Chemosphere 202 (2018) 788e796

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Integrated use of histological and ultrastructural biomarkers for

assessing mercury pollution in piranhas (Serrasalmus rhombeus) from
the Amazon mining region
Ana Carolina Borges a, *, Caroline Da Silva Montes a, Liziane Amaral Barbosa a,

Francisco Berre
Maria Auxiliadora Pantoja Ferreira b, Jose ^do c, Rossineide Martins Rocha a
a
- Rua Augusto Corr^
Cellular Ultrastructure Laboratories - Biological Sciences Institute, Federal University of Para ea, 01 - Guama
. CEP 66075-110, Bel

em,
Para
, Brazil
b
Laboratory of Immunohistochemistry and Developmental Biology, Biological Sciences Institute, Federal University of Para
- Rua Augusto Corr^
ea, 01 -
Guama
. CEP 66075-110, Bel

, Brazil
em, Para
c
Coordination of Earth Sciences and Ecology (CCTE), Museu Paraense Emílio Goeldi - Av. Perimetral, 1901, Terra Firme. CEP 66077-530, Bel

, Brazil
em, Para

h i g h l i g h t s

MUCC is a protected area known for encompasses the largest mineral reserve in the world.

The local geology and hidrology have favor Hg entry in Itacaiúnas River.

THg concentrations in the bottom sediment did not represent risks to the aquatic biota from Itacaiúnas River.

Histological changes suggest a biological damage degree of the Itacaiúnas River.

Histological and ultrastructural biomarkers provide reliable data for assessement aquatic ecossistems pollution.

a r t i c l e i n f o

Article history: amounts of the aforementioned metal are released in different

Received 19 November 2017 Amazonian abiotic and biotic compartments (Bastos et al., 2006).
Received in revised form However, since mercury vapor (Hg0) presents stable chemical
7 February 2018 species such as its volatile form in the atmosphere, it can be
Accepted 26 February 2018
Available online 21 March 2018
transported on global scale and affect remote natural areas located
far from occasional contamination sources (Driscol et al., 2013).
Handling Editor: A. Gies Mercury may be found in its solution form and/or in suspension
in its inorganic forms Hg0 and Hg2þ, as well as associated with
other chemical species such as iron and manganese oxyhydroxides,
sulfates and carbonates, and complexed with organic matter. In
addition, it may reach relatively high and toxic concentrations in
water columns. Mercury may also precipitate and accumulate on
1. Introduction the bottom sediment depending on the physico-chemical and
biological properties of the water column, which may work as both
Mercury (Hg) found in aquatic environments in the Amazon mercury reservoir and source. When it comes to the aquatic biota,
derives from the transport phenomena and atmospheric deposi- mercury mainly accumulates in fish (Magalha ~es et al., 2015; Morel
tion, from lateritic soils in the region, and from deforestation, et al., 1998; Selin, 2009). The mercury methylation process results
burning and flooding events in extensive forest areas, as well as in organic derivatives such as methylmercury (CH3Hg) - its most
from the gold mining activity (Lechler et al., 2000; Malm, 1998; toxic form - and significantly increases the bioavailability, bio-
Roulet and Lucotte, 1995; Wasserman et al., 2003). The gold mining accumulation and biomagnification of such derivatives along the
activity stands out because, for decades, it has been using mercury trophic chain (Dabrowska et al., 2012). These derivatives interact
for gold amalgamation purposes. Consequently, substantial with living organisms and lead to multiple changes that may have
severe consequences, from the molecular to the ecosystem level.
These consequences change depending on the contamination level
* Corresponding author. and on exposure time (Adams et al., 1999; Brabo et al., 2003).
E-mail addresses: rmrocha@ufpa.br (A.C. Borges), auxi@ufpa.br (M.A.P. Ferreira), The main mercury-contamination route in humans lies on fish
^do), rmrocha@ufpa.br (R. Martins Rocha).
berredo@museu-goeldi.br (J.F. Berre

