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Acute Effects of Microcystis aeruginosa from the Patos Lagoon Estuary, Southern
Brazil, on the Microcrustacean Kalliapseudes schubartii (Crustacea: Tanaidacea)
W. Montagnolli, A. Zamboni, R. Luvizotto-Santos, J. S. Yunes
Unidade de Pesquisas em Cianobacte´rias (UPC), Departamento de Quimica, Fundac¸a˜o Universidade Federal do Rio Grande (FURG), Cx Postal 474,
CEP 96201-900, Rio Grande/RS, Brazil
Received: 12 January 2003/Accepted: 6 September 2003
Abstract. Toxic blooms of the cyanobacterium Microcystis
aeruginosa, a microcystin producer, have been observed in the
past two decades in the Patos Lagoon estuary (southern Brazil).
This cyanobacterium reaches the estuary from northern waters
and accumulates as toxic blooms in the shallow margins of the
environment. Microcystins are phosphatase (PP1 and PP2A)
inhibitors and cause animal death via alteration of the liver cell
cytoskeletons and intrahepatic hemorrhage. The massive accu-
mulation of toxic material affects the survival of several ben-
thonic estuarine local organisms. The tanaidacea Kalliapseudes
schubartii is a benthonic estuarine species which occurs at high
densities throughout the year in mixohaline areas of the Patos
Lagoon. This microcrustacean is of high ecological relevance
and plays an important role in the estuarine food web, as it is
consumed on a large scale by estuarine fish. This work verifies
the acute toxicity of aqueous extracts of M. aeruginosa
RST9501 and of sediments spiked with lyophilized material of
the same strain on K. schubartii; it also evaluates the sublethal
effects on tanaidacean oxygen consumption rates and glycogen
levels under acute exposure to M. aeruginosa aqueous extracts.
The strain M. aeruginosa RST9501 was cultured in BGN/2
medium. The aqueous extracts were prepared using the lyo-
philized material from the strain cultures. Acute tests were
performed over 96 h at a salinity of 15, at six toxic concentra-
tions, and resulted in an average 96-h LC50 of 1.44 mg ml
⫺1
.
The spiked sediment tests were performed with a 10-day du-
ration, using the lyophilized material in three proportions of
powder/sediment and showed an average LC50 of 1.79 mg
ml
⫺1
. Oxygen consumption was determined after 24 and 48 h
of incubation in adult organisms exposed to sublethal aqueous
extract concentrations and showed a significant increase at the
highest concentrations. This suggests alterations in the organ-
ism’s metabolism by exposure to the cyanobacterium extract.
The glycogen levels were determined with a commercial kit
(Glicox 500; DOLES Ltd.); after 24 and 48 h the dosages were
administered in the same organisms utilized in the oxygen
consumption test and did not demonstrate significant differ-
ences. The results demonstrate the possible risks of intoxica-
tion to which the natural populations of K. schubartii were
exposed in the environment and emphasize the importance of
studies involving sublethal concentrations of M. aeruginosa to
other organisms of the trophic web in this aquatic system.
Cyanobacterial blooms have several consequences for water
quality and their collapse frequently causes high mortality
among aquatic animal populations (Vasconcelos et al. 2001).
Among many species of cyanobacteria that can develop toxic
blooms, Microcystis aeruginosa is one of the most common
and is a matter of great concern (Chorus and Bartram 1999).
The cyanobacterium Microcystis aeruginosa produces toxins
called microcystins, monocyclic heptapeptide molecules, of
which over 65 structural variants are known. Most of the
variants are potent hepatotoxins and tumor promoters in mam-
mals (Codd et al. 1999; Sivonen and Jones 1999). At the
molecular level, microcystins are serine/threonine phosphatase
PP1 and PP2A inhibitors, resulting in hyperphosphorylation of
proteins, affecting intracellular signaling, cell growth, and dif-
ferentiation processes, and inducing cell disturbance (Toivola
and Eriksson 1999).
