217

Neotrop. Helminthol., 8(2), 2014

2014 Asociación Peruana de Helmintología e Invertebrados Afines (APHIA)

ISSN: 2218-6425 impreso / ISSN: 1995-1043 on line

ORIGINAL ARTICLE / ARTÍCULO ORIGINAL

CHEMICAL ANALYSIS TROUGH “ENERGY-DISPERSIVE SPECTROSCOPY (EDS)” OF

DIGENEA METACERCARIAE FOUND INFESTING SPECIMENS OF HOPLOSTERNUM

LITORALLE (HANCOCK, 1828) (SILURIFORMES: CALLICHTHYDAE), CAPTURED IN

MANAUS POLLUTED IGARAPES

CARACTERIZACIÓN QUÍMICA EN TREMATODOS DIGENÉTICOS PARASITOS DE

HOLPOSTERNUM LITORALLE (HANCOCK, 1828) (SILURIFORMES: CALLICHTHYDAE)

USANDO LA ESPECTOSCOPIA DE ENERGÍA DISPERSIVA (EDS) CAPTURADOS EN

IGARAPES CONTAMINADOS DE MANAUS

1,2 1

Aprigio Mota Morais & José Celso de Oliveira Malta

Abstract

Keywords: Amazon - chemical elements - fishes - Platyhelminthes - pollution.

Suggested citation Aprigio M. Morais& Malta J.C.O.2014. Chemical analysis trough “Energy-Dispersive Spectroscopy (EDS)”

of digenea metacercariae found infesting specimens of Hoplosternum litoralle (Hancock, 1828) (Siluriformes: Callichthydae),

captured in Manaus polluted Igarapes. Neotropical Helminthology, vol. 8, n°2, jul-dec, pp. 217-225.

1INPA – Instituto Nacional de Pesquisas da Amazônia, Laboratório de Parasitologia e Patologia de Peixes, Manaus, AM.

2 Bolsista do PNPD da CAPES – Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

Corresponding author: e-mail aprigio.mota@inpa.gov.br CEP 69011-970 Phone (55) (092) 3643-3378. or aprigiomota@yahoo.com.br

We examined 82 Hoplosternum littorale captured in two Manaus polluted igarapes, namely, São

Raimundo and Educandos, in October and November 2010. Fish specimen's standard length and

weight showed to be 14.87±71 cm and 116.53±24.01 g, respectively. We carried out the chemical

analyses of tissues with the aid of an Energy-Dispersive Spectroscopy (EDS) system in

conjunction with a Scanning Electron Microscope (SEM). We determined the punctual qualitative

and semi-quantitative chemical composition of the metacercariae of Clinostomum marginatum,

Tylodelphis destructor and of the pro-genetic metacercaria Herpetodiplostomum caimanicola, by

using the characteristic X-ray emission. Microanalyses by EDS determined the presence of

carbon (C); oxygen (O); iron (Fe); zinc (Zn); aluminum (Al); siliceous (Si); phosphorous (P);

sulfur (S); potassium (K); calcium (Ca); chlorine (Cl); copper (Cu); lead (Pb); manganese (Mn);

platinum (Pt); tungsten (W) and cobalt (Co). The detected heavy metals Cu, Pb and W indicated

that igarapes is heavily polluted, and that Digenea metacercariae are highly tolerant to the

accumulation of these elements.

Morais & Malta

Chemical analysis of Digenea metacercariae

218

Resumen

Palabras clave: Amazonía - elementos químicos - peces - Platyhelminthes - polución.

Fueron examinados 82 Hoplosternum littorale capturados en dos arroyos contaminados en la

ciudad de Manaus: São Raimundo y Educandos, entre octubre a noviembre del 2010. La longitud

y peso promedio de los peces analizados fueron de 114,87±71cm y 116,53±24,01g,

respectivamente. Los análisis químicos de los tejidos fueron realizados con ayuda de un sistema

de Espectroscopía por Dispersión de Energía (EDS) en conjunto con un Microscopio Electrónico

de Barrido (MEB). Fue determinada la composición química puntual cualitativa y semi-

cuantitativa en larvas de Clinostomum marginatum, Tylodelphis destructor y de la metacercaria

progenética de Herpetodiplostomum caimanicola, utilizando la emisión de rayos X

característicos. Los microanálisis por EDS determinaron la presencia de carbono (C); oxígeno

(O): hierro (Fe); Zinc (Zn); aluminio (Al); silicio (Si); fósforo (P); azufre (S); potasio (K); calcio

(Ca); cloro (Cl); cobre (Cu); plomo (Pb); manganeso (Mn); platino (Pt); tungsteno (W) y cobalto

(Co). Los metales pesados detectados Cu, Pb y W son indicadores de polución en los arroyos, y las

metacercarias de Digenea son altamente tolerantes a estos elementos de acumulación.

occurring on that aquatic environment. Fish

parasite species hold abilities, which enable

them to be bio-indicators and markers (Gunkel,

1994; Sures, 2004, 2008; Buron et al., 2009).

