Ecotoxicology (2007) 16:263–270
DOI 10.1007/s10646-006-0126-9
Evaluation of cyanobacteria toxicity in tropical reservoirs using
crude extracts bioassay with cladocerans
Denise Tieme Okumura Æ
Rosana Barbosa Sotero-Santos Æ
Renata Akemi Takenaka Æ Odete Rocha
Accepted: 17 October 2006 / Published online: 28 November 2006

Springer Science+Business Media, LLC 2006
Abstract During a cyanobacterial bloom in a eutro-
phic environment, particularly at the end when
decomposition occurs, toxic compounds such as the
cyanotoxins and the lipopolysaccharides can be
released in high concentrations into the water
column damaging aquatic organisms. In this work,
the effects of this release of toxic compounds during
a cyanobacterial bloom were investigated. The acute
and chronic toxicity of cyanobacterial crude extracts
from two natural blooms in the Barra Bonita and
Ibitinga reservoirs (Middle Tietê River, São Paulo
State, Brazil) and of a toxic strain cultured in the
laboratory were tested. The cladocerans Daphnia
similis, Ceriodaphnia dubia and Ceriodaphnia silves-
trii were used as test organisms. In the chronic
toxicity tests, only a native cladoceran found in
Brazilian freshwaters, Ceriodaphnia silvestrii, was
used. Microcystins were detected in all cyanobacte-
rial samples. The acute toxicity tests showed that the
crude bloom material extract from the Ibitinga
D. T. Okumura
R. B. Sotero-Santos
O. Rocha
Department of Ecology and Evolutionary Biology,
Biological and Health Sciences Center, Federal University
of São Carlos, Rodovia Washington Luis, km 235,
CEP 13565-905 São Carlos, SP, Brazil
R. A. Takenaka
Engineering School of São Carlos, University of São Paulo,
Avenida Trabalhador São Carlense, 400, CEP 13566-590,
São Carlos, SP, Brazil
R. B. Sotero-Santos (&)
Great Lakes Institute for Environmental Research,
University of Windsor, 401 Sunset Avenue, N9B 3P4
Windsor, Ontario, Canada
e-mail: rmabarb@yahoo.com.br
Reservoir (48-h EC50 values between 32.6 and
35.8 lg microcystin g–1 of freeze-dried material)
exhibited higher toxicity to cladoceran than did the
crude bloom material extract from Barra Bonita
Reservoir (48-h EC50 values between 46.0 and
80.2 lg microcystin g–1 of freeze-dried material).
The chronic toxicity test data showed that the three
extracts reduced the fecundity of C. silvestrii, and the
crude extract of Barra Bonita Reservoir bloom
material also affected the survival of this cladoceran.
Both acute and chronic tests effectively prognostica-
ted possible changes in the cladoceran population,
and probably other components of the biota due to
cyanobacterial blooms in natural aquatic ecosystems.
Keywords Cyanobacterial blooms
Aquatic toxicity

