Environ Monit Assess (2009) 152:209–222

DOI 10.1007/s10661-008-0309-3

An assessment of acute biomarker responses in the demersal

catfish Cathorops spixii after the Vicuña Oil Spill
in a harbour estuarine area in Southern Brazil
A. Katsumiti & F. X. Valdez Domingos &
M. Azevedo & M. D. da Silva & R. C. Damian &
M. I. M. Almeida & H. C. Silva de Assis &
M. M. Cestari & M. A. F. Randi &
C. A. Oliveira Ribeiro & C. A. Freire

Received: 6 November 2007 / Accepted: 9 April 2008 / Published online: 14 May 2008
# Springer Science + Business Media B.V. 2008

Abstract The Vicuña oil tanker exploded in Para- orops spixii, comparing a contaminated (at the spill
naguá Bay (South of Brazil), during methanol site) and a reference site inside the Bay. Data were
unloading operations in front of Paranaguá Harbour, compared to previous measurements, taken before the
on November 15th, 2004, releasing a large amount of accident, in the same species, from the same sites.
bunker oil and methanol. Two weeks after the The physiological biomarkers were the ones that best
accident, the acute effects of the Vicuña Oil Spill reflected acute effects of the spill: plasma osmolality,
(VOS) were evaluated in the demersal catfish Cath- chloride, calcium, magnesium, and potassium. Mor-
phological (liver and gill histopathology) and genetic
(piscine micronucleus and DNA strand breaks) bio-
markers revealed that damage was already present in
A. Katsumiti : F. X. V. Domingos : M. A. F. Randi :
fishes from both reference and contaminated sites
C. A. O. Ribeiro inside the Bay. Thus, the reference site is not devoid
Departamento de Biologia Celular, of contamination, as water circulation tends to spread
Setor de Ciências Biológicas, the contaminants released into other areas of the Bay.
Universidade Federal do Paraná (UFPR),
CEP 81531-990 Curitiba, Paraná, Brazil
Acute field surveys of oil spill effects in harbour areas
with a long history of contamination should thus be
M. Azevedo : C. A. Freire (*) viewed with caution, and whenever possible previous
Departamento de Fisiologia, Setor de Ciências Biológicas, evaluations should be considered for proper appraisal
Centro Politécnico,
Universidade Federal do Paraná (UFPR),
of biomarker sensitivity, especially in mobile bio-
CEP 81531-990 Curitiba, Paraná, Brazil indicators such as fish.
e-mail: cafreire@ufpr.br
Keywords Biomarkers . Cathorops . Estuary .
M. D. da Silva : H. C. S. de Assis
Departamento de Farmacologia, Setor de Ciências
Harbour . Hydrocarbons . Oil spill . Paranaguá Bay
Biológicas, Universidade Federal do Paraná (UFPR),
CEP 81531-990 Curitiba, Paraná, Brazil

R. C. Damian : M. I. M. Almeida : M. M. Cestari

Introduction
Departamento de Genética, Setor de Ciências Biológicas,
Universidade Federal do Paraná (UFPR), In November 15th, 2004, the Chilean ship Vicuña
CEP 81531-990 Curitiba, Paraná, Brazil exploded after the unloading of around 9 million liters
210 Environ Monit Assess (2009) 152:209–222