https://doi.org/10.1016/j.chemosphere.2018.02.169
0045-6535/© 2018 Elsevier Ltd. All rights reserved.
 A.C. Borges et al. / Chemosphere 202 (2018) 788e796
intake (WHO, 1990). Several studies have reported that more than
85% of the THg found in different fish species is bioaccumulated in
their muscles as methylmercury (Durrieu et al., 2005; Kasper et al.,
2009; Mieiro et al., 2009; Lacerda and Pfeiffer, 1992). The highest
trophic level species tend to accumulate the highest mercury
concentrations (Castilhos and Bidone, 2000), as well as larger
species (Do
rea et al., 2004; Lacerda et al., 1994). This fact that leads
to potential risks to human health, mainly to Amazonian riverine
populations that have fish as their main protein source (Bastos
et al., 2006). Therefore, these species are often used as water
quality bioindicators in environmental monitoring programs
(Arockia et al., 2013; Kroon et al., 2017).
Histology provides us with medium-term responses to sub-
lethal stressors in fish, representing a rapid method for detecting
toxic effects in several organs, which may be used as exposure
biomarkers (Bernet et al., 2004; Dalzochio and Gehlen, 2016; Van
Dyk et al., 2012). The liver is one of the organs most commonly
used as biomarker (Abdel-Moneim et al., 2012; Dabrowska et al.,
2012). It is the organ where the detoxification and excretion of
toxic substances takes place (Hinton and Lauren, 1990). In addition,
it plays a key role in the bioaccumulation of pollutants, besides
hosting a series of biotransformation enzymes (Van der Oost et al.,
2003).
The Caraja
s Mosaic of Conservation Units (MUCC - Mosaico de
Unidades de Conservaça ~o de Caraja
s) is a protected area of
approximately 520,000 ha that comprises five conservation units
(CUs) (Souza-Filho et al., 2016). The area, which holds significant
iron, copper, manganese and gold deposits, is the largest mineral
reserve in the world (Damous et al., 2002; Galarza et al., 2008).
Currently, the main exploitation form adopted in the area lies on
the industrial mining activity, which includes opencast extraction
processes applied to different ores (Ruivo and Sales, 1989; Souza-
Filho et al., 2016). In addition to the mining activity, different
land use forms such as pastures and urbanized areas can be found
around the protected site (Souza-Filho et al., 2016). In the 1980s and
1990s, the Eastern area surrounding the Mosaic was intensely
exploited by gold mining activities (Verde et al., 2014). Despite its
critical importance to the maintenance of ecosystem services, little
is known about the anthropic impacts on the area, such as mercury
contamination.
The aim of the present study was to assess the environmental
quality of Itacaiúnas River, the main geographical accident in the
region of the MUCC, by analyzing THg concentrations on bottom
sediments, as well as in the liver and muscles of S. rhombeus species
living in the river, and by associating them with histological and
ultrastructural changes in the liver of S. rhombeus individuals.
2. Materials and methods
2.1. Study site
The present study was carried out in Itacaiúnas River (Fig. 1),
which is the main geographical accident in the Caraja
s region
(390 km long). Its tributaries receive drainage from different mines;
consequently, its watershed is considered an area under the influ-
ence of the mining activity in the area.
2.2. Sampling sites
Samples were collected during the dry season (in 2011 and
2012) from three sampling sites along Itacaiúnas River. Site 1, S1
(5 590 68''e5 590 9200 S; 50 430 3600 e50 440 5200 W), is located in one
of the most preserved domains of MUCC, although it is affected by
the drainage of a decommissioned gold mine. Site 2, S2
(5 540 62''e5 540 0900 S; 50 330 2500 e50 3301900 W), was influenced
789
by one gold (decommissioned) and one copper mine. Site 3, S3
(5 51040''e5 500 7900 S; 50 270 2500 e50 260 7300 W), receives drainage
from manganese and copper mines. Two (2) km were randomly
selected in each site for sampling purposes. S1 is approximately
30 km away from S2, which, in its turn, is approximately 15 km
away from S3. There are geographical barriers such as rapids and
knickzones between sampling sites. The potential risk of mine areas
as mercury contamination sources was the criterion adopted in the
current study to define the herein selected sampling sites.
2.3. Physico-chemical parameters of the water
Temperature ( C), pH, electrical conductivity (mS cm1), total
dissolved solids (ppm) and dissolved oxygen (ppm) were measured
for each site. A previously calibrated HANNA® multi-parameter
probe (model HI9828) was herein used.
2.4. Fish collection
Fish were caught through the line fishing technique; the animals
were immediately anesthetized and euthanized, according to the
