Toxicon 55 (2010) 1244–1254

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Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Chronic effects of cyanobacterial toxins on Daphnia magna and

their offspring
Thanh Son Dao a, b, Lan-Chi Do-Hong c, Claudia Wiegand b, d, *
a
Institute for Environment and Resources, 142 To Hien Thanh Street, District 10, Ho Chi Minh City, Vietnam
b
Leibniz Institute of Freshwater Ecology and Inland Fisheries, Mueggelseedamm 301, 12587 Berlin, Germany
c
Vietnam National University- HoChiMinhCity, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam
d
University of Southern Denmark, Institute of Biology, Campusvej 55, 5230 Odense M, Denmark

a r t i c l e i n f o a b s t r a c t

Article history: The zooplankton grazer Daphnia magna endures living in water bodies up to moderate
Received 10 July 2009 densities of cyanobacteria, such as Microcystis spp., known for producing toxic secondary
Received in revised form 14 December 2009 metabolites. Although daphnids are affected via decreased food filtering, inhibition of
Accepted 26 January 2010
digestive proteases and lethality, development of tolerance against cyanobacterial toxins
Available online 2 February 2010
has also been observed. Aim of our study was to investigate in detail chronic effects of
cyanobacterial toxins, with emphasis on microcystin, on D. magna. The animals were
Keywords:
exposed chronically for two generations to either microcystin-LR in 5 or 50 mg L1, or to
Chronic effects
Daphnia magna cyanobacterial crude extract containing the same amount of total microcystin, starting at
Microcystin neonate stadium. Survival, growth, maturation and fecundity were observed for the first
Cyanobacterial crude extract generation during two months. In the offspring survival, maturation, and growth were
Life traits followed for the first week.
Malformation Low concentration of microcystin-LR slightly affected the growth and reproduction of
parent daphnids. Survivorship decreased during chronic exposure with increasing
microcystin concentration. Age to maturity of the offspring increased and their survival
decreased after parent generation was exposed to the toxin, even if the offspring were
raised in control medium. Besides, cessation of the eggs/embryos was observed and
malformation of neonates caused by cyanobacterial toxins was firstly recorded.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Banker and Carmeli, 1999; Rohrlack et al., 2003). Most of

In nature, cyanobacteria are not only low nutrient food
for zooplankton (Lampert, 1977; Müller-Navarra et al.,
2000; Von Elert et al., 2003) but also difficult to consume
due to commonly big colonial or long filamentous forma-
tion and mucilage production (Rohrlack et al., 1999; Ebert,
2005). Furthermore, cyanobacteria are capable of
producing toxic metabolites and bioactive compounds such
as microcystin (MC), anatoxin-a, cylindrospermopsin,
microviridin J or microcin SF608 (Sivonen and Jones, 1999;
* Corresponding author at: University of Southern Denmark, Institute
of Biology, Campusvej 55, 5230 Odense M, Denmark. Tel.: þ45 6550 2785.
E-mail address: wiegand@biology.sdu.dk (C. Wiegand).
those bioactive metabolites are contained inside the cells
and released into the water during cell lysis. Zooplankton
directly suffer from toxic cyanobacteria as they are primary
consumers feeding on suspended particles, and phyto-
plankton including small cyanobacteria. Zooplankton
accumulated MC at concentrations ranging from 0.3 to
16.4 mg g1 dried weight (DW) which is much higher than
present in natural water, from undetectable 5.8 ng g1
DW (Ferrão-Filho et al., 2002a). The negative correlation
between the density of cladoceran and the biomass of
cyanobacteria producing MC was recorded in the field
reflecting the nutritional and toxin effects on the animals
(Ferrão-Filho et al., 2002b). Similarly, Hansson et al. (2007)
found a clear negative correlation between MC in water and