Cyanotoxins
Microcystins
Cladocerans
Introduction
The eutrophication of lakes and reservoirs of Europe
and North America, up until the middle of the 20th
century, had been recognized as a local problem related
to pollution in the more industrialized countries. Now-
adays, however, it is a worldwide phenomenon that has
led to the deterioration of aquatic environments, preju-
dicing the many uses to which the water is put (Bartram
et al. 1999). Besides high nutrient concentrations, high
temperatures also favor microalgal proliferation, espe-
cially in tropical regions. Many cases of massive devel-
opment of cyanobacteria have been recorded and there
is common concern about this because of the likely
effects due to global warming, especially regarding
those species that produce toxins (Aboal and Puig 2005).
123
264
Dozens of cyanobacterial species produce toxic sec-
ondary metabolites that are harmful to humans and
other components of aquatic biota, through direct or
indirect exposure. In principle, every water surface can
act as a habitat for cyanobacteria (Dietrich and Hoeger
2005). Even inhospitable environments such as very arid
(Wynn-Williams 2000), semi-arid (Pouria et al. 1998;
Domingos et al. 1998) and very cold climates (Hitzfeld
et al. 2000) may harbour cyanobacteria. Some features
such as the trophic status, climate and morphology of the
water-body, determine the composition of the cyano-
bacterial species in the phytoplankton community.
The occurrence of cyanobacterial blooms is a typical
phenomenon in eutrophic environments, affecting the
zooplankton community and interfering in the energy
transference to upper trophic levels (Rocha et al. 1997;
Tundisi et al. 2002; Hanazato 2000). There is evidence
that cyanobacteria have deleterious effects on zoo-
plankton but these effects are highly variable between
the genera and species, and even clones of individual
zooplankton species. One of the main questions yet to
be resolved is whether the observed inhibitory effects
are due to the known cyanotoxins, to other unidenti-
fied compounds, or to the putative poor nutritional
value of cyanobacteria (Sivonen and Jones 1999).
Some studies, which were developed in laboratory
conditions, investigated the relations between cyano-
bacteria and zooplankton; however, the majority of
studies have focused on the effects of cyanobacterial
cultivated strains on cladocerans from temperate cli-
mate (Lampert 1981; Gilbert 1990; DeMott et al. 1991;
Reinikainen et al. 1995; Thostrup and Christoffersen
1999; Rohrlack et al. 2003; Gustafsson and Hansson
2004). There is a lack of knowledge regarding the
effects of natural cyanobacterial blooms and their
toxins on tropical zooplankton species (Sotero-Santos
et al. 2006). Daphnids are excellent representatives of
Cladocera, a key group of organisms in freshwater
systems, and disturbance of this population may have
effects throughout the aquatic food-web. These organ-
isms are commonly used in toxicity tests worldwide. In
the present study, we investigated the acute toxic
effects of crude extracts of natural cyanobacterial
blooms and also that of a known toxic strain of
Microcystis aeruginosa on the survival of three species
of cladocerans (Ceriodaphnia silvestrii, Ceriodaphnia
dubia and Daphnia similis). The chronic effect on the
fecundity of Ceriodaphnia silvestrii, a native cladoceran
in Brazilian freshwaters was also investigated. Studies
regarding the effects of cyanobacterial extracts on the
fecundity of cladocerans are very rare, especially
concerning tropical species that might be important as
an early warning for water management authorities.
123
D. T. Okumura et al.
Materials and methods
Biological samples
Two sources of material were used: natural cyanobac-
terial bloom scum with a predominance of Microcystis
spp and laboratory cultures from a toxic strain of
Microcystis aeruginosa (NPLJ-4). Cyanobacterial scum
was collected with a 25 lm mesh phytoplankton net in
November 2002 at Barra Bonita and Ibitinga reservoirs
(Middle Tietê River, São Paulo State, Brazil). Cyano-
bacteria belonging to Microcystis spp were dominant in
the field at this time.
NPLJ-4 is a toxic Microcystis aeruginosa strain
isolated from Jacarepaguá Lagoon, Rio de Janeiro,
Brazil and supplied by Dr. Sandra M.F.O. Azevedo
from the Núcleo de Pesquisas de Produtos Naturais/
CSS/UFRJ (Federal University of Rio de Janeiro,
Brazil). The NPLJ-4 strain produces four types of
microcystins; microcystin-LR, the main congener, rep-
resents 80% of the total (Soares et al. 2004). To obtain
sufficient biomass for the bioassays and analyzes, the
NPLJ-4 strain was cultivated in 2 l Erlenmeyer flasks
containing 1.5 l ASM-1 medium (Gorham et al. 1964),
under 0.018 lmol m–2 s–1 with a 12/12 h light–dark
cycle and a constant temperature incubator at
21.0 ± 0.2C. Cultures were harvested at the late
exponential phase of growth by centrifuging; the cell
material was lyophilized and then stored at –20C.
All material used for extract preparation (Micro-
cystis aeruginosa culture cells and natural bloom
materials) were freeze-dried at –80C (Lyophilizer