guidelines of the National Council of Animal Experimentation
Control (CONCEA). Individuals were measured (cm) and weighed
(g). An abdominal incision was made after the biometry in order to
identify the sex of the fish and to remove liver fragments for his-
topathological analysis. Muscle and liver fragments (22-to-28 cm
long) were removed from the fish for mercury analysis purposes.
2.5. Bottom sediment collection
A stainless-steel Van-venn dredger was used to collect three
bottom sediment samples from each sampling site. The sediment
samples were collected in low-depth sediment deposition zones,
which presented low hydrodynamic flow speed. After the bottom
sediment collection, sediment samples were placed in plastic bags,
which were previously identified and conditioned in thermal boxes
until the beginning of the analytical procedure.
2.6. Total mercury (THg) determination
Bottom sediment samples and biological tissues were subjected
to acid digestion through the addition of 2 ml HNO3 and HClO4
(1:1), 5 ml H2SO4 and 1 ml H2O, on a heating plate at 230e250  C,
for 20 min. The samples were cooled at room temperature,
measured in 50 ml volumetric flasks and homogenized. The mer-
cury available in ionic solution was analyzed through cold vapor
atomic absorption spectrophotometry (CVAAS), in a Mercury
Analyzer HG-3500, according to the method by Akagi et al. (1995).
All analyses were run in duplicate and followed by quality control
using certified reference material CRM DOLT-3 with analytical re-
covery 99.1% (3.340 ± 0.343).
2.7. Treatment applied to bottom sediment data
Freshwater Sediment Quality Guidelines (SQGs) set by Canadian
Council of Ministers of the Environment (CCME, 2002) were used as
evaluation criteria to analyze the likelihood of deleterious effects
on the mercury-exposed biota. According to such likelihood, the
lowest threshold e (TEL e Threshold Effect Level) - lies on the
concentration below which adverse effects on aquatic organisms
are rarely expected to happen. On the other hand, the highest
threshold e PEL (Probable Effect Level) e concerns the concentra-
tion above which the aforementioned adverse effects on aquatic
organisms are often expected to take place. The mercury range
between TEL and PEL (0.17 mg kg1 and 0.486 mg kg1,
790 A.C. Borges et al. / Chemosphere 202 (2018) 788e796
Fig. 1. Map showing the study site location in Itacaiúnas River; sampling sites: S1 (minimum risk), S2 (medium risk) and S3 (maximum risk).
respectively) comprises values occasionally expected to trigger
such effects.
2.8. Histological procedures
2.8.1. Light microscopy
Liver samples were fixed in Bouin's solution for 24 h and
embedded in paraffin, according to standard histological tech-
niques (Prophet et al., 1995). Liver sections (5 mm) were collected
and stained with hematoxylin and eosin (HE) for analysis purposes.
Photomicrographs were taken through a Nikon optical microscope
(Nikon DS-Ri1) equipped with the NIS-Elements BR4.00.07
software.
2.8.2. Histopathological index of the liver (HIL)
Histological changes in the liver were qualitatively identified
according to descriptions by Hibyia (1982). These changes were
semi-quantitatively assessed according to the (adapted) protocol by
Bernet et al. (1999), in which an importance factor was attributed to
each change by taking into consideration the following tissue injury
levels: (1) minimum e easily reversible change; (2) moderate -
reversible if the stressor is neutralized; and (3) severe - often
irreversible, thus leading to decreased liver function. It was possible
attributing an occurrence value to the changes after they were
identified; this value corresponded to the level and extent of the
tissue changes, namely: (0) unchanged; (2) occasional; (4) mod-
erate and (6) severe (diffuse lesion). The histopathological index of
the liver (HIL) of each individual was set; the index was calculated
by multiplying the importance factor by the occurrence value of
each change.
The frequency percentage of the histological changes was
calculated based on the occurrence of a specific change in all the
analyzed fish and in each sampling site.
2.8.3. Fish health
The classification by Zimmerli et al. (2007) was used to analyze
fish health; histopathological index were adopted to classify liver
injury severity as class 1 (CL1) - index < 10, mild changes; class 2
(CL2) - index ranging from 10 to 25, moderate changes; class 3 (CL3)
e index ranging from 26 to 35, significant changes; and class 4
(CL4) e index > 35, severe changes. Thus, it was possible calculating
the prevalence rate of severity classes per sampling site.
2.8.4. Transmission electron microscopy (TEM)
Liver fragments were fixed in Karnovsky's solution (4% para-