0041-0101/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2010.01.014
 T.S. Dao et al. / Toxicon 55 (2010) 1244–1254
total zooplankton biomass. In addition, big cladoceran had
an apparently negative response while smaller cladoceran
seemed to have a weakly positive response to MC. This was
assumed as the effects of toxic cyanobacteria which
induced a shift in zooplankton size and community
composition in nature (Hansson et al., 2007).
Considerable numbers of laboratorial studies investi-
gated acute effects of cyanobacterial toxins on daphnids
concerning survival, food filtering, molting or enzyme
activities of the animals. Survival of Daphnia spp. was
rapidly reduced by toxic Microcystis aeruginosa (Nizan et al.,
1986; Ferrão-Filho et al., 2000). The 48-h lethal concen-
tration (LC50) of purified microcystin-LR (MC-LR) for three
Daphnia species ranged from 9.6 to 21.4 mg MC-LR
mL1(DeMott et al., 1991). Similarly, cyanobacterial crude
extract containing MC varied in concentrations of 48-h
LD50 or 48-h effective concentration (EC50) ranging from 36
to 162.45 mg mL1 or 34.2 to 1380 mg MC g1 DW in Daphnia
pulicaria and Daphnia similis respectively (Jungmann and
Benndorf, 1994; Sotero-Santos et al., 2006; Okumura
et al., 2007). Hietala et al. (1997a) showed a discrepancy
among the 48-h EC50 concentration of toxic M. aeruginosa
to ten clones of Daphnia pulex, varying from 0.022 to
>2.61 mg C L1. Hence, susceptibility to cyanobacterial
toxins differs not only between zooplankton species but
also within clones of Daphnia.
The food filtering rate of Daphnia spp. was inhibited when
the organisms were exposed to toxic cyanobacteria and
purified MC-LR (Lampert, 1982; Nizan et al., 1986; DeMott,
1999; Ferrão-Filho et al., 2000; Ghadouani et al., 2004).
Toxic M. aeruginosa caused sudden stops in swimming and
filtering activity in daphnids (Rohrlack et al., 2001). Defor-
mation of carapace and interruption of molting process of
daphnids were induced by the cyanobacterial metabolite
microviridin J (Kaebernick et al., 2001; Rohrlack et al., 2004).
Physiological responses included elevated activities of
the biotransformation enzyme glutathione S-transferase in
D. magna after exposure to MC-LR or Cylindrospermopsis
raciborskii but inhibition after exposure to microcin SF608
or Aphanizomenon issatschenkoi (Wiegand et al., 2002;
Nogueira et al., 2004a,b). In addition, the digestive
enzyme trypsin of daphnids was inhibited by cyanobacte-
rial metabolites obtained from a widely distributed cya-
nobacterial genus Planktothrix (Rohrlack et al., 2005).
In chronic and semi-chronic exposures, toxin accumu-
lation and detrimental influence on the animals such as
survival, growth, maturation and fecundity were observed.
Exposed to toxic M. aeruginosa, daphnids accumulated MC
at very high concentrations, up to 0.0712 mg MC per
Daphnia and 24.5 mg MC g1 DW of daphnids respectively
(Thostrup and Christoffersen, 1999; Mohamed, 2001).
Similar to acute exposures, survival of Daphnia was
decreased by toxic M. aeruginosa, both at species and clone
levels (DeMott et al., 1991; Hietala et al., 1995, 1997b;
Rohrlack et al., 2001).
Body length of daphnids was significantly reduced
when they were fed with M. aeruginosa (Lürling, 2003;
Lürling and Van der Grinten, 2003; Trubetskova and
Haney, 2006). Consequently, maturation and time to first
reproduction of daphnids were delayed if they fed on toxic
M. aeruginosa (Hietala et al., 1995; Laurén-Määttä et al.,
1245
1997). Toxic M. aeruginosa also caused aborted eggs
(Gustafsson et al., 2005) and reduced the fecundity of
D. magna (Thostrup and Christoffersen, 1999; Lürling and
Van der Grinten, 2003; Trubetskova and Haney, 2006) or
even completely inhibited the reproduction of D. pulex
(Hietala et al., 1997b). In contrast, dissolved MC-LR at the
concentration of 3.5 mg L1 had no effect on growth and
reproduction of D. magna (Lürling and Van der Grinten, 2003).
By continuous exposure to low densities of M. aeruginosa over
generations, D. magna increased their tolerance to the toxic
cyanobacterium, had higher fitness and earlier reached the
maturity age and first clutch of reproduction. These adapta-
tions were assumed as the result of maternal effects
(Gustafsson and Hansson, 2004; Gustafsson et al., 2005).
Overall, toxic cyanobacteria and their toxins severely
impacted daphnids in both acute and chronic exposures.