Previous works have shown that microcystin can be conju-
gated with the tripeptide glutathione (GSH), through a conju-
gation reaction catalyzed by glutathione-S-transferase (GST)
(Pflugmacher et al. 1998; Takenaka 2001; Vinagre et al. 2003).
The microcystin–GSH complex has a less inhibitory effect on
phosphatases, clearly showing that this conjugation reaction is
involved in detoxification processes (Metcalf et al. 2000). The
enzyme (GST) is reported to be involved in detoxification
reactions of several kinds of pollutants, such as metals, fungi-
cides, and pesticides (Freedman et al. 1989; Gallagher et al.
1992; Maracine and Segner 1998).
Different aquatic organism species from all trophic levels in
the web are susceptible to microcystins, these include bacteria,
protozoa, macrophytes, green algae and diatoms, and micro-
and mesozooplankton, as well as invertebrates and fish (Christ-
offersen 1996). A number of reports have demonstrated the
sensitivity of different species of marine and freshwater crus-
tacea to Microcystis aeruginosa cells and microcystins
(Delaney and Wilkins 1995; Salomon et al. 1996; DeMott
Correspondence to: J. S. Yunes; email: dqmsarks@furg.br
Arch. Environ. Contam. Toxicol. 46, 463–469 (2004)
DOI: 10.1007/s00244-003-2304-6
ARCHIVES OF
Environmental
Contamination
and
Toxicology
© 2004 Springer-Verlag New York LLC
1991; Hietalla et al. 1997; Yogui 1999; Vasconcelos et al.
2001).
Microcystis aeruginosa blooms have been observed in the
past two decades in the Patos Lagoon estuary (RS-Brazil)
(Yunes et al. 1996, 1998), and their toxicity and microcystin
contents have been reported previously (Yunes et al. 1992).
This cyanobacterium reaches the estuary of the Patos Lagoon
from northern waters and accumulates toxic blooms and scums
in the shallow margins of the estuary. This massive accumu-
lation of toxic material in the sediment puts the survival of
several local benthonic estuarine organisms at risk.
The Patos Lagoon estuary has a diversified fauna and is an
important nursery for marine and limnic species. Kalliapseudes
schubartii Man˜e-Garzon 1949 (Crustacea–Tanaidacea) is
among the most interactive benthonic organisms (Fig. 1). This
tanaidacean is a benthonic tube-forming filter-feeding species,
which occurs at high densities year-round in mixohaline re-
gions of the Patos Lagoon (Zamboni 2000). The organism
carapace encloses the branchial chamber along the thorax.
Respiration through the gills and filter feeding on detritus and
plankton (McLaughlin 1980) are the possible routes for the
uptake of cyanotoxins. The species plays an important role in
the estuarine food web, as it is consumed on a large scale by
estuarine fish (Micropogonias furnieri, Odontesthes bonar-
ienses, etc.) and estuarine crustacea (Farfantepenaeus paulen-
sis, Callinectes sapidus). All of them are very important fishing
resources in the Patos Lagoon estuary. The easy adaptability of
the species Kalliapseudes schubartii to laboratory maintenance
has led other authors (Costa 1998; Zamboni 2000) to use this
estuarine tanaidacean as a test organism for heavy metal tox-
icity tests and water quality bioassays.
The objective of this work was to determine the acute tox-
icity of an aqueous extract of Microcystis aeruginosa RST9501
(isolated from the Patos Lagoon estuary in 1995 and kept as an
axenic strain at the Unit of Research on Cyanobacteria of
FURG, Rio Grande, Brazil) and the toxicity of sediments
spiked with lyophilized material obtained from the same strain
cultures, by measurements in Kalliapseudes schubartii. It also
aimed to evaluate the sublethal effects of acute exposure to
Microcystis aeruginosa aqueous extracts on the tanaidacean
oxygen consumption rates and glycogen levels.