formaldehyde, 2% glutaraldehyde in sodium cacodylate buffer
0.1 M, pH 7.3) for 2 h. Next, they were post-fixed in 1% osmium
tetroxide solution buffered with sodium cacodylate (0.1 M, pH 7.3)
for 2 h at room temperature, and contrasted in block with 1% uranyl
acetate. Subsequently, samples were dehydrated in a graded
acetone series, as well as infiltrated and embedded in Epon 812.
Ultrathin sections were cut, contrasted with uranyl acetate and lead
citrate, examined and photographed in a LEO 906E TEM.
2.9. Statistical analysis
The physico-chemical parameters of the water, biometric data,
HIL and THg concentrations were tested for normality and homo-
geneity through the Kolmogorov-Smirnov and Levene tests,
respectively. After the HIL and the physico-chemical parameters of
the water had presented homogeneous variance, they were sub-
jected to one-way analysis of variance (ANOVA) in order to analyze
the differences between groups of samples. The non-parametric
Kruskal-Wallis test was applied to the other data set. It was fol-
lowed by H-tests, which were applied to statistically significant
results in order to compare the sampling sites. Correlations be-
tween data pairs were calculated through Spearman correlation r.
Statistically significant results were set at p < 0.05. Statistical
 A.C. Borges et al. / Chemosphere 202 (2018) 788e796
analysis was performed in the Statistica 8.0 software.
3. Results
3.1. Physico-chemical parameters of the water
The physico-chemical parameters of the water did not present
significant differences between sampling sites (ANOVA: p > 0.05).
These variables presented variation ranges, as well as their
respective mean and standard deviations, as follows: pH, between
6.95 and 7.57 (7.38 ± 0.21); temperature, between 25.69 and
29.08  C (27.49 ± 1.05); electrical conductivity, between 30 and
85 mS cm1 (67.27 ± 14.87); total dissolved solids, between 15 and
47 ppm (37.8 ± 9.6); and dissolved oxygen, between 3.84 and
13.18 ppm (8.22 ± 2.2).
Table 1 shows the mean values and standard deviations recor-
ded for the three sampling sites. Significant correlations were
found between water temperature and electrical conductivity
(p < 0.05), as well as between electrical conductivity and total
dissolved solids in the water (p < 0.0001). There was no correlation
between these data and the THg concentrations in the bottom
sediment.
3.2. Fish analysis
Seventy-three (73) fish were collected, in total; 33 were female,
32 were male, and 8 presented undetermined sex. The length of the
specimens ranged from 18 to 44 cm (26.7 ± 5.73), their body mass
from 150 to 2000 g (495.4 ± 416.9), and their liver mass from 8 to
70 g (23.2 ± 13.9). The mean fish length in S3 was significantly
lower than that recorded in S1 and S2 (KeW: p < 0.05, for both).
The mean body mass of fish in S3 was significantly lower than that
recorded for fish in S1 (KeW: p < 0.05). There was significant
(p < 0.05) correlation between fish length and body mass, between
liver length and mass, and between body mass and liver mass, in
each sampling site. There was no correlation between fish length
and liver mass in S3 (r ¼ 0.41; p > 0.05).
3.3. Total mercury in the bottom sediment and in the fish
The bottom sediment predominantly consisted of silty-sandy
material in the three sampling sites. The mean (standard devia-
tion) THg concentration in S1 was 0.077 (±0.019) mg kg1; whereas
in S2 and S3, it was 0.066 (±0.001) mg kg1.
Table 2 presents data about fish subjected to THg analysis. These
results concern individuals whose length ranged from 22 to 28 cm.
There was significant correlation (r ¼ 0.74, p < 0.05) between the
weight and length of these fish. Individuals in S3 were significantly
smaller in weight and size than the ones in S1 (KeW: p < 0.05).
The highest mean THg concentration in the muscle was found in
S3 individuals, who were followed by individuals in S2 and S1.
However, the mean THg concentration in the liver followed the
reverse order: S3 < S2 < S1 (Fig. 2) and presented significant dif-
ferences in S3 in comparison to S1 and S2 (ANOVA: p < 0.05). No
Table 1
Mean and standard deviation of physicochemical parameters of the water in S1, S2
and S3.
Parameter S1 S2 S3
pH 7,2 ± 0,25 7,4 ± 0,11 7,46 ± 0,16
Temperature ( C) 27,17 ± 1,38 27,04 ± 1,15 27,54 ± 0,79
Conductivity (mS cm1) 67,83 ± 3,76 67,25 ± 25,17 66,75 ± 12,74
TDS (ppm) 40 ± 3,46 38,5 ± 15,86 37,63 ± 8,63
Dissolved Oxygen (ppm) 7,34 ± 3,09 8,68 ± 1,79 8,10 ± 1,28
791
significant correlation (r ¼ 0.068; p > 0.05) was found between
THg concentration in the muscle and in the liver.
3.4. Histopathology of the liver
The healthy liver of S. rhombeus individuals presented homo-
geneous structure and reddish-brown shade. The organ was
divided in three lobes, namely: one ventral lobe and two (right and
