

different exposure time is shown in Table 2.

Discussion

Acute toxicity

The exposition of fish to sublethal concentrations of

heavy metals has correlation generally with an accumu-
lation, more or less linear, in target organs, producing at
the same time, histomorphological alterations. The LC50
values showed a gradual decrease with the increase of
the exposure time. In general, the increase in percent
mortality was related to both time and copper concen-
trations. The highest mortality occurred at 48 h in the Fig. 2. (A) Histological section of liver in Solea senegalensis
highest concentration tested of the metal. specimen. Normal aspect of hepatic parenchyma. Hepatocyte
The sensitivity of S. senegalensis was found to fall (h) and sinusoid (s) (hematoxylin/VOF  40). (B) Blood
stagnation in a hepatic capilar of a specimen exposed to
within the range of other fish species reported in
0.01 mg/L copper during 96 h (hematoxylin/VOF  25). (C)
literature. Only the concentrations used of 0.01 and Hepatic vacuolization in S. senegalensis exposed to 0.1 mg/L
0.1 mg/L copper could be considered sublethal because copper at 96 h exposure (hematoxylin/VOF  25). (D) Vacuo-
those concentrations did not produce the death of the lization grade increase in S. senegalensis exposed to 1 mg/L
organisms. Arellano et al. (1999) observed the same copper at 96 h exposure (hematoxylin/VOF  10).
effects in adults of S. senegalensis exposed to 0.1 mg/L
copper. Ortiz (1997) observed no mortality in Fundulus
heteroclitus exposed to 0.2 and 0.8 mg/L copper. mortality of 26.6% at 24 h, 83.3% at 48 h, 96.6% at 72 h
In the copper test with juvenile S. senegalensis, the and 100% at 96 h exposure. Torres et al. (1987) did
juveniles exposed to 1 mg/L copper showed a corrected not observe any mortality in Scyliorhimus canı´cula exposed
 ARTICLE IN PRESS
118 M. Oliva et al. / Experimental and Toxicologic Pathology 61 (2009) 113–121
to 2 mg/g copper but 4 mg/g of copper produced a
mortality of 50% at 48 h exposure. Hansen et al. (2002)
observed, in rainbow trout juveniles exposed to 0.05 mg/L
copper, a mortality of 47.8% at 56 days exposure.
Using Spearman–Karber method, we observed a
decrease of the LC50 with the increase of the exposure
time and an LC50 of 0.35 mg/L copper at 96 h exposure.
The finding indicates the existence of a metal accumu-
lative process in the tissues and due to this process, the
apparition of irreversible histopathological alterations
that affect to physiological health of the organisms
producing the mortality of them.
The relative importance of acute toxicity test with fish
to derive sound water quality criteria has been stressed
by different author and organizations (i.e. NAS/NAE,
1973; EEC, 2000). In most of the predictions, the
application factor (0.1–0.0001) is actually used along
with LC50 values. In order to save the life of marine
organisms, maximum permissible levels of the heavy
metals were established by employing an application
factor of 0.01 to 96 h LC50 values (Ahsunullah et al.,
1981). Accordingly, the calculated safe concentration for
copper in juvenile S. senegalensis was 0.0032 mg/L.
Andalusia is a region located in the south of Spain,
between the Atlantic Ocean and the Mediterranean Sea.
The regional government of Andalusia has approved
two water-quality objectives (WQO) for copper (II)
(Order 14.02.1997). For normal, open, and non-enclo-
sure waters or waters of a special ecological value, the
WQO is 0.02 mg/L, while for limited waters, estuaries,
bays, and any other enclosure waters (hatchery zones of
this fish), the WQO is 0.04 mg/L. The safe concentration
for copper obtained in this work demonstrates than the
criterion for limited waters do not protect totally the
early stages of S. senegalensis due to the histopatholo-
gical alterations observed at 0.01 mg/L of copper and
the safe concentration value calculated in this study.
Unless the great difference between the mortality
percentages of the three concentrations employed, the
realization of other bioassays with other intermediate
concentrations for the determination with more preci-
sion of LC50 values and the safe concentration value for
copper in this species would be necessary.
Histopathology
Copper exerts a wide range of physiological and
histopathological effects on fish (Playle et al., 1993; Teh
et al., 1997; Capeta da Silva, 1997; Ortiz, 1997). This
metal affects gills and liver that are target organs for
aquatic pollutants.
Gills
As gills are the main site for gas exchange and other
important functions such as ionic and osmotic regulation
and acid–base equilibrium, histopathological changes in
