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Marine Pollution Bulletin 50 (2005) 956–964
Cyanobacterial blooms in estuarine ecosystems: Characteristicsand effects on Laeonereis acuta (Polychaeta, Nereididae)
Carlos E. da Rosa a,b, Marcio S. de Souza c, Joao S. Yunes d, Luis A.O. Proenca e,Luiz E. M. Nery a,b, Jose M. Monserrat a,b,*
a Departamento de Ciencias Fisiologicas, Fundacao Universidade Federal do Rio Grande (FURG), R. Eng Alfredo Huch 475,
96201-+55 53900, Rio Grande, Brazilb Programa de Pos-graduacao em Ciencias Fisiologicas, Fisiologia Animal Comparada (PGCF-FAC), FURG, Brazil
c Laboratorio de Fitoplancton e Microorganismos Marinhos, Departamento de Oceanografia (FURG), Brazild Unidade de Pesquisa em Cianobacterias, Depto. de Quımica, FURG, Brazil
e Universidade do Vale de Itajaı (UNIVALI), Centro de Ciencias Tecnologicas da Terra e do Mar (CTTMar), Santa Catarina, SC, Brazil
Abstract
In January of 2003, a cyanobacterial bloom in the Patos� Lagoon (Southern Brazil) (32�05 0S–52�12 0W) was observed. Water sam-
ples were taken to identify the composition and abundance of the bloom, as well as the occurrence of toxins. The effects of this
occurrence on the estuarine worm Laeonereis acuta (Polychaeta, Nereididae) was also evaluated. Predominance of cyanobacteria,
particularly Anabaena trichomes (�2.5.106 individuals per liter) was observed, and low concentrations of microcystins and antich-
olinesterasic toxins were detected. Augmented levels of lipid hydroperoxides (LPO) and glutathione-S-transferase activity, and
lowering of total protein content were also observed in organisms collected during the bloom event. Although non-toxic, the cyano-
bacterial bloom could augment the cycle of hyper-oxygenation and hypoxia in the water. During hyperoxia, L. acuta, an oxycon-
former, should consume more oxygen, thus augmenting the rate of reactive oxygen species generation. A repeated cycle of hyper-
oxygenation and hypoxia would finally induce oxidative stress, as evidenced by the high levels of LPO and glutathione-S-transferase
activity.
� 2005 Published by Elsevier Ltd.
Keywords: Cyanotoxins; Patos� Lagoon; Laeonereis acuta; Oxidative stress; Antioxidant enzymes; Cholinesterase
1. Introduction
The presence of cyanobacterial blooms in natural and
artificial water bodies has been frequently reported
around the world (Christoffersen, 1996). These photoau-totrophic microorganisms are well represented in a wide
range of habitats, where they reach an overwhelming
dominance (Vance, 1965; Reynolds, 1997). The bloom-
forming cyanobacterial events can have potential health
hazards to humans and aquatic fauna. Harmful effects
0025-326X/$ - see front matter � 2005 Published by Elsevier Ltd.
doi:10.1016/j.marpolbul.2005.04.004
* Corresponding author. Tel.: +55 53 2338695; fax: +55 53 2338680.
E-mail address: [email protected] (J.M. Monserrat).
extend up to the ecosystem level by changing species
interaction and community structure (Lehtonen et al.,
2003). Some cyanobacteria produce toxins as secondary
metabolites. Molecular structures of about 60 toxin
variants produced by brackish and freshwater cyano-bacteria are known to be hepatotoxic (microcystin and
nodularin), neurotoxic (anatoxin-a, anatoxin-a(s), saxi-
toxin), or to cause allergenic reactions or irritation
(LPS) (Codd, 1995). The toxicity of hepatotoxins is ex-
erted by effective inhibition of serine/threonine phospha-
tases (PP1/PP2A), resulting in a hyperphosphorylation
of many kinds of hepatic functional proteins (Lehtonen
et al., 2003). This hyperphosphorylation causes cyto-
C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964 957
skeletal deformation in hepatocytes, collapse of liver
architecture, profuse hemorrhage and necrosis (Lyu
et al., 2002). The neurotoxins are toxic through several
mechanisms. The anatoxin-a is an analogue of the neu-
rotransmitter acetylcholine, which cannot be degradated
by acetylcholinesterase (Charmichael, 1994). The ana-toxin-a(s) is a natural organophosphate which exerts
their toxicity by the inhibition of acetylcholinesterase
(Charmichael, 1994; Monserrat et al., 2001).