LB 5000 TT—TERRONI-FAUVEL) until full dehy-
dration. Freeze-dried material was weighed in analyt-
ical scale (10–4 g) and preserved at –20C until extract
preparation.
Cladoceran cultures
Stock cultures maintained for several years at the
Ecotoxicology Laboratory of the Federal University of
São Carlos, Brazil, were used in acute toxicity tests.
Daphnia similis, Ceriodaphnia silvestrii and Ceriodaph-
nia dubia (Cladocera, Crustacea) were cultured in 2 l
glass beakers in an incubator kept at 25C, with an
12/12 h light–dark cycle. The culture medium (modi-
fied from Environment Canada 1990a, b; ABNT 2003)
was reconstituted water (pH 7.0–7.6; conductivity
160 lS cm–1 and hardness 40 to 48 mg CaCO3 l–1).
Daphnids were fed with an algal suspension of
Pseudokirchneriella subcapitata (previously known as
Selenastrum capricornutum) at 5 · 105 cells ml–1 and a
Evaluation of cyanobacteria toxicity in tropical reservoirs
mixture of baker’s yeast and fermented trout chow
(Environment Canada 1990a, b; ABNT 2003, 2004).
Preparation of crude cyanobacterial extract
Lyophilized natural bloom material (50 mg of freeze-
dried cells) was dispersed in distilled water (100 ml) and
subjected to a freeze/thaw cycle four times. After the last
cycle, the thawed material was ultrasonicated for 10 min
(a procedure adapted from Oberemm et al. 1999; Pietsch
et al. 2001). Finally, debris was removed by centrifuging
at 3,500 rpm for 30 min. Similarly, crude extracts from
NPLJ-4 strain was prepared as described above. The
concentrations adopted in this study were based on
preliminary tests (data unpublished).
Acute toxicity test
Acute toxicity tests were carried out with cladocerans
(Daphnia similis, Ceriodaphnia silvestrii and Cerio-
daphnia dubia) to analyze their sensitivity and
survival during exposure to aqueous crude extracts of
Microcystis spp. The concentrations are given as
lg microcystin g–1 dry weight of freeze-dried material.
The basic design for the experiments consisted of
exposing five neonates (<24-h old) of each cladoceran
species in separate glass tubes containing 10 ml of
aqueous crude extracts at microcystin concentrations
of 7.8, 15.5, 31.1, 62.2 and 124.4 lg g–1 (Barra
Bonita Reservoir material); 6.6, 12.2, 26.2, 53.0 and
106.0 lg g–1 (Ibitinga Reservoir material) and 936,
1,872, 3,750, 7,500 and 15,000 lg g–1 (cultured NPLJ-4
strain). Crude extract was diluted in reconstituted
water. Three replicate tubes per treatment were used.
Organisms were maintained in an incubator at 25C,
without food. Dissolved oxygen and pH were checked
at the beginning and end of bioassays. Mortality was
reported after 48 h. Experimental data were summa-
rized and analyzed by the Trimmed Spearman-Karber
test to estimate median lethal concentrations in the
toxicity bioassay (Hamilton et al. 1977).
Chronic toxicity test
Neonates of Ceriodaphnia silvestrii (<24-h old) were
also used in the chronic tests with diluted cyanobac-
terial crude extracts. Tests were carried out at 25C in
30 ml nontoxic plastic beakers, with 10 replicates and
one control per sample. Each beaker contained 15 ml
of extract diluted in reconstituted water, seeded with
one daphnid. Sub-lethal concentrations were defined
based on the results obtained from acute toxicity
tests. Concentrations of microcystin (lg microcystin
265
g–1 dry weight of freeze-dried material) in crude
extracts were 3.7, 7.8, 15.5 and 31.1 lg g–1 (Barra
Bonita Reservoir material); 0.8, 1.6, 3.2 and 6.6 lg g–1
(Ibitinga Reservoir material) and 60.0, 120.0, 240.0
and 480.0 lg g–1 (cultured NPLJ-4 strain). In the
control only reconstituted water was used, the same
as in daphnid cultures. During the 7-days or 10-days
exposure, the medium with cyanobacterial crude
extract was renewed three times per week, by using
Pasteur pipettes. Neonates were counted and re-
moved, to prevent the build-up of excretion products
in test beakers. Food was then added to each beaker.
The cladocerans were fed with the green alga Pseud-
okirchneriella subcapitata and a yeast solution, as in
the daphnid stock culture. The test was finished when
at least 60% of daphnids in the control had produced
a third brood. Results were expressed as total
fecundity and mortality (%). Water quality parame-
ters (pH, electrical conductivity and water hardness)
were measured soon after the water renewal, three
times per week (Environment Canada 1990a, b;
ABNT 2003).
Sensitivity tests with reference substance
The sensitivity of the cladoceran (D. similis, C. silvestrii
and C. dubia) from stock cultures to the reference
compounds potassium dichromate (K2Cr2O7) and
sodium chloride (NaCl) were investigated (Environ-
ment Canada 1990a, b; ABNT 2003).
Statistical analysis
Fecundity (chronic test) and mortality (acute test) data
were tested for normality and homogeneity of variance
(v2 and Hartley tests) and then submitted to Dunnett
and Tukey parametric tests if the distribution was
normal, or Kruskall-Wallis non-parametric, if not. The
computer program TOXSTAT 3.3 (Gulley et al. 1991)
was used to perform these analyses (USEPA 1993).
Sensitivity test data were summarized and analyzed
