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1

Toxicity of complex cyanobacterial samples and their fractions in
Xenopus
laevis

embryos and the role of microcystins


Blanka Burýšková
a
, Klára Hilscherová
b
, Pavel Babica
b
, Dagmar Vršková
c,d
, Blahoslav
Maršálek
b

, Luděk Bláha
b,

*


a
RECETOX


Resear
ch Centre for Environmental Chemistry and Ecotoxicology, Masaryk
University, Kamenice 3, CZ 625 00 Brno, Czech Republic

b
Centre for Cyanobacteria and their Toxins; Institute of Botany, Czech Academy of Science
and RECETOX, Masaryk University, Kamenice 3,
CZ 625 00 Brno, Czech Republic

c

Department of Biology and Wildlife Diseases,
University of Veterinary and Pharmaceutical
Sciences, Palackého 1
-
3, CZ 612 42 Brno, Czech Republic

d
Department of Pharmacology, Faculty of Medicine, Masaryk University, Komensk
ého nám.
2, CZ 662 43 Brno, Czech Republic



*
Corresponding author:
blaha@recetox.muni.cz






Phone: +420

549

493

194





Fax: +420549 492 840





2

Abstract

The object of this work was to evaluate the effects of various cyanobac
terial fractions
in Frog Embryo Teratogenesis Assay Xenopus (FETAX) with African clawed frog embryos.
Fractions were prepared from five biomasses with different dominant genera (
Microcystis
,
Aphanizomenon
,
Anabaena
,
Planktothrix
) and different microcystin
content. Effects of
following fractions were investigated: (I) homogenate of complex cyanobacterial biomass, (II)
cell debris (pellet) after centrifugation of complex biomass, (III) supernatant after
centrifugation of complex biomass (= crude aqueous extra
ct), (IV) permeate after passing of
crude extract through C
-
18 column (fraction devoid of microcystins), (V) eluate from C
-
18
column (containing microcysins, if present). Besides classical parameters evaluated in 96 h
FETAX (mortality, growth inhibition, m
alformations), we have also assessed the effects on
biochemical markers of oxidative stress and detoxification (glutathione pool, GSH; activity of
glutathione peroxidase, GPx, and glutathione reductase, GR, activity of glutathione S
-
transferase, GST). Comp
lex biomass (I) and aqueous extract (III) were generally the most
toxic fractions in terms of mortality and growth inhibition, whereas eluates containing
microcystins (V)

were generally less toxic. On t
he other hand, the same fraction (eluates)
induced sig
nificant malformations in low concentrations but the effects were not related to the
content of microcystins. Biomarkers were affected in variable manner but no significant effect
or clear relation to microcystin content was observed. Our data support the
hypothesis that
microcystins are not the only or major toxic compounds in the complex cyanobacterial
samples and that more attention should be paid to other components of complex
cyanobacterial biomass including
non
-
specific parameters such as oxygen conte
nt or toxic
ammonia released during bacterial decay of organic material.


Key words:
FETAX; Xenopus laevis; Malformations; Cyanobacterial fractions; B
iomarkers



3


1.

Introduction


Along with continuous worldwide anthropogenic water eutrophication,
th
e appearance
of massive water blooms has become a worldwide problem in the last decades. Blooms
dominated by cyanobacteria
(blue
-
green algae)
have severe impacts on ecosystem health and
also on water quality as many cyanobacterial species synthesize a wide

range of odours or
undesirable tastes.
Additionally, cyanobacteria produce various other biologically active
compounds such as toxins, attractants, antibiotics or hormone
-
like

chemicals
(Nowak et al.,
1988; Falconer, 1989
;

Renstrom and Bergman, 1989
)
. Cya
notoxins are of growing
environmental as well as health concern and were shown to cause serious adverse effects in
human as well as aqu
atic organisms

(Codd et al., 1989).

Cyanobacterial toxins can be
categorized according to the effect as neurotoxins, hepa
tot
oxins, cytotoxins,

dermatotoxins,
genotoxins and
irritant toxins

and they were also shown to play an important
role in the
carcinogenesis
(Svrcek

and Smith
, 2004)
.