Although the molecular targets of several cyanotox-
ins are well established, other alternative mechanisms
of toxicity can be considered, such as oxidative stress
(Ding et al., 1998a,b). Pflugmacher et al. (1998) have
demonstrated the existence of a microcystin–glutathioneconjugate formed enzimatically by glutathione-S-trans-
ferase activity. This process can induce a depletion of
the cellular glutathione (GSH) pool, favoring oxidative
stress, since GSH is the main non-enzymatic antioxidant
defense and constitutes the first line of defense against
reactive oxygen species (Sies, 1999). These mechanisms
of conjugation have been described in various aquatic
organisms, ranging from plants to fish (Pflugmacheret al., 1998; Beattie et al., 2003). Some works have dem-
onstrated that oxidative stress and/or antioxidant
responses are induced by cyanobacterial toxins (Ding
et al., 1998b; Guzman and Solter, 1999; Vinagre et al.,
2003). However, cyanobacterial blooms can exert other
toxicological effects, related to variations in oxygen con-
centration. Seki et al. (1979) have observed that, during
a bloom event, the profiles of dissolved oxygen in thewater column augmented its amplitude, ranging from
0 (at night) to 190% (at day) of normal saturation.
Under such conditions, the biota in these regions must
cope with cycles of anoxia–hyperoxia resembling the
well-known physiological process of ischemia-reperfu-
sion (Halliwell and Gutteridge, 1999).
Several animal species present well-developed mecha-
nisms to avoid the deleterious effects caused by varia-tions on oxygen availability. These known mechanisms
include metabolic rate depression (Hochachka and
Somero, 1984; Storey, 1996a; Hochachka and Lutz,
2001), and maintenance and/or increase of antioxidant
defense systems previous to the re-oxygenation period
(Hermes-Lima et al., 1998; Lushchak et al., 2001).
In the Patos� Lagoon, the largest lagoonal system in
South America, the occurrence of cyanobacterialblooms, dominated by the genus Microcystis, have been
registered irregularly during the last years (Odebrecht
et al., 1987; Yunes et al., 1998). A typical inhabitant
of this environment is the estuarine benthonic worm
Laeonereis acuta (Polychaeta, Nereididae), a selective
deposit feeder, with high abundance and biomass,
occurring in the Atlantic coast of South America from
Recife (Northeastern Brazil) to Penınsula de Valdez(Southern Argentina) (Omena and Amaral, 2001). A
previous study showed that this animal is susceptible
to oxidative stress when exposed to metals (Geracitano
et al., 2002), showing different antioxidant profiles in
populations sampled at unpolluted and polluted sites
(Geracitano et al., 2004).
The objective of the present study was to characterize
the cyanobacterial bloom occurred in January 2003 inthe Patos� Lagoon (Southern Brazil). The putative
harmful effect of the bloom on Laeonereis acuta was ac-
cessed considering the pro-antioxidant balance. Also, the
activity of the enzyme cholinesterase (ChE) was mea-
sured as a biomarker of the presence of anatoxin-a(s)
in the water.
2. Material and methods
2.1. Samples collection
Water and animals samples were collected after 7, 10
and 16 days of the beginning of the bloom. Previously to
this and after the end of bloom (�35 and 77 days,
respectively), additional animal samples were collected.Water samples from surface layer were divided in two
aliquots: one fixed in formaldehyde (10%) and other
stored to �20 �C for posterior chemical analysis. The
samples were collected at one station located at ‘‘Saco
do Justino’’ in the Patos� Lagoon (32�C05 0S–
52�C12 0W) (Fig. 1).