using the Trimmed Spearman-Karber test to estimate
median lethal concentrations in the toxicity bioassay
(Hamilton et al. 1977).
Microcystin analysis
Microcystin content was quantified by immunoassay
with an Envirogard microcystin plate kit (Strategic
Diagnostics, Newark, USA). The results were
expressed as microcystin-LR equivalents (in lg g–1
dry weight freeze-dried cyanobacteria). The analyti-
cal procedure used was that described by Chu et al.
(1990).
123
266
Results
The results of acute toxicity tests in which the
cladocerans Daphnia similis, Ceriodaphnia silvestrii
and Ceriodaphnia dubia, were exposed to crude
extracts of natural cyanobacterial blooms and of the
laboratory strain NPLJ-4 are summarized in Table 1.
The results show that the crude bloom material extract
from Ibitinga Reservoir had higher toxicity to cladoc-
erans than the crude bloom material extract from
Barra Bonita Reservoir. The values of 48-h EC50 for
the material obtained from Barra Bonita Reservoir
(73.1 and 80.2 lg microcystin g–1 of freeze-dried mate-
rial for Ceriodaphnia dubia and C. silvestrii, respec-
tively) were about two times higher than those
obtained for Ibitinga Reservoir (32.6 and 35.8 lg micr-
ocystin g–1 of freeze-dried material for C. dubia and
C. silvestrii, respectively), revealing a higher toxicity in
the material from Ibitinga Reservoir. Also, it was
evident that Daphnia similis had higher sensitivity
(48-h EC50 = 46.0 lg microcystin g–1 freeze-dried
material) to Barra Bonita material than both Cerio-
daphnia species tested.
Regarding the toxicity of extracts from laboratory
cultures of Microcystis aeruginosa (NPLJ-4 strain), all
cladoceran species tested had very similar sensitivity
with 48-h EC50 values varying between 1,380 and
1,470 lg microcystin g–1 of freeze-dried material.
These values were unexpectedly higher than those
obtained with the extracts of natural cyanobacterial
blooms, indicating a higher toxicity of the latter.
Table 2 shows 48-h EC50 values for Daphnia similis,
Ceriodaphnia dubia and Ceriodaphnia silvestrii ex-
posed to reference substances. All 48-h EC50 values
obtained were in the acceptable level for these species.
The measured concentration of microcystins from the
freeze-dried samples of Barra Bonita and Ibitinga
reservoirs bloom material and from the cultured strain
NPLJ-4 are shown in Table 3.
Chronic effects of cyanobacterial extracts on the
fecundity and survival of Ceriodaphnia silvestrii are
summarized in Figs. 1–3 and Table 4, respectively.
Table 1 Estimated 48-h EC50 (lg microcystin g–1 dry weight of freeze-dried cells) values for cladocerans exposed to crude extract of
cyanobacterial bloom samples from Barra Bonita and Ibitinga reservoirs and the cultured cyanobacterial strain NPLJ-4
Source of cyanobacteria 48-h EC50 (lg g–1)
D. similis
Barra Bonita 46.0 (42.0–50.4)a
Ibitinga 34.2 (28.9–40.5)
NPLJ-4 strain 1,380 (1,260–1,560)
a
Values between parentheses correspond to 95% confidence interval
123
D. T. Okumura et al.
Barra Bonita bloom extract significantly affected the
survival of cladocerans at the three highest microcy-
stin concentrations tested (7.8, 15.5 and 31.1 lg g–1),
causing mortalities from 20% to 80% (Table 4). The
lower concentrations of the Barra Bonita extract, the
sample from Ibitinga reservoir, and the NPLJ-4 did
not affect the survival of aquatic organisms through
exposure to cyanobacterial extracts and were not
different from the control (p = 0.05), with a minimum
of 90% surviving.
The fecundity of C. silvestrii exposed to the crude
extract of Barra Bonita Reservoir bloom material was
significantly lower than that of the control (p = 0.05,
Tukey test) for the three highest concentrations tested
(Fig. 1). Some decrease in fecundity was observed in
cladocerans exposed to the extracts from Ibitinga
Reservoir bloom material and to the crude extract of
cultured strain NPLJ-4 (Figs. 2 and 3), however the
effects were less evident.
Discussion
Cyanobacterial blooms are a recurring feature of
hydroelectric reservoirs of the Middle Tietê River
(SP, Brazil), according to earlier investigations (Ko-
márek et al. 2002; Calijuri et al. 2002). In the case of
the Reservoir at Barra Bonita, microcystins have also
been isolated, identified and quantified in phytoplank-
ton samples (Törökne et al. 2000). Nevertheless, such
toxicological data are still rare for this type of water
body. Recently, however, toxic cyanobacterial blooms
have been demonstrated to occur at Barra Bonita,
through bioassays performed on both cladocerans and
mice (Sotero-Santos et al. 2006).
Data obtained in the current acute toxicity test have
shown that C. silvestrii and C. dubia were less sensitive
to the crude extracts of natural blooms than Daphnia
similis reinforcing the necessity of using more than a
single test organism. In general, it appears that
cyanobacteria may have a harmful effect on aquatic
organisms however the effect is highly variable
C. dubia C. silvestrii
73.1 (62.5–85.5) 80.2 (71.0–90.6)
32.6 (25.7–41.3) 35.8 (26.8–48.0)
1,470 (960–2,340) 1,440 (918–2,358)
Evaluation of cyanobacteria toxicity in tropical reservoirs 267