Materials and Methods
Sampling and Adaptation of Organisms
Using a shovel, test organisms were collected on the sandy margins of
the Patos Lagoon estuary (southern Brazil), and organisms and sedi-
ments were separated with a 500-m mesh net. The organisms were
immediately transferred to the laboratory and separated into three
groups: adults, juveniles, and females with eggs. Only adult organisms
measuring between 0.7 and 1 cm long were used. Depending on the
salinity of the site from which the sample was taken, the adaptation to
salinity 15 (‰) was made by gradually adding brine, obtained from
melting filtered seawater, or with filtered freshwater (1‰every 2 h).
Thus, these adults were adapted to salinity 15 and maintained in plastic
containers approximately 10 L in volume, containing a 2-cm-deep
layer of sediment sifted through a 500-m mesh net, with a maximum
of 100 organisms in each recipient. They were maintained under a
controlled temperature (22°C), a light/dark cycle of 12:12 h, and
constant aeration for a minimum of 5 days and a maximum of 7 days
prior to the toxicity tests. Animals were fed with a solution of two
commercial rations for trout and fingerling (Alcon). For each lot of 100
organisms, 2 ml of this solution was supplied every 48 h.
Cyanobacterial Toxic Extract Preparation
The strain M. aeruginosa RST9501 (UPC Culture Collection, Brazil)
was grown in batch cultures in BGN/2 (BG11 with half the total N)
medium (Rippka et al. 1979) under constant conditions (salinity 0;
temperature, 25°C; radiation, 63 Em
⫺2
s
⫺1
; and pH 8.0) in 5-L
flasks. These unialgal and axenic cultures had a growth rate ()of
⫹0.2 day
⫺1
and were lyophilized on reaching 1 ⫻10
7
cells ml
⫺1
. The
aqueous extracts were prepared using lyophilized material from the
strain cultures and sterilized water at a salinity of 15 ppm. Samples
were frozen and thawed three times and sonicated (50 mHz; Soniprep,
USA) at ⫺10°C for 10 min. This suspension was centrifuged at 14,000
rpm on a bench centrifuge (Eppendorf, Germany) at room temperature
for 10 min and the supernatant was separated for the tests and stored
at ⫺20°C. The microcystin content of the extracts was determined
using a commercial enzyme-linked immunoassay (ELISA) with poly-
clonal antibodies (EnviroLogix Inc., Portland, ME, USA), which has a
detection limit of 0.01 gL
⫺1
.
Acute Tests with the Reference Substance
For the toxicity tests presented in this work, many samples of organ-
isms in the environment were necessary. To evaluate whether the
sensitivity of the animals collected was the same, they were tested
according to Zamboni (2000) using the standard reference toxic sub-
stance (CdCl
2
). Sampling and adaptation of organisms were done, and
the water used in the tests was prepared as described above. All tests,
following the same procedure described for the toxic extracts, were
static, acute over 96 h, at salinity 15, temperature of 22°C, and a
light/dark cycle of 12:12 h, and without feeding and aeration. The
organisms were tested at five toxic concentrations (0.5, 0.7, 0.98, 1.37,
and 1.92 mg L
⫺1
and controls), including four replicates at each.
Acute Tests Applying the Microcystis aeruginosa Aqueous
Extracts
Three acute tests were performed over 96 h, at a salinity of 15 ppm,
without feeding, at six toxic concentrations (0.65, 0.84, 1.09, 1.42,
1.85, and 2.4 mg lyophilized material/ml water, plus controls), with
four replicates of each. The microcystin concentration in the aqueous
extract was 1094.07 gg
⫺1
. The concentrations used were established
in previous tests, which determined a range defined by the highest
concentration at which no mortality was observed and the lowest
concentration at which a mortality of 100% was observed. The water
Fig. 1. Adult form of Kalliapseudes schubartii
464 W. Montagnolli et al.
used in the tests was sterilized in an autoclave for 40 min at 120°C.