2.2. Algal identification and enumeration
The phytoplanktonic organisms were observed under
a transmitted and inverted light microscope (ZEISS
Axiovert 135). They were mainly identified and counted
at genus level using modified sedimentation chambers
with 10 ml aliquots (Utermohl, 1958; Sournia, 1978) at
400· to study the community composition and abun-
dance. For identification, specific taxonomic informa-tions were used (Bourrelly, 1972; Drebes, 1974;
Komarek and Anagnostidis, 1989; Round et al., 1990;
Anagnostidis and Komarek, 1996; Hasle and Syvertsen,
1996; Komarek and Anagnostidis, 2000). The density of
the organisms is expressed as individuals/l (ind./l). An
individual was considered an isolated unit of cells, such
as cyanobacteria trichomes, colonies and cenobia, of
certain chlorophytes and other cyanobacteria. Unidenti-fied centric and pennate diatoms were counted using size
classes.
2.3. Microcystin detection assays
Water samples were frozen, thawed three times and
then centrifuged (12,000 · g) at room temperature, for
10 min. The supernatant was collected and microcystinscontent determined using a commercial enzyme-linked
immunoassay (ELISA) with policlonal antibodies
Fig. 1. Site of Laeonereis acuta collection during the cyanobacterial bloom at Patos Lagoon estuary (Southern Brazil; 32�05 0S–52�12 0W).
958 C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964
(EnviroLogix Inc., Portland, ME), according to Vinagre
et al. (2003).
2.4. Animal tissues homogenates preparation
Whole animals were homogenized (25% W/V) in cold
buffer containing Tris 20 mM, EDTA 1 mM, dithio-
threitol (DDT) 1 mM, sucrose 500 mM, KCl 150 mMand phenylmethylsulfonyl fluoride 0.1 mM, with pH ad-
justed to 7.60 (Geracitano et al., 2002). After a centrifu-
gation at 9000 · g at 4 �C during 45 min the supernatant
was stored (�80 �C) for posterior enzymatic assays.
2.5. Biochemical measurements in tissue homogenates
Total protein content of tissue extracts was deter-mined using a commercial diagnostic kit (Doles Reagen-
tes LTDA, Goiania, GO, Brazil) based in Biuret reagent.
The determinations were done at least in duplicate, at
550 nm.
All enzyme assays were conducted as previously de-
scribed (Geracitano et al., 2002), except for cholinester-
ase (ChE).
Briefly, the activity of catalase (CAT) was measuredby following the initial rate of 50 mM H2O2 (Merck)
decomposition at 240 nm (Beutler, 1975). The results
were expressed in CAT units/mg protein and CAT
units/g wet weight, were one unit is the amount of en-
zyme hydrolyzing 1 lmol of H2O2 per minute and per
g of protein or wet weight (ww), at 30 C and pH 8.00.
Superoxide dismutase (SOD) activity was determined
according to McCord and Fridovich (1969). In this as-
say superoxide anion is generated by the xanthine/xan-
thine oxidase system and the reduction of cytochrome
c monitored at 550 nm. Enzyme activity is expressed
as SOD units/mg of protein and SOD units/g of ww,where one unit is defined as the amount of enzyme
needed to inhibit 50% of cytochrome c reduction per
minute and per g of protein or ww at 25 �C and pH 7.80.
Glutathione S-transferase (GST) activity was
measured by monitoring the formation of a conjugate
between 1 mM GSH and 1 mM 1-chloro-2, 4-dinitro-
benzene (CDNB, from Sigma) (at 340 nm) (Habig
et al., 1974; Habig and Jakoby, 1981). The results are ex-pressed in GST unit/mg of protein and GST unit/g wet
weight, where one unit is defined as the amount of
enzyme that conjugate 1 lmol of CDNB per minute
and per mg of protein or ww at 30 �C and pH 7.4.
ChE activity was measured according to Ellman et al.
(1961) and adapted to worms by Rao et al. (2003), using
DTNB (5,5 0-dithio-bis (2-nitrobenzoic acid); 0.5 mM,
from Sigma) and acetylthiocoline iodide (7.5 mM, fromSigma) as substrate, monitoring the change of ab-
sorbance at 412 nm at 25 �C and pH 7.2. Results are ex-
pressed in nmoles of acetylthiocholine iodide hydrolyzed
per minute and per mg of protein or g of ww. The acet-
C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964 959
ylthiocoline iodide concentration employed in L. acuta
ChE activity determination was selected after kinetic as-
says with different substrate concentrations in the same
conditions previously described.