of methanol to the marine terminal of the Cattalini ermen that directly depend on the marine resources
company in Paranaguá Bay, southern Brazil (Figs. 1 for their survival, and who were obviously directly
and 2). The Vicuña tanker was loaded with approx- affected by the contamination of the Bay (ABDL/ISA
imately 1,265,000 l of bunker oil, 173,000 l of diesel 2005).
oil and around 4,079 tons of methanol before the Biomarkers have been previously used in sentinel
explosion (IAP 2005). The oil spill reached the species of fish after other oil spill accidents. Follow-
Paranaguá, Antonina and Guaraqueçaba Bays at a ing the Braer spill on the coast of Shetland (Scotland)
distance of more than 30 km from the explosion site, in 1993, hepatic ethoxiresorufin O-deethylase
in front of Paranaguá Harbour (Figs. 1 and 2). The (EROD) activities in livers of inshore demersal fish
largest Harbour in Southern Brazil, Paranaguá Har- species at various sites were found to be elevated ~7–
bour has the loading and unloading of grains and 9-fold, 3 months after the accident (George et al.
containers among its most important operations. 1995). Two weeks and 3 months after the Sea
Some major companies have private terminals, such Empress oil spill that affected South West Wales in
as that of Fospar (fertilizers), PETROBRÁS (flam- 1996, EROD activity in fish (Kirby et al. 1999), and
mables), Dibal (vegetable oil), Becker (acids), and DNA-adduct levels in mussels and fish (Lyons et al.
Cattalini (soy oil, methanol and petroleum deriva- 1997, Harvey et al. 1999) were able to discriminate
tives; Paranaguá and Antonina Harbour Administra- between areas directly polluted by the spill and
tion, retrieved from www.portosdoparana.com.br in unpolluted locations. Most data concerning the use
July 2007). Important conservation areas such as the of biomarkers to assess biological effects of oil spills
Superaguí National Park and the Ecological Station of have been obtained after the Exxon Valdez oil spill
Guaraqueçaba, located in the estuarine complex of (EVOS), which affected Northern Prince William
Cananéia-Iguape-Paranaguá, also received oil from Sound (Alaska) in March 1989. One year after the
the Vicuña spill around 5 days after the explosion accident, histopathological alterations as focuses of
(Fig. 2). Ten days after the accident, birds, turtles and hepatic lipid were found in Sebastes ruberrinus
dolphins, and many invertebrates were found dead, (Marty et al. 2003); increase in macrophage aggregate
covered with oil (IAP 2005). Therefore, we can scores were reported for Sebastes maliger (Khan and
consider that all aquatic organisms were indeed Nag 1993), hepatic necrosis in Clupea pallasi (Marty
exposed to VOS hydrocarbons (IAP 2005). This et al. 1999), and genetic damages were also registered
estuarine area shelters about 4,000 independent fish- in larvae of C. pallasi (Norcross et al. 1996; Hose et

Fig. 1 Map showing the

Paranaguá Bay complex in
the Southern coast of Brazil,
depicting the localization of
the Vicuña Oil Spill and the
reference and contaminated
sites. The gray shaded area
illustrates the numerical sim-
ulation of the spread of the
oil spilled from the accident,
at the day the catfish C.
spixii have been obtained
(December 1st, 2004; Goobi
et al. 2004; image retrieved
from www.simepar.br in
December 2004)
Environ Monit Assess (2009) 152:209–222 211

al. 1996; Hose and Brown 1998) after the accident, all
associated with oiled sites.
No studies, to our knowledge, have so far proposed
an evaluation of the short-term effects of an oil spill
accident with a time reference for the same species
and sites. In addition, the current study poses the
question: Are those biomarkers (biochemical, physi-
ological, genetic, and morphological), when applied
to a demersal fish species, suitable for the evaluation
of the effects of an oil spill in a harbour area a few
weeks after the accident?

Materials and methods

Animals

Cathorops spixii (Ariidae, Actinopterygii) is a tropical

demersal catfish thoroughly distributed along the
coast of South America, from Colombia to Uruguay
(www.fishbase.org, version 02/2007). This species is
found in shallow coastal marine waters and brackish
estuaries, lagoons and river mouths, as well as in
hypersaline waters (Cervigón et al. 1992), feeding
mainly on invertebrates and small fishes (Le Bail
et al. 2000).
Adult specimens were sampled from Contaminated
(C) and Reference (R) sites in Paranaguá Bay (Fig. 1) in
February, 2004 (before the explosion), and again in
December, 2004 (2 weeks after the explosion).
Significant seasonal differences should not be expected
between these two dates, as they are within the
Summer season, the rainiest season in Southern Brazil,
with minor temperature differences (Technological
Institute SIMEPAR, www.simepar.br, retrieved on
April 2005; Lana et al. 2000; Valdez Domingos et al.
2007). The Contaminated (C) site (25°19′430″S; 48°
32′398″W) was located in front of the Harbour closer
to the accident area. This site had visible oil patches at
the second sampling date for this study (December 1st,
Fig. 2 Photographs depicting the Vicuña oil spill in front of 2004). Besides the spilled oil, this area regularly
Paranaguá Harbour. A One day after the explosion (November receives runoffs from Harbour, local industrial and
16th, 2004), showing barriers placed around the ship; B 11 days
after the explosion (November 26th, 2004) – 5 days before the
agricultural activities, as well as domestic sewage. The
sampling date for this study – oil unrestrained by the barriers; C Reference site (R) (25°31′271″S; 48°29′690″W) was
1 day after our sampling date (December 2nd, 2004), fishermen located in Laranjeiras Bay, away from the influence of
boats with absorption barriers helping to prevent further spread harbor activities in a relatively well preserved area of
of the oil. From IAP (2005)
the estuarine complex (Fig. 1).
212 Environ Monit Assess (2009) 152:209–222