gill structure will involve respiratory anomalies and
electrolytic balance disruption (Evans, 1987).
The exposure of fish to copper is associated with
structural damages in gill epithelia. Lauren and McDo-
nald (1985) demonstrated that the acute exposure of
rainbow trout to copper is correlated with a decline in
blood Na+ and Cl concentrations.
Arellano et al. (1999) observed lifting and peeling of
the lamellar epithelium as common lesions in the
Senegal sole gills after copper exposure. Lamellar
fusion, hyperplasia, rupture of capillaries and, conse-
quently, release of erythrocytes also occurred.
Ortiz et al. (1999) observed in F. heteroclitus exposed
to copper an increase in mucus secretions in the gills as
well as hypertrophy in the chloride cells, with signs of
blood extravasation.
Studies of the morphological changes induced by
copper in fish gill tissue have shown two types of
responses: defensive (inflammatory) and compensatory
(cell proliferation, mucus secretion) responses (Mallatt,
1985; Takashima and Hibiya, 1995). Both responses
help in hindering the entry of toxic substances or
pollutants and prevent them from reaching the blood-
stream and, to a lesser extent, prevent damages caused
by the direct effects of copper (necrosis, peeling of gill
filament) (Cerqueira and Fernández, 2002). These
modifications can produce adverse effects on the health
of fish, and may increase their susceptibility to
secondary infectious diseases and cause even death
(Hawkins et al., 1984). This susceptibility may increase
with undernourished individuals since generally higher
copper levels are detected (Collvin, 1984; Segner, 1987;
Ortiz et al., 1999).
Light microscopy studies on the effects of copper
accumulation on fish gills revealed pathological altera-
tions after copper treatments ranging from 0.01 to 1 mg/L
(Bunton and Fraizier, 1989; Arellano et al., 1999; Ortiz
et al., 1999; Cerqueira and Fernández, 2002).
The histopathological changes in S. senegalensis gills
following acute exposure to copper were similar to those
reported by Arellano et al. (1999) for the adult specimen
of the same species and toxic agent. Lifting and peeling
of the lamellar epithelia were common lesions in adult S.
senegalensis gills during copper exposure (0.1 mg/L).
Additional effects include lamellar fusion, hyperplasia,
rupture of capillaries, and consequently, release of
erythrocytes (Arellano et al., 1999).
Ortiz et al. (1999) observed, in F. heteroclitus exposed
to 0.8 mg/L of copper, histopathological alterations as
hyperplasia with lamellar fusion, epithelial hypertropro-
phy, edemas with epithelial separation of the basal
membranes, generalized necrosis, and epithelial flaking.
Cerqueira and Fernández (2002) observed, in Prochi-
lodus scrofa exposed to 0.02 mg/L of copper, severe
histopathological changes in gill epithelia. Hypertrophy
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M. Oliva et al. / Experimental and Toxicologic Pathology 61 (2009) 113–121
of pavement and chloride cells, hyperplasia of filament
epithelia, and lifting of the lamellar epithelia decreased
the interlamellar distance. Vascular changes were
characterized by aneurysms and rupture of the blood
vessels and the epithelial cell layers and were usually
accompanied by hemorrhages. Excessive cell prolifera-
tion in the filament epithelia caused partial or total
lamellar fusion.
Similar alterations have been described for other
species contaminated with copper and other heavy
metals such as Zn or Hg (Daoust et al., 1984; Sola
et al., 1995; Krishnani et al., 2003).
These epithelial changes appear to be a general
response to most irritating agents in fish gill tissue.
Most gill changes caused by copper in S. senegalensis
were characterized by an inflammatory response of the
tissue. These responses may be progressive under
unchanged environmental conditions, leading to irre-
parable damage of gill morphology and function and
even to fish mortality. In the case of removable polluting
agents, damage may become regressive (Cerqueira and
Fernández, 2002).
Heavy metals produce osmorregulatory, acid–base, or
hemodynamic malfunctions. Inflammatory responses
and flaking of epithelial tissue could be related to a
decrease of the gill Na+, K-activated ATPase, and with
a decline in blood Na+ and Cl concentrations
(Arellano et al., 1999). Cu2+ can compete with Ca2+
for biological ligands (Neiboer and Richardson, 1980),
producing membrane alterations. Lamellar fusion was
found in sole contaminated specimens. This change
could be a protective response in order to diminish the
amount of vulnerable gill surface area (Mallatt, 1985).
An increase of the number and proportion of the