2.6. Lipid peroxidation assay
Lipid peroxidation was measured according to Mon-
serrat et al. (2003). Whole L. acuta were homogenized in
methanol (10% W/V) and centrifuged at 1000 · g, for
10 min. Lipid hydroperoxides were determined using
FeSO4 (0.25 mM) prepared immediately before use,
H2SO4 (0.25 mM), xylenol orange (1 mM, from Sigma).
Samples absorbance (580 nM) were measured in micro-plate reader after 1 h of incubation at room temperature
and quantified in terms of cumene hydroperoxide (CHP,
from Sigma) equivalents, which was used as standard
(5 nmol/ml).
2.7. In vitro acetylcholinesterase inhibition by bloom
water samples
The effect of bloom water samples on eel (Electropho-
rus electricus) electrogenic organ purified acetylcholin-
esterase (AChE) (V-S type, from Sigma) was determined
in vitro in order to estimate the percentual inhibition of
enzymatic activity, according to Monserrat et al.
(2001). The purified enzyme (50 ll of a 0.25 U/ml solu-
tion) was dilluted in 950 ll of phosphate buffer
(50 mM) containing 20% of glycerol at pH 7.4. Todetermine the AChE activity DTNB (0.4 mM) and acety-
lthiocoline iodide (0.8 mM) as substrate were employed.
In order to evaluate the presence of anticholinesterasic
compounds in bloom water samples, several aliquots,
ranging from 1.25% to 5% of the final reaction volume
(1 ml), were used. Samples were incubated for 1 h at
25 �C. After that, the substrate and DTNB were added,
and enzyme activity measured as previously described.In addition, eserine inhibition tests were conducted in
order to evaluate the responsiveness of the purified eel
acetylcholinesterase to a known inhibitor. For this
purpose, the bloom water samples were substituted for
eserine solutions in a final concentration of 5.6 and
1.2 · 10�6 M and incubated 1 h at 25 �C. The acetylcho-
linesterase activity was determined as described above.
Results are expressed as relative inhibition (percentual)in respect to the control group (AChE activity in absence
of water samples).
2.8. Determination of oxygen consumption rates (VO2)
by L. acuta at different water oxygen concentration
Oxygen consumption rate of individuals specimens of
L. acuta, collected in August 2003 in ‘‘Saco do Justino’’,weighting 83.37 ± 5.1 mg (mean wet weight ± S.E.), were
measured according to Nithart et al. (1990). Respiration
chambers, with a volume of 10 ml, containing brackish
water (10 &) and different oxygen concentrations, rang-
ing from 3.7 to 19 mgO2/l, were maintained at 20 �C.
Oxygen concentrations were measured with an oxymeter
(Digimed). VO2 is expressed as mgO2/h/gww.
2.9. Statistical analysis
Values for all enzymatic determinations were com-
puted as means ± standard error (±SE). Statistical ana-
lysis was performed by means of analysis of variance
followed by Newmann–Keuls test or polynomial con-
trasts (a = 0.05). Normality and variance homogeneity
were previously verified (Zar, 1984).
3. Results
During the bloom period, of approximately 3–4
weeks, the mean (±SE) values of pH and temperature
of the water during the sampling time (10:00–14:00 h)
were 9.55 ± 0.59 and 27 ± 1.53 �C, respectively.Analysis of fixed water samples revealed a clear
predominance of certain cyanobacterial genera during
the bloom period (Table 1). The genera Anabaena,
Aphanocapsa,Merismopedia and Snowella were the most
predominant photoautotrophic constituents, with maxi-
mum abundance values of 2.96, 4.60, 7.74 and
2.41.106 ind./l, respectively. Specimens of Anabaena
spp. were characterized by more than 20 lm long,straight or spiral filamentous (trichomes) structures,
while Aphanocapsa spp., Merismopedia spp. and Snow-
ella spp. were only observed forming smaller colonies.
Cyanobacteria were characterized by frequency val-
ues between 75.9% and 83.5%, followed by chlorophytes
(13.4% and 21%) and diatoms (3.1% and 4.5%) (Table 1).
Cyanobacteria and chlorophytes stood out among the
other phytoplanktonic groups, i.e. diatoms and eugle-nids, either in number of taxa or by their higher fre-
quency values in the samples.
The detection tests of microcystin in water samples
reveal low levels of this toxin during the bloom period.