Fish parasite species have shown to be good

indicators of pollutant accumulation, because

they provide valuable information on the

environment's chemical status, both by pointing

out the presence or lack of pollutants, and the

ability to concentrate them in their tissues (Sures

et al., 2000; Buron et al., 2009).

The utilization of fish parasites as pollutant

concentrators is justified by their much greater

ability than that of hosts or environment

themselves to bio-accumulate or bio-

concentrate heavy metals (Sures & Siddall,

1999; Sures, 2003). That bio-accumulative

characteristic of the parasite is not the result of a

slow accumulation process, but rather a

relatively quick adhesion to a leveled status

(Sures et al., 2000).

Currently, several techniques, such as Energy-

Dispersive Spectroscopy (EDS), detect

chemical elements in fish parasites. EDS

analysis: electrons interacting with the sample's

atoms generate signals from these interactions,

Rising population density and heavy anthropic

activity surrounding urban water bodies, has

affected the water quality along with its

biodiversity. Changes in trophic structure and

elimination of spawning and recruiting sites,

strongly affect the aquatic fauna, thus

diminishing diversity and consequently

affecting the ecosystem as a whole (Silva, 1995).

Biological monitoring is a water quality

evaluating method, which takes into account the

biological community responses to the extant

environmental conditions (Whitfield & Elliott,

2002; Goulart & Callisto, 2003). Bio-indicators

serve as indicators of the level of contamination,

and the changes in the number and distribution

of individuals and species, may point out

environmental deterioration (Karydis & Tsirtsis,

1996; Harrison & Whitfield, 2004; Buron et al.,

2009).

There have been studies carried out on a large

variety of organisms to evaluate their ability to

serve as bio-indicators, bio-monitors or

biological indicators, as well as on their use as

markers pointing out any kind of pollution

INTRODUCTION

Neotrop. Helminthol., 8(2), 2014

219

done by using the Energy-Dispersive

Spectroscopy (EDS) system in conjunction with

a FEI Quanta 200 model Scanning Electron

Microscope (SEM). The characteristic X-rays

emission identifying elements chemically

present in Digenea metacercariae trematodes.

Microanalyses were done in the Electronic

Microscopy and Microanalyses Laboratory at

“Universidade Estadual de Londrina (UEL)”.

EDS microanalyses: Twenty (20) fixed parasites

were washed three times in 0.1M cacodylate

buffer for 10 min; dehydrated in an ascending

series of ethyl alcohol; dried to a critical point in

a BAL-TEC CPD 030 model drier, and mounted

in metal cylinders (stub). Afterwards, they were

covered with carbon film in a BAL-TEC SCD

050 model “Sputter coater” metalizer.

The spectra were obtained with the aid of an

INCA X-ray secondary electron detector system

(155 eV resolution) using the INCA-Analyzer

software for identifying the specific peaks for

the elements of interest, mainly the heavy metals

present in the samples, since the main purpose of

EDS ananylis is to to detect all known chemical

elements present within a sample during a

predefined time, unlike any special element per

run as other techniques do. Therefore, our

quantitative results were based on each

element's peak height in the generated spectrum,

and 300 s was how long it took for each

specimen to be analyzed. The EDS system was

very efficient it detected minute amounts of 17

chemical elements present in the tissues from a

sample comprised by in the three metacercariae

species: Clinostomum marginatum (Rudolphi,

1819), Herpetodiplostomum caimanicola

(Dollfus, 1935) and of Tylodelphis destructor

(Szidat & Nani 1951).

Taxonomical identification is in accordance

with Travassos et al. (1969) and Kohn et al.

(1995). Vouchers specimens' C. marginatum

(INPA 639), H. caimanicola (INPA 640) and of

T. destructor (INPA 641) were deposited in the

collection of the Instituto Nacional de Pesquisas

da Amazônia (INPA).

which provide information on the sample's

properties. When the beam hits the samples their

atoms are exited, as they return to their

fundamental status, they emit photons with the

atom's characteristic energies, (characteristic X-

rays). Photons identified in terms of their energy

and counted by the X-rays detector, located

within the vacuum chamber (Heckmann et al.,

2007).

The joint system's hardware and software, stores

information generating the spectrum relative to

the number of counts according to kev energy

(kilo electron volt), identifying the chemical

elements present in the sample (Heckmann et

al., 2007). Compared with the analysis by

wave-length dispersion, the EDS allows

detecting all chemical elements present in a

sample during a single run, instead of only one

element per run (Whallon et al., 1989; Johnson,

1 9 9 3 ) . U n l i k e a t o m i c a b s o r p t i o n

spectrophotometry, it is a practically non-

destructive method for chemical composition

microanalysis, and minute-sized samples can be

analyzed (Vaughn, 1989; Johnson, 1993).

