Effects of a saxitoxin-producer strain of Cylindrospermopsis raciborskii (cyanobacteria) on the swimming movements of cladocerans.

Abstract

This study evaluated the effects of a saxitoxin-producer strain (T3) of the cyanobacteria species Cylindrospermopsis raciborskii on the swimming movements of three cladoceran species (Daphnia gessneri, D. pulex, and Moina micrura). Acute toxicity bioassays were designed to access the effects of T3 strain, of a nonsaxitoxin producer strain (NPLP-1) of the same species and of a raw water sample from Funil reservoir (Rio de Janeiro, Brazil), that contained this and other cyanobacteria. In the acute bioassays, animals were exposed to C. raciborskii filaments or Funil water for 24-48 h and then transferred to food suspensions without cyanobacterial filaments for a further 48 h. During the exposure time to T3 strain filaments there was a decrease in the number of swimming individuals, with animals showing progressive immobilization. The same effect was observed with Funil water sample. Animals stayed alive on the bottom of the test tube and recovered swimming movements when transferred to food suspensions without toxic cells. This effect was not observed with the strain NPLP-1. The cladoceran D. pulex showed to be extremely sensitive to T3 strain and to Funil water containing C. raciborskii filaments, showing complete paralysis after 24-h exposure to T3 cell densities of 10(3) and 10(4) cells mL(-1), and after 24-h exposure to only 10% of raw water. However, D. gessneri was not sensitive to both T3 and to Funil water, whereas M. micrura was intermediate in sensitivity. This is the first report on the effects of cyanobacterial saxitoxins on movements of freshwater cladocerans, showing also difference in sensitivity among closely related Daphnia species.

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Effects of a Saxitoxin-Producer Strain of
Cylindrospermopsis raciborskii (Cyanobacteria)
on the Swimming Movements of Cladocerans
Aloysio da S. Ferra ˜o Filho,1Simone M. da Costa,2Manuel Gustavo Leita ˜o Ribeiro,2
Sandra M. F. O. Azevedo2
1Departamento de Biologia, Instituto Oswaldo Cruz, FIOCRUZ, Laborato ´rio de Avaliac ¸a ˜o e
Promoc ¸a ˜o da Sau ´de Ambiental, Av. Brasil 4365, Manguinhos, Rio de Janeiro,
RJ 21045-900 Brazil
2Instituto de Biofı ´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do
Funda ˜o, Cidade Universita ´ria, Rio de Janeiro, RJ 21949-900 Brazil
Received 6 July 2007; accepted 8 July 2007
ABSTRACT: This study evaluated the effects of a saxitoxin-producer strain (T3) of the cyanobacteria spe-
cies Cylindrospermopsis raciborskii on the swimming movements of three cladoceran species (Daphnia
gessneri, D. pulex, and Moina micrura). Acute toxicity bioassays were designed to access the effects of
T3 strain, of a nonsaxitoxin producer strain (NPLP-1) of the same species and of a raw water sample from
Funil reservoir (Rio de Janeiro, Brazil), that contained this and other cyanobacteria. In the acute bioassays,
animals were exposed to C. raciborskii filaments or Funil water for 24–48 h and then transferred to food
suspensions without cyanobacterial filaments for a further 48 h. During the exposure time to T3 strain fila-
ments there was a decrease in the number of swimming individuals, with animals showing progressive
immobilization. The same effect was observed with Funil water sample. Animals stayed alive on the bot-
tom of the test tube and recovered swimming movements when transferred to food suspensions without
toxic cells. This effect was not observed with the strain NPLP-1. The cladoceran D. pulex showed to be
extremely sensitive to T3 strain and to Funil water containing C. raciborskii filaments, showing complete
paralysis after 24-h exposure to T3 cell densities of 103and 104cells mL21, and after 24-h exposure to
only 10% of raw water. However, D. gessneri was not sensitive to both T3 and to Funil water, whereas
M. micrura was intermediate in sensitivity. This is the first report on the effects of cyanobacterial saxitoxins
on movements of freshwater cladocerans, showing also difference in sensitivity among closely related
Daphnia species.#2008 Wiley Periodicals, Inc. Environ Toxicol 23: 161–168, 2008.
