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Establishment and analytical quality control of
laboratories for Hg determination in biological
and geologicalsamplesin the Amazon, Brazil
WANDERLEY R. BASTOSI , OLAF MALMI,
WOLFGANG C. PFEIFFERI, DAVID CLEARy2
lLaboratório de Radioisótopos EPF, Instituto de Biofisica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21940-900 and 2Laboratório de Mercúrio,
Fundação Esperança, Santarém, PA 68040-100, Brasil
During the past 20 years significant amounts of Hg have been released in the Amazonian environment due to goldmining activities. Several Brazilian and foreign research groups have
been studying the region, always facing difficulties regarding preservation and transfer of the
collected samples from the field to the analytical laboratories. Aiming to solve these problems two high quality Hg analytical laboratories were established in the region. One is 10cated at Fundação Esperança (FE, Santarém, PA), a medical assistance foundation and the
other at the Universidade Federal de Rondônia (UNIR), Porto Velho, RO. Their main equipment is: Atomic absorption spectrophotometers for Hg analysis (Flow Injection Mercury
System-FIAS 400 with automatic samples-AS90-Perkin Elmer) and a microwave oven digestion system (MDS-2000-CEM). Technicians and researchers were trained in several aspects
like environmental and human sampling, digestion methods and Hg analysis for biotic and
abiotic materiaIs. This training as well as the installation of the mentioned laboratories were
the responsibility of the Universidade Federal do Rio de Janeiro (UFRJ). Intercalibration
exercises were performed between several laboratories: Environmental Medicine of Odense
University (Denmark), Centre de Toxicologie du Québec, Canada and Occupational Health
Sciences (Health-Canada). In the last 18 months the FE laboratory joined the Hg urine analysis intercalibration program of the Laboratory of the Centre of Toxicology in Québec, Canada,
reaching accurate results. To date both Amazonian laboratories are able to analyze several
different materiaIs: Sediment, soil, fish, urine, hair and blood. The FE laboratory is becoming
more specialized in human samples such as urine, blood and hair, while UNIR laboratory deals
with environmental samples such as fish, sediment and soil.
A Amazônia vem sendo, nos lÍltimos 20 anos, alvo de significativos
lançamentos de merclÍrio no ambiente devido à atividade dos garimpos de ouro. Vários grupos de pesquisa do Brasil e do exterior
vem estudando esta região, deparando-se sempre com problemas
de p,'eservação e transporte das amostms coletadas pam os laboratÓI'ios analíticos, pois estes ficam muito distantes. Em jimção disto.
e principalmente, para transferência de conhecimentos cientificos e
formação de pesquisadores da região. projetou-se a instalação de
dois laboratórios de alto nível técnico de qualidade analítica. um
na Fundação Esperança em Santarém, PA e outro na Universidade
Federal de Rondônia em Porto Velho, RO. Tendo estes como equipamentos principais: Espectl'ofotômetro de absorção atômica dedicado para análises de merclÍrio-FIMS (Flow Injection Mercury
System-FIAS 400 com amostrador automático-AS90-Perkin Elmer),
Correspondence to: Wanderley Rodrigues Bastos, Centro de Ciências da Saúde, Laboratório de Radioisótopos EPF, Instituto de Biofisica
Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21940-900, Brasil
E-mail: [email protected]
Volume 50(4)
.
July/ August
1998
Ciência e Cultura Journal of the Brazilian Assoc/at/on for the Advancement of Sc/ence. 255
......................
. . . . . . . . . . . . . . . . . . . . . . .Technical
- Review.
Sistema de Digestão por Microondas
(MDS-2000-CEM).