left) lateral lobes presenting equivalent sizes. Liver parenchyma is
formed by the centrilobular vein (CLV), which are sinusoid capil-
laries between the anastomosed hepatocyte cords. The hepatocyte
presented polygonal shape with spherical and centralized nucleus
(Fig. 3a). Diffuse pancreatic exocrine tissue (Fig. 3b) was observed
near the hepatic veins and arteries; there was lack of portal triad,
whereas bile ducts and hepatic arteries were scattered throughout
the parenchyma.
Hepatic changes comprised hepatocyte cord disorganization,
cellular hypertrophy, leukocyte infiltration (Fig. 3d), circulatory
disorders characterized by vascular congestion and bleeding
(Fig. 3e), steatosis with substantial number of lipid droplets
(Fig. 3f), and cell vacuolization and necrosis (Fig. 3g). The bile duct
presented hypertrophy in the coating epithelial tissue and
increased connective tissue (Fig. 3c).
With respect to the frequency of hepatic changes, the S1 fish
presented the greatest variety of changes, whereas the S2 fish
presented the smallest one. However, severe changes happened
more often in S3 fish: more than 94% of the individuals presented
necrosis and 100% presented leukocyte infiltration.
The histopathological index of S. rhombeus livers followed the
magnitude order S2<S1<S3, they presented mean values 15.1, 20.3
and 23.1, respectively. However, there were no significant differ-
ences (p ¼ 0.104) between sampling sites.
3.5. Liver ultrastructure
Healthy animals presented varying hepatocyte shapes; the
cytoplasm showed several spherical and elongated mitochondria
with homogeneously-distributed tubulovesicular cristae and rough
endoplasmic reticulum (Fig. 4a). The nucleus presented oval shape,
usually located in the center of the cell, as well as granular chro-
matin, condensed heterochromatin in the periphery of the nuclear
membrane, and prominent nucleolus. The perinuclear area in the
cytoplasm was rich in organelles, whereas the peripheral area
showed predominant glycogen (Fig. 4b). Erythrocytes were
observed in the sinusoid capillaries (Fig. 4c).
Ultrastructural changes are related to the morphology and dis-
tribution of organelles. Mitochondria presented heterogenous
shape and size, as well as reduced mitochondrial cristae (Fig. 4g).
Rough endoplasmic reticulum dilation, glycogen depletion, and
cellular vacuolization were also observed (Fig. 4d, e and f). Some
cells presented irregularly shaped nucleus and reduced size, as well
as significant deformation in their membrane and heterochromatin
accumulation (Fig. 4g).
4. Discussion
Itacaiúnas River presents hydrodynamic and hydrochemical
features typical of clear water rivers (Sioli, 1975), as well as pH close
to neutrality. Studies indicate that metals such as Hg precipitate as
oxides and hydroxides at alkaline pH; moreover, these metals
become less bioavailable and less toxic (Cornelis and Nordberg,
2007). Ruivo and Sales (1989) studied the water resources in the
Caraja
s Iron Project (Projeto Ferro Caraja
s) site and recorded acidic
pH during the rainy season. It may have happened mainly due to
soil leaching through rainwater, which transports humic and fulvic
792 A.C. Borges et al. / Chemosphere 202 (2018) 788e796
Table 2
Mean, standard deviation, minimum and maximum weight and length, and THg concentrations in the muscle and liver of S. rhombeus.
Site Weight (g) Length (cm)
S1 343.9 ± 57.4a 25.6 ± 1.2a
(n ¼ 9) 250e450 24e28
S2 325.4 ± 122.3 25 ± 2.5
(n ¼ 12) 185e525 22e28
S3 251.8 ± 42.9 23.3 ± 0.9
(n ¼ 11) 200e350 22e25
p 0.023 0.019
a
Significant differences in comparison to S3 (p < 0.05).
Fig. 2. Mean THg concentrations in the muscle and liver of S. rhombeus in S1, S2 and
S3. *Significant differences in comparison to S3 (p < 0.05). The error bar represents 95%
confidence interval.
acids to the water resources. Ribeiro et al. (2017) suggested that
hydrological flows affected the Hg concentration in the water, since
their results indicated higher Hg concentrations in Xingu River
water during the rainy season than during the dry season.
Mean THg concentrations in the bottom sediment of Itacaiúnas
River were below the TEL limit and showed concentration decrease
trend in the magnitude order S1>S2S3. According to the indices
adopted in the current study, the possibility of having the herein
recorded THg concentrations affecting the aquatic biota of Ita-
caiúnas River is virtually inexistent. However, it is necessary
monitoring them, since confined mercury can remain active as the
substrate available for methylation for many years, even when the
source is eradicated (Bastos et al., 2006; Driscol et al., 2013). Factors
such as the ionic strength of the medium, water hardness, organic
