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.br2 Department of Geochemistry, UFF, Rio de Janeiro, Brazil.

3 FIOCRUZ- Brazilian Ministry of Health, Rio de Janeiro, Brazil4 UNIDO, United Nation Industrial Development Organization, Austria

ABSTRACTAn 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.

INTRODUCTIONMercury (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.

EXPERIMENTALFish 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 DISCUSSIONThe 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 Scientific name FH São Chico Creporizinho Total

Acari Hypostomus sp D 1 9 10Arraia Potamotrygon motoro C 3 - 3Bocudo Ageneiosus brevifilis C - 1 1Candiru Hemicetopsis candiru O - 4 4Cará Cichlasoma spectabile M 3 3 6Curimatã Prochilodus nigricans D 6 4 10Sairu Cyphocharax sp MF - 11 11Ituí Sternopygus macrurus I 4 - 4Mandi Pimelodus blochii O 2 2 4Pacu Myleus sp H 1 - 1Piau Anostomoides laticeps H 1 13 14Piranha Serrasalmus rhombeus C 2 13 15Pirarara Phractocephalus hemioliopterus C 1 - 1Surubim Pseudoplatystoma fasciatum C 1 - 1Traíra Hoplias malabaricus C 19 9 28Total 44 69 113

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 Weight g Lengthcm Mercury µg/g AChE

µmoles/min.mgBChE

µmoles/min.mgHypostomus sp 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)

P. motoro 3400.0±953.9 (3) 37.0±2.6 (3) 0.63±0.27 (3) 0.18±0.10 (3) 0.18±0.03 (2)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)

C. spectabile --- 8.8±0.6 (6) 1.06±0.38 (6) 0.26±0.15 (6) 0.15±0.13 (2)P. nigricans 212.5±62.9 (4) 14.4±4.2 (10) 0.16±0.04 (10) 0.79±0.43 (10) ---

C. sp --- 9.1±0.7 (11) 0.25±0.06 (11) 0.27±0.12 (11) 0.05 (1)S. macrurus 150.0±40.8 (4) 38.8±6.0 (4) 0.28±0.04 (4) 0.75±0.40 (4) 0.08 (1)P. blochii 725.0±742.5 (2) 22.8±13.0 (4) 0.95±0.61 (4) 0.75±0.41 (4) ---Myleus sp --- 12.0 (1) 0.09 (1) 0.34 (1) ---A. laticeps 195.8±39.6 (12) 18.6±2.2 (14) 0.09±0.07 (14) 0.36±0.15 (14) 0.11±0.04 (5)

S. rhombeus 200.0±177.5 (11) 14.9±4.5 (15) 0.42±0.27 (15) 0.57±0.27 (13) 0.09±0.04 (5)P.hemioliopterus 3,500 (1) 58.0 (1) 0.28 (1) 0.48 (1) ---

P. fasciatum 7,800 (1) 90.0 (1) 1.2 (1) 0,13 (1) 0.1 (1)H. malabaricus 267.3±308.5 (26) 20.6±6.1 (28) 4.62±5.53 (28) 0.61±0.35 (28) 0.10±0.08 (10)

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 AChEµmoles/min.mg

BChEµmoles/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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