Grupos
foram treinados em procedimentos
como: Amostragens. métodos
de digestão e análises de amostras biológicas e geológicas. O Laboratório
de Radioisótopos
(LREPF)
da
UFRJ
é o principal
res-
ponsável no
desenvolvimento de técnicas de preparo de amostras e
no controle de qualidade analítico desses laboratórios em implantação. O laboratório
de Santarém. nos riltimos 18 meses. participa
T
he main sourceof mercwy (Hg) inthe BrazilianAmazonian environrnent during the past decades is from
goldmining activities. Its use began at the end of
the 50's in the Tapajós river basin and during the
70's, an intense gold rush spread over other river basins inc1udingthe Madeira river basin.
Mercury is used in gold prospecting for the amalgamation of alluvial gold, reaching the environment mainly
through water and air. The total amount of Hg used during
the whole process depends on the technique applied but it is
about 1.3 kg ofHg per kg of gold produced (1).
Since the 80's several research groups have been working on the Hg problem in the Amazon, trying to understand
its cyc1eand its fate in tropical areas. Factors such as preservation and transfer of samples, have always been a limiting
factor for more detailed follow-up studies, but also for results
with analytical quality.
To solve these problems, establishing regionallaboratories and training local people in the technical requirements
ofHg analysis is important, both regarding the public health
aspects and the development of local research groups about
other different environrnental issues.
Two new generation analyticallaboratories for Hg determination were established in the Amazon, near the main
affected areas: One at a nongovernrnental medical assistance
organization, Fundação Esperança (FE) in Santarém, PAand
the other at the Universidade Federal de Rondônia, in Porto
Velho, RO (UNIR). A third laboratory at the Universidade
Federal do Rio de Janeiro (UFRJ) provides the methodologies and controls the quality of analysis.
Digestion methods of environrnentaland human samples
using microwave in c10sedsystems, were developed, making
the process less time consuming and almost free of contamination risk. These methods were adapted to a new automated
flow injection Hg analysis system, controlled by a PC computer with aWindows software programoInterlaboratorycomparison programswere implementedfor analyticalqualitycontrol in several different matrices.
Amazon laboratories
A research project supported by the European Union, began in 1994 with the establishment ofthe laboratories in the
Amazon.
Fundação Esperança situated in the central part of
Amazonia in Pará state, has provided medical and social assistance to the Tapajós riverine population for more than 20
years, therefore the Hg laboratory was established at this
foundation in Santarém at the beginning of 1994. This city
256. Ciência e Cultura Journal of the Brazllfan Assoclatlon
for the Advancement
de um programa de intercalibração com o Laboratório do Centro
de Toxicologia de Québec. Canadá, obtendo resultados excelentes.
AtlIalmente esses laboratórios estão aptos à rotina analítica em
matrizes como: Sedimento. solo. peixe, urina. cabelo e sangue. O
laboratório instalado na Fundação Esperança em Santarém tem se
especializado nas amostras humanas, e o Laboratório da UNIR em
amostras ambientais.
was an important gold trading center but is also a strategic
site for the follow-up studies of Hg contamination in the
Tapajós river basin. It can also serve other local areas such as
the Tocantins river basin.
The UNIR laboratory, was installed at the end of 1994,
when analytical Hg determination training began. This site
was selected since Porto Velho city is located on the border
of the Madeira river, southwest Amazon and could provide
analytical support also for areas like the Negro river. During
15 years (1978-1993) the Madeira river area was a heavy mining area and is one of the most studied regions in Amazonia
regarding Hg contamination.
Sample digestion: Traditional method
Traditional digestion of different biological and nonbiological
samples for total Hg analysis can last a couple of hours to a
few days before achieving complete solubilization. These
processes are mainly performed in open systems without an
effective temperature control, increasing risks of volatilization losses and sample contamination as well as exposing the
laboratory to acid vapors. Equipments such as hot baths,
digestion blocks, hot plates, teflon digestion bombs and others are used. These are efficient but time consuming processes that have been widely used for many years.
Those methods have some advantages: The sample mass
is not limited; reagents and sample are easily introduced; it is
possible and sometimes easy to monitor the process; and it
has a low cost. But it also has some disadvantages: Time
consuming; higher risk of contamination; risk of volatilization or losses; release of acid vapours and higher consumption of reagents.
