BIOMARKERS OF MERCURY EXPOSURE IN AMAZONIAN FISH

BIOMARKERS OF MERCURY EXPOSURE IN AMAZONIAN FISH, PARÁ, BRAZIL
RODRIGUES, ANA PAULA DE CASTRO1,2; RAMOS, ALINNE DOS SANTOS1; CASTRO, ALINE
MACHADO1; LIMA, CRISTIANE ANDRADE DE1; CASTILHOS, ZULEICA CARMEN1; VIANA, THIAGO
AUGUSTO PIMENTA3; DE ALBUQUERQUE, CARLA3; INÁCIO, ALAN FERREIRA 3; NOVO, LEONARDO
AGOSTINI3; LINDE, ANA ROSA3; RODRIGUES-FILHO, SAULO 1; VILLAS-BÔAS, ROBERTO 1; VEIGA,
MARCELO 4; BEINHOFF, CHRISTIAN4
1
CETEM, Center for Mineral Technology, Av. Ipê, 900, 21941-590, Rio de Janeiro, Brazil
– arodrigues@cetem.gov.br
2
3
4
Department of Geochemistry, UFF, Rio de Janeiro, Brazil.
FIOCRUZ- Brazilian Ministry of Health, Rio de Janeiro, Brazil
UNIDO, United Nation Industrial Development Organization, Austria
ABSTRACT
An Environmental and Health Assessment was being accomplished in two small scale
gold mining areas in the Brazilian Amazon – municipality of Itaituba – by CETEM with
collaboration of the Evandro Chagas Institute and the Association of Miners of Tapajós for
the Global Mercury Project, under the general coordination of UNIDO. In ecological risk
assessment is common to use the biochemical parameters like enzyme activities to
express any disturbance in animal’s physiology, as a consequence of contaminants
exposure. The objective of this work was to evaluate the relationship between mercury
levels
in
muscles
and
biochemical
parameters
(acetylcholinesterase,
butyrylcholinesterase) in Amazonian fish. A total of 113 fish specimens of 15 species [acari
(10), arraia (3), bocudo (1), candiru (4), cará (6), curimatã (10), joão duro (11), lampréia
(4), mandi (4), pacuí (1), piau (14), piranha (15), pirarara (1), surubim (1), and traíras (28)]
were collected in August 2003, at two study areas: 44 at São Chico and 69 at
Creporizinho. These specimens were measured and weighted and muscles sampled. For
correlation analysis, the specimens were separated by species. As a result, traíras from
São Chico and Creporizinho showed a significant correlation between total Hg and AChE
(0.77 p<0.001 n=19; 0.78 p<0.05 n=9), which may indicate an increasing of enzyme
activity with increasing of Hg levels in muscles. These results were surprising, since AChE
and BChE activities usually decrease with the increase of pollutants exposure. However, it
should take into account the kinetic form of the studied AChE.
INTRODUCTION
Mercury (Hg), especially its organic form (methylmercury- MeHg), is a very toxic
substance for humans and superior organisms. MeHg accumulates and biomagnifies
mainly in aquatic food chain, where carnivorous fish species accumulate mercury at a
higher rate. For the detection of organisms’ responses for mercury exposure, effect
biomarkers as dosage of enzymes activities like acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) have been used in order to identify effects at a tissue/organ
before they are apparent at a clinical/pathological level in organism.
The objective of this work was to evaluate the relationship between mercury levels in
muscles and biochemical parameters (acetylcholinesterase, butyrylcholinesterase) in
Amazonian fish from small-scale gold mining areas.
EXPERIMENTAL
Fish sampling was conducted in August 2003, at two study areas: at São Chico and at
Creporizinho mining sites. These areas are located inside of the Mineral Tapajós Reserve,
State of Para, between the cities of Jacareacanga and Itaituba, where the mining sites are
distributed alongside the tributaries of the Tapajós River. However, these two areas belong
to two distinct hydrographic basins: Jamanxin river basin and Crepori river basin,
respectively. A total of 113 fish specimens of 15 species were collected: 44 at São Chico
and 69 at Creporizinho. These specimens were measured, weighted and muscles
sampled and frozen.
Hg was analyzed in the fish muscle through Atomic Absorption Spectrophotometer
(KK.Sanso SS) using a Vapor Generation Accessory-VGA (CVAAS). The sample was
digested using an acid humid solution. The digested sample solution was introduced in the
Automatic Mercury Analyzer Hg 3500.
The determinations of the AChE and of the BChE activities (Oliveira Silva, 2000), were
quantified based in Ellman (1961). The enzymatic activities were measured in Shimadzu
UV 1601 (λ = 412 nm).
RESULTS AND DISCUSSION
The fifteen species colleted had a heterogeneous distribution in sampling areas. The
absolute frequency of the species is shown in Table I. Fish with higher frequency were
Hoplias malabaricus (n=28) and Serrasalmus rhombeus (n=15). The results organized by
fish species for length, weight, Total Hg, AChE and BChE activities are showed in Table II.
Table I. Absolute frequency of the fifteen fish species collected in Tapajós Region.
Popular name
Acari
Arraia
Bocudo
Candiru
Cará
Curimatã
Sairu
Ituí
Mandi
Pacu
Piau
Piranha
Pirarara
Surubim
Traíra
Total
Scientific name
FH
São Chico
Creporizinho
Total
Hypostomus sp
Potamotrygon motoro
Ageneiosus brevifilis
Hemicetopsis candiru
Cichlasoma spectabile
Prochilodus nigricans
Cyphocharax sp
Sternopygus macrurus
Pimelodus blochii
Myleus sp
Anostomoides laticeps
Serrasalmus rhombeus
Phractocephalus hemioliopterus
Pseudoplatystoma fasciatum
Hoplias malabaricus
D
C
C
O
M
D
MF
I
O
H
H
C
C
C
C
1
3
3
6
4
2
1
1
2
1
1
19
44
9
1
4
3
4
11
2
13
13
9
69
10
3
1
4
6
10
11
4
4
1
14
15
1
1
28
113
carnivorous and/or icthyophagous=C; herbivorous=H, insectivorous=I; microphagous=M; omnivorous=O;
macrofauna=MF; food habit = FH; sampling sites = A.