Eight days after the explosion, the sum of 16 Physiological biomarkers

isomers of petroleum hydrocarbons (among which,
naphtalene, fluorene, anthracene, etc) in the area Plasma osmolality and ion concentrations
around the explosion site was between 33 and
480 ng/g sediment (IAP 2005). In this area, Plasma osmolality was read in undiluted samples
sediment levels remained high 35 days after the using the vapor pressure micro-osmometer Wescor
explosion (December 20th): 234–330 ng/g sediment 5520 VAPRO. Sodium and potassium concentrations
(IAP 2005). One day after our sampling date for this were read in duplicates through flame photometry
study (December 2nd), oils and greases were (Digimed DM 61) in appropriately diluted samples
detected in the water, not only next to the Harbour (1:1,000). Chloride, calcium and magnesium concen-
(contaminated, C site), but also inside Baía das trations were read through colorimetric methods
Laranjeiras, here considered our reference (R) site, (Labtest kits, Lagoa Santa, Brazil), after appropriate
and at the Ilha do Mel, at the entrance of the whole dilutions of the samples in ultrapure water (1:3 for Cl−
estuarine system, compatible with the predicted and Ca2+, 1:10 for Mg2+).
spread of the spilled oil by SIMEPAR (Fig. 1 and
IAP 2005). Genetic biomarkers
Sampled fish were transported (1 h) in tanks
(100 l) with aeration to the Fish Laboratory of the Piscine micronucleus test
Center for Marine Studies of the Federal University of
Paraná (CEM/UFPR), located in Pontal do Paraná. The micronucleus test was performed on a thin smear
Fish were then anesthetized with benzocaine (0.04%) of a drop of fish blood, on glass slides. The smear was
for the following procedures. After complete anesthe- air-dried for 1–2 h, fixed with absolute methanol
sia, blood was sampled for the piscine micronucleus (Merck) for 1 h, and stained for 20 min with 5% (w/v)
test and comet assay, and plasma osmolality and ion Giemsa in phosphate buffer (Merck), pH 6.8 (Ferraro
determinations. Muscle samples (~2–4 g) were col- et al. 2004). Erythrocytes (2,000 cells) were exam-
lected for cholinesterase activity, and the gills (two ined, for each fish, under 1,000×-magnification, and
right branchial arches) and liver (~2–4 g) for light were scored for the presence of both typical micro-
microscopy procedures. nuclei (MN) or nuclear alterations (NA), characterized
as changes in the normal elliptic shape of the nucleus
Biochemical biomarker (Ayllon and Garcia-Vazquez 2000). These two fea-
tures were considered as nuclear abnormalities and
Cholinesterase (ChE) activity were scored together.

Axial muscle samples were homogenized (5% w/v, Comet assay

Potter-Elvehjem) in 2 ml phosphate buffer (0.1 M,
glycerol 20%, pH 7.5). Homogenates were centri- For the comet assay, we followed the Speit and
fuged for 20 min at 12,000×g (4°C). Cholinesterase Hartmann (1999) procedure. A blood sample (10 μl)
activity was determined using the colorimetric tech- was diluted in fetal calf serum (1 ml). The slides for
nique described by Ellman et al. (1961), and later microscopy were prepared with the cell suspension in
adapted by Monserrat et al. (2002). Acetylthiocholine low melting point agarose (120 μl) at 37°C, followed by
iodide (ATC) 9 mM was used as substrate and 5,5′- incubation in lysis solution for 1 h in the dark, at 8°C.
dithio-bis (2-nitrobenzoic) acid (DTNB) as the color After lysis, the slides were placed in buffer (pH>13) for
reagent. Optical density was measured using an 25 min to allow unraveling of the DNA. Electrophoresis
ELISA plate reader (TECAN) at 415 nm. ChE (25 min at 25 V and 300 mA) was performed, and slides
activity was expressed per mg protein. Protein were neutralized for 15 min in 0.4 M Tris, fixed in
concentrations were measured at 595 nm using the absolute ethanol (Merck) for 10 min, and stained with
Bradford method (1976), with bovine serum albumin ethidium bromide (0.02 μg/ml). DNA strand breaks
as standard. were scored using a Leica epifluorescence microscope
Environ Monit Assess (2009) 152:209–222 213

at a magnification of 400×. For each fish, 100 cells were was significantly higher compared to the same site
visually analyzed according to the method of Kobayashi before the Vicuña Oil Spill (VOS, 54.8±4.8 ηmol
et al. (1995). min−1 mg prot.−1; Fig. 3). No differences were noted
with respect to the oil spill in fish obtained from the
Morphological biomarkers contaminated (C) site. In addition, either before or
after the VOS, no differences were detected between
Light and scanning microscopy fish from C and R sites (Fig. 3).