The organisms were not only suppressed in their survival
but impaired in development as well. As mentioned above,
investigations of the chronically detrimental influence on
daphnids have been conducted with cyanobacterial
cultures or pure cyanobacterial toxin. In most of the
mentioned chronic studies, exposures were performed
with one generation of daphnids and lasted for around
three weeks. Gustafsson et al. (2005) were conducting
transgenerational experiments in which the F1 daphnids
produced up to seven clutches; hence for the F1 generation
the exposure lasted for around one month.
In this study, chronic effects of dissolved MC-LR and
a cell free cyanobacterial crude extract containing MC on
life history of D. magna were investigated over their entire
life duration of two months. Their offspring were raised in
either toxic or control medium until maturation to follow
consequences of maternal exposure. The observed life traits
of the daphnids were survival, growth, maturation, time to
first reproduction and fecundity. The investigation on
chronic effects was carried out with two concentrations of
MC (5 and 50 mg L1) which would fall within the range of
dissolved MC in natural waters in the world, from trace
concentration to 200 mg L1 (Sivonen and Jones, 1999) and
have already impacted enzyme activities in D. magna
(Wiegand et al., 2002). In all exposure, Scenedesmus spp.
were provided as food source to exclude effects due to
malnutrition, hence to be able to attribute effects to cya-
nobacterial toxin or toxic compounds.
2. Materials and methods
2.1. Microcystin-LR and cyanobacterial crude extract
Microcystin-LR and a cyanobacterial crude extract con-
taining MC were used for the exposures. The MC-LR was
purchased from Axxora (Germany), and the cyanobacterial
crude extract was produced from our batch cultures of toxic
M. aeruginosa. The cyanobacterium, strain AB 2005/47, was
isolated from Chaohu Lake in China in 2005 and has been
cultivated as single strain in the laboratory of the Leibniz
Institute of Freshwater Ecology and Inland Fisheries, Berlin,
Germany since then. The crude extract was prepared
according to Fastner et al. (1998) with modification. Briefly,
the biomass of cultures on GF/C filters (Whatman) were
homogenized and firstly extracted over night in 70% MeOH
1246 T.S. Dao et al. / Toxicon 55 (2010) 1244–1254
(Carl Roth, Germany) containing 5% acetic acid (Merck,
Germany) and 0.1% triflouracetic acid (TFA; Merck,
Germany) followed by 2  60 min in MeOH 90% containing
5% acetic acid and 0.1% TFA with 30 s sonication during the
last extraction. Each extraction step was followed by
centrifugation (4500 rpm, 10 min, 4  C) The supernatants of
all extractions were pooled, dried at 35  C, re-dissolved in
MeOH (100%) and centrifuged at 14 000 rpm, 1  C for
10 min. The supernatant was kept at 20  C prior to high-
performance liquid chromatography (HPLC) analysis.
Microcystin content was analyzed according to
Pflugmacher et al. (2001) by high-performance liquid chro-
matography (HPLC, Waters Alliance, Eschborn, Germany)
system equipped with a reverse phase column (RP 18; 5 mM
LIChroSpher 100) and photodiode array detection. Injection
volume of the samples was 80 mL. Separation was achieved at
40  C by a gradient of Milli-Q water and acetonitrile (ACN,
Rathburn, Walkerburn, UK), both enriched with 0.1% (v/v)
TFA. The flow rate was 1 mL min1, starting at 35% ACN,
linearly increasing to 55% ACN within 15 min, followed by
a cleaning step of 100% ACN and 10 min equilibration to start
conditions. MC-LR was used as the reference toxin. The HPLC
analysis shown that there were several different variants of
MC and their total concentration in the crude extract was
6.92 mg mL1 MC-LR equivalent.
Mass spectrometer measurements for the cyanobacte-
rial crude extract were performed using an API 165 mass
spectrometer (Applied Biosystems, Foster City, CA, USA)
with an atmospheric pressure ionization source operating
in turbo ion-spray mode. Quantitative determination of the
toxins was carried out by comparing peak areas in sample
chromatograms with the corresponding peak areas
obtained from the pure toxins. Proportion of the main
cyanobacterial toxins in the crude extract was as follows,
MC-RR, 71.09%; MC-LR, 24%; MC-LA, 3.09%; MC-YR, 1.79%
and MC-LF, 0.03% Krüger et al., (submitted for publication).