Microwave oven digestion
The use of microwave energy for acid digestion was first
demonstrated approximately 20 years ago (2). The use of a
microwave oven for acid digestion of solid samples is welldocumented (3). Afterwards, several different companies developed various adaptations with open and c10sed teflon
vessels, with pressure and temperature controI. Other types
ofvessels made of glass, quartz and plastics can also be used
with acids like HN03, HCl, ~SO 4'
In general accuracy and precision can be greatly affected
by digestion efficiency. The presence ofpartic1es in mineralized organic matter can give unrealiable results or cause interference.
For trace metaIs, and specifically for Hg, analysis usof Sclence
Volume50(4). July/August1998
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,
Detection
ing mineralization methods in a microwave oven is being
more widely used, and is considered a very competitive procedure (4).
Those methods have some advantages: Can be performed in a short time; digestion can be done at 270°C in
closed systems; acids do not evaporate; drastic reduction of
The detection limit (DL) ofFIMS-400 provided by Perkin- Elmer
Corporation is ofO.O1 ppb (parts per billion). For practical reasons, the DL for each specific technique is ca1culated as:
risks of contamination and losses; control of pressure and
temperature; on the other hand, the main disadvantage is the
high cost of the equipment.
-
DL (llg gl orllg VI) =men
The present paper aims to present the methodological improvements and results of intercalibration programs, which
guaranteed the analytical quality control of Hg concentrations measured by two laboratories installed in a tropical area,
mainly the Amazon basin.
Establishment of two high levellaboratories in the Amazonregion,traininglocalpeopleinsampling,preparationand
determinationof Hg in human (hair,urine and blood) and
environmental (sediment, soil and fish) samples.
Toimproveandadaptnewmethodsformineralizationof
different matrices using a microwave oven coupled to the
very recent automated flow injection mercury system.
Implementation of an analytical intercalibration exercise
I
5.0 mL of H20milli-Q + 5.0 mL HCI:HNOa
(3:1)
JJ
Hot water bath (60°C) during 5 mino
JJ
50.0 mL HP milli-Q + 15.0 mL KMn04 5%
JJ
Hot water bath (60°C) during 30 mino
JJ
control programo
Overnigth
..
I
I
I
I
,
"S
vl.:
JJ
between the three laboratories as well as with other Brazilian
The three laboratories, besides the infrastructure (analytical balances-AB204 Mettler, water purification systemsmilli-Q Millipore, hoods and others) where provided with:
Microwave oven CEM-:MDS-2000(630i: 50 W), with an exhaustion system and microprocessed control of energy and
pressure. Each laboratory received 3 complete turntables with
12teflon (PTFE)line devicevessels (LDV)with 120ml capacity each, and atomic absorption spectrophotometers for Hg
analysis with a flow injection system (FIMS-400) and
autosampler (AS-90), both from Perkin-Elmer.This system is
fully controlled by a PC-486, 433-DX from Digital; software
Winlab (perkin-Elmer) running under Windows.
LJ
mass(g)orvolume(rnL)a
2.0 9 of sediment or soi! «200 mesh)
and internationalreferencelaboratoriesas part of a quality
i
ael!!.
Figure 1presents the traditional technique for Hg analysis in sediment and soil samples. Figures 2 to 6 present the
analytical methods developed for urine, blood, hair, soil and
sediment,and fish samples.Detectionlimit (DL) for eachtechnique is included in the Figures.
Objectives
o
limit of the methods
Add drops of NHPCI12%
JJ
Filter inWhatman 42
final volume 100mL
JJ
Analysis on CVAAS".
Figure 1. Traditionalanalyticalsediment and soi! techniquesused at
the UFRJ laboratOlY. DL = 15.0 J.!gklf'/ (5).