matter, pH, redox potential, iron, sulfur, among others, favor Hg
remobilization from the bottom sediment to the water column, as
well as its incorporation to the aquatic biota (Magalha ~es et al., 2015;
Morel et al., 1998). In addition, studies indicated that remote re-
gions receiving Hg inflows from widely global sources often show
long-term Hg concentration increase (Brabo et al., 2003; Drevnick
et al., 2012).
Mercury concentration in fish depends on their trophic position
in the food chain, on the specimen size/age and physiological fac-
tors, as well as on the physico-chemical parameters of the water
and on mercury speciation (Beltran-Pedreros et al., 2011; Dalzochio
and Gehlen, 2016; Lacerda et al., 1994; Silva et al., 2011). Many
studies already proved the susceptibility of predatory fish to
accumulate mercury and other metals (Akagi et al., 1995; Do
rea
et al., 2004; Lacerda et al., 1994; Silva et al., 2011). The herein
studied species, Serrasalmus rhombeus, has non-migratory habit, is
predominantly carnivorous, and is considered a top-chain species
(Goulding, 1980; Lowe-McConnell, 1987); therefore, it reflects the
environmental quality of the aquatic ecosystem.
Mean THg concentrations in S. rhombeus muscle tissues were
THg muscle (mg kg1) THg liver (mg kg1)
0.189 ± 0.039 0.210 ± 0.056a
0.130e0.265 0.134e0.329
0.210 ± 0.093 0.202 ± 0.096a
0.098e0.437 0.066e0.432
0.283 ± 0.134 0.114 ± 0.043
0.085e0.525 0.074e0.189
0.168 0.006
similar to those found in other species belonging to genus Serra-
salmus living in Amazonian rivers (Akagi et al., 1995; Brabo et al.,
2000; Castilhos et al., 1998; Do
rea et al., 2004; Lacerda et al.,
1994). The mean THg concentration in the muscle represented
the magnitude order S1<S2<S3, which was the opposite of that
found in the liver (S3<S2<S1). However, no sampling site showed
significant correlation between fish size and THg concentration in
both the muscle and the liver.
The liver tends to accumulate higher THg concentration than the
muscle tissue; it happens because the liver shows higher metabolic
activity (Kasper et al., 2009; Mieiro et al., 2009). The continuous
demethylation process in the liver reduces mercury toxicity; thus,
mercury (in its methylmercury form) begins to accumulate, pref-
erably in the muscle (Lacerda and Pfeiffer, 1992). Therefore, the
liver tissue is subjected to the most prolonged (chronic) effects of
the accumulated metal (Cerqueira and Fernandes, 2002).
The liver of fish collected in the S3 site presented lipid degen-
eration and steatosis associated with necrotic foci. These severe
changes have been associated with fish exposure to contamination
by metals such as mercury (Dabrowska et al., 2012; Marchand et al.,
2009; Raldúa et al., 2007).
The most significant hepatic changes were recorded in S1 and S3
fish. S3 individuals presented the highest histopathological index of
the liver. According to Van Dyk et al. (2012), histopathological index
recorded in C. gariepinus have successfully demonstrated the
pollution record of rivers in different South African regions.
Therefore, the frequency and severity histological changes, in as-
sociation the histopathological index observed in S. rhombeus
livers, allowed setting the biological damage degree of the water in
Itacaiúnas River at magnitude order S2S1<S3.
The ultrastructural changes observed in the hepatocyte, such as
nuclear changes typical of apoptosis and reduced mitochondrial
cristae, indicated the existence of a stress-response mechanism and
suggested that the energy-production was compromised by the
oxidative route (Gallis et al., 2011). Thus, glycogen depletion in the
cell led to greater cytosolic glycolysis activation to ATP conversion
(Green and Reed, 1998; Panepucci et al., 2001; Gallis et al., 2011).
Ultrastructural results suggested the activation of stress response
mechanisms in the sampled-fish cells.
The Amazonian soil is rich in iron and the high mercury level
found in the Amazonian rivers may be related to the formation of
iron-mercury complexes, thus suggesting that the mercury found
in these environments has natural rather than anthropogenic origin
(Roulet and Lucotte,1995). The Caraja
s region holds large ore re-
serves such as iron and manganese (Galarza et al., 2008). The
mining activity developed in the region helped increasing the total
amount of dissolved solids and particulates in the water column
(Damous et al., 2002; Ruivo and Sales, 1989). Mercury is strongly
adsorbed in suspended solids, including iron and manganese ox-
ides (Balogh et al., 1997; Brabo et al., 2003; Gasparatos, 2013;
Magalha ~es et al., 2015). Low THg concentrations in bottom sedi-
ments and in S. rhombeus individuals found in Itacaiúnas River, in
 A.C. Borges et al. / Chemosphere 202 (2018) 788e796 793