The mean toxin value during the sampling period was
0.29 ± 0.14 lg/l, with the highest value of 0.46 lg/l re-
corded at day 77.
The total protein content of tissue extracts of L. acutavaried during the sampling period, being highest before
the beginning of the bloom (�35 days) (23.57 ± 1.78 mg/
ml) returning to mean (17.17 ± 0.54 mg/ml) values in the
next sampling periods. For this reason, the enzyme
determinations realized in all the homogenates were ex-
pressed in terms of the total protein content and in wet
weight basis.
The whole body homogenates of L. acuta reveals nosignificant differences (p > 0.05) in ChE activity during
the sampling period, remaining stable. Mean values
Table 1
Composition and succession of cyanobacterial blooms in ‘‘Saco do
Justino’’ (Patos Lagoon, Southern Brazil; January 2003)
Organisms 7 10 16
Cyanobacteria
Anabaena spp. 2.96 (34.1) 1.93 (16.9) 2.79 (11.0)
Anabaenopsis sp. 0.04 (61) 0.14 (610) 0.83 (610)
Aphanizomenon sp. 0.04 (61) 0.13 (610) 0.34 (610)
Aphanocapsa spp. 2.11 (24.3) 4.21 (36.8) 4.6 (18.1)
Cyanodictyon sp. 0.13 (610) 0.39 (610) 0.09 (61)
Gomphosphaeria spp. 0.09 (61) 0.21 (610) 0.43 (610)
Merismopedia spp. 0.69 (610) 0.52 (610) 7.74 (30.4)
Planktolyngbya
limnetica
0.08 (61) 0.09 (61) 0.4 (610)
Pseudanabaena sp. 0.11 (610) 0.04 (61) 0.26 (61)
Snowella spp. 0.3 (610) 1.29 (11.3) 2.41 (610)
Spirulina sp. 0.02 (61) 0.01 (61) 0.02 (61)
Other cyanobacteria 0.01 (61) 0.6 (610) 0.87 (610)
Diatomophyceae
Aulacoseira spp. – 0.1 (61) 0.45 (610)
Cyclotella sp. >20 lm – 0.002 (61) 0.08 (61)
Cyclotella spp. <20 lm 0.21 (610) 0.17 (610) 0.17 (61)
Unindentified
pennates 10–100 lm0.05 (61) 0.01 (61) 0.27 (610)
Other centrics 0.01 (61) – 0.1 (61)
Other pennates – 0.08 (61) 0.07 (61)
Euglenophyceae – – 0.006 (61)
Chlorophyceae
Dichotomococcus cf.
hoefleri
0.13 (610) 0.13 (610) 0.01 (61)
Eudorina elegans – 0.002 (61) 0.01 (61)
Golenkinia radiata 0.13 (610) 0.09 (61) 0.21 (61)
Kirchneriella spp. 0.47 (610) 0.39 (610) 0.82 (610)
Lagerheimia spp. – 0.13 (610) 0.13 (61)
Micractinium cf.
pusillum
0.21 (610) 0.04 (61) –
Monoraphidium spp. 0.08 (61) 0.12 (61) 0.29 (610)
Oocystis lacustris 0.21 (610) – 0.13 (61)
Scenedesmus spp. 0.17 (610) 0.24 (610) 0.84 (610)
Schroederia spp. 0.18 (610) 0.12 (61) 0.3 (610)
Tetraedron spp. – 0.21 (610) 0.39 (610)
Tetrastrum
triacanthum
0.13 (610) 0.04 (61) 0.17 (61)
Other clorophytes 0.11 (610) 0.02 (61) 0.25 (61)
Number of cells is expressed as 106 individuals/l. Percentage of counted
organisms for each sampling date is expressed in parentheses and, the
most abundant phytoplanktonic organisms are in bold.