Liver and gills from C. spixii were fixed in ALFAC Physiological biomarkers
fixative solution (ethanol 80% – 85 ml, formaldehyde
40% – 15 ml and glacial acetic acid – 5 ml) for 16 h, Plasma osmolality and ion concentrations
were dehydrated in a graded series of ethanol, and
embedded in Paraplast Plus resin (Sigma). Five- Plasma osmolality in catfishes decreased after the
micron sections were stained with hematoxylin and VOS in fishes obtained from both sites (Fig. 4A).
eosin, and images were obtained and recorded Fishes from the R site, after the VOS, displayed the
through a Zeiss Axiophot Photomicroscope. Free lowest plasma osmolality values (344.73 ±
melanomacrophages and melanomacrophage centers 3.51 mOsm/kg H2O) when compared with the other
in liver were counted as described by Rabitto et al. groups (Fig. 4A). Plasma osmolality decreased in the
(2005) and other lesions in liver were quantified as C site after the VOS (375.3±5.2 mOsm/kg H2O),
described by Bernet et al. (1999). when compared to the respective value measured in
fish from the same site before the accident: 399.4±
Statistical analysis 7.7 mOsm/kg H2O. Plasma chloride values were
higher in fish from the C site after the VOS (185.6±
Piscine micronucleus and comet assay test results are 7.2 mM) than in fish from the same site before the
expressed as median±1 quartile, and differences were accident (159.0±5.2 mM), or fish from the R site after
localized using the Kruskal–Wallis test followed by the VOS (157.6±7.6 mM; Fig. 4B). Plasma potassi-
the Student–Newman–Keuls test. Results from bio- um concentration showed the lowest values in fishes
chemical and physiological procedures are displayed from the C site after the VOS (6.8±0.4 mM), a value
as mean±SEM. Data were tested for normality using markedly lower from values obtained for fishes from
the Shapiro–Wilk W-test. Cholinesterase activity and the R site before VOS (10.1±1.2 mM; Fig. 4C).
plasma concentrations were analyzed by One-Way Plasma calcium increased after the VOS, from 2.6±
ANOVAs followed by the post hoc test of Tukey. 0.1 mM in fishes from the R site before the VOS, to
Morphological evaluation of free melanomacroph- 3.5±0.1 mM in fish from the R site and 3.3±0.2 mM
ages, melanomacrophage centers and necrosis count-
ing was performed using a One-way ANOVA. For all 120
b
a,b
Cholinesterase activity

tests, a level of significance of 0.05 was adopted.

ηmol.min-1.mg prot.-1)

(10)
(10)
Other morphological parameters are displayed as 90 a,b
a (10)
percentages, and were treated only qualitatively. (10)
60

30
Results
0
R C R C
(η

Biochemical biomarker
before after

Cholinesterase activity Fig. 3 Biochemical biomarker. Cholinesterase activity in

muscle of C. spixii from Paranaguá Bay before and after the
Vicuña Oil Spill, for fish obtained from either the reference (R)
Cholinesterase activity in the reference (R) site after or the contaminated (C) site. Bars Mean±SE. Bars that do not
the oil spill accident (85.7±7.7 ηmol min−1 mg prot.−1) share a lower case letter are significantly different (P<0.05)
214 Environ Monit Assess (2009) 152:209–222

Fig. 4 Physiological biomarkers. Plasma osmo-ionic concen- share a lower case letter are significantly different (P<0.05). A
trations of C. spixii from Paranaguá Bay before and after the Osmolality; B chloride; C potassium; D calcium; E sodium; F
Vicuña Oil Spill, for fish obtained from either the reference (R) magnesium plasma concentrations
or the contaminated (C) site. Bars Mean±SE. Bars that do not