The most common and the most widely investigated cyanotoxins are microcystins that
are p
roduced by species within genera
Anabaena, Microcystis, Nostoc, Oscillatoria,
Planktothrix
et
c.
(Sivonen and Jon
es, 1999)
.
The

toxicology and ecotoxicology of

microcystins have been investigated in detail

(Dawson, 1998; Duy et al., 2000; Wiegand

and
Pflugm
acher
, 2005
; Zurawell et a
l., 2005)
but several recent findings indicate that
cyanobact
erial water blooms contain as
-
yet
-
unidentified

components that can evoke more
pronounced toxic effects than microc
ystins
or other well
-
recognized cyanotoxins

(Jungmann
a
nd Benndo
rf, 1994
;

Oberemm et al., 1997; Oberemm et al., 1999; Pietsch et al., 2001
)

In natural environments, surface aggregations of some cyano
bacteria may accumulate
to scum

with high cell density and toxin concentrations. This phenomen
on

often occur
s

in

shallow littoral areas, which are the primary environments for the early life stages of aquatic

4

vertebrates. Such extreme conditions can lead to significant exposures and various chronic
effects or event deaths of aquatic organisms
(Sivonen and Jones, 199
9; Chorus, 2001).
Early
-
life stage development plays a crucial role in the ontogenesis of aquatic organisms and the
embryolarval tests that model these steps of development were successfully used to study
cyanobacterial toxicity in fish as well as amphibia
ns. One of such assays, Frog Embryo
Teratogenesis Assay Xenopus
(
FETAX) is established and highly reproducible assay for
evaluating embryotoxicity and teratogenicity of environmental contaminants in amphibians

(Bantle et al., 1996).

Besides the traditiona
l toxicological parameters, biochemical markers might be used to
investigate sub
-
lethal toxic effects

and they can give a good indication of potential sub
-
lethal
toxicity and inform about possible mechanism of action.
Oxidative stress, i. e. pathological
p
rocesses related to overproduction of reactive oxygen species (ROS) is one of important
toxicity mechanisms of many xenobiotics

(Klaunig et al., 1998).
Several oxidative stress
biomarkers have been studied and they include: (i) direct assessment of ROS ove
rproduction
after exposure to tested chemicals (suitable mostly for in vitro studies due to short lifetime of
ROS),
(ii) measurement of
concentrations of non
-
enzymatic antioxidants
(such as glutathione,
GSH) or
activity of
detoxification

or
antioxidant enz
ymes

(i.e. glutathione
-
S
-
transferase,
GST;

glutathione peroxidase, GPx

or glutathione reductase, GR),

(iii)
determination

of
oxidation products of biological molecules such as
measurement of lipid peroxidation product
malondialdehyde (MDA).

Assessment

of
the

biomarkers as early warning of adverse changes
and damage was shown to be a suitable tool in various

organisms including fish
(Van der
Oost et al., 2003; Blaha et al., 2004)
or amphibians and their embryos

(Cavas and Tarhan,
2003; Venturino et al., 200
3).

As there are numerous contradictory information on the toxicity of cyanobacterial
samples
containing cyanotoxins
to early life stages of aquatic organisms, we have investigated

5

ecotoxicological effects of various fractions prepared from several comple
x cyanobacterial
blooms that varied in composition and in the content of microcystins on mortality,
malformations, growth inhibition and changes in biochemical markers in African clawed frog
(
Xenopus laevis
).
Our results demonstrate highly significant toxi
cities of complex
cyanobacterial samples to frog embryos but they indicate only minor role of microcystins.


2.
Material and methods


2.1.
Samples


Five different natural cyanobacterial water blooms were collected with plankton net
from natural reservoirs
and stored frozen at
-
18°C. Origin of the samples and the details on
the dominant cyanobacteria are given in Table 1. The concentrations of microcystins were
determined by HPLC according to the method described by
Lawton et al.

(
1994)
and
previously used i
n

our laboratory
(Babica et al.
, in press
)
.

Content of microcystins (µg/L) in
individual fractions is shown in Table 2.
C
omplex cyanobacterial bloom was homogenized by
sonication

and the biomass homogenate including fragments of cells was used as fraction
I for
further experiments (complex homogenized biomass). A portion of the homogenized biomass
was centrifuged and
other two fractions were separated
and stored frozen: fraction II
containing cell debris (pellet with for example cell bound lipopolysaccharid
es) and fraction III
-

crude aqueous extract (supernatant). A portion of the aqueous extract was further
fractionated by solid phase extraction (SPE) using a C18 column (Waters SepPak 35cc 10g).
Permeate from the cartridge, i.e. polar fraction devoid of mi
crocystins was collected (fraction
IV, "permeate"). Methanolic eluate from SPE containing less polar compounds including
microcystins was evaporated under vacuum and the residue was re
-
dissolved in the distilled