2.2. Experimental organisms
The test organism was D. magna Straus, obtained from
MicroBioTests Inc. Belgium and has been maintained in the
laboratory of the Leibniz Institute of Freshwater Ecology
and Inland Fisheries, Berlin, for several generations. The
Daphnia medium provided by MicroBioTests Inc. Belgium
consisted of CaCl2, KCl, NaHCO3 and MgSO2. The living
green algae Scenedesmus spp. were used as the sole food for
D. magna. The algae were cultivated in Z8 medium (Kotai,
1972) with continuous aeration. Both culturing of Scene-
desmus and Daphnia as well as the exposures were con-
ducted at a temperature of 20  1  C and a photoperiod of
14 h light and 10 h dark.
Prior start of the experiments, fifteen adolescent female
D. magna were incubated in a 500 mL beaker and fed with
Scenedesmus spp. for 2–3 weeks. Offspring from the second
to fifth clutch of these D. magna were used for experiments
and hereafter referred to as mother daphnids. Thirty
neonates less than 24 h old were randomly selected for
each chronic exposure (Adema, 1978) and individually
incubated in 50 mL beakers containing 20 mL of medium.
The animals were fed 4  106 Scenedesmus spp. cells per
daphnid per day equaling a cell density of 2  105 cells
mL1 (approximately 3 mg C L1). Density of Scenedesmus
cells was adjusted daily. The food density and availability
were chosen according to Lampert (1977) and Gustafsson
et al. (2005) respectively, in which individual animal was
fed with 6  104 Scenedesmus cells mL1 in 60 mL beakers,
equaling 3.6  106 cells per daphnid per day.
2.3. Experimental setup
For exposure, MC-LR or cell free cyanobacterial crude
extract containing MC was added into Daphnia medium. In
total, there were four different exposures: 5 and 50 mg L1 of
purified MC-LR, and 5 and 50 mg L1 of total MC in crude
extract, hereafter referred as M5, M50, E5 and E50 respec-
tively. The control was implemented twice (C1 and C2)
because the food was changed between exposure M5 and the
others. Daphnids of C1 and M5 treatments were fed with
green alga Scenedesmus vacuolatus. Due to unavailability of S.
vacuolatus, daphnids were fed with another Scenedesmus
species, Scenedesmus armatus for the other exposures (M50,
E5 and E50 respectively). Consequently a new control (C2)
was conducted in which daphnids were fed with S. armatus
in order to get the same supply of nutrient quality. Results of
daphnids incubated in the same food regime were compared
only (C1–M5; C2–M50, E5 and E50). Twice a week, half of
toxic and non-toxic Daphnia medium was renewed whereas
food density was kept constant on a daily basis. Each expo-
sure and control experiment lasted for two months.
2.4. Life history traits
For life history study, the survival, growth, maturation,
time to first reproduction and fecundity of mother Daphnia
were observed daily for two months. Simultaneously, the
survival, growth and maturation of their offspring were
also recorded for one week. Death of the animal was
defined as the stop of heartbeat confirmed by microscopic
observation (Olympus TH3). Maturation of Daphnia was
defined as time point of first egg occurrence in the brood
chamber. The time to first reproduction was at first
offspring release from brood chamber during molting.
Fecundity of animals was recorded as the number of
clutches and number of offspring per clutch produced by
every mother Daphnia during exposure time. In case of
release of decomposed eggs, embryos or neonates, the
offspring number of that clutch was assumed as zero.
Twice a week, the growth of test organisms was moni-
tored by measuring the distance from top of the head to the
base of tail spine (body length) by photographing the
animals under a microscope (Olympus SZX7) equipped
with a digital camera (Olympus XC50). The body length of
Daphnia was measured exactly to 1 mm based on the soft-
ware DT5 analysis (Soft Imaging System – Olympus).
Neonates (<24 h old, called F1 Daphnia) were randomly
selected for survival, growth and maturation study of the
offspring. F1 Daphnia from control experiment were raised
in either non-toxic or in the four different kinds of toxic
media. Similarly, F1 daphnids from toxin exposed mother
D. magna, were split in two groups: a) raised under
continuous exposure in the same toxic medium and
b) raised in control medium (Fig. 1). One hundred F1
 T.S. Dao et al. / Toxicon 55 (2010) 1244–1254 1247