in fish from the C site after the VOS (Fig. 4D). Genetic biomarkers
Plasma sodium showed increased values in fish from
the C site, but only before the VOS: 221.8±10.2 mM, Piscine micronucleus test and comet assay
against 181.8±6.5 mM in fish from the R site, also
before the accident (Fig. 4E). As with sodium, plasma The piscine micronucleus test showed a high occur-
magnesium increased sharply in fishes from the C rence of nuclear alterations in fishes from the C site,
site, but only before the VOS: from 3.1±0.4 mM in both before and after the VOS. When compared to its
fishes from the R site (before the VOS) to 8.0± respective R site, the difference was significant only
0.9 mM in the C site. However, after the VOS, values after the VOS (Fig. 5A). Similar results were found
of plasma magnesium remained high, in fishes from through the comet assay test, which revealed high
both sites (Fig. 4F). levels of DNA strand break scores, either before or
Environ Monit Assess (2009) 152:209–222 215

before the accident (compare with healthy hepatic

tissue in Fig. 6C), and leukocyte infiltrations were
equally abundant either before or after the VOS
(Table 2). After the oil spill, liver parasites were
frequent in both sites, but hemorrhages (Fig. 6F) were
observed only in livers of individuals from the C site
(Table 2). The occurrence of melanomacrophage
centers (Fig. 6D,E), free melanomacrophages and
necrosis (Fig. 6G) in liver was not different between
both studied sites, either before or after the VOS
(Table 3).

Discussion

Cholinesterase activity is widely used as a biomarker

in biomonitoring studies to detect sublethal effects of
xenobiotics as organophosphates and carbamates
(Weiss 1958; Abou-Donia and Menzel 1967; Babu
et al. 1989; Magnotti et al. 1994; Thompson et al.
1995; Silva de Assis 1998). Other contaminant
Fig. 5 Genetic biomarkers. Genotoxicity detected in C. spixii classes such as metals, hydrocarbons and paper mill
from Paranaguá Bay before and after the Vicuña Oil Spill, for
effluents are also associated with cholinesterase
fish obtained from either the reference (R) or the contaminated
(C) site. Bars Mean±SE. Bars that do not share a lower case inhibition (Labrot et al. 1996; Guilhermino et al.
letter are significantly different (P<0.05). A Piscine micronu- 1998; Galgani and Bocquene 1990, Payne et al.
cleus (MN) test. NA Nuclear alterations; B DNA strand breaks 1995). According to Kaufer et al. (1999), capture
scored under the Comet Assay
stress can alter ChE activity. Pavlov et al. (1994)
described an increase in the ChE activity after
after the VOS in fishes from the C site, and also in exogenous adrenaline injection in fish. The present
fishes from the R site before the VOS (Fig. 5B); results indicated an increase in ChE activity in the R
fishes from the R site after the VOS presented the site, after the VOS, a result which is difficult to
lowest scores of damage (64.5±28; Fig. 5B). associate either with chronic pollution of the area, or
with the oil spill, specifically. Thus, this biomarker is
Morphological biomarkers apparently not ideal for an acute evaluation of the
effects of an oil spill in demersal fish species.
Light microscopy of gills and liver Osmotic and ionic homeostasis of the extracellular
fluid of the catfishes was distinctly affected by the
A high incidence of branchial epithelium lifting was general long-term harbour contamination of Para-
observed in fishes from both sites either before or naguá Bay, as well as by the exposure to oil from the
after the VOS (Fig. 6A) (Table 1). Hyperplasia in oil spill. Before the accident, salt secretion by the
secondary lamellae was higher (qualitative evaluation catfish gills and kidneys was suggested to be
only) after the VOS in fishes from both sites (Table 1), disturbed in fish from the C site, resulting in increased
followed by aneurisms (Fig. 6B). Lamellar fusions sodium and magnesium plasma levels (Fig. 4E,F).
and lamellar disorganizations presented a high occur- However, a different pattern emerged after the oil
rence in fishes from the R site before the VOS spill, with hampered osmotic and chloride homeosta-
(Table 1). After the VOS, the presence of branchial sis (Fig. 4A,B). A certain hypokalemia also devel-
parasites was higher in fishes from the C site, and oped after the oil spill in the C site, when compared to
lamellar bifurcations were observed only in this site the R site before the spill (Fig. 4C). As K+ is
(Table 1). Vacuolization in liver tissue was higher essentially an intracellular cation, either K+ regulation
216 Environ Monit Assess (2009) 152:209–222
Fig. 6 Histopathological
findings in C. spixii
obtained from the reference
(R) and contaminated (C)
sites, before and after the
VOS. A Normal structure
(ne) of gills and branchial
epithelial lifting (el); B gills
aneurisms (arrows); C nor-
mal liver structure; D mela-
nomacrophage center (mm);
E vacuolization of liver tis-
sue and melanomacrophage
centers (mm); F, Hemor-
rhagic area (ha); G area of
necrosis (na)