The present work studies the chemical

composition of Digenea metacercariae

trematodes parasites of H. littorale the only fish

species that was captured in the studied

environment. It analyzes heavy metal

accumulation in pollution-exposed parasites.

We performed necropsies on 82 H. littorale

specimens captured from October to November

2010. Fish averaged 14.87±71 cm and

97.49±24.01g. They had been captured in

polluted streams within the city of Manaus, in

the State of Amazonas Figure (1). These streams

were, São Raimundo (3º 09' 47"S and 59º 54' 29"

W) and Educandos (3º 04' 16"S and 59º 55' 62"

W). The collected Digenea metacercariae were

fixed in cold AFA (alcohol, formaldehyde, acetic

acid) solution with and without compression.

Digenea metacercariae qualitative and semi-

quantitative composition determination was

MATERIAL AND METHODS

Morais & Malta

potassium (K); calcium (Ca); chlorine (Cl);

copper (Cu); lead (Pb); manganese (Mn);

platinum (Pt) and tungsten (W) (figure 2). In

addition to these 16 chemical elements, cobalt

(Co) was also present in Digenea metacercariae

captured in igarapé Educandos (figure 3).

EDS chemical elements semi-quantitative

distribution shows percentile differences

between elements in the studied sites. Igarapé

Educandos presents a higher contribution of

heavy metals, such as, Cu, Pb, Pt and Zn, as

compared to igarapé São Raimundo (figure 4).

The parasites were morphologically identified

as metacercariae of C. marginatum, H.

caimanicola (progenetic metacercaria) and T.

destructor. There was no detected chemical

element difference for different parasite species

analyzed by DES in the two collecting sites.

EDS microanalyses recorded the presence of 16

chemical elements in Digenea metacercariae

captured in igarapé São Raimundo: carbon (C);

oxygen (O); iron (Fe), zinc (Zn), aluminum (Al),

siliceous (Si); phosphorous (P); sulfur (S);

RESULTS

Figure1. Manaus city map showing the collecting sites. Igarapé São Raimundo (SR) and igarapé Educandos (ED).

220

Chemical analysis of Digenea metacercariae

CONVENÇÕES

Igarapés

Sample points

CONVENÇÕES

Igarapés

Sample points

Neotrop. Helminthol., 8(2), 2014

221

Figure 2. EDS - generated spectrum for Hoplosternum littorale metacercariae from igarapé São Raimundo.

Igarapé São Raimundo

Figure 3. EDS - generated spectrum for Hoplosternum littorale metacercariae from igarapé Educandos.

Igarapé São Raimundo Igarapé Educandos

222

Figure 4. EDS found the chemical element semi-quantitative distribution in Hoplosternum littorale Digenea metacercariae

captured in igarapés São Raimundo and Educandos. Yet, Carbon (C) and oxygen (O) were overlooked because of being common

in the analyzed metacercariae tissues.

Morais & Malta

sulfur (S); potassium (K); calcium (Ca); chlorine

(Cl); copper (Cu); lead (Pb); manganese (Mn);

platinum (Pt) and tungsten (W). In addition to

these 16, cobalt (Co) was also present in Digenea

metacercaria from igarapé Educandos.

The EDS system was very efficient; it detected

minute amounts of 17 chemical elements present

in the tissues from a sample comprised by three

metacercariae species: C. marginatum, H.

caimanicola and T. destructor. It determined the

spatial distribution of elements chemical in

every sample analyzed. It generated X-ray

composition maps showing its practicality for

analyses of anthropogenic-impacted

environments.

Heavy metal contamination, mainly by: zinc

(Zn); cobalt (Co); nickel (Ni); copper (Cu); iron

(Fe) and lead (Pb) threatens the whole world. It

can contaminate soil, water and accumulate in

tissues of living organisms (Loumbourdis,

1997). Determination of low concentrations of

heavy metals present in the samples requires the

use of sufficiently susceptible and versatile

techniques. The present work shows the EDS

system technique to be very efficient in analyses

determinations.

The energy-dispersive spectroscopy system

(EDS) is a valuable tool in studying and

characterizing microscopic materials. It is much

employed in petrography characterization and

petrological studies in geosciences. It can

determine the chemical constituents of living

organism, i.e., heavy metals present in fish

parasite tissues (Heckmann et al., 2007).

The coupled EDS- SEM system determined the

chemical constitution of proboscis and hooks of

f i v e A c a n t h o c e p h a l a s p e c i e s :

Neoechinorhynchus qatarensis Amin, Saoud &

Alkuwari, 2002; Acanthocephalus dirus (Van

Cleave 1931); Neoechinorhynchus idahoensis

Amin and Heckmann 1992; Echinorhynchus

s a l m o n i s ( M ü l l e r 1 7 8 4 ) a n d

Pseudoacanthocephalus sp. High sulfur levels

were found (Heckmann et al., 2007).