Table 2 Sensitivity test data with reference substances using the to the cyanotoxins varies even within related groups of
three species of cladocerans organisms (DeMott et al. 1991; Sotero-Santos et al.
Organism 48-h EC50 Acceptable Reference 2006), as found here for daphnids.
level substance At similar microcystin concentrations the freeze-
dried bloom material from Ibitinga Reservoir had
D. similis 0.04 (0.03–0.05)a 0.002–0.122 K2Cr2O7 (mg l–1)
C. silvestrii 1.47 (1.38–1.57) 1.2–2.0 NaCl (g l–1) higher acute toxicity than the material from Barra
C. dubia 1.51 (1.38–1.64) 1.63 NaCl (g l–1) Bonita Reservoir. The toxicity of different natural
a
Values between parentheses correspond to 95% confidence
blooms can vary greatly since the blooms themselves
interval vary in their species composition and so in the type and
amounts of toxins. Codd and Bell (1985) pointed out
that cyanotoxin production per unit mass of cyanobac-
Table 3 Microcystin-LR concentrations (in lg g–1 dry weight
teria is extremely variable in natural waters and in
freeze-dried cyanobacteria) in natural cyanobacterial blooms reservoirs.
from Barra Bonita and Ibitinga reservoirs (Middle Tietê River) Within a bloom composed of several species of
and cultured strain NPLJ-4 cyanobacteria, there may be a mixture of toxic and
Sample Concentration lg g–1 atoxic strains and a variety of different toxins. The
microcystin quantification performed in our study most
Barra Bonita 311
Ibitinga 265
probably do not reflect the total cyanotoxicity if other
NPLJ-4 strain 6,000 toxins were present. Additionally, other contaminants
contribute to the total toxicity in the reservoirs. Barra
Bonita and Ibitinga reservoirs both belong to Tietê
River basin, largely contaminated by domestic and
between genera and species, and even between clones industrial sewage discharges.
of individual zooplankton species (Sivonen and Jones The combined effect of different toxicants (cyano-
1999). Earlier studies have reported that the sensitivity toxins, metals and polycyclic aromatic hydrocarbons