by the osmoregulatory epithelia (Singh et al. 2002), or anisms may be the cause of hypokalemia. Hypokale-
intracellular potassium regulation (or both) are prob- mia from exposure to insecticides has been reported
ably being disturbed. In addition, an indirect effect for the catfish Clarias batrachus and from exposure
from a disturbance of cell volume regulatory mech- to cadmium for the flounder Platichthys flesus (Singh
Environ Monit Assess (2009) 152:209–222 217

Table 1 Morphological alterations (% of occurrence) in gills of PAHs have been discussed as a cause for genotoxic
C. spixii from reference (R) and contaminated (C) sites before
effects in fish or fish cells detected with the comet
and after the Vicuña Oil Spill
assay (Kammann et al. 2001; Akcha et al. 2003). In
Before After addition, the process of PAH biotransformation is
R C R C often associated with an intracellular elevation of
reactive oxygen species (ROS) via redox cycling,
Lamellar fusion 70 50 50 40 some of which, mainly the hydroxyl radicals, are
Aneurisms 10 10 30 47 highly aggressive to DNA. These metabolites bind to
Branchial epithelium lifting 80 100 80 100
DNA via CYP transformation, particularly through
Lamellar disorganization 50 30 na na
the isoform 1A (Stegeman and Lech 1991; Goksøyr
Hyperplasia 0 30 60 60
Parasites na na 20 40 and Förlin 1992), to arene oxides, forming DNA
Lamellar bifurcation na na 0 7 adducts, that are more effective sites for the produc-
Lamellar hyperthrophy na na 0 0 tion of strand breaks. A high prevalence of unrepaired
DNA lesions may lead to incomplete transcription,
na Not assayed
cellular dysfunction, growth inhibition, aging, weak-
ened immunity and diseases in the organism itself
et al. 2002). These plasma alterations reflect pollu- (Woo et al. 2006), also potentially affecting food
tants responses, as estuarine fishes are excellent chains and ecosystems (Kurelec and Gupta 1993).
osmoregulators (Plaza-Yglesias et al. 1988; Cataldi The piscine micronucleus test has been previously
et al. 1995, Plaut 1998, Prodocimo and Freire 2001; demonstrated as an efficient tool, responsive to crude
Freire and Prodocimo 2007), even when exposed to a oil exposure, to different PAHs (Maria et al. 2002;
wide range of salinities; the salinity difference Gravato and Santos 2002, 2003; Teles et al. 2003), to
between both sites was negligible. Osmoregulatory petroleum derivatives (Pacheco and Santos 2001), or
blood parameters are indeed considered useful bio- other contaminants (Pacheco and Santos 1998; Ayllon
markers of pollution in estuarine environments, also and Garcia-Vazquez 2000, 2001; Gravato and Santos
when coupled to the analysis of impairment of the 2002). In accidents with oil spills, Frenzilli et al.
activity of key enzymes from the osmoregulatory (2004) showed a high level of DNA damage in
organs, such as the Na+, K+-ATPase or the carbonic Zoarces viviparus from a site impacted by an oil spill
anhydrase (reviewed in Monserrat et al. 2007). in the Göteborg harbour, 3 weeks after the accident.
Several studies report extracellular fluid ionic dis- Our data confirm the results obtained by this previous
turbances upon exposure of fish to metals (Wood et study, and the rapid fish genotoxic response from oil
al. 1999; Rogers et al. 2003) or insecticides (Singh et spills. The quantification of damage to genetic
al. 2002). It appears that the chronic pollution in material was indeed a good biomarker for the acute
Paranaguá Bay resulted in higher plasma sodium and effects of the oil spill, being enhanced in the site next
magnesium levels, while the acute effects of the oil to the accident (Fig. 5). However, the previous results
spill on the catfish osmoregulation were mostly obtained from those sites and that same species make
reflected on osmolality and chloride. It should be the picture a bit more complex. Nuclear abnormalities
pointed out that high plasma magnesium and calcium
levels were measured in fishes caught both at the R Table 2 Morphological alterations (% of occurrence) found in
and the C sites after the VOS (Fig. 4D,F). This liver of C. spixii from reference (R) and contaminated (C) sites
before and after the Vicuña Oil Spill
observation adds to the already discussed enhance-
ment of ChE activity in the reference site, after the Before After
VOS. It may well be that more labile and volatile R C R C
compounds had already reached the site considered as
reference (R) (Figs. 1 and 2). Still, these physiological Vacuolization 70 60 20 20
data thus are suggested to be useful (and easy) Leukocyte infiltration 90 90 100 93
parameters to be monitored immediately following Hemorrhages na na 0 53
Parasites na na 60 73
an oil spill, even in already contaminated areas such
as Harbour areas. na Not assayed
218 Environ Monit Assess (2009) 152:209–222

