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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: jose@octopus.furg.br (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& salinity

by 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. Discussion

The 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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