Keywords: zooplankton; cladocerans; cyanobacteria; saxitoxins; toxicity bioassays
INTRODUCTION
Saxitoxins (STXs) are potent alkaloid neurotoxins that act
by blocking sodium ion channel in neurons, impairing
impulse propagation, and leading to a lack of stimulus in
muscle cells. These toxins were first isolated in marine
shellfish contaminated with marine dinoflagellate species
(Anderson, 1994), and have been involved in the death of
humans (Bricelj et al., 1990). The intoxication syndrome in
humans is known as paralytic shellfish poisoning (PSP) and
the toxins involved are known as paralytic shellfish toxins
(PST) produced by many genera of red tide forming dino-
flagellates such as Alexandrium sp., Pyrodinium sp., and
Gymnodinium sp. (Daranas et al., 2001), and by some fresh-
water cyanobacteria such as Anabaena circinalis (Negri
Correspondence to: Aloysio da S. Ferra ˜o Filho; e-mail: aloysio@ioc.
fiocruz.br
Published online 23 January 2008 in Wiley InterScience (www.
interscience.wiley.com). DOI 10.1002/tox.20320
?
C 2008 Wiley Periodicals, Inc.
161

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and Jones, 1995), Aphanizomenon flos-aquae (Mahmood
and Carmichael, 1986), and Cylindrospermopsis raciborskii
(Lagos et al., 1999).
STXs are known to accumulate in several marine organ-
isms such as mussels and in zooplankton, which act as vec-
tors of these toxins to higher trophic levels such as fish and
whales (White, 1981; Boyer et al., 1985; Turrif et al., 1995;
Teegarden and Cembella, 1996; Turner et al., 2000; Chen
and Chou, 2001; Durbin et al., 2002; Hamazaki et al.,
2003).
Despite the great number of saxitoxin studies in marine
ecosystems, there are few studies that have been done with
freshwater organisms. The study of Negri and Jones (1995)
demonstrated the accumulation of STXs produced by A.
circinalis in freshwater mussels Alathrya condola, and
Nogueira et al. (2004) have shown the accumulation of
STXs from A. issatschenkoi in D. magna. Since no adverse
effects have been reported on these filter-feeders, the accu-
mulation of STXs can be attributed to resistance of these
organisms to such toxins.
However, adverse effects of STXs in aquatic organisms
includes a variety of responses including reduced ingestion
rates caused by ‘‘physiological incapacitation’’ (Ives, 1985,
1987), avoidance of toxic cells by chemosensory means in
copepods (Huntley et al., 1986; Sykes and Huntley, 1987;
Teegarden and Cembella, 1996), reduced somatic growth,
size at maturity, and fecundity (Dutz, 1998; Colin and
Dam, 2004). Haney et al. (1995) have reported a reduction
in the thoracic appendages beating rate and an increase in
rejection rate of particles by the postabdomen of D. cari-
nata when exposed to a filtrate of A. flos-aquae and to puri-
fied saxitoxin. Nevertheless, to date no other adverse effects
of STXs have been reported in freshwater cladocerans.
In this study, we present the first report on the effects of
a saxitoxin-producer strain of C. raciborskii on the swim-
ming behavior of freshwater cladoceran species. We also
perform bioassays with a nontoxic strain of C. raciborskii
and with a water sample from a reservoir with occurrence
of this cyanobacterium.