Polyethylene flasks were used containing 100 ml of test solution, and
10 organisms were put in each flask. No effects due to the volume of
the culture and the number of organisms were observed. Tests were
maintained with constant aeration, at a controlled temperature (22 ⫾
2°C), and under light/dark cycles of 12:12 h. The pH was checked at
the beginning and end of each experiment and each toxic concentra-
tion. The 96-h LC50 was calculated by the trimmed Spearman–Karber
method (Hamilton et al. 1977).
Acute Tests Using an Aqueous Extract of Aphanotece
microsco´pica
To confirm whether the mortality observed was provoked by the
presence of microcystins, acute tests were prepared as were the pre-
vious tests, using an aqueous extract of the cyanobacterium strain
Aphanotece microsco´pica RSMan 9401, previously characterized as a
nontoxic strain using mouse tests and microcystin analysis (Yunes et
al. 1996).
Acute Tests with the Sediment Spiked with M. aeruginosa
Two spiked sediment tests were performed using the lyophilized
cyanobacteria at three proportions of powder/sediment. Thus, 110 g of
dry sediment was used with 354, 510, or 735 mg of M. aeruginosa
RST9501 dry lyophilized powder at each test concentration, respec-
tively. These concentrations were based on the CL50 value obtained
with the aqueous extract. The microcystin concentration in the lyo-
philized material was 1086.41 gg
⫺1
. Sediment was collected at the
same site as the organisms, sifted in a 500-m mesh, and dried at 40°C
in the laboratory. The methodology used for sediment tests applied to
Kalliapseudes schubartii followed Zamboni (2000) and consists of
acute tests, without water or sediment renewal, and with constant
aeration. Adult organisms were exposed for 10 days to the sediment
previously spiked with the M. aeruginosa powder, with each concen-
tration having four replicates. Tests were done in 400-ml beakers
containing 110 g of spiked sediment at the respective concentrations,
300 ml of sterilized water (salinity, 15 ppm), and 10 organisms. Thus,
considering the volume of tests, the tested concentrations were 2.45,
1.70, and 1.18 mg ml
⫺1
, plus the control. The sediment and flasks used
in the tests were previously sterilized at 121°C during 40 min for
elimination of microorganism contamination. During the experiment,
the medium was maintained with constant aeration, light/dark cycles
of 12:12 h, a temperature of 22°C, and feeding every 48 h using the
same maintenance diet. The organic fraction was determined by the
Strickland and Parsons (1972) method. The LC50 value was calculated
by the trimmed Spearman–Karber method.
Oxygen Consumption of Kalliapseudes schubartii
Oxygen consumption was determined after 24 and 48 h of incubation
of four average adult organisms exposed to sublethal extract concen-
trations (0.36, 0.8, and 1.2 mg ml
⫺1
and controls), with four replicates
each. The microcystin concentration in the lyophilized material was
304.84 gg
⫺1
. The water used in the tests was previously autoclaved
for 40 min at 120°C. Organisms were exposed in polyethylene flasks
containing 100 ml of test solution and 10 organisms. Tests were
performed over 48 h, at a salinity of 15 ppm, with constant aeration,
a controlled temperature (22 ⫾2°C), and a light/dark cycle of 12:12
h, and without feeding. Adult organisms of Kalliapseudes schubartii
having an average weight of 7.3 ⫾1.4 mg were used. Four Clark-type
estatic-system digital respirometers (Rank Brothers, England) (Fig. 2)
were used to perform simultaneously four tests at the same concen-
tration and, consequently, four replicates. Following this procedure,
the four digital respirometers used were tested for all concentrations,
thus avoiding equipment differences. Each system contained a micro-
cathode oxygen electrode within a closed glass jacket connected to an
oxygen meter display. At each exposure period of 24 and 48 h, one
organism from each of four replicate flasks was transferred to each of
four respirometer chambers containing 1.5 ml of aerated test solution.