6

water to reach the volume of the original sa
mple before SPE (fraction V, "eluate"). Effects of
all fractions prepared from one of the biomass sample "A" (see Table

3
) were evaluated in
detail. For other biomass samples (B
-
E, Table 4), only fractions III (aqueous extract) and V
(SPE eluate containing

microcystins) were chosen for toxicological testing. Equivalent
amounts of homogenates from the leaves of spinach (
Spinacea oleracea
) were assessed as a
reference complex sample (fraction "biomass control") to have a control on possible non
-
specific effec
ts such as
decrease in oxygen content or formation toxic ammonia by bacterial
degradation of the biomass.
Final concentrations of tested samples were

expressed in
equivalents of initial cyanobacterial biomass on the dry weight basis (mg dw/L).


2.2.
Bioas
say


Frog embryos were obtained from adult pairs of
Xenopus laevis

injected with human
chorionic gonadotropin (HCG; N.V. Organon, Oss, Holland) in the dorsal lymph sac
(females: 300IU; males: 150IU). Amplexus normally ensued within 2 to 6 h and the
deposit
ion of eggs occurred from 9 to 12 h after injection. Embryo toxicity

tests were
conducted using the standard guide for the Frog Embryo Teratogenesis Assay


Xenopus

(ASTM, 1998)
. Only mid
-
blastula (stage 8) to early gastrula (stage 11) embryos
(Nieuwkoop
a
nd Faber, 1994)

were selected for testing. Groups of 25 embryos were randomly placed in
covered 60
-
mm plastic Petri dishes in 10 mL of standard FETAX solution (625 mg NaCl, 96
mg NaHCO
3
, 30 mg KCl, 15 mg CaCl
2
, 60 mg CaSO
4
∙2H
2
O, and 75 mg MgSO
4

per L of
di
stilled water; pH 7.6
-
7.9) and exposed to the samples of interest. The dilutions of individual
experimental fractions were prepared in distilled water and applied at concentrations
corresponding to 50, 100, 200, 400 and 800 mg biomass dry weight .L
-
1
. Each

concentration
was tested in three parallels. Control group (C
-
FETAX) was exposed to the standard FETAX

7

medium only. Homogenate of spinach leaves was used as a reference material (non
-
toxic
biomass control) at the two highest concentrations 400 and 800 mg
biomass dry weight .L
-
1
.
The assay was performed at 23°C±1°C for 96 h, exposure solutions were changed every 24 h,
and dead embryos were recorded and removed. At the end of the assay (96 h) surviving
embryos were fixed in 3% v/v formaldehyde, assessed for

morphological abnormalities under
the dissecting microscope and the length of embryos was determined by a ruler. For
biochemical analyses portion of embryos was kept at
-
80°C and they were then homogenized
on ice in phosphate buffer saline (PBS, pH 7.2).
Supernatant was collected after

centrifugation (5 min at 2500 g at 4°C), and stored

at
-
80°C prior to analysis. All treatment
variants were assessed for mortality, malformations and growth inhibition. Effects on
biochemical markers were determined only for

biomass A for which detailed investigation of
all fractions was available.


2.3.
Biochemical analyses


Glutathione S
-
transferase activity was measured
spectrophotometrically using 1

mM 1
-
chloro
-
2,4 dinitroben
zene (CDNB) as a substrate and 2

mM GSH in phos
phate buffer saline
(PBS, pH 7.2), according to t
he method of
Habig et al. (1994)
. Specific activity was expressed
as nmoles of formed product per minute

per milligram protein.

Concentra
t
ion of glutathione was determined accordin
g to the method of
Ellman
(1959)
,
using DTNB (5,5´
-
dithiobis
-
2
-
nitrobenzoic acid) as a substrate. Samples were treated with
trichloracetic acid (TCA, 25 % w/v) and centrifuged (6000 g for 10 min).
50 µL of
s
upernatant was mixed with
230 µL of
TRIS
-
HCl buffer (0.8 M TRIS/HCl, 0.02 M

EDTA,
pH 8.9) and
20 µL of
0.01 M DTNB. Reaction mixture was incubated for 5 min at a room

8

temperature, absorbance of GSH
-
DTNB conjugate was determined at 420 nm and
concentrations (nmol GSH/mg protein) calculated according to the standard calibration.