MATERIALS AND METHODS
Origin and Culture of Organisms
Two Brazilian strains of C. raciborskii Woloszynska (T3
and NPLP-1) were cultured in ASM-1 medium (Gorham
et al., 1964), pH 8.0, 23 6 28C, and 40–50 lE m22s21and
12:12 h light–dark cycle. The strain T3, isolated from Bill-
ings Reservoir, City of Sa ˜o Paulo (SP, Brazil), was firstly
reported to produce STXs by Lagos et al. (1999), and its
toxins include neosaxitoxin (NEO), decarbamoyl neosaxi-
toxin (dcNEO), saxitoxin (STX), and decarbamoyl saxi-
toxin (dcSTX) (G. K. Eaglesham, Queensland Health Sci-
entific Services, Coopers Plains, Austra ´lia; pers. comm.).
The strain NPLP-1 was isolated from Paranoa ´ Lake, Brası ´-
lia D. C. (GO, Brazil), and was nontoxic in mouse bioassay.
Both C. raciborskii strains were grown in the form of
straight trichomes of[100 lm in length.
Two cladocerans species, D. gessneri Herbt and Moina
micrura Kurs, were isolated from an oligotrophic reservoir
(Lajes) in Rio de Janeiro State, Brazil, with no occurrence
of toxic blooms. A temperate clone of D. pulex Leydig was
obtained from Carolina Biological Supply, NC. Cladocer-
ans were cultured in mineral commercial water, with a
hardness of 77.2 mg CaCO3L21, pH of 7.4, at 23 6 28C
and at dim light. For D. gessneri and M. micrura, 20% of
filtered (glass fiber membrane, Sartorius AG 37070) water
from Funil reservoir was added to mineral water. This fil-
tered reservoir water was free from cyanobacteria and was
supposed to supply some organic matter (i.e., vitamins),
necessary as a growth factor for these species. Cladocerans
were fed Ankistrodesmus falcatus Braun at a density of 1.6
3 104cells ml21(?0.5 mg C L21).
Acute Toxicity Bioassays
These assays were conducted under the same conditions as
the cladoceran cultures, using 30-mL flat-bottom test tubes.
The experimental design consisted of two phases: (1) Expo-
sure phase: when animals were exposed to different fila-
ment densities of each strain of C. raciborskii plus nutri-
tious alga (A. falcatus) and (2) Recovery phase: when ani-
mals were transferred to new suspensions containing only
the nutritious alga. The exposure phase usually lasted 48 h,
except for D. pulex in the experiment with raw water from
Funil reservoir which lasted 24 h. The recovery phase
always lasted 48 h. For strain NPLP-1, exposure phase
lasted 48 h and there was no recovery phase. There were 10
neonates (\24 h) in each test tube and four replicates per
treatment. Every day, during both phases, animals were
transferred to new algal suspensions and the number of
immobile, dead, or swimming individuals were counted.
Controls consisted of animals exposed only to the nutritious
alga.
Two experiments were performed with strain T3.
Experiment 1 was performed with D. pulex only, and
Experiment 2 was performed with both D. gessneri and M.
micrura. Although exposure concentrations were based on
cell densities (10–10 000 cells mL21), toxin concentrations
varied between experiments since cyanobacterium culture
was used in different phases (12 days apart). The experi-
ment with strain NPLP-1 was performed at the same time
as Experiment 2 with strain T3.
Another toxicity bioassay was carried out with a raw
water sample from Funil Reservoir (40 km2size and 70 m
maximum depth), located near Resende City (228 300S e
448 450W, RJ, Brazil). This reservoir was built for water
supply and electricity generation purposes, but it is also
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FERRA˜O FILHO ET AL.
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used for recreation, fishing and for the regulation of floods
in the surrounding areas. Its drainage basin is highly
impacted because of erosion processes and discharge of
domestic and industrial wastes. The large input of nutrients
has turned this reservoir hypereutrophic, with recurrent
blooms of cyanobacteria. The water sample was taken close
to the dam in August 2003 (spring). This assay had the
same experimental design as the previous assays except
that treatments with raw water consisted of dilutions of this
sample in mineral water.