Fig. 3. Serrasalmus rhombeus liver: [a] Normal structural architecture of hepatic tissue showing central vein (V), hepatocytes (He) and sinusoid capillaries (arrow); [b] exocrine
pancreatic tissue; [c] increased connective tissue (arrowhead) and nuclear change in the bile duct (arrow); [d] leukocyte infiltrate (*) near the hepatic artery (arrow); [e] central vein
(V) and sinusoid capillaries (arrow) showing blood cell congestion and leukocyte infiltrate (*); [f] lipid deposits characterizing steatosis (fatty degeneration); [g] necrosis to the left
of the dotted line and vacuolated hepatocytes to the right. Stained with H&E.

association with the pH of the water, suggested that THg was
mainly bound to the suspended particulate material.
However, although the mercury concentrations in the muscle
tissue were within the limit for human consumption (0,5 mg g1)
set by the World Health Organization (WHO, 1990), the health of
the collected fish was already compromised. It was evidenced by
the frequency and severity of structural and ultrastructural changes
recorded in the liver tissue of specimens from the three sampling
sites. Therefore, these findings allowed inferring that the compro-
mised health of S. rhombeus impos sublethal survival conditions to
them, fact which also represents a long-term risk to the health of
riverine populations living in the region, since they consume
distinct species belonging to this genus on a daily basis (Brabo et al.,
2000; Lacerda et al., 1994).
According to Souza-Filho et al. (2016), the MUCC is a successful
model of public-private partnership between Brazilian environ-
mental agencies and a multinational mining company, and it forms
large forest blocks that work as “green barrier”. On the other hand,
the aforementioned authors emphasized that the land-cover and
land-use changes in the surroundings of the protected area led to
deforestation and to the formation of a new landscape dominated
by pastures, and to the consequent 85% increase in water discharge
in Itacaiúnas River, over 40 years. Deforestation, fire events and
pastures also influence the Hg entry in aquatic ecosystems (Lechler
794 A.C. Borges et al. / Chemosphere 202 (2018) 788e796