A
ctivities of glutathione peroxidase
(Flohé and Gunzler, 1984)

and glutathione
reductase
(Carlberg and Mannervik, 1986)
were measured as the decrease of the absorbance
at 340nm due to oxidation of NADPH.

GPx activity was assayed in microplates with final
co
ncentrations of 3 mM GSH, 1

U/mL GR, 0.15 mM NADPH and 1.2 mM BH
P (t
-
Butyl
hydroperoxide) in 0.02

M potassium phosphate
/0.2

mM EDTA buffer (pH 7). GR activity
was assayed with 0.05 M

potassium phosphate/1 mM EDTA buffer (pH 7), 1 mM oxidized
glutathione (G
SSG) and 0.1 mM NADPH.
Specific activities of both GPx and GR were
expressed as nmoles NADPH oxidized per minute per milligram protein.

Protein concentrations were determined according to the method of
Lowry et al. (1951)
using bovine serum albumin as a st
andard. The assay was modified for use on a microplate
spectrophotometer and absorbance at 680 nm was measured.


2.4.
Statistical evaluation


Concentrations causing 50% lethality (LC
50
) and concentrations eliciting
malformations in 50% of surviving embryo
s (EC
50
) were estimated with probit model.
Differences among the total embryo lengths were evaluated by ANOVA and Dunnett test.
The homogeneity of variances prior to ANOVA was assessed by the Levene’s test. The 95%
confidence intervals were used as the mea
sure of variability. The teratogenic index (TI) was
calculated as a ratio of LC
50

and EC
50

for tested samples. Differences in frequencies of
malformations were compared by chi
-
square test.
P values less than 0.05 were considered
statistically significant
.



9

3.
Results


3.1. Mortality and malformations


Signi
ficant toxicity and teratogenicity in

frog embryos were observed and they
differed with respect to various cyanobacterial fractions and amount of microcystins. The
LC
50
values are shown in Table 3 (biomas
s A and its fractions) and in Table 4 (biomasses B,
C, D and E) and the concentration dependent effects of biomass A on frog embryo mortality
are presented in Fig. 1
A
. The most toxic fractions of biomass A were the complex biomass
(fraction I) and aqueous
extract (fraction III) with the LC
50
values of 150 mg dw/L and 235
mg dw/L, respectively. Only weak lethal effects were recorded at fractions II (pellet) and IV
(permeate). In general, very low nonsignificant mortality was observed for eluate containing
mi
crocystins
(
fraction V) in all tested biomasses (up to 10 %, see Tables 3 and 4). Control
variant with the non
-
toxic complex biomass (spinach leaves) showed only weak toxicity in
400 mg dw/L concentration but mortality about 45
%
was determined in the highe
st tested
concentration 800 mg dw/L

(Fig. 1A)
.

As for malformations, all fractions of biomass A significantly induced developmental
abnormalities (the LOEC for all fractions was the lowest tested concentration 50 mg dw
/L;
Table 3
, Fig. 1B
).

The strongest
effects had the fraction IV
(
permeate without detectable
microcystins
;
EC
50

= 150 mg dw/L; Table 3). Both tested fractions (aqueous extract and
eluate) of other biomasses
(
B, C, D, E
)

caused significant malformations with
the exception of
t
he eluate of bio
mass B (Table 4). Also the complex non
-
toxic biomass lead to malformations
at 66
% o
f
embryos in the highest tested concentration 800 mg dw/L

(Fig. 1B)
. Oedema,
adverse curving of backbone, eye and mouth deformities were the most often observed
malformation
s at cyanobacterial extracts. According to FETAX methodology, teratogenic

10

indexes (TI) were calculated as ratios of LC
50

and EC
50

and three significant values
(TI

>

1.5
)
were

derived for permeate of biomass A (Table 3) and aqueous extracts of biomasses D a
nd E
(Table 4).


3.2. Growth inhibition


Significant growth reductions were recorded after exposure to all fractions of biomass
A except the eluate (fraction V; Table 3). Testing of other biomasses confirmed some
inhibitory effects at several aqueous extra
cts but only minor effects of eluates,
i.e. fractions
containing microcystins (Table 4).
However, concentrations causing growth inhibitions were
relatively high and comparable to the LC
50
values (compare LOEC for growth inhibition with
LC
50

in Tables 3 and

4).


3.3 Alterations of biomarkers


Slight changes in biochemical markers were recorded after exposure of frog embryos
to cyanobacterial samples but there was no significant and clear concentration dependent
effect. Activities of detoxification and antiox
idant enzymes as well as levels of glutathione
were both decreased or increased at different exposures. As an example, the effects of
aqueous extract, permeate and eluate of biomass
A on biomarkers

are shown in Fig.
2.