Toxin Analysis
For STXs, 1 mL from C. raciborskii cultures or 10 mg of
the lyophilized raw water (2 L) sample were extracted with
1 mL of HCl 0.1 N (1:1 v/v) for 1 h. Samples were then
centrifuged at 10 800 3 g for 10 min and the supernatant
stored at 2188C until analyzed. Before the analysis, sam-
ples were oxidized with periodate solution according to
Lawrence and Me ´nard (1991), and then analyzed by high
performance liquid chromatography (HPLC) using a Shi-
madzu/CLASS VP apparatus with fluorescence detector
(RF-10A XL), adjusted to 330 nm of excitation and 400 nm
of emission, using a 20-lL loop, reverse column Merck
LC-18 (Lichrocart1150 mm 3 4.6 mm Ø, 5 lm). The mo-
bile phase consisted of acetonitrile:ammonium formate
0.1 M (v/v), adjusted to pH 6.0, with a gradient varying
from 0 to 1% in the first 15 min and from 1 to 4% in the
last 15 min at a flow rate of 1 mL min21. Standard solutions
of saxitoxin (STX), neosaxitoxin (NeoSTX), and gonyau-
toxins (GTX 1 and 4 e GTX 2 and 3) were purchased from
the National Research Council (NRC), Institute for Marine
Biosciences (Canada), and solutions of C toxins (C1 and
C2) and B toxin (B1) were kindly provided by Dr. Y. Osh-
ima, Tohoku University, Japan. Those standard of toxins
were used to identification and quantification of toxins pres-
ent in the sample, by direct comparison to standards carried
through the oxidation periodate procedure and the sum of
all peak areas of toxins found was expressed as concentra-
tion equivalents of STX.
Microcystins were analyzed from 1 L of raw water sam-
ple of the reservoir, filtered through borosilicate filters (Sar-
torius), extracted three times with butanol:methanol:water
(5:20:75 v/v). The extract was evaporated to 1/3 of its
initial volume and then passed through a C18 cartridge
(500 mg/6 mL Bond-Elut, Varian). This cartridge was
washed and eluted with distilled water, with methanol 20%
and then with methanol 100%. This last fraction was dried
and stored at 2208C for subsequent analyses. The micro-
cystin analysis was performed by HPLC (Shimadzu/
CLASS VP with UV detector PDA M10A VP), using a
Lichrospher 100 RP-18 column (150 3 4.6 3 5 lm3
Merck), and a 100-lL loop. Chromatography was con-
ducted under isocratic conditions as follows: mobile phase
acetonitrile and 20 mM of ammonium acetate in pH 5.0
(28:72 v/v) for 10 min, at a flow rate of 1 mL min21, UV
detection at 238 nm, and the spectrum of absorbance of each
peak was analyzed between 190 and 300 nm. A standard of
microcystin-LR (Sigma) was used as an external standard for
microcystin quantification. The concentration of microcystin
was estimated by the sum of all peak areas presenting an
absorption spectrum 95% of similarity index with the standard
microcystin-LR and was expressed as microcystin-LR equiva-
lents, as described in Chorus and Bartram (1999).
Because of the presence of Anabaena sp. in the reservoir
water, lyophilized water sample (2 L) was used to detect
anatoxin-a(s), a potent acetylcholinesterase (AChE) inhibi-
tor (Henriksen et al., 1997). In addition, the anatoxin-a(s)
producer strain ITEP-024 (A. spiroides; Molica et al., 2005)
was used as a positive control. Both lyophilized water sam-
ple and the ITEP-024 strain were extracted with 0.5–
1.0 mL ethanol:acetic acid 1 N (20:80 v/v) for 3–4 h, and
centrifuged at 12 400 3 g for 10 min, being the supernatant
utilized in a AChE activity inhibition assay according to
Nunes-Tavares et al. (2002) [adapted from Ellman et al.