2.0 mL of urine
JJ
3.0 mL HNOfiH2S04(1:1)
Development of the digestion method for samples
4.5 mL KMn04 5%
JJ
Microwave (urine Hg program) 20 mino
A methodology with a minimum of sample handling to avoid
contamination and losses was developed adapting the experience of the laboratory of environmental medicine at Odense
University, Denmark (with a long experience ofmicrowave
After cooling to room tempoadd drops of
NHPCI12%
digestion), aiming to reduce the number of steps. Some modifications were introduced in the microwave instruction manual
for the same matrices, to adapt the system to specific Hg
properties.
Internal standard addition ofHg was initially used during the development of the techniques. Afterwards certified
reference materiaIs and other interlaboratory
reference
samples were established for continuous routine analysis.
Volume50(4) o July/August1998
Ciência
e Cultura
JJ
JJ
Final volume 10 mL
analysis on FIMS*
Figure 2. Digestion of urine samples in a microwave oven for total Hg
determination. DL = 1.5 J.!gL-/.
a
Average mass value (g) was used for fish, hair, sediment and soil
samples, while volume (mL) was used for blood and urine.
Journal
of the Brazillan
Assoclation
for the Advancement
of Science
o 257
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-- - -1.0 mL of blood
500 mg of fish
U
U
3.0 mL HN03cone.
1.0 mL H202 cone.
5.5 mL KMn04 5%
3.0 mL H2S04:HN03(1:1)
,-
U
U
U
U
5.0 mL KMn045%
Microwave (blood Hg program) 35 mino
U
U
.
Microwave(fishHgprogram)30 mino
U
Aftercoolingto roomtempoadddropsof
NHPCI12%
After cooling to room tempoadd drops of
NHPCI12%
U
Final volume 10 mL:=} analysis on FIMS*
~
Figure 3. Digestion of blood samples in a microwave oven for total
Hg determination. DL = 3.0 !1gL-I.
Final volume 10 mL:=} analysis on FIMS*
Figure 6. Digestion of jish samples in a microwave oven for total Hg
determination. DL = 10.0 !1g kg-1.
50 mL of hair
(washed with EDTA0,01%)
U
METHODOLOGY COMPARISON
3.0 mL HN03:H2S04(1:1)
U
MERCURYIN SOILANDSEDIMENT( ~g.g-1)
8
::-7
6.0 mL KMn04 5%
U
y
~6
C>
Microwave (Hair Hg program) 40 mino
'::""5
~4
After cooling to room tempoadd drops of
NHPCI12%
U;
3
J
fE2
=>
1
O J.
O
Final volume 10 mL:=} analysis on FIMS*
Figure 4. Digestion of hair samples in a microwave oven for total Hg
determination. DL = 35.0 !1g Kg-l.
- 0.0197
R2 = 0.9966
N=9
U
U
= 1.1411x
2
6
3
4
5
UFRJ-VGA (~g.g.1)
7
8
Figure 7. Correlation between traditional (hot bath. continuous flow
(VGA)) and new digestion (microwave. flow injection (FlMS)) and
Hg determination in sediment samples. at the UFRJ laboratory.
100 mg of soi! or sediment
U
1.0mLHP milli-Q
U
6.0 mL HN03:HCI (1:1)
U
3.0 mL KMn04 5%
U
Microwave (sediment Hg program) 30 mino
U
After cooling to room tempoadd drops of
NHPCI12% and filterWhatman 42
U
Final volume 10 mL:=} analysis on FIMS*
Figure 5. Digestion of soi! or sediment samples in a microwave oven
for
258.
total
Hg
determination.
DL
=
4.0
!1g kg-I
Ciência e Cultura Journal of the Brazlllan Associatlon
for the Advancement
INTERLABORATORY COMPARISON PROGRAM
MERCURY IN FISH ( ~g.g'1)
::- 1.2
b>
y = 1.2601x - 0.0156
ÓJ 1
.::....
R2 = 0.9847
~ 0.8
N=22
o
"§ 0.6
o
ii!
...I 0.4
9;;
z
=>
.