Table 2
Inhibition of purified eel acetylcholinesterase (AChE, V–S type) activity aft
bloom samples
Final dilution or concentration Samples
7 10
5% Ni 7.15 ± 7.5
3.75% Ni 3.92 ± 2.18
2.5% 19.7 ± 7.8 3.22 ± 5.89
1.25% 14.8 ± 1.8 7.45 ± 6.59
5.6 · 10�6 M – –
1.2 · 10�6 M – –
The values are expressed in % of activity inhibition ± S.E. compared to contr
during the bloom (see the text for definitions). The eserine concentrations em
-35 7 10 16 770.0
0.1
0.2
0.3
nmols/(min x mg of protein)nmols/(min x g of wet weight)
0
500
1000
1500
2000
a
Aa
a a
aA
AA A
Cholinesterase
Time (days)
Fig. 2. Activity of Laeonereis acuta cholinesterase (ChE) during the
bloom period. Enzymatic activity was normalized in both, tissue
extract total protein content and wet weight basis. Equal letters
indicate absence of significant difference between means (p > 0.05).
Data is expressed as mean ± 1 S.E. of at least four samples per
sampling time. Left axis indicates enzyme activity per mg of proteins.
Right axis represents enzyme activity per g of wet weight.
960 C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964
(±SE) varied between 0.096 ± 0.013 and 0.156 ±
0.003 nmoles/min/mg protein (751.17 ± 15.15 and
1135.55 ± 323.78 nmoles/min/g ww) (Fig. 2). The inhibi-
tion of purified eel AChE was very low, about
4.85 ± 6.84 % (mean values ± SE), and do not follow a
dose response relationship for the dilutions of all water
samples. The inhibition registered with a known AChE
inhibitor like eserine was higher than 95% and con-firmed the responsiveness of the purified eel AChE
(Table 2).
Also, during all the sampling period CAT and SOD
activities remained almost constant (p > 0.05), ranging
from (mean values ± SE) 2.06 ± 0.17 to 3.46 ± 0.97 U
CAT/mg of protein (132.15 ± 10.82–257.41 ± 45.78 U
CAT/g ww) and 17.70 ± 1.90 to 30.183 ± 5.26 U SOD/
mg of protein (1354.71 ± 363.83–1852.55 ± 320.6 USOD/g ww), respectively (Fig. 3).
GST activity fitted to a second-order function, show-
ing a peak in sampling day 10 (p < 0.05). At the end of
the sampling period (77 days), GST activity was similar
er 1 h exposure to different dilution of aqueous extracts of the water
16 77 Eserine
3.36 ± 5.02 Ni –
Ni Ni –
2.20 ± 6.25 Ni –
12.40 ± 6.66 4.06 ± 2.54 –
– – 97.29 ± 0.09
– – 96.05 ± 0.78
ol activity. Seven, 10, 16 and 77 are the four different sampling periods
ployed were 5.6 and 1.2 · 10�6 M. Ni: no inhibition registered.
O2 consumption(mgO2 x h x g)
0 5 10 15 20 250.0
0.4
0.8
1.2
[O2] in water (mg/l)
Fig. 4. Rates of oxygen consumption [mg O2/(h · g ww)] versus
-35 7 10 16 770
1
2
3
4
5
0
80
160
240
320
400
U/g of proteinU/g of wet weight
A
A
A
AA
aa
a
aa
Catalase
Time (days)-35 7 10 16 77
0
10
20
30
40
0
500
1000
1500
2000
2500U /g of wet weightU /mg of protein
a
A
A
A
A A
aa
aa
Time (days)
Superoxide dismutase
-35 7 10 16 770.00
0.01
0.02
0.03
0.0
0.3
0.6
0.9
1.2
1.5
U/mg of proteinU/g of wet weight
AA
aa
BB
B
b
b
b
Glutathione S-transferase
Time (days)-35 7 10 16 77
0
250
500
750
a a
a
a
b
Lipid peroxidation
nmoles CHP/g of wet weight
Time (days)
Fig. 3. Activities of catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) and lipid peroxidation (LPO) in the worm
Laeonereis acuta during the bloom period. Enzymatic activities were normalized in both, tissue extract total protein content and wet weight basis.
Equal letters indicate absence of significant difference between means (p > 0.05). Data is expressed as mean 1 S.E. of at least four samples per
sampling time. Left axis indicates enzyme activities per mg of proteins. Right axis represents enzyme activities per g of wet weight.