Table 3 Morphological alterations/mm2 (mean±SEM) found in liver of C. spixii from reference (R) and contaminated (C) sites before
and after the Vicuña Oil Spill

Before After

R C R C

Free melanomacrophages 6.4±1.1 6.3±0.9 5.6±3.3 4.7±3.6

Melanomacrophage centers 11.0±2.8 9.5±1.5 7.9±5.2 13.5±7.0
Necrosis 0.27±0.07 0.18±0.02 0.12±0.12 0.10±0.05

were equally affected by the chronic contamination of These lesions hamper gas exchange and osmoregula-
Paranaguá harbour, as well as by the oil spill (Fig. 5). tion, essential functions for fish survival. Hyperplasia
The high levels of genetic alterations displayed by indicate tissue disruption, observed after chronic expo-
fishes obtained from the reference site again may sure to PAHs, chloride pesticides, PCBs and others (Ali
indicate chronic effects of contamination also in the and Sreekrishnan 2001; Shailaja and D’Silva 2003).
reference site, or, alternatively, recent migration of Morphological signs of exposure to chemical stress,
fish from more contaminated sites to cleaner areas, resulting in hepatic melanomacrophage centers, free
where they may have been sampled. It is known that melanomacrophages and necrosis, were frequent in
breaks detected by comet assay can be transient both reference and contaminated sites, either before or
lesions, either indicating high damage or an ineffi- after the VOS (Table 3). Melanomacrophage centers in
cient repair process (Collins et al. 1997). Although the liver are related to a higher phagocytotic activity, as
genetic material was sensitive to the oil spill, and its part of the immune response to tissue lesions in
damage effective to distinguish the contaminated exposed organisms (Couillard et al. 1999, Oliveira
from the reference site after the spill, again the Ribeiro et al. 2005, Rabitto et al. 2005). Hemorrhage
chronic contamination of the Bay may have clouded foci have been found only in the contaminated site
the observations. after VOS (Table 2). These alterations probably reflect
Gill and liver lesions as found in the present study an acute exposure.
have been previously reported in fish from estuarine However, previous analyses (1998–2000) of water
environments (e.g., Lyons et al. 2004, Noreña- and sediment in areas close to the reference and
Barroso et al. 2004, Oliveira Ribeiro et al. 2005), contaminated sites surveyed here revealed the pres-
and some of the lesions found in branchial tissue in ence of copper in the water (~0.065 mg/l, IAP –
the present study are considered as acute effects Environmental Institute of Paraná, available online,
(Zodrow et al. 2004). However, the occurrence of www.pr.gov.br/meioambiente/iap, retrieved in August
lamellar fusion was detected in fishes from both sites 2007), slightly above the limits set by CONAMA
before and after the accident. Lamellar fusion was (National Environmental Council, Resolution number
also reported for fish from areas contaminated by 20, of June 18, 1986, class 7) for brackish waters
sources such as paper mill effluents (Pacheco and (0.05 mg/l). No significant levels of contamination
Santos 2002; Valdez Domingos 2001), heavy metals either in the water or in the sediment were reported
such as copper (Arellano et al. 1999) and urban for cadmium, lead, chrome, mercury or zinc (IAP –
effluents with secondary treatment (Coutinho and Environmental Institute of Paraná, available online,
Gokhale 2000; Valdez Domingos 2001). Branchial www.pr.gov.br/meioambiente/iap, retrieved in August
epithelium lifting was found in all fishes obtained in 2007). Thus, some of the indications of damage
front of the harbour, either before or after the VOS. reported before the VOS may at least partially be
This alteration had been previously described in ascribed to the presence of copper. In addition, high
Arellano et al. (1999) and Fanta et al. (2003) in concentrations of arsenic were recently detected in
fishes exposed respectively to copper and organo- Paranaguá Bay (22.5 μg/l), including in the proximity
phosphate pesticides. As gills are directly exposed to of our reference site (14.4 μg/l; Anjos 2006). These
the external environment, these branchial alterations values are normally related to impacted estuarine
can represent a relatively acute answer to the oil spill. environments (Seyler and Martin 1990), such as
Environ Monit Assess (2009) 152:209–222 219