Fig. 4. Ultrastructural organization of Serrasalmus rhombeus hepatocytes: [a] normal hepatic parenchyma with sinusoid cord (S) separating hepatocytes (He); [b] normal hepatocyte
presenting spherical nucleus (black arrow), nucleolus (white arrow) and organelles homogeneously distributed throughout the cytoplasm; [c] erythrocytes (arrow); [d] change in
the spherical nuclear shape (arrow), with heterochromatin formation; [e] mitochondria presenting different shapes and sizes (arrowhead) and rough endoplasmic reticulum in
vesicles (*); (f) hepatocyte in apoptosis with condensed chromatin tending to the nuclear periphery (to the left), loss of mitochondrial cristae (arrow) and rough endoplasmic
reticulum in vesicles (*); (g) evidence of rough endoplasmic reticulum dilatation and vesiculation (*) and loss of mitochondrial cristae (arrow).

et al., 2000; Malm, 1998; Roulet and Lucotte, 1995; Wasserman
et al., 2003).
Even without anthropogenic influence, rivers and streams in the
Caraja
s region often carry many suspended sediments due to their
erosion power and to the geological features of the lands they run
through, mainly during the rainy season (Damous et al., 2002;
Galarza et al., 2008; Ruivo and Sales, 1989). Ribeiro et al. (2017)
conducted a study under the same climatic conditions and inves-
tigated hydrochemical features similar to those found in the cur-
rent study. They confirmed contamination by heavy metals in the
 A.C. Borges et al. / Chemosphere 202 (2018) 788e796
abiotic compartments of Xingu River, close to Triunfo do Xingu
Environmental Protection Area. On the other hand, the aforemen-
tioned authors found low metal levels in the muscles of Myloplus
rubripinnis and Cichla melaniae individuals, and it indicated low
metal bioavailability to the aquatic biota. In contrast, high Hg
concentrations were found in 81% of the fish sampled in the
Tumucumaque National Park, which is located in Amapa
State
(Brazil) and is considered the largest contiguous fragment of
tropical rainforest in the world. The Hg concentrations were above
the limit set by WHO mainly for predator fish (Venturieri et al.,
2017).
Results indicated chronic effects on top-chain fish and evi-
denced mercury contamination in the aquatic ecosystem of a le-
gally protected area, which is focused on retaining the
anthropogenic pressure and on assuring safeguard conditions to
aquatic ecosystems. These results suggested the natural origin of
the mercury, as well as that it may lead to long-term risks to the
aquatic biota in Itacaiúnas River and to riverine populations living
in the region. Thus, future studies should investigate biomarkers
more sensitive to Hg exposure, as well as their origin and
bioavailability mechanisms in the region.
5. Conclusion
The current study evidenced that the environmental quality of
Itacaiúnas River is strongly influenced by geological and hydro-
logical factors favoring Hg entry in aquatic systems, as well as that
such entry is intensified by surface runoff and erosion resulting
from anthropogenic processes such as mining, deforestation,
burnings and pasture. The low THg concentrations in the bottom
sediment and in S. rhombeus individuals, as well as the physico-
chemical conditions of the water, along with the input of ores
mined in the area (Fe, Mn, Cu, among others), indicate that Hg is
mainly bound to suspended particulate matter and presents low
bioavailability to the aquatic biota. However, the histological and
ultrastructural changes observed in the liver of S. rhombeus in-
dividuals provide reliable information on the environmental con-
ditions of the study area, indicating responses to non-specific sub-
lethal stressors present in the Itacaiúnas River.
Declarations of interest
I declare that all authors have read and approved the manu-
script, are aware the submission for publication and agree, and
there is not conflicts of interest.
Acknowledgment
We are especially grateful to the National Council for Scientific
and Technological Development (CNPq) (Beneficiary's processes:
132192/2011-0), for granting a scholarship; to Evandro Chagas
Institute (IEC), for the mercury analysis; and to Chico Mendes
Institute for Biodiversity Conservation (ICMBio), for granting the
research license for the study field and for the logistical field
support.
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