C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964 961
to that registered at the beginning (�35 days). Meanvalues ± SE varied between 0.006 ± 0.001 and 0.016 ±
0.004 U GST/mg of protein (0.51 ± 0.11 and 1.12 ±
0.2 U GST/g ww) (Fig. 3).
The lipid hydroperoxides content in L. acuta reveals a
similar pattern to that of GST activity A peak (p < 0.01)
after 10 days of the beginning of the bloom respect to
the other periods was registered. The values varied bet-
ween 87.24 ± 14.75 (�35 days) and 508.33 ± 76.65 (10days) nmol CHP/g ww (Fig. 3).
Finally, the oxygen consumption (VO2) profile of
L. acuta under different [O2] in water, reveals that this
animal is an oxyconformer. The VO2 varies from
0.0460.006 mg O2/(h · g ww) under 3.7 mg O2/l to
0.85 ± 0.11 mg O2/(h · g ww) under 19.16 mg O2/l
(Fig. 4).
oxygen concentration in the water (mg O2/l) at 20 �C and 10& salinityby Laeonereis acuta. Data are the mean ± 1 S.E. of oxygen consump-
tion of five animals per [O2] sampled. The line indicates the linear-
regression curve (R2 = 0.96).
4. DiscussionThe community composition and abundance ob-
served in this study are different from others carried
out in the same, or close, areas during previous summer
periods (Jesus and Odebrecht, 2002; Bergesch and
962 C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964
Odebrecht, 1997; Persich et al., 1996; Yunes et al., 1994,
1998). These authors cited high density (up to 106
cells l�1) of small-unidentified cyanobacteria (<5 lm),
pennate diatoms, nanoflagellates and a different phyto-
planktonic group–dinoflagellates (Jesus and Odebrecht,
2002) and/or indicated that the highest biomass and den-sity of cells belonged to the species Microcystis
aeruginosa.
Although the cyanobacterial bloom occuring in the
Patos Lagoon includes potent toxin producer genera
in its community composition (Table 1), low levels of
microcystin and anatoxin-a(s) were detected. It is known
that the genus Anabaena, the predominant phytoplank-
tonic species in this bloom, produces both types of tox-ins. (Codd, 1995). In the present study microcystin
concentrations in water samples lower than 0.5 lg/l were
registered. In Brazil, the maximum allowed microcystin
concentration is 1.0 lg/l, in water bodies destined to
human consumption (Fundacao Nacional De Saude,
2001), indicating that the detected levels were in fact
very low. In previous blooms in the same area, domi-
nated by Microcystis, higher levels of this toxin (up to265.1 lg/l) were registered (Minillo et al., 2000).
It is well known that the cyanobacterial neurotoxin
anatoxin-a(s) is a potent inhibitor of the enzyme acetyl-
cholinesterase (Charmichael, 1994), and episodes of
massive death of domestic and wild animals by their
effects have been reported (Mahmoud et al., 1988; Ono-
dera et al., 1997). Monserrat et al. (2001) observed that
Anabaena spiroides aqueous extracts induced in vitroinhibition of AChE activity in fish (Odontesthes argen-
tinensis), crab (Callinectes sapidus) and purified eel
AChE. In the present study, no differences were ob-
served in worm cholinesterase activity during the bloom
period. In the in vitro assays, when purified eel AChE
was assayed with bloom water samples, the maximum
observed inhibition value was 19.7%. The low inhibition
results do not follow a dose–response curve, a result ex-pected for water samples containing anatoxin-a(s).
Both, the results of cholinesterase worm activity and
in vitro assays, indicate that the analyzed water samples
were free, or with low concentrations, of neurotoxins
like anatoxin-a(s). Previous studies (Barros et al.,
2004) have shown that alkaline pH (8.5) induces loss
of inhibitory potency of aqueous extracts of A. spiroides.
The higher pH values registered during the bloom sug-gest that if this toxin was released from cyanobacteria,
its chemical stability should be compromised.