Fig. 1. Experimental setup for the Daphnia magna from mothers (n ¼ 30) to offspring (n ¼ 100) incubated in non-toxic and toxic medium. Mother Daphnia
exposure to: C1 and C2, controls; M5 and M50, to 5 or 50 mg L1 of MC-LR; E5 and E50, to 5 or 50 mg L1 of total MC from cyanobacterial crude extract respectively.
Offspring (F1) exposure: neonates from each mother Daphnia were split into groups and either continuously exposed as their mothers or raised in control
medium (C1 or C2). Offspring from control mother Daphnia were split and those from C1 raised in either C1 or M5, whereas from C2 raised in C2 or M50, E5 or
E50.

daphnids were selected for each treatment in which every
ten neonates were transferred into a beaker containing
100 mL of exposure or control medium. Half of the medium
was renewed twice a week. The animals were fed with
Scenedesmus ad libitum. The survival and body length of
the offspring were monitored until maturation. The
experiment terminated when the last offspring matured.
2.5. Statistical analysis
Sigmastat, version 2.0, SPSS Inc. was used for data
treatment. One-way Analysis of Variance (ANOVA) and
Tukey test Post Hoc were applied to calculate statistically
significant difference of the body length of adult D. magna.
Kruskal–Wallis was applied for calculation of statistically
significant difference of the maturation, time to first
reproduction and body length of offspring D. magna due to
lacking normality or distributed homogeneity of the
samples.
first but then they developed slower than the control
animals, C1 and C2. It resulted in significantly longer bodies
of the M5, M50 and E50 treated daphnids compared to
their controls during the first 4, 15 and 32 days respectively
(ANOVA followed by Tukey’s test, p < 0.05; Fig. 3A,C,D).
During the age from 15 to 57 days daphnids in M5 were
smaller with some fluctuation compared to those in control
(Fig. 3A). The animals in M50 and E50 treatments stayed
significantly smaller than the control from the age of 39
and 43 days to the end of incubation (ANOVA followed by
Tukey’s test, p < 0.05; Fig. 3C,D). Contrastingly, the body
length of one day old animals in E5 was smaller than that in
C2. However, three days later to the end of exposure the
daphnids in E5 grew faster and were significantly bigger
than the C2 (ANOVA followed by Tukey’s test, p < 0.001;
Fig. 3B). The data of E5, 32 days old and M50, 11 days old
were missed due to camera failure.