0.2
O
O
0.2
0.4
0.6
0.8
1.2
FE Laboratory (~g.g-1)
Figure 8. Correlation between FE and UNIR laboratories regarding
Hg analysis in jish samples.
of Sclence
Volume 50(4).
July/August
1998
~
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......................
--Review.
- INTERLABORATORY COMPARISON PROGRAM
MERCURY IN HAIR ( ~g.g")
INTERLABORATORY COMPARISON PROGRAM
MERCURY IN URINE ( ~g.L-1)
350
~ 300
.:.J
P 250
y = 0.9172x- 1.9177
R2= 0.9839
N = 18
~
~ 200
o
150
25
.
~ 15
16
210
~o
(\3
...J
fà 100
...J
~
it
50
O
50
100
150
200
CTQ-Canada (~g.L-')
250
300
30
.
y = 0.7964x + 2.7471
R2= 0.857 N = 27
UFRJ lab.
20
.
-g
:g 15
eu "C
o
it 16 10
u.
o
y = 0.7989x + 2.0566
R2= 0.8577 N = 27
FE lab.
~
fà 5
::> ...J
O
O
5
10
15
20
Odense Universitet Lab. (~g.L-')
25
30
Figure 10. Correlation between three laboratories: Odense. UFRJ
and FE regarding Hg analysis of urine.
35
30
y = 0.93x - 0.8612
R2= 0.9996
N=05
P 25
~
~
20
16
(; 15
.Q
(\3
...J
w 10
u.
5.
5
10
15
20
25
CTQ-Canada (~g.L-')
30
35
Figure 11. Intel'laboratory comparisonprogram of Hg blood analysis
between Fundação Esperança Laboratory (FE) and the Centre de
Toxicologie dll Québec. Canada (CTQ).
Volume 50(4) -July/August 1998
5
10
15
OHS-Canada (~g.g")
20
25
Figure 12. lnterlaboratory comparisonprogram of Hg hair analysis
between UFRJ Laboratory and Occllpational Health Sciences-Health.
Canada (OHS).
During the development ofthe methods, mercury determinations were performed using'a modern flow injection system
(FIMS-400) and compared with the traditional methods and
analytical systems of our laboratory provided with a cold
vapor atomic absorption spectophometer (CV-AAS-Varian
VGA-76 andVarian-1475).
Internal standard addition was initialIy used for testing
recovery during-the development of the techniques. Afterwards certified reference materiaIs were used for checking
accuracy. For routine use in monitoring analysis we prepared
at our laboratory homogenized freeze-dried fish and homogenized sediment samples and determined their Hg concentration. Those samples were used regularly in the three laboratories. International intercalibrationprogram were established
jointly with the Centre de Toxicologie du Québec, Canada for
urine blood and hair samples. AlI analysis were done in triplicate including analytical blanks.
A complete solubilization of the samples was obtained
in all the developed methods'.Recovery ofHg in internal standard experiments was between 90 and 105%.
Table 1presents the results ofHg determinations in several different certified and internaI reference materiaIs performed by the three laboratories.
Interlaboratory calibration exercises with a greater number of samples present a very good analytical correspondence between the participant laboratories regarding soil and
sediment (Fig. 7), fish (Fig. 8), urine (Figs. 9 and 10), blood
(Fig. 11)and hair (Fig. 12).Correlationcoefficients(R2)ranged
from 0.8570-0.9997, and angular coefficients (a) between
INTERLABORATORY COMPARISON PROGRAM
MERCURY IN BLOOO ( ~g.L")
~
O
Hg determinations and quality control
INTERLABORATORY COMPARISON PROGRAM
MERCURY IN URINE SAMPLES ( ~g.L-1)
~ 25
.:.J
o
350
Figure 9. lnterlaboratory comparisonprogram of Hg urine analysis
between Fundação Esperança Laboratory (FE) and the Centre de
Toxicologie du Québec. Canada (CTQ).
~
5
u.
::>
O
~t
y = 1.0872x - 0.4108
R2= 0.9997
N=6
~20
~
0.7964-1.2601.