Table II. Results of weight, length, total Hg in fish muscle, AchE and BchE activities (wet
weight) for all fifteen studied species.
Fish Species
Hypostomus sp
P. motoro
Weight
g
Length
cm
Mercury µg/g
AChE
BChE
µmoles/min.mg
µmoles/min.mg
100.0±0.0 (5)
11.9±0.9 (10)
0.06±0.02 (10)
0.57±0.84 (9)
0.09±0.05 (6)
0.18±0.03 (2)
3400.0±953.9 (3)
37.0±2.6 (3)
0.63±0.27 (3)
0.18±0.10 (3)
A. brevifilis
200.0 (1)
20.5 (1)
0.27 (1)
0.66 (1)
H. candiru
100.0±0.0 (4)
16.8±0.3 (4)
0.74±0.21 (4)
0.68±0.21 (4)
0.08±0.01 (1)
8.8±0.6 (6)
1.06±0.38 (6)
0.26±0.15 (6)
0.15±0.13 (2)
14.4±4.2 (10)
0.16±0.04 (10)
0.79±0.43 (10)
9.1±0.7 (11)
0.25±0.06 (11)
0.27±0.12 (11)
0.05 (1)
0.08 (1)
C. spectabile
P. nigricans
C. sp
--212.5±62.9 (4)
---
---
---
S. macrurus
150.0±40.8 (4)
38.8±6.0 (4)
0.28±0.04 (4)
0.75±0.40 (4)
P. blochii
725.0±742.5 (2)
22.8±13.0 (4)
0.95±0.61 (4)
0.75±0.41 (4)
---
12.0 (1)
0.09 (1)
0.34 (1)
---
18.6±2.2 (14)
0.09±0.07 (14)
0.36±0.15 (14)
0.11±0.04 (5)
14.9±4.5 (15)
0.42±0.27 (15)
0.57±0.27 (13)
0.09±0.04 (5)
58.0 (1)
0.28 (1)
0.48 (1)
90.0 (1)
1.2 (1)
0,13 (1)
0.1 (1)
20.6±6.1 (28)
4.62±5.53 (28)
0.61±0.35 (28)
0.10±0.08 (10)
Myleus sp
A. laticeps
--195.8±39.6 (12)
S. rhombeus
200.0±177.5 (11)
P.hemioliopterus 3,500 (1)
P. fasciatum
7,800 (1)
H. malabaricus
267.3±308.5 (26)
---
Fish from São Chico (3.08±4.85 µg/g; n=44) showed close to ten (10) times higher total
Hg levels than fish from Creporizinho (0.35±0.37 µg/g; n=69) (t-Test, p<0.001; n=113).
Considering the fish species, H. malabaricus specimens from São Chico areas are
different of the specimens from Creporizinho (Mann-Whitney U, p<0.05; n=28), showing
higher Hg levels, as showed in Table III. Although the Hg levels in H. malabaricus from
São Chico are higher than Creporizinho, the AChE and BChE activities showed no
significant differences.
Table III. Results of total Hg in H. malabaricus fish muscles (arithmetical mean±standard
deviation; wet weight) from different sites of São Chico and Creporizinho garimpo’s areas.
Garimpo area
Weight
g Length
cm Mercury µg/g
AChE
BChE
µmoles/min.mg
µmoles/min.mg
São Chico
276.3±353.7 (19)
21.9±5.9 (19)
6.42±5.93 (19)
0.64±0.37 (19)
0.11±0.09 (7)
Creporizinho
242.9±142.7 (7)
18.3±6.2 (9)
0.84±0.58 (99)
0.55±0.33 (9)
0.07±0.03 (3)
For correlation analysis, the specimens were separated by species. H. malabaricus
showed a strong correlation between Hg levels and AChE (0.63; p<0.001; n=26) and S.
rhombeus, between length and AChE (-0.74; p<0.005; n=13) and length and Hg levels
(0.71; p< 0.005; n=15). All the other thirteen species had non-significant correlation or a
small number of specimens (less than 5). When correlations were investigated inside each
area, H. malabaricus showed the same correlation mentioned before (Hg levels and
AChE) in São Chico (0.77; p<0.001; n=19) and in Creporizinho (0.78; p<0.05; n=9). These
results may indicate an increasing of enzyme activity with increasing of Hg levels in
muscles. This was not an expected result, since it was supposed that AChE activity would
be in inhibition process. Nevertheless, Souto (2004) found the same positive correlation in
Amazonian fish from Tapajós River and Tortelli (2004a; 2004b) associated this to biologic
diversity and differences in the AChE kinetic form. In addition, S. rhombeus from
Creporizinho showed a significant correlation between length and Hg levels (0.57; p<0.05;
n=13). None correlation was found with BChE activity, what may be explained by the low
number of muscle samples that were available to analyze.
Concluding, fish from São Chico area presented higher total Hg in muscle than fish
from Creporizinho area. The H. malabaricus was present in both areas with higher
absolute frequency. This species showed a positive and significant correlation between Hg
levels and AChE activity (0.63; p<0.001; n=26). This result was surprising, since AChE
and BChE activities usually decrease with the increase of pollutants exposure. However, it
should take into account that distinct species may express the biomarker effects in
different ways and cholinesterases would have differences in kinetic form, which should be
further distinguished.
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