4.
Discussion

and conclusion


One of

the major objective
s of this work was to provide
detailed information on a role
of microcystins in ecotoxicity of complex cyanobacterial blooms. Although there has been a

11

significant amount of research on toxicity of microcystins including the effects on
aquatic
organisms, the results of various experimental setups are
often contradictory

and several
studies demonstrated microcystin
-
independent toxicities
(Oberemm et al., 1997; Best et al.,
2001; Pietsch et al., 2001)
.

In our experiments, we tried to simul
ate ecosystem situation when
developing stages of aquatic organisms are exposed to complex cyanobacterial blooms. We
studied the effects of complex cyanobacterial biomasses and their fractions (varying by
composition of dominant cyanobacteria and the conte
nt of microcystins) on development of
Xenopus laevis
embryos at concentrations typical for the naturally occurring cyanobacterial
water blooms in the Czech Republic
(Marsalek et al., 2001).

Cyanobacteria often cumulate in
littoral zones and create thus exp
osure situations for early
-
life stages of amphibians and fish
(Wiegand et al., 1999)
that might be more affected than juveniles or adults due to their lower
mobility and limited ability to avoid contamination
(Oberemm et al., 1999).

Our experiments showed
high mortalities of embryos exposed to complex biomass and
to crude aqueous extract of cyanobacterial water blooms. On the other hand, other fractions
(cellular pellet, permeate and eluate) had generally minor effects on viability of tadpoles (s
ee
Fig. 1
A
and LC
50

values in Tables 3 and 4). Besides lethal effects, cyanobacteria also
significantly induced malformations in surviving embryos and inhibited growth. However, in
most cases, non
-
lethal effects occur
r
ed in concentrations comparable to LC
50

(Fig. 1A
and 1
B,
Table 4) and significant teratogenic indexes (TI

>

1.5) could be calculated for only three
samples (Tables 3, 4). Taken together, our results indicate that cyanobacteria significantly
affect both viability and development of frog embryos but there
is lower risk of some specific
mechanisms leading to "
teratogenic" effects that prece
d
e

lethality at lower concentrations.

Our studies also indicate that microcystins in general play only minor role in both
lethal and sub
-
lethal effects of cyanobacteria.
There was no clear relationship between the
effects of aqueous extracts from biomasses A
-
E differing in content of microcystins (Tables 2,

12

4). Further, as shown at Table 3 and Fig. 1 for a model biomass "A", there was no elevated
toxicity of eluate (i.e. f
raction with concentrated microcystins prepared by SPE) in
comparison with the permeate fraction (extract devoid of microcystins). Similarly, the effects
of eluates containing microcystins prepared from other biomasses were much less pronounced
than the ef
fects of corresponding complex crude aqueous extract and they were independent
from the microcystin content (Table 2 and 4).
Our observat
ions correspond to the study of
Fischer and Dietrich (2000)
who observed no mortality, malformations or growth inhibiti
ons
in
Xenopus laevis

embryos exposed for 96h to purified microcystins up to 2000 µg/L.
However, prolonged exposures to microcystins (that included feeding periods of larvae) lead
to the uptake of toxins demonstrating thus necessity of the oral uptake for
the toxicity of
microcystins
(Fischer and Dietrich, 2000).
Further,
Oberemm et al. (1999)
observed only a
delay in feeding of axolotl larvae (
Ambystoma

mexicana
) exposed to 5 and 50 µg/L of
microcystin
-
LR. In their study no mortalities or morphological dev
elopmental changes were
observed at axolotl as well as other amphibian species
-

smooth newt (
Triturus vulgaris
) and
marsh frog (
Rana ridibunda
). Similarly, no developmental tox
icity of microcystin
-
LR (up to
20

000 µg/L) was observed in experiments with to
ad
Bufo arenarum

(Chernoff et al., 2002).
Partially contradictory results were reported by
Dvorakova et al. (2002)
who observed
developmental malformations at
Xenopus

laevis
embryos exposed to 25
µ
g/L (and higher
concentrations) of purified microcystin
-
LR.