T
he digestion of samples is better and faster using a microwave oven in all types of studied matrices.
The developed methods are more sensitive, efficient and
Ciência e Cultura Journal of the Brazifian Association
for the Advancement
of Science - 259
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clean, reducing contamination risks due to the closed system
digestion and less manipulation of samples during analysis
(automated).
The established laboratories in Amazonia present excellent
performances,succeedingin overcomingphysical
isolation regarding materiais and technical and human resources.
.
-
Table 1 Average Hg concentration in reference samples: Different
analytical methods and different laboratories.
Standard
samp/es
UFRJ
FIMS
UFRJ
VGA-76
Hair (Jlg g-I)
IAEA397
11.80
11.76
2.98
3.06
3.10
3.19
2.85 (IPEN)
2.94 (CENA)
Fish (Jlg g-')
APPX2960"
5.03
4.98
4.84
4.68
4.72 (IPEN)
4.98 (CENA)
Blood (Jlg VI)
SERONORM
8.80
10.60
9.00
(SERONORM)
Soil(Jlgg-I)
GMSL-167"
0.23
0.23
Sediment pond
(Jlgg-I)
1.22
\.16
Fish (Jlg g-I)
APPX2958"
Lab
UNIR
Lab
FE
Certificatel
Reference va/ue
12.30
(IAEA)
Q.21
0.23
(GKSS)
1.30
(No. 2-NIES)
" Reference sample of UFRJ laboratory.
IAEA = Intemational Agency of Energy Atomic, USA; IPEN = Instituto
de Pesquisas Energéticas e Nucleares, Brazil; CENA = Centro de Energia
Nuclear e Agricultura, Brazil; GKSS = GKSS Research Center, Germany; NIES = National Institute for Environment Sciences, Japan;
SERONORM = Seros AIS, Norway; UFRJ = Universidade Federal do
Rio de Janeiro, Brazil; UNIR = Universidade Federal de Rondônia,
Brazil; FE = Fundação Esperança, Brazil; FIMS = Flow Injection Mercury System, Perkin-Elmer; VGA = Vapor Generation Accessory, Varian.
References and notes
,
1. Pfeiffer WC, LD Lacerda 1988 Mercury inputs into the Amazon
region, Brasil. Environ Techno/ Lett 9: 325-330
2. Heltai G, K Peresich 1994 Moderated pressure microwave digestion system for preparation of biological samples. Elsevier Sei
Talanta 41: 1067-1072
3. Zhou CY, MK Wong, LL Koh, YC Wee 1995 Orthogonal array
design for the optimization of closed-vessel microwave digestion
parameters for the determination of trace metais in sediments.
Anal Chim Acta 314: 121-130
4. Welz B, LD Tsalev, M Sperling 1992 On-line microwave sample
pretreatment for the determination of mercury in water and urine
by flow-injection cold-vapour atomic absorption spectrometry.
Chim Acta 261: 91-103
5. Malm O, WC Pfeiffer, WR Bastos, CMM Souza 1989 Utilização
do acessório de geração de vapor frio para análise de mercúrio em
investigações ambientais por espectrofotometria
de absorção
atômica. Ci Cult 41: 88-92
6.Acknowledgements:
The authors wish to thank the Fundação
Esperança (FE), Universidade Federal de Rondônia (UNIR) and
Universidade Federal do Rio de Janeiro (UFRJ) for local support,
and Fernando Pinto (UFRJ), Doralice A Leão (FE), Prof Ene
Glória and Priscila Gali (UNIR) for technique assistance, Profs
Miriam Brugnara and Luciana Andrade for reviewing manuscript.
This research was supported by the European Union (Contract
no. B7-5041/1/93/15) and CNPq.
-
-
Received 04 July 1997
Accepted 10 August 1997
,.
260. Ciência e Cultura Journal ofthe Brazillan Association
for the Advancement
of Sclence
Volume 50(4). July/August 1998