harbour areas. Those elevated levels of arsenic could in situ exposure to genotoxic compounds. Mutation
Research, 534, 21–32.
result from industrial activities such as the industry of
Ali, M., & Sreekrishnan, T. R. (2001). Aquatic toxicity from
fertilizers, petroleum handling activities, or even pulp and paper mill effluents: a review. Advances in
residual fractions from the VOS accident. Environmental Research, 5, 175–196.
In conclusion, the present study discloses that Anjos, V. E. (2006). Especiação de cobre e arsênio no
complexo estuarino da Baía de Paranaguá. (in Portuguese:
caution is necessary when acutely evaluating bio- Copper and arsenic speciation in the estuarine complex of
markers after a severe oil spill in an already Paranaguá Bay). Masters Thesis, Federal University of
contaminated region, such as an estuarine complex Paraná, Curitiba, Brazil
that houses an important commercial harbour. Further, Arellano, J. M., Storch, V., & Sarasquete, C. (1999). Histolog-
ical changes and copper accumulation in liver and gills of
the evaluation of biomarkers using fish as bioindica-
the Senegales Sole, Solea senegalensis. Ecotoxicology and
tors poses an additional problem, due to their Environmental Safety, 44, 62–72.
mobility, in that it is not possible to know how long Ayllon, F., & Garcia-Vazquez, E. (2000). Induction of micro-
they have in fact been exposed to the contamination nuclei and other nuclear abnormalities in European
minnow Phoxinus phoxinus and mollie Poecilia latipinna:
(or else present in the “reference” area). On the other
an assessment of the fish micronucleus test. Mutation
hand, the results suggest that, due to chronic chemical Research, 467, 177–186.
disturbance by a complex mixture of deleterious Ayllon, F., & Garcia-Vazquez, E. (2001). Micronuclei and other
chemicals present over a period of time the living nuclear lesions as genotoxicity indicators in rainbow trout
Oncorhynchus mykiss. Ecotoxicology and Environmental
organisms in those environments probably developed
Safety, 49, 221–225.
compensatory mechanisms. Still, the function of Babu, P. R. A., Reddy, G. R., Babu, G. R. V., & Chetty, C. S.
osmoregulation has been shown to be reasonably (1989). Recovery of Benthiocarb-Inhibited AChE in fish
sensitive for an acute evaluation of such oil spills. brain: an in vitro study. Ecotoxicology and Environmental
Safety, 17, 317–322.
And, despite the fast spread of oil to the whole Bay,
Bernet, D., Schmidt, H., Meier, W., Burkhardt-Holm, P., &
including the site considered as Reference (Figs. 1 Wahli, T. (1999). Histopathology in fish: proposal or a
and 2), the biomarkers employed were in general protocol to assess aquatic pollution. Journal of Fish
useful in disclosing differences between sites of Diseases, 22, 25–34.
Bradford, M. M. (1976). A rapid and sensitive method for the
distinct distance from the epicenter of an oil spill.
quantification of microgram quantities of protein utilizing
the principle of protein-dye binding. Analytical Biochem-
Acknowledgements We acknowledge the financial support istry, 72, 248–254.
by CNPq (Recos Project, Institutos do Milênio), National Cataldi, E., Ciccotti, E., Di Marco, P., Di Santo, O., Bronzi, P.,
Brazilian Council for Scientific and Technological Develop- & Cataudella, S. (1995). Acclimation trials of juvenile
ment, and DAAD, German Academic Exchange Service. The Italian sturgeon to different salinities: morpho-physiolog-
authors are grateful to Dr. Henry L. Spach from the Marine ical descriptors. Journal of Fish Biology, 47, 609–618.
Studies Center of the Federal University of Paraná for all Cervigón, F., Cipriani, R., Fischer, W., Garibaldi, L., Hen-
support during field work, and to Dr José E Gonçalves from drickx, M., Lemus, A. J. et al. eds. (1992). Fichas FAO de
SIMEPAR for information about the model predicting the identificación de especies para los fines de la pesca. Guía
spread of the oil after the explosion, cited in Fig. 1. de campo de las especies comerciales marinas y de aquas
salobres de la costa septentrional de Sur América. Rome:
FAO.
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