(1961)]. Membrane fractions rich in AChE were obtained
from the electric tissue of Electrophorus electricus (Somlo ´
et al., 1977). The method is based on the measurement of
the rate of production of thiocholine as acetylthiocholine
(ATCh) that is hydrolyzed at 258C in the presence of 5-50-
dithiobis-2-nitrobenzoic acid (DTNB), producing its yellow
anion, which can be detected at 412 nm (Hitachi spectro-
photometer model U-3300). Control consisted of Tris-HCl
50 mM pH 8.0, MgCl25.0 mM, ATCh 0.4 mM, DTNB
0.125 mM, and 10.0 mg of total protein. Specific activity
was expressed in micromole of ATCh hydrolyzed per mi-
nute per milligram protein. Protein concentration was deter-
mined according to the method of Lowry et al. (1951).
Data Analysis
For testing the effect of the C. raciborskii strains and reser-
voir water on cladocerans’ swimming activity, during the
exposure and recovery phases, a two-way ANOVA was
performed, determining mobility as the dependent variable
and species and concentration as independent variables. We
also used one-way ANOVA for testing differences between
controls and treatments with each strain and reservoir water
in the mortality rates at the end of each experiment. The
statistical software utilized was Systat V.9.0, 1998 (SPSS,
Chicago, IL).
RESULTS
STXs concentration varied between experiments, being
about 10 times higher in Experiment 2 (Table I). No STXs
were found in NPLP-1 strain.
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EFFECTS OF SAXITOXIN ON THE SWIMMING MOVEMENTS OF CLADOCERANS
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In bioassays with the strain T3, we observed a rapid
immobilization of animals in the presence of toxic fila-
ments (Fig. 1). The symptom was characteristic of neuro-
toxicity, with animals showing immobilization after a few
hours (beginning after ?30 min) but staying alive on the
bottom of the test-tube during the exposure phase. When
transferred to fresh nutritious food suspensions without
toxic filaments, some animals recovered movements and
started swimming again after a few hours (1–2 h; data not
shown), with most recovering after 48 h (Fig. 1). Species
responded differently to the toxic filaments, with D. pulex
being more sensitive than the other two species. Although
immobilization in D. pulex started at 10 cells mL21
(0.000846 ng eq STX L21), this effect was shown by M.
micrura only at 1000 cells mL21(0.937 ng eq STX L21).
Nevertheless, D. gessneri mobility was not affected at all
by the strain T3.
The ANOVA performed on the data of the exposure
phase showed a significant effect of species and concentra-
tion on the mobility of cladocerans, and also a significant
interaction between these factors (Table IIA).
Mortality rates of animals exposed to strain T3 ranged
from 0 (control) to 20% for D. pulex, 10% for D. gessneri,
and 5% for M. micrura during the experiment, but no sig-
nificant differences were found between controls and treat-
ments (Table IIB).
The nonsaxitoxin producer strain NPLP-1 did not cause
any effect on the swimming movements of cladocerans.
However, mortality rates with this strain ranged from 5 to
15% for D. pulex, 0–20% for D. gessneri, and 0–5% for M.
micrura, but no significant differences between treatments
were found for any species (ANOVA; P[0.05).
The water sample from the Funil reservoir included
C. raciborskii (5350 cells mL21), present as straight tri-
chomes, as well as two other potentially toxic cyanobacte-
ria, Anabaena sp. (2317 cells mL21) and Microcystis sp.
(14770 cells mL21), present as spiral trichomes ([100
lm), and colonies (variable size), respectively.
Reservoir water contained 24.6 ng eq STX L21(Table
III) and the concentration of microcystins in reservoir water
was 1.34 lg eq MC-LR L21. Also, the reservoir water sam-
ple caused 28% inhibition of the AChE activity relative to
control. The strain ITEP-024, however, caused a 100% in-
hibition in the AChE activity. Specific activities for control,
reservoir water, and ITEP-024 strain were 5700, 4150, and
0 lmol ATCh hydrolyzed per minute per milligram protein,
respectively.