The animal was placed and kept above the magnet stirrer by means of
a plastic net adapted to the internal dimensions of the chamber. Each
toxic concentration was measured for 30 min, the first 5 min being for
adaptation. Thereafter, all organisms were frozen at ⫺20°C for pos-
terior total glycogen analysis. After each experiment, the chamber was
soaked with alcohol, then rinsed thoroughly with distilled water to
reduce microbial growth and accumulation inside the chamber. The
estimated effect of organism weight on oxygen consumption was
verified. No correlation between organism weight and oxygen con-
sumption (for body mass) (p⫽0.607 and R
2
⫽0.0021) was observed.
Total Glycogen Analysis
Since phosphatase inhibition maintains the glycogen phosphorylase
enzyme activity, causing a collapse in the organism energy supply
(MacKintosh and MacKintosh 1994), glycogen concentration mea-
surements in Kalliapseudes schubartii were made. All organisms,
including the control, used for total glycogen analysis were previously
submitted to the oxygen consumption tests described above. The
glycogen analyses, made using a commercial kit (Glicox 500; DOLES
Ltd, Brazil), were based on glucose equivalents and followed the
oxidase glucose method. The methodology for microcrustacea was
described by Nery and Santos (1993).
Statistical Analysis
Data were submitted to analysis of variance (ANOVA) and previous
requirements for analysis (normality and homoscedasticity) were also
verified. Treatments that demonstrated significance differences (p⬍
0.05) were submitted to the LSD test. All statistical analyses and
correlations were made using the program Statistics for Windows
v.5.0. LC50 values for the toxicity tests with Kalliapseudes schubartii
Fig. 2. Respirometer schematic design. (1) Oxygen meter; (2) bath
chamber; (3) respirometer chamber and stirrer; (4) electrode; (5) bath
water flow
Effects of Microcystis aeruginosa on a Microcrustacean 465
were calculated using the trimmed Spearman–Karber program (Ham-
ilton et al. 1977).
Results
Establishing Microcystin Concentrations
In the acute toxicity tests done with the aqueous extract and
sediment spiked with lyophilized M. aeruginosa, the microcys-
tin concentration was estimated as 1094.07 and 1087.43 g
g
⫺1
dw, respectively. However, in the sublethal tests of oxygen
consumption, the toxic extracts contained 304.84 gg
⫺1
dw of
microcystins.
Reference Substance
The organism sensitivity tests for a reference substance
(CdCl
2
) resulted in an average 96-h LC50 of 0.89 gml
⫺1
. The
data confirm the equal sensitivity of different lots of organisms
collected in the wild (Table 1).
Acute Tests with Microcystis aeruginosa
Kalliapseudes schubartii exposed to toxicity experiments with
the Microcystis aeruginosa RST9501 aqueous extract re-
sulted in a 96-h LC50 varying from 1.41 to 1.50 mg ml
⫺1
with an average of 1.44 mg ml
⫺1
. The pH values were
between 7.5 and 8 throughout the test. The 96-h LC50
values of all three tests are listed in Table 2.
Acute Tests with Aphanothece microsco´pica
Tests with the cyanobacterium Aphanotece microscopica RS-
Man9401 showed a low mortality for Kalliapseudes schubartii.
The mortality percentages for every 24 h are listed in Table 3.
Acute Tests with Lyophilized M. aeruginosa-Spiked
Sediment
After 10 days of organism exposition to the sediment spiked
with M. aeruginosa RST9501, the average LC50 was 1.79 mg
ml
⫺1
(approximately 537 mg of lyophilized M. aeruginosa in
110 g of sediment). The pH values were 6.5–7.5 and 6.7–8at
the beginning and end of the tests, respectively. The organic
fraction in the sediment was 1.53%. The results obtained in the
two tests are listed in Table 4.