Fig. 1 Fecundity of 300

Ceriodaphnia silvestrii (sum
239
of neonates produced by all 250
surviving females) in the 205
Total fecundity

chronic test with 200

cyanobacterial crude extract
from Barra Bonita Reservoir 150 125

100
55
50
8
0
0.0 3.7 7.8 15.5 31.1
-1
Concentration g g

Fig. 2 Fecundity of 300

Ceriodaphnia silvestrii (sum 239
of neonates produced by all 250
206
Total fecundity

surviving females) in the

chronic test with 200 171
cyanobacterial crude extract 133
150 116
from Ibitinga Reservoir
100

50

0
0.0 0.8 1.6 3.2 6.6
-1
Concentration g g

123
268 D. T. Okumura et al.

Fig. 3 Fecundity of 300

Ceriodaphnia silvestrii (sum
239
of neonates produced by all 250
surviving females) in the 207 209

Total fecundity
chronic test with 200 166
cyanobacterial crude extract
of cultured strain NPLJ-4 150 116
100

50

0
0.0 60.0 120.0 240.0 480.0
-1
Concentration g g

Table 4 Mortality data (%) from chronic toxicity tests with non-existent or very rare. The mortality results in the
cyanobacterial crude extracts from Barra Bonita and Ibitinga chronic toxicity tests indicated that only Barra Bonita
reservoirs and cultured strain NPLJ-4
bloom extract affected the survival of cladocerans at the
Sample Concentration lg g–1 Mortality (%) highest concentrations of extract. With the samples
from Ibitinga reservoir and NPLJ-4, the mortality was
Barra Bonita 0.000 0.0
3.7 0.0 within the acceptable limit for control groups (USEPA
7.8 20.0a 1993; ABNT 2003). Some research has been published
15.5 50.0a on the question of the harmful effects of cyanobacteria
31.1 80.0b on cladoceran fecundity but such works have been
Ibitinga 0.000 0.0
0.8 0.0 based on use of cyanobacterial cultures as food-source
1.6 0.0 (Gustafsson and Hansson 2004; Guo and Xie 2006).
3.2 10.0a The fecundity data obtained here in chronic toxicity
6.6 10.0a tests with natural bloom extracts from the Barra Bonita
NPLJ-4 strain 0.000 0.0
60.0 0.0 and Ibitinga reservoirs show clearly that as the extract
120.0 0.0 concentration rises, neonate production reduces. With
240.0 10.0a the cultured strain extract, a tendency for the number of
480.0 10.0a neonates to decrease with increasing extract concen-
a
No significant difference from control according to Kruskal- tration is still apparent but it is less marked than in the
Wallis test (p = 0.05) case of the natural blooms of the Middle Tietê River
b
Statistically significant difference from control according to reservoirs. Extrapolating our results to the real aquatic
Kruskal-Wallis test (p = 0.05)
ecosystem, during an episode of cyanobacterial bloom
or even during the collapse of a bloom when decom-
position is the predominant process, the zooplankton
(PAHs)) is also the most probable explanation for the community would suffer harmful effects that can affect
higher acute toxicity found (on a dry-weight basis) the whole system negatively.
when crude extracts from the reservoirs were com- Lastly, another relevant finding from this work is
pared with crude extracts from the toxic NPLJ-4 strain, the fact that cyanobacterial bloom material with
despite one order of magnitude higher microcystin Microcystis spp. being the dominant species had a
concentrations in the latter. The bioavailability and much lower amount of detectable microcystin by the
toxicity of metals and PAHs in the water and sediment ELISA immunoassay but higher toxicity than toxic
of Tietê River reservoirs were recently assessed NPLJ-4 strain. We can think of two possible expla-
(Fracácio et al. 2003; Silvério et al. 2005; Araújo et al. nations, hypothetically: (a) Although microcystin
2006) as part of an institutional effort to deal with the concentrations in bloom material is lower, the toxicity
fast water quality deterioration of important water was higher because other toxic compounds known to
resources in São Paulo State, Brazil. occur in Tietê reservoirs, such as metals and PAHs,
Data on the chronic effects of cyanobacterial extracts could be accumulated by cyanobacteria and algae; (b)
on the fecundity and mortality of cladocerans are Microcystin quantification on bloom material was

123
Evaluation of cyanobacteria toxicity in tropical reservoirs
underestimated because it suffered interference from
other compounds (possibly metals). This fact was
shown to occur regarding microcystins and metals
present in water treatment coagulants alum and iron,
mainly (Oliveira et al. 2005). Consequently, much
research needs to be done to unravel complex
questions, both ecological and toxicological imposed
by cyanobacterial blooms in eutrophic reservoirs.
Future research would have to encompass not only
the cyanotoxins but also other potentially toxic
substances.
Acknowledgements We are grateful to Fundação de Amparo à
Pesquisa do Estado de São Paulo (Processos 03/00352-2, 01/
13213-5 and 02/08341-7) for financial support and fellowships
during the course of this work.
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