There was also a pronounced effect of the reservoir
water sample on the swimming movements of cladocerans
(Fig. 2). The effect was even faster in D. pulex, with virtu-
ally all animals being immobilized after 24-h exposure to
only 10% (2.5 ng eq STX L21) of reservoir water. For this
reason we decided to transfer the individuals of D. pulex
to cyanobacteria-free medium after 24-h exposure. The
TABLE I. Cell densities and corresponding saxitoxins
concentrations (ng eq STX L21) in experiments 1 and 2
with the strain T3
Cell Density
(cells mL21)
Experiment 1
(August 28, 2003)
D. pulex
Experiment 2
(September 9, 2003)
D. gessneri 1 M. micrura
10
100
1000
10 000
0.000846
0.00846
0.0846
0.846
–
0.0937
0.937
9.37
Fig. 1. Percent mobility of D. pulex and M. micrura during
the exposure phase (0–48 h) to T3 strain and during the re-
covery phase (48–96 h). The arrow represents the beginning
of the recovery phase. Controls consisted of animals
exposed to only mineral water with food and treatments con-
sisted of animals exposed to different concentrations of T3
strain (cells mL21) mixed with a fixed amount of food (0.5 mg
C L21).
164
FERRA˜O FILHO ET AL.
Environmental Toxicology DOI 10.1002/tox

Page 5

same effect was experienced by M. micrura only with
50% (12.3 ng eq STX L21) after 48-h exposure and with
100% (24.6 ng eq STX L21) of reservoir water after 24 h.
As in the assay with T3 strain, the animals recovered their
movements after being transferred to fresh nutritious food
suspensions. Species rank in sensitivity was the same as in
the previous bioassay, with D. pulex being the most sensi-
tive and D. gessneri not being affected by the reservoir
water.
The results of the two-way ANOVA for the exposure
to reservoir water showed a significant effect of species
and concentration on mobility of cladocerans, and a sig-
nificant interaction between these factors (Table IVA).
During the exposure to reservoir water no mortality was
observed for D. gessneri, whereas mortality rates ranged
from 20 to 50% for D. pulex and from 25 to 35% for M.
micrura (not significantly different from controls for both
species; Table IVB). There was also some mortality in the
controls for D. pulex and M. micrura (\10% for both
species).
DISCUSSION
Our results show strong evidence that STXs can cause a
decrease in the swimming movements of some species of
freshwater cladocerans. The effect is suggestive of a neuro-
toxicosis, with rapid immobilization and rapid recovery af-
ter the animals were removed from the water medium con-
taining toxic filaments. This effect, however, is dependent
on the test-organism (cladoceran species), on the density of
toxic filaments in the water and also on the exposure time.
The fact that cladocerans did not show any alteration in the
swimming behavior in the presence of the nonsaxitoxin-
TABLE III. Percentage of raw water from Funil reservoir,
cell densities of C. raciborskii and saxitoxins
concentrations in experiment 3
Raw
Water (%)
Cell Density
(Cells mL21)
Saxitoxins
(ng eq STX L21)
10
25
50
100
535
1337
2675
5350
2.5
6.2
12.3
24.6
Water sample collected in Aug. 8, 2003.
Fig. 2. Percent mobility during the exposure phase to raw
water sample from Funil reservoir: 0–24 h for D. pulex and
0–48 h for M. micrura; and during the recovery phase: 24–
72 h for D. pulex and 48–96 h for M. micrura. The arrow
represents the beginning of the recovery phase. Controls
consisted of animals exposed to only mineral water with
food and treatments consisted of animals exposed to
different dilutions of raw water in mineral water with food.
TABLE II. (A) Results of two-way ANOVA for the acute
toxicity experiment with the T3 strain. (B) Results of
one-way ANOVA for testing differences between
controls and treatments with strains T3
Factor df MSF
P
(A) Effects on mobility at the end of exposure period
Species
Concentration
Species 3 concentration
Error
(B) Effects on mortality rates at the end of the experiment
D. gessneri
Concentration
Error
D. pulex
Concentration
Error
M. micrura
Concentration
Error
2
4
8
8346.7
13523.3
1638.3
224.4
37.19
60.25
7.30
\0.0001
\0.0001
\0.0001
45
391.7
58.3
1.570.248
12
4200.0
186.7
1.070.405
15
3 66.7
83.3
0.800.517
12
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EFFECTS OF SAXITOXIN ON THE SWIMMING MOVEMENTS OF CLADOCERANS
Environmental Toxicology DOI 10.1002/tox

Page 6

producer strain NPLP-1 also corroborates the hypothesis
that STXs are the cause of immobilization.