However, more pronounced effects were
observed after exposures to complex cyanobacterial extracts regardless of microcystin
content. Our observations of generally minor role of microcystins in overall toxicity to aquatic
vertebrates are also supported by
a study of
Best et al. (2001)
with
brown trout

alevins

(
Salmo
trutta
). Authors showed significant effects of complex aqueous extracts of the
Microcystis

sp.
strain on larval cardiac development but no changes were recorded at variants with purified
microcy
stin only.
As reported by

Oberemm et al. (1997, 1999)
, exposure to crude

13

cyanobacterial extracts containing microcystins had generally far more pronounced effects
on
fish and amphibian embryos (malformations or

mortalities)
, if

compared to those obtained
w
ith pure toxins.
Furthermore, Keil et al. (2002)

showed that toxicity of
Planktothrix
extracts
to larvae of
Danio

rerio
did not correspond to microcystin content and other compounds were
responsible for
the
observed effects.
Several studies also described
embryotoxicity and/or
teratogenicity of cyanobacterial species or strains non
-
producing microcystins at all, e.g.
Aphanizomenon flos
-
aquae
(Papendorf et al., 1997),
Planktothrix
sp. FP
-
1
(Prati et al., 2002)
,
Planktothrix agardhii

NIVA
-
34
(Keil et al., 200
2)
, or
Fischerella ambigua

(Wright et al., in
press
)
.

The general lack of the stronger effects can be caused by limited uptake of microcystin
by embryos, because severe impairment of embryonal development has been observed in fish
eggs after microinjection

of microcystins
(Jacquet et al., 2004; Wang et al., 2005)
.
On the
other hand, other studies with fish indicate that
also immersion exposure to
microcystins
might affect development of these aquatic vertebrates. For example, in the study with zebra
fish (
D
anio rerio
)
Oberemm et al. (1997)
reported retarded larval growth already at 0.5 µg/L
of purified microcystin
-
LR and decreased survival was further observed at embryos exposed
to 5 and 50 µg/L.
Liu et al. (2002)
observed increase of developmental abnormali
ties and
mortality together with hepatotoxicity and cardiotoxicity in embryos and larvae of loach
Misguruns mizolepis
exposed to dissolved microcystin
-
LR, whereas juveniles were less
sensitive
. Fish embryotoxicity and/or teratogenicity of microcystin
-
produ
cing cyanobacteria
have been clearly demonstrated by several studies (Ojaveer et al., 2003
; Palikova et al., 2003;
Palikova et al., 2004)
, but the responsibility of microcystins for observed effects remains
disputable.

However, aquatic organisms are not na
turally exposed to purified toxins but to
complex water blooms, and other hydrological factors (not solely related to cyanobacteria) are

14

known to have severe impacts such as low concentrations of dissolved oxygen depleted by
bacterial metabolism of decompo
sing biomass
(Barica, 1978; Snyder et al., 2002; Vos and
Roos, 2005)
.

In our study, the non
-
toxic biomass control (extract of spinach leaves) induced
significant toxicities and malformations at h
igher tested concentrations as

indicates that
effects associa
ted with bacterial decay of general organic substrate can play a significant role
in the toxicity of complex cyanobacterial samples. Correspondingly, the results of some
previous studies that related toxic effect directly to microcystins but they were base
d on
testing complex cyanobacterial extracts
or biomass (Vinagre et al., 2003;

Palikova et al.,
2003; Palikova et al., 2004; Pichardo et al., 2006)

might be affected by such parameters
. In
summary, aside from the presence of numerous secondary cyanobacteri
al metabolites, basic
water quality parameters such as dissolved oxygen, pH or ammonia concentrations should
carefully be considered when testing toxicities of complex water blooms
(Seymour, 1980;
Kopp and Hetesa, 2000).

Our study also indicated possible
synergistic effects among the components of
complex aquatic extract. After SPE fractionation of the aqueous extract significant decrease in
toxicity was observed as no significant mortalities were observed at eluate or
most of
permeate fractions (Table 3,
Fig. 1
A
). Although removal of some toxic compounds during the
SPE cannot fully be excluded (such as degradation of
unstable
chemicals or weaker efficiency
of eluting solvent), possible synergistic interactions among compounds present in both
fractions coul
d explain this observations. Similar to our results,
Wright et al. (in press
)
observed loss of embryotoxicity in
Danio rerio

exposed to fractions prepared from exudates
of terrestric cyanobacterium
Fischerella ambigua
. Further, modulation of microcystin ef
fects
by lipopolysaccharides was also demonstrated by co
-
exposure experiments with embryos of
Danio rerio

(Best et al., 2002)
.