Although the toxin levels seemed to be low, the
population of L. acuta at ‘‘Saco do Justino’’ suffered a
dramatic reduction in density (field observations), indi-
cating that the cyanobacterial bloom exerted some harm-
ful effect. One possibility is to consider that the bloom
exerted its toxicity to L. acuta through oxidative stress.Commonly, a disturbance between the production of
the reactive oxygen species (ROS) and the antioxidant
defenses system favoring the first, lead organisms prone
to suffer oxidative damage (Halliwell and Gutteridge,
1999). Nevertheless, no difference was observed in enzy-
matic antioxidant defense system (CAT and SOD) re-
lated to interception and degradation of the reactive
oxygen species H2O2 and O��2 . Concomitant to the lack
of induction of antioxidant enzymes, higher levels of oxi-
dative damage in terms of lipid hydroperoxides was ob-
served in animals collected during the middle of the
bloom (10 days). Interestingly, GST activity also showed
a peak in the same sampling period. This enzyme is
involved in the conjugation of oxidative products, like
4-hydroxyalkenals (membrane peroxides) and/or base
propenals, resulting from the DNA oxidative degrada-tion (Leaver and George, 1998). So, the high activity of
GST registered can be ascribed to phase II reactions in
order to conjugate products derived from oxidative dam-
age. The fact that the higher values of GST and lipid
hydroperoxides were found in the middle of bloom and
not at its end can be associated with the intense mortality
observed in the field. At the end of the bloom probably
only resistant worms survived, perhaps more protectedagainst oxidative stress.
These situations can be related to the hyperoxia/
anoxia induced by the cyanobacterial through photo-
synthetic and respiratory processes, as previously
observed under a cyanobacterial bloom dominated by
the genera Microcystis and Anabaena, among others
(Seki et al., 1979). In that situation, a disturbance in
the normal dissolved oxygen curves, reaching up to190% of saturation at noon, and close to 0% at the mid-
night, was observed. This resembles the ischemic/reper-
fusion process. After a reduction of oxygen flow
(ischemia), the turn back of oxygen in the reperfusion
causes an increase in the production of reactive oxygen
species, leading to oxidation of cellular components
including proteins, membrane lipids and DNA (Lush-
chak et al., 2001). The electron carriers of the mito-chondrial respiratory chain are reduced during ischemia,
whereas immediate re-oxygenation of these carriers
takes place after the reperfusion, leading to oxyradical
overproduction (Halliwell and Gutteridge, 1999).
According to Storey (1996b), the ROS generation rate
was closely related to the oxygen consumption and the
amount of mitochondrion in the tissue. So, if an animal
is an oxyconformer (O2 consumption varies accordingwater oxygen availability; McMahon, 1988), like L. acu-
ta, in a hyperoxic situation its oxygen consumption will
be higher, thus augmenting ROS generation. Other
authors have observed augmented levels of oxidative
damage products in some animal species (reptile, mol-
lusk, fish) when submitted to anoxia/hyperoxia cycles
(Storey, 1996a; Pannunzio and Storey, 1998; Lushchak
et al., 2001). In general, this response is accompaniedby an elevation of antioxidant defense, an anticipatory
mechanism to cope with oxidative stress (Hermes-Lima
C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964 963
et al., 1998). In L. acuta, no variations of antioxidant de-
fenses were observed, and the higher levels of lipid
hydroperoxides evidenced a situation of oxidative stress.
Whether this situation leads to a lowering of worm den-
sity along the bloom event remains to be studied.
Finally it cannot be discarded that an oxidative stresssituation can be a consequence of ROS (O��
2 and H2O2)
produced by phytoplanktonic organism (Kim et al.,
2000). In a culture of Chattonella marina, the levels of
H2O2 production in a cellular suspension (104 cells/ml)
were 1.07 nmol/l/min (Kim et al., 2002). During a
bloom, the density of planktonic organisms is much
higher, which should increase the ROS concentration
in the water column.In conclusion, the cyanobacterial bloom, composed
mainly by trichomes of Anabaena spp., exerted harmful
effects in the L. acuta population. These effects a priori
cannot be linked to cyanotoxins but to a hyperoxic/
anoxic cycle amplification generated by the photosyn-
thetic/respiratory process, which, in turn, caused oxida-
tive stress, as evidenced by the higher levels of LPO.
Acknowledgements
C.E.R. is a post-graduation student financed by
CAPES. J.M.M. and J.S.Y. are research fellows from
Brazilian CNPq. The research was partially supported
by PPGCF-FAC (FURG). Authors also acknowledged
the assistance of F.R. Piedras and R.B. Robaldo.Authors also acknowledged the aid of Prof. Dr. Eucly-
des Santos in the English revision of the manuscript.
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