3. Results

3.1. Mother D. magna life traits

3.1.1. Survival
After two months of exposure, the concentration of
5 mg L1 MC-LR or total MC in crude extract slightly
decreased the survival of adult D. magna, at about 10% of
total animals (Fig. 2). However, those of 50 mg L1 caused
the reduction of 60 and 55% of total daphnids in M50 and
E50 treatments respectively. The number of daphnids in
M50 and E50 were quite stable within the first three weeks
of incubation but quickly descended afterwards. The
number of daphnids in C1 decreased 13% whereas that in
C2 did 37% during two months. There was little difference
between the survival of C1 and M5 treatments. Unexpect-
edly, the number of daphnids in C2 reduced more than
those in E5 by the end of exposure. However, survival
proportion of M50 and E50 incubations was 23 and 17%
respectively lower than that of C2 incubation (Fig. 2).

3.1.2. Growth performance

Animals in C1 and C2 were fed with different Scene-
desmus and those in C2 grew somewhat larger (Fig. 3). Fig. 2. Survival of mother Daphnia magna during exposure time (n ¼ 30 at
Daphnids of the treatments M5, M50 and E50 grew faster at the start). Abbreviation as in Fig. 1.
1248 T.S. Dao et al. / Toxicon 55 (2010) 1244–1254

Fig. 3. Body length of mother Daphnia magna (mean value  SD of n ¼ 30 at the start) during the incubation. Asterisks indicate significant differences by ANOVA
followed by Tukey’s test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Abbreviation as in Fig. 1.
 T.S. Dao et al. / Toxicon 55 (2010) 1244–1254
3.1.3. Maturation
D. magna exposed to pure toxin or crude extract at both
5 and 50 mg MC L1 reached their maturity around the age
of 6 days, significantly earlier than the control, which took
around 7 days (Kruskal–Wallis test, p < 0.01; Fig. 4A).
Subsequently, the toxin exposed animals were significantly
younger at first reproduction (Kruskal–Wallis test,
p < 0.001) except that those of M5 and C1 had a similar age
at first reproduction (Fig. 4B).
3.1.4. Fecundity
During two months of incubation, the mother D. magna
produced 16–17 clutches of offspring. The accumulative
number of neonates from the control daphnids was highest
in the end (Fig. 5). Compared to the control accumulative
offspring, those from the M5, E5 and E50 raised higher
within the first 8, 4 and 12 clutches respectively followed
by a slower development. In the E50 treatment, reproduc-
tion only marginally proceeded after the 10th clutch
(Fig. 5). In total, the concentration of 5 mg L1 MC-LR or both
concentrations of MC in crude extract resulted in a reduc-
tion of around 10% neonates compared to the control. The
concentration of 50 mg L1 MC-LR caused a decline of more
than 60% of offspring (Table 1).
3.1.5. Malformation
Exposures to the pure toxin or crude extract induced the
abortion of eggs/embryos via decomposition and death