Taken together, there are clear evidences that various

15

components of complex cyanobacterial water blooms might elicit synergistic

toxic effects and
further research could provide detailed insight into the cyanobacterial toxicity.

One of important toxicity mechanisms playing a role
in
cyanobacterial toxicity is
oxidative stress, i.e. consequences of reactive oxygen species (ROS) ove
rproduction. Most of
available studies focused on responses of antioxidant system or detoxification enzymes afte
r
exposure

of
fish
(Blaha et al., 2004; Jos et al., 2005; Prieto et al., 2006; Cazenave et al., 2006)
to purified microcystins
or microcystin
-
co
ntaining cyanobacteria
but to the best of our
knowledge there is no information on oxidative stress induced by cyanobacteria in
amphibians. Some of the documented effects related to oxidative stress induced by purified
microcystins or crude cyanobacterial
extracts were depletion of intracellular glutathione
(Li et
al., 2003)
,

changes in mitochondrial function
and apoptosis induction
(Ding and Ong, 2003)
,
lipid peroxidation and cell damage
(Ding et al., 1998)
or
modulations of

glutathione S
-
transferase, cata
lase, superoxiddismutase and glutathione peroxidase
(Pietsch et al., 2001; Li
et al., 2003).
In our study we examined
modulation of specific biomarkers related to oxidative
stress and detoxification (i.e. concentrations of glutathione and activities of
glu
tathione S
-
transferase, glutathione peroxidase

and glutathione reductase) in
Xenopus

laevis
embryos
exposed to samples of
Microcystis aeruginosa
domina
ted water bloom (Fig. 2
). However, our
results did not reveal an
y
significant changes in studied biomarke
rs and there were only slight
alterations in measured parameters (both inductions and inhibitions). Literature also shows
variable results in biomarkers responses to cyanobacterial samples. For example, in vitro
studies with carp hepatocytes have shown sig
nificant increases in ROS production, elevations
in activities of superoxidismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and
decrease in glutathione (GSH) levels but authors observed no modulations of GST activity
(
Li
et al., 2003)
. Only w
e
ak responses of GST to microcystin
-
LR were observed in early life
stages of zebra fish
Danio reiro

(Wiegand et al., 1999). Pietsch et al. (2001)
reported

16

significant inductions of GST in zebra fish (
Danio reiro
) after exposures to the pure
microcystins but

suppressions of enzymes activities were observed after exposures to the
crude
cyanobacterial
extract

of
M
icrocystis

aeruginosa
dominated bloom, probably due to
presence of lipopolysaccharides
. On the contrary, reduction of

GST activities in liver of
juven
ile goldfish (
Carassius auratus
)

after
intraperitoneal injection of

microcystin
-
LR was
observed in the study of
Malbrouck et al. (2003)
. In summary, complex cyanobacterial
samples as well as purified microcystins apparently affect detoxification and antiox
idant
responses in aquatic vertebrates but there is a significant

variability depending on various
parameters such as species and the life stage investigated, exposure scenario etc. Further,
there is still limited and controversial information on
functions
, activities and induci
bilities

of
detoxification ap
p
aratus during the early development stages
(Sparling et al., 2000).

In conclusion, our results demonstrate significant embryotoxicities of various
cyanobacterial fractions in
Xenopus laevis

embryos. How
ever, the effect
s

were not related to
the amount of micr
ocystins and other factors seem

to play important role in the toxicity of
complex cyanobacterial samples. Our study indicates possible synergistic interactions among
various components of cyanobacteri
al blooms as demonstrated by significant decrease in
toxicity after SPE fractionation. Assessment of control non
-
toxic biomass also pointed out
importance of non
-
specific water quality parameters that affect toxicity results such as
depletion of oxygen or
release of toxic ammonia during bacterial decay of external organic
substrate. Our results support a hypothesis that cyanobacterial blooms might be a factor
contributing to worldwide
amphibian decline.






17

Acknowledgements


Research was supported by the G
rant Agency of the Czech Academy of Sciences
(KJB6005411) and the project IM6798593901 of the Ministry of Education, Czech Republic.