Oxygen Consumption
Acute exposure of Kalliapseudes schubartii to Microcystis
aeruginosa RST9501 aqueous extract at sublethal concentra-
tions caused a significant increase (p⫽0.001023, F⫽
7.52443) in oxygen consumption at the two highest concentra-
tions (0.8 and 1.2 mg ml
⫺1
) in both the 24-h and the 48-h
periods (Fig. 3).
Total Glycogen
Total glycogen results of exposed Kalliapseudes schubartii
after 48 h at different sublethal concentrations of Microcystis
aeruginosa aqueous extracts are presented in Figure 4.
ANOVA did not indicate significant differences (p⬍0.05) in
the total glycogen levels determined during the exposure time
of the four tested concentrations. The values are expressed as
milligrams per gram.
Table 1. The 96-h LC50 values (gml
⫺1
) and respective 95%
confidence intervals of Kalliapseudes schubartii collected in the
wild and exposed to the reference substance (CdCl
2
)
Test 96-h LC50
I 0.70 (0.85–0.58)
a
II 0.70 (0.95–0.53)
III 0.94 (1.34–0.66)
Average 0.89
a
Confidence interval in parentheses.
Table 2. The 96-h LC50 values (mg ml
⫺1
) and respective 95%
confidence intervals of Kalliapseudes schubartii exposed to Micro-
cystis aeruginosa RST9501 aqueous extract (microcystin concentra-
tion in extract, 1094.07 gg
⫺1
)
Test 96-h LC50
I 1.42 (1.34–1.53)
a
II 1.50 (1.40–1.60)
III 1.41 (1.24–1.61)
Average 1.44
a
Confidence interval in parentheses.
Table 3. Average percentage mortality of Kalliapseudes schubartii
exposed to Aphanothece microsco´pica RSMan 9401 aqueous extract
at different concentrations (mg ml
⫺1
), n⫽40
Time (h) Control 0.65 0.84 1.09 1.42 1.85 2.45
24 0 00 5000
48 7.5 0 2.5 10 2.5 2.5 2.5
72 7.5 2.5 2.5 15 10 7.5 5
96 10 7.5 5 15 15 15 10
Table 4. LC50 values (mg ml
⫺1
) and respective 95% confidence
intervals of Kalliapseudes schubartii exposed for 10 days in sedi-
ment spiked with lyophilized Microcystis aeruginosa RST9501 (mi-
crocystin concentration in lyophilized material, 1086.41 gg
⫺1
)
Test 10-day LC50
I 1.71 (1.50–1.96)
a
II 1.86 (1.68–2.07)
Average 1.79
a
Confidence interval in parentheses.
466 W. Montagnolli et al.
Discussion
The results obtained for the toxicity tests with aqueous extracts
of Microcystis aeruginosa RST9501 showed that Kalliap-
seudes schubartii is equally sensitive (96-h LC50 ⫽1.44 mg
ml
⫺1
) compared with other crustacean species tested with the
same strain. The decapod crustacean Farfantepenaeus paulen-
sis has also been acutely tested with M. aeruginosa RST9501
extracts and showed a 24-h LC50 of 2.96 mg ml
⫺1
, while the
brine shrimp Artemia salina presented an 18-h LC50 of 4.73
mg ml
⫺1
(Yogui 1999).
The low mortality observed in the tests with the nontoxic
extracts of the cyanobacterium Aphanotece microsco´pica RS-
Man9401 confirms the toxic action of the Microcystis aerugi-
nosa toxins in the organism and also indicates the low sensi-
tivity of Kalliapseudes schubartii to other cell components
such as pigments and membrane lipopolysaccharides present in
the extract.
When the microcystin concentrations of the cyanobacterial
extracts were considered, a 96-h LC50 of 1.58 gml
⫺1
was
calculated for Kalliapseudes schubartii. This value is close to
the 48-h LC50 of 2.25 gml
⫺1
in the pink shrimp Farfante-
penaeus paulensis postlarvae reported by Salomon et al.