The possibility that microcystins present in the raw
water sample from Funil reservoir could have caused the
immobilization is less likely since other studies have
reported effects of these toxins other than in swimming
behavior of cladocerans (DeMott et al., 1991; Ferra ˜o-Filho
et al., 2000; Rohrlack et al., 2005). The effect observed is
more compatible with the mechanism of action of neurotox-
ins than with that of hepatotoxins. On the other hand, ana-
toxin-a(s) is a potent AChE inhibitor having also effects on
neural cells (Mahmood and Carmichael, 1986; Henriksen
et al., 1997). Therefore, the slight inhibition of AChE by
the reservoir water sample in the inhibition assay indicates
that anatoxin-a(s) could be implicated in the immobiliza-
tion of animals. However, due to its big size and spiral
shape, Anabaena filaments were probably inedible to most
cladocerans tested (Porter and Orcut, 1980; DeBernardi and
Giussani, 1990), and thus it is unlikely that anatoxin-a(s)
alone have caused the immobilization of cladocerans in res-
ervoir water. However, we can not exclude the possibility
that some smaller colonies of Microcystis and fragments of
Anabaena filaments have been eaten by cladocerans. Thus,
the strongest immobilization of both cladocerans and the
higher mortality observed during the bioassay with reser-
voir water may be attributed to the higher concentration of
STXs in reservoir water (about 2.6–29 times higher than
the maximum concentrations of T3 strain) and to a syner-
gistic effect of other cyanobacterial toxins.
Most of the studies done with STXs are in marine sys-
tems (Landsberg, 2002). Some studies have shown the
effect of dinoflagellates’ STXs on the feeding behavior and
reproduction of marine copepods (Ives, 1985; Huntley
et al., 1986; Sykes and Huntley, 1987; Teegarden and Cem-
bella, 1996; Dutz, 1998). However, no other study has dem-
onstrated that STXs can exert inhibitory effects on the
swimming movements of freshwater cladocerans.
The only study dealing with effects of STXs in cladocer-
ans was that of Haney et al. (1995), who reported a reduc-
tion in the thoracic appendage beating rate and an increase
in rejection rate of particles by the postabdomen of D. cari-
nata when exposed to a filtrate of a saxitoxin-producing
strain of A. flos-aquae and to purified STX. As in our study,
they showed that animals recovered thoracic appendages
beating and postabdominal rejection rates after the medium
containing STXs was changed to control medium. Simi-
larly, they found also the same effect using filtered water
from a pond with a bloom of A. flos-aquae. Differently,
they did not use intact filaments during their bioassays.
Also, they showed a significant effect of incubation time of
A. flos-aquae medium in these parameters, suggesting that
products released by this cyanobacterium were responsible
by the effects on D. carinata. The fact that purified STX
caused inhibition of thoracic appendage beating, which was
rapid and reversible and comparable in strength to the inhi-
bition caused by the pond water containing A. flos-aque,
lead the authors to conclude that these chemicals play an
important role in the feeding behavior of freshwater herbi-
vores such as Daphnia. Because of the mixed responses (in-
hibition of thoracic appendages and stimulus of postabdo-
men) of Daphnia they also concluded that these chemicals
act as a chemosensory cue causing behavioral change in
feeding activities rather than as a direct inhibition of motor
activity.