mucous cells and hypersecretion of branchial mucus
appears to be a defense mechanism against pollutant
exposures (Benedetti et al., 1989) and was observed in
gills of other fish species exposed to copper (Arellano
et al., 2000; Ortiz et al., 1999; Cerqueira and Fernández,
2002). In this work, such effect has not been observed
even a decrease of the mucus secretion and empty
mucous cell was observed. This effect could be due to
the modification in the mucous molecular composition
produced by copper and the fact that the young
organisms were starved during the assay.
Liver
Light microscopy studies on the effects of copper on
fish liver revealed pathological alterations after copper
treatments ranging between 1 and 3.2 mg/L (Baker,
1969; Eisler and Gadner, 1973; Bunton and Fraizier,
1989).
The liver is a target organ for copper (Collvin, 1984).
Buckley et al. (1982) observed a significant correlation
between the environment copper levels and its accumu-
lation in the liver. Nevertheless, this situation can be
119
more complex since the fish are able to regulate the
incorporation of metals (Segner, 1987).
In S. senegalensis exposed to copper, a generalized
disorganization of hepatic parenchyma was observed,
this disorganization was related to the increase of the
copper exposure time.
At 0.01 mg/L of copper, the pathological alterations
were not important, being observed a certain disorga-
nization of hepatic parenchyma and signs of vascular
degeneration. At 0.1 and 1 mg/L of copper, the injury
degree was elevated being observed necrotic zones, a
disorganization of total hepatic parenchyma, blood
stagnation, and expansion of the capillaries. Also, a
vacuolization of the tissue that increases with the
concentration of toxic metal was observed.
The hepatocytes presented a diffuse degeneration of
the cytoplasm and in some cases, although of moderate
form, some zones of the tissue with steatosis were also
observed.
Steatosis could possibly indicate an alteration of lipid
metabolism or a partial change in the morphology of
liver. In fish treated with different heavy metals,
morphological changes in lysosomes were observed by
Segner (1987) and Segner and Storch (1985). Arellano
et al. (1999) observed that the endothelial lining of the
sinusoids and the microvilli of the hepatocytes were often
disrupted and membranous inclusions near the sinusoids
were observed in the hepatic endothelia of S. senegalensis
during the contamination and depuration periods.
In fish, the liver tends to concentrate metals and
exhibits relatively high potential for bioaccumulation
and detoxification (Roesijadi and Robinson, 1994).
Excessive copper loading leads to the synthesis of
metallothionein in the liver, which is related to
detoxification mechanisms (Bremmer, 1991).
Ortiz (1997) observed, in F. heteroclitus exposed to
sublethal concentrations of 0.2 mg/L copper, a gradual
increase of the copper levels in the liver during the
exposure time with no great differences in the hepatic
copper levels between the fed and starved organisms. In
fishes exposed to 0.8 mg/L copper, the same effect with
the difference that copper levels in liver were higher in
the fed specimens compared to the starved specimens
was observed.
In Chanos chanos exposed to 0.1 mg/L of copper,
Segner and Braunbeck (1990) did not observe serious
hepatic alterations. Nevertheless, Ortiz et al. (1999), in
F. heteroclitus exposed to concentrations of 0.8 mg/L
copper, observed a general disorganization of the
hepatic structure, focal necrosis, and erythrocyte accu-
mulation in the hepatic parenchyma and the vascular
system.
Arellano et al. (1999) observed in adults of S.
senegalensis exposed to 0.1 mg/L copper vacuolization
in the hepatic parenchyma and steatosis being observed
gradual copper accumulation in liver during the
 ARTICLE IN PRESS
120 M. Oliva et al. / Experimental and Toxicologic Pathology 61 (2009) 113–121
exposure time of the organisms to sublethal copper
concentrations.
The present study demonstrated that sublethal con-
centrations of 0.01–0.1 mg/L of copper cause histologi-
cal alterations in liver and gills and due to the
importance of S. senegalensis in the Andalusia littoral,
we recommend the revision of the actual WQC and the
election of new objectives according to the sensibility of
this species.
Acknowledgments
The authors would like to extend their gratitude to
Mr. Santos for technical assistance.
This work was supported by Project SEAPAT:
‘‘Biomarkers and histopathology in gilthead seabream,
Sparus aurata, and Senegal sole, Solea senegalensis’’
(CICYT, REN 2003-09361-CO3-03).
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