already in the brood chamber of D. magna (Fig. 6A,B).
Furthermore, it caused the malformation of neonates
including the incomplete development of the antennae and
un-rejected tail spine (Fig. 6C). The proportion of mal-
formed and dead neonates was higher in the M50 (7.1% and
0.7% respectively) and E5 (6.6% and 0.5%) incubations
Fig. 4. A, Maturation and B, Time to first reproduction of mother Daphnia
magna (mean value  SD of n as indicated in the columns), asterisks indicate
significant difference by Kruskal–Wallis test (**, P < 0.01; ***, P < 0.001).
Abbreviation as in Fig. 1.
1249
Fig. 5. Accumulative fecundity of mother Daphnia magna during exposure
time as proportion of neonates in relation to the total neonates of control.
Abbreviation as in Fig. 1.
compared to the M5 (0.3% and 0.09%) and E50 (0.05% and
0%) exposures (Table 1). The abortion proportion of
exposed D. magna was highest in M50 (21.9%), followed by
E5 (15.1%), E50 (9.8%) and lowest in M5 (0.7%). Similarly,
the percentage of aborting females was highest in M50
(83.3%), E5 (80%), E50 (66.7%) and lowest in M5 (10%).
3.2. Offspring Daphnia magna life traits
3.2.1. Survival
The survival of offspring from control mother D. magna
ranged from 97 to 100% in both control and toxin expo-
sures. However, those of exposed mother daphnids
suffered from mortalities within their first week post natal
(death proportion of 6–47%), regardless, if raised in control
medium or under continuing exposure (Fig. 7). Survival
proportion was lower if the offspring of pure toxin expe-
rienced mothers were continuously incubated in toxin
medium compared to raised in control medium. In detail,
the survival proportion of M5M5 (86%) was 8% lower than
that of M5C1 (94%). Similarly, the survival proportion of
M50M50 (53%) was 7% lower than that of M50C2 (60%)
(Fig. 7A). However, the E50E50 offspring survived slightly
better than the E50C2 ones, 94% compared to 88% (Fig. 7B).
Only offspring of E5 mothers did not significantly decrease
their survival after eight days of exposure (Fig. 7B).
Microcystin-LR had stronger negative effects on the survi-
vorship of the offspring than the crude extract did.
Mortality rate of daphnids correlated directly to toxin
concentration (Fig. 7).
3.2.2. Growth performance
Offspring of D. magna previously exposed to pure toxin
or crude extract containing MC dealt with a significant
decrease of their body length during the first week of
incubation in non-toxic or toxic medium (Kruskal–Wallis
test, p < 0.001; Fig. 8). In addition, the growth of offspring
of mother daphnids previously exposed to 50 mg MC L1
was more inhibited than that of mother exposed to
5 MC mg L1 (Kruskal–Wallis test, p < 0.05; Fig. 8).
3.2.3. Maturation
Offspring from D. magna exposed to 5 and 50 mg L1 of
MC-LR or total MC from crude extract significantly delayed
1250 T.S. Dao et al. / Toxicon 55 (2010) 1244–1254