(1996). In addition, a value of 3.75 gml
⫺1
for 24-h LC50 was
found for Artemia salina when pure microcystin-LR (L-Leu
and L-Arg in positions 2 and 4) was used (Delaney and Wilkins
1995). Close values were observed in toxicity tests with mi-
crocystin-LR on the cladocera Daphnia pulicaria, D. hyalina,
and D. pulex (48-h LC50 of 21.4, 11.6, and 9.6 gml
⫺1
,
respectively) by DeMott et al. (1991). The same authors ob-
tained a microcystin-LR 48-h LC50 between 0.45 and 1 g
ml
⫺1
in tests with the copepod Diaptomus birgei.
Although all crustacea have a distinct liver, no research has
yet been done on the effects of hepatoxins and their functions.
To evaluate the effects of microcystins on organisms subjected
to either acute or chronic tests, the uptake mode of such toxins
must be considered. Microcystins are relatively polar mole-
cules with a low cellular membrane permeability and, there-
fore, require a transport system via bile acids to reach the
hepatocytes and liver protein phosphatases (Runnegar et al.
1995).
Protein phosphatases serve an important role in maintaining
homeostasis in cells (Cohen 1989). In liver cells, intermediate
filaments of the cytoskeleton can become hyperphosphory-
lated, leading to cellular disruption. The progressive loss of the
cell architecture disrupts cytoskeleton hepatocytes, leading to
the detachment of adjacent cells and sinusoidal capillaries.
Thus, blood accumulation in the liver causes a hemorrhagic
shock (Falconer and Yeung 1992; Carmichael 1994; Torvola
and Eriksson 1999).
Moreover, phosphatase inhibition maintains the glycogen
phosphorylase enzyme activity, causing a collapse in the or-
ganism’s energy supply (MacKintosh and MacKintosh 1994).
The total glycogen content in organisms exposed to sublethal
concentrations of Microcystis aeruginosa did not vary signifi-
cantly. These values in Kalliapseudes schubartii (average gly-
cogen concentration, 5.41 ⫾0.94 mg g
⫺1
) are very close to
those found in the postlarvae of Fanfantepenaeus paulensis
(average glycogen concentration, 3.62 ⫾0.47 mg g
⫺1
) (Pinho
2000) using the methodology described by Nery and Santos
(1993).
However, alterations were expected in the glycogen levels of
organisms exposed to Microcystis extracts, due to a possible
inhibition of protein phosphatases and, consequently, an in-
crease in glycogen breakdown. However, considering the low
toxin concentrations in the oxygen consumption tests, phos-
phatase inhibition by microcystins may have been insufficient
to unbalance the phosphorylation and desphosphorylation ra-
tios. It is not known how many protein phosphatase molecules
have to be inactivated by microcystins in a given incubation
period to cause alterations in cellular functions. It is also not
known whether there is further control of the synthesis and
degradation rates of phosphatase molecules. It may be, how-
ever, that the microcystin–phosphatase complex accumulates
in the tissues or is degraded (MacKintosh and MacKintosh
1994).
Studies on respiratory rates can reveal important aspects of
the metabolic energy states and internal equilibrium in crusta-
cea. In fact, oxygen consumption measurements can be very
useful indicators of the sublethal effects of compounds such as
cyanotoxins for purposes of validation and toxicology, espe-
Fig. 3. Oxygen consumption of K. schubartii exposed to sublethal
concentrations of M. aeruginosa extract during 24- and 48-h tests
Fig. 4. Glycogen levels of K. schubartii exposed to sublethal concen-
trations of M. aeruginosa extracts for 24 and 48 h
Effects of Microcystis aeruginosa on a Microcrustacean 467
cially because compounds like microcystins have never had
their effects tested on crustacea respiratory rates.