Our results, however, strongly suggest that chemicals
produced by C. raciborskii do act as a direct inhibitor of
motor activity, as shown by the inhibition effect on mobil-
ity of cladocerans. This effect is probably caused by STXs
produced by this strain, since the other nonsaxitoxin-pro-
ducing strain of C. raciborskii did not cause any effect on
mobility. The mechanism involved in such effect is in
agreement with that described in squid and crayfish giant
axons when exposed to STX, leading to blocking of sodium
channels and interference with nerve impulse conduction,
with such effect being completely reversible (Adelman
et al., 1982). Thus, in cladocerans, its is likely that STXs
are acting on the nerves cells that control the muscles of the
second antennae, leading to inhibition of nerve impulse
conduction and paralysis of this appendage.
Therefore, contrary to the conclusions of Haney et al.
(1995), our results suggest a toxicosis response rather than
a behavioral response, since the response in cladocerans
mobility was proportional to the density of toxic cells dur-
ing both the exposure and recovery phase. Although we did
not measure the feeding activity of our animals, we
observed that they kept thoracic beating during the experi-
ments. However, we cannot exclude the possibility of a be-
havioral inhibition of the feeding activity. This inhibition
would be, however, beneficial for the animals since they
would decrease the ingestion of toxic filaments. This
TABLE IV. (A) Results of two-way ANOVA from the acute
toxicity experiment with the Funil reservoir water. (B)
Results of one-way ANOVA for testing differences
between controls and treatments with reservoir water
Factor dfMSF
P
(A) Effects on mobility at the end of exposure period
Species
Concentration
Species 3 concentration
Error
(B) Effects on mortality rates at the end of the experiment
D. pulex
Concentration4
Error15
M. micrura
Concentration3
Error 12
2
3
6
28258.3
11066.7
2991.7
27.8
1017.3
398.4
107.7
\0.0001
\0.0001
\0.0001
36
870.0
360.0
2.420.094
891.7
975.0
0.9150.463
166
FERRA˜O FILHO ET AL.
Environmental Toxicology DOI 10.1002/tox

Page 7

behavior would have an adaptative value for cladocerans
living in aquatic environments with occurrence of toxic
blooms, which would favor the capacity of rapid sensing
and response to these chemical cues (Haney et al., 1995).
Resistance of freshwater zooplankton to toxic blooms of
cyanobacteria have been observed in some studies (DeMott
and Moxter, 1991; Ferra ˜o-Filho and Azevedo, 2003) and
local adaptation (resistance) of historically exposed cope-
pod populations to STXs -producing dinoaflagellates have
been reported (Colin and Dam, 2004). Copepods have gen-
erally been reported as selective filter-feeders while clado-
cerans are considered nonselective (DeMott, 1990) and are
indeed able to discriminate between toxic and nontoxic
cyanobacteria (DeMott and Moxter, 1991). However, data
on food ingestion of Notodiaptomus inheringi exposed to a
natural phytoplankton from Funil reservoir showed that this
copepod preyed efficiently on small colonies of Microcystis
and also on C. raciborskii (Panosso et al., 2003). Two of
the cladocerans tested (D. gessneri and M. micrura) are
commonly found in Brazilian reservoirs, with D. gessneri
being the most frequently found in Funil reservoir. The re-
sistance shown by D. gessneri to C. raciborskii may explain
the coexistence between these species in Funil reservoir.
Although we cannot measure if the resistance of D. gess-
neri is behaviorally or physiologically mediated, this sug-
gests that different susceptibilities of cladocerans to STXs
may be of adaptative value, shaping the structure of plank-
ton communities in freshwater lakes.
The rapid and sensitive response of cladocerans, espe-
cially D. pulex, to C. raciborkii chemicals in water can be
of great value in biomonitoring of water bodies, especially
public water supply reservoirs dominated by these cyano-
bacteria. The chemical analyses of cyanotoxins are expen-
sive, requiring sophisticated equipment and highly qualified
technicians. Thus, specific standardized bioassays with
these organisms for the detection of STXs in water samples
should be developed to ensure, in a first instance, if water is
free of these toxins, before a chemical analysis can be
made.
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