In this work by using the same procedure we

detected 16 chemical elements in Digenea

metacercariae from igarapé São Raimundo:

carbon (C); oxygen (O); iron (Fe), zinc (Zn),

aluminum (Al), siliceous (Si); phosphorus (P);

DISCUSSION

Chemical analysis of Digenea metacercariae

223

Neotrop. Helminthol., 8(2), 2014

studied. But for cobalt (Co), which only

occurred in metacercariae from igarapé

Educandos.

Analyses of Digenea metacercariae collected in

the two igarapés presented quantitative

differences. Samples from igarapé Educandos

presented higher heavy metal concentration

values. This is due to the continuous inflow of

industrial pollutants into this igarapé.

Chemical elements as copper (Cu); iron (Fe);

manganese (Mn); cobalt (Co); selenium (Se);

einsteinium (Es); iodine (I) and chrome (Cr) are

considered to be essential to the metabolism of

living organisms. Cadmium (Cd), mercury (Hg),

lead (Pb), nickel (Ni), aluminum (Al), platinum

(Pt), tungsten (W) and Argonne (Ar) exert no

known biological function and are usually toxic

to a large number of organisms (Roesijadi &

Robinson, 1993; Heath, 1995).

Igarapés contaminated through anthropic action

present a reduction on dissolved oxygen

contents, increase of pH and show high

concentrations of heavy metals (Zn, Co, Ni, Cu,

Fe, and Pb). There occur at high fecal and total

coliform rates, much above those acceptable for

human consumption or recreation (Silva, 1995;

Melo et al., 2005; Santana & Barroncas, 2007).

Digenea metacercariae trematodes, parasites of

H. littorale from igarapés São Raimundo and

Educandos contained lead (Pb), platinum (Pt),

aluminum (Al) and tungsten (W) in their tissues.

All this contamination came about through the

inflow of toxic effluents originating from the

Manaus Industrial District factories sewage.

That contamination determined the death of

living organisms inhabiting the water bodies

within these ecosystems.

Pollutants cause a significant impact on the host-

parasite system in aquatic environments. By

assessing this relationship one concludes the fish

parasite fauna, mainly digenetic trematode

species, to be important parameters in

monitoring pollutant-contaminated water

quality (Sures, 2008; Vidal-Martinez et al.,

2009; Khalil et al., 2009).

The EDS system was utilized in species of

Acanthocephala N. qatarensis, A. dirus, N.

i d a h o e n s i s , E . s a l m o n i s a n d

Pseudoacanthocephalus sp. Following 60 s of

exposition we detected the presence of: sodium

(Na); aluminum (Al); magnesium (Mg);

siliceous (Si); potassium (K); sulfur (S); calcium

(Ca) and phosphorous (P) (Heckmann et al.,

2007).

In this study, we increased, this exposition time

in each Digenea metacercariae sampled point, to

300 seconds. This resulted in the detection of:

lead (Pb); tungsten (W); copper (Cu) and

platinum (Pt). Therefore we recommend the fish

parasites samples exposition time in EDS to be

higher than 60 seconds, so as to run no risk of not

detecting low-concentrated elements present in

the sample.

Manaus city igarapés ranked as being

environments presenting diversified

characteristics, ranging from pristine to

degraded, there occurring polysaprobic

environments presenting heavy pollution

(Lopes et al., 2008).

The biological diversity differs in igarapés São

Raimundo and Educandos. São Raimundo

presented higher macro-invertebrate diversity

than that of Educandos. The latter is subjected to

the inflow from chemical effluents

contaminated with copper (Cu); nickel (Ni) and

zinc (Zn) originating from the Manaus Industrial

District (Cleto-Filho, 1998).

Igarapé Educandos presents cleared banks and

secondary vegetation along some of its stretches.

Its upper course gets the sewage from the

Manaus Industrial District, receiving the inflow

from around 400 chemical; thermoplastic;

metallurgic; mechanical and electronic

industries, the main polluting agents of which,

happen to be heavy metals (Lopes et al., 2008).

The present study showed the chemical

composition found in Digenea metacercariae

trematodes tissues of C. marginatum, H.

caimanicola and T. destructor, parasites of H.

littorale to be the same on the two igarapés

224

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T h e A u t h o r s t h a n k C o n s e l h o d e

Aperfeiçoamento de Pessoal de Nível Superior

(CAPES) for scholarship. Célia G.T. de Jesus

Andrade, Ângela Teresa Silva-Souza and

Oswaldo Capello of LMEM/PROPPG/UEL for

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Henrique Scheneider for donating the fish used

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PIATAM project, INPA/MCT.

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