The presence of Microcystis aeruginosa extracts causes an
increase in the Kalliapseudes schubartii oxygen consumption
rate. The significant increase in oxygen consumption rates
observed in organisms exposed for 24 and 48 h to sublethal
concentrations of M. aeruginosa suggests an increase in the
total organismic energy expenses, probably due to the expen-
diture of homeostatic and enantiostatic mechanisms.
When an animal is exposed to a toxic agent, physiological
processes go into action to compensate for the toxic stress (the
detoxification process). As a consequence, an alteration in the
organism’s normal health may occur (Depledge 1989). In re-
lation to microcystins, detoxification would be related to the
tripeptide glutathione (GSH), through a conjugation reaction
catalyzed by GST (Pflugmacher et al. 1998). This conjugate
with microcystin covalently bound to glutathione has less in-
hibitory action on protein phosphatases and may be a charac-
teristic of the detoxification process (Metcalf et al. 2000). In
the same manner, the oxygen consumption rate increase after
48 h of exposure could be a result of activation caused by
higher concentrations of Microcystis aeruginosa extract. Pos-
sibly, the synthesis of new proteins during the detoxification
process, as well as the repair of toxin-damaged tissues, in-
creases the total organism energy expenditure, thus reflecting
the oxygen consumption.
The lyophilized Microcystis aeruginosa RST9501-spiked
sediment was highly toxic to the tanaidacean Kalliapseudes
schubartii (10-day LC50 of 1.79 mg ml
⫺1
). In the environment,
sediment contamination by cyanotoxins may occur at bloom
termination, when cyanobacterial cells can be deposited on the
bottom and margin sediment, and microcystins of intact cells
may persist for several months. This process of toxic debris
accumulation and persistence in sediment may be hazardous to
the benthonic biota. Once deposited on the sediment, cells are
subjected to bacterial action, lysis, and toxin transfer (Chorus
and Bartran 1999).
A sediment analysis in the environment of material collected
7 cm deep in the sediment surface at the Baia do Casamento
(northeast of Patos Lagoon, Brazil), for example, revealed the
presence of a cyanobacterial layer deposited several seasons
before. The sediment contained up to 0.12 gg
⫺1
of micro-
cystin contamination (Yunes 2000), a level not far from the
LC50 value obtained for the acute tests in the present work.
Considering that M. aeruginosa toxic blooms and scums accu-
mulate in the narrow mouth of the Patos Lagoon estuary, it is
probable that benthic organisms in the environment are sub-
jected to toxic cell deposition and bottom sediment toxicity.
Although this process has received only slight attention from
the scientific community, studies on microcystin interaction
with sediments and their respective biota and toxin presence
are fundamental to benthic community maintenance as well as
to environmental health control in regions like the Patos La-
goon estuary.
Despite the limitations involving extrapolation of laboratory
toxicity to the environment, the effects verified in this work are
from brief acute-mode tests compared to the organism life
cycle, however, they can be expected to happen in situ in the
environment. Lethal and sublethal effects resulting from
chronic exposure to the microcystin toxin might be expressed
for lower concentrations than those applied in the acute tests.
Matthiensen (1996) reported extracellular microcystin concen-
trations in Patos Lagoon estuary waters of up to 0.245 gml
⫺1
during Microcystis aeruginosa blooms. These data show that
extracellular toxin concentrations in the environment can reach
levels within the same range as those used for laboratory tests
during oxygen consumption determinations (0.366, 0.244, and
0.110 gml
⫺1
). These concentrations caused sublethal effects
in the organisms and were not so far from the 96-h LC50 value
of the 1.58 gml
⫺1
obtained in the acute tests.
The results demonstrate the potential risks of intoxication to
natural populations of Kalliapseudes schubartii in the environ-
ment, when heavy blooms are present in the estuary. This
tanaidacean is a very important organism in the Patos Lagoon
estuary trophic web, and if these and other similar species are
compromised by cyanobacterial toxins in the sediments, other
important fish and crustacea of economic importance to local
fisheries may also be affected.
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