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25

Table 1

Characterization of
cyanobact
erial samples used in the study



Biom
ass

Locality

Date

Dominant species

Content of
microcystins
(MCs)

A

Brno reservoir

1/10/1999

Microcystis aeruginosa
(98%)

Microcystis wesenbergii
(2%)

2600 µg/g dw
(MC
-
LR 48%;
MC
-
YR 9%;
MC
-
RR 7%; MC
-
LW 1%;
unidentified MCs
35%)

B

Fraumuhln

29/7/1996

Micr
ocystis wesenbergii
(85%)

Microcystis
sp. (15%)

140 µg/g dw
(MC
-
RR 100%)

C

Jedovnice

19/8/1998

Anabaena spiroides

free of
microcystins

D

Skalka

14/8/1997

Aphanizomenon flos
-
aquae
(70%)

Microcystis viridis
(30%)

715 µg/g dw
(MC
-
LR 80%;
MC
-
YR 20%)

E

Dubic
e

8/9/2004

Planktothrix aghardii

2600 µg/g dw
(unidentified
MCs)




26

Table 2


Concentrations of microcystins (µg/L) in individual fractions (equivalents corresponding to
t
he highest tested concentration

800 mg dw.L
-
1
)



Fractions

Biomass No.

complex
bioma
ss (I)

pellet (II)

aqueous
extract (III)

permeate (IV)

eluate (V)

A

2048

179

1761

< LOD*

1622

B

56

< LOD*

22

< LOD*

56

C

< LOD*

< LOD*

< LOD*

< LOD*

< LOD*

D

572

126

397

< LOD*

465

E

1832

89

1396

< LOD*

1697


* LOD (limit of detection) =

13 µg/L




27

T
able 3


LC
50

values (mg dw/L), EC
50

values (mg dw/L), LOEC for malformations (mg dw/L), LOEC
for growth inhibition (mg dw/L) and TI (teratogenic index) for individual fraction
s of biomass
A (Brno reservoir);

n.s.


non significant (TI < 1.5)


Fraction

LC
50


(mg dw/L)

EC
50


(mg dw/L)

LOEC for
malformations
(mg dw/L)

LOEC for
growth
inhibition

(mg dw/L)

TI
-

teratogenic
index

(LC
50
/EC
50
)

complex
biomass (I)

150

250

50

100

n.s.

pellet (II)

> 800

> 800

50

400

n.s.

aqueous
extract (III)

235

324

50

400

n.s.

permeate (IV)

> 800

150

50

800

> 5.3

eluate (V)

> 800

652

50

> 800

n.s.




28

Table 4

LC
50

values (mg dw/L), EC
50

values (mg dw/L), LOEC for malformations (mg dw/L), LOEC
for growth inhibition (mg dw/L) and TI (teratogenic index) for fractions aqueous extr
act and

eluate of all tested biomasses;

n.s.


non significant (TI < 1.5)


Biomass

Fraction

LC
50

(mg dw/L)

EC
50

(mg dw/L)

LOEC for
malformations
(mg dw/L)

LOEC for
growth
inhibition

(mg dw/L)

TI
-

teratogenic
index

(LC
50
/EC
50
)

Brno
reservoir
(A)

aqueous
e
xtract

235

324

50

400

n.s.

eluate

> 800

> 800

50

> 800

n.s.

Fraumuhln
(B)

aqueous
extract

278

332

400

400

n.s.

eluate

> 800

> 800

> 800

> 800

n.s.

Jedovnice
(C)

aqueous
extract

186

205

50

200

n.s.

eluate

> 800

> 800

50

100

n.s.

Skalka (D)

aqueous
extract

358

100

50

200

3.6

eluate

> 800

> 800

50

> 800

n.s.

Dubice (E)

aqueous
extract

352

209

100

400

1.7

eluate

> 800

> 800

50

> 800

n.s.



29






Fig.
1


A

B


30





Fig.
2











A

B

C

D


31

Fig. 1.
Effects of various

cyanobacterial samples on mortality (A) and number of
malformations (B) in
Xenopus

laevis
embryos in 96h FETAX. Presented are the results of
testing fractions from biomass "A" (Brno reservoir dominated by
M. aeruginosa
).

Bars
represent means ± standard de
viation from two independent experiments, each performed in
three parallels. Asterisks (*) indicate statistically significant increase in mortality in
comparison with the control (Chi
-
square, P
<
0.05).



Fig. 2. Levels of reduced glutathione (GSH) (A), ac
tivity of glutathione S
-
transferase (GST)
(B), activity of glutathione peroxidase (GPx) (C) and activity of glutathione reductase (GR)
(D) in tissue homogenates from
Xenopus laevis

embryos after 96h exposure to cyanobacterial
fractions (biomass "A" from Brno reservoir dominated by
M. aeruginosa
).