Table 1
Fecundity of mother Daphnia magna and malformation and abortion of their offspring.

Exposure Total neonates Malformed Dead neonates Number of decomposed clutches Aborting females
relative to control neonates
M, Me Mn
C1 100% 0 0 0 0 0
M5 89.3% 0.3% 0.09% 3M 0 10%
C2 100% 0 0 0 0 0
M50 38.2% 7.1% 0.7% 64 M þ 6 Me 8 Mn 83.3%
E5 90% 6.6% 0.5% 64 M þ 5 Me 1 Mn 80%
E50 92.1% 0.05% 0 39 M 0 66.7%

M, empty carapace; Me, removed carapace with decomposed eggs/embryos inside; Mn, removed carapace with dead neonates.

maturity age compared to their mothers, by 1–4 days
(Kruskal–Wallis test, p < 0.001; Fig. 9).
4. Discussion
In this study, daphnids in each treatment were fed with
either S. vacuolatus or S. armatus at 4  106 Scenedesmus
spp. cells per daphnid per day (around 3 mg C L1), that is
a density providing surplus food, as cells were not
completely consumed every day. Unexpectedly, differences
between the control groups occurred, as C2 grew bigger but
suffered lower survival rates for unknown reasons. Other
differences were not significant. Therefore, life trait differ-
ences between control and toxin exposed D. magna were
compared within the groups fed with the same species of
Scenedesmus only and could then be attributed to the cya-
nobacterial toxins applied.
The molecular mechanism of MC-LR toxicity is mediated
via inhibition of protein phosphatases 1 and 2A
(MacKintosh et al., 1990) thereby leading to deregulation in
cells. Furthermore, MC-LR has been found binding to ATP
synthetase beta-subunit affecting the energy sustaining
ability of the cells (Mikhailov et al., 2003). Another more
general effect of cyanobacterial compounds is via
provoking of oxidative stress (Wiegand and Pflugmacher,
2005). Cyanobacterial bioactive metabolites caused the
suppression of trypsin-like activities, which together with
chymotrypsin enzymes account for 75–83% of proteolytic
activities in Daphnia sp. gut contents (Von Elert et al., 2004;
Czarnecki et al., 2006). Furthermore, MC-LR inhibited D.
pulicaria filtering activity, hence impaired food uptake
(Ghadouani et al., 2004). Summing up, cyanobacteria and
their compounds decrease the energy re-establishing
process in daphnids at various points: by malnutrition, by
impairment of feeding and digestion, by cellular processes;
all in turn will sum up to lower energy availability for
maintenance, growth and reproduction (scope for growth).
Under exposure to toxin, additional energy is spent for
physiological reactions, such as biotransformation and
antioxidant enzyme activities, toxin excretion, or mecha-
nisms of repairing damages. In our study a lower scope of
growth was evidenced by growth inhibition, fecundity
failure and mortality increase.
Survival of daphnids in treatments with low toxin
concentration was not much affected in our study. This is in
agreement with the investigation of Lürling and Van der
Grinten (2003) in which D. magna exposed to 3.5 mg L1
dissolved MC-LR showed no adverse effect. However, at
a ten times higher concentration (50 mg MC L1) survival of
mother daphnids was strongly decreased by either pure
MC-LR or cyanobacterial crude extract (Fig. 2B). These
records were in line with previous studies (Hietala et al.,
1995, 1997b; Lürling, 2003; Semyalo et al., 2009) in which
daphnids were treated with toxic cyanobacterial cultures.
Hence, chronic exposure to a concentration of 50 mg MC L1,
a concentration that is about 400 times lower than the 48-h

Fig. 6. Malformation of eggs/embryos and offspring Daphnia magna (arrows). A, decomposed eggs/embryos; B, dead neonates; C, normal neonate (left) and
malformed neonate with incomplete development of the antennae and un-rejected tail spine (right).
 T.S. Dao et al. / Toxicon 55 (2010) 1244–1254 1251

dominant, gaining 71.09% and 24% of total MC respectively.

Fig. 7. Survival of offspring Daphnia magna during the first week of incu-
bation, starting with 100 neonates for each exposure. Abbreviation as in
Fig. 1.
LC50 values (9.6–21.4 mg MC-LR mL1 DeMott et al., 1991), is
sufficiently potent to cause a declined survival over the life
span of D. magna.
In cyanobacterial crude extracts not only variants of MC
but also other cyanobacterial bioactive compounds
adversely affected daphnids (Reinikainen et al., 2001;
Wiegand et al., 2002; Rohrlack et al., 2004). In our study,
cyanobacterial crude extract contained five different MC
congeners of which MC-RR and MC-LR were mainly
The daphnid survival was concentration dependently
decreased by the cyanobacterial crude extract used, but not
as much as by the MC-LR treatment due to the lower
toxicity and higher hydrophilicity of MC-RR compared to
MC-LR (Atencio et al., 2008). Despite the concentrations of
50 mg L1 dissolved MC used in our study adversely affected
daphnids’ biotransformation and antioxidant enzymes
(Wiegand et al., 2002) the detrimental impact on their life
history was only revealed after three weeks of exposure
(M50 and E50 treatments). In contrast, during toxic
Microcystis cell treatments Daphnia pulex, Daphnia long-
ispina, D. pulicaria, D. magna and Daphnia lumholtzi started
to significantly decrease their survival already after the first
week (Lampert, 1981; Hietala et al., 1995, 1997b;
Trubetskova and Haney, 2006; Semyalo et al., 2009).
However, if it could be considered that the average toxin
content per cell of Microcystis is 0.2 pg (Falconer et al., 1999)
and 1 mg C L1 of this species corresponds to around
1.4  105 cells mL1 (Gustafsson and Hansson, 2004) the
concentration of MC in the cyanobacterial cultures used for
exposures of previous investigations ranged from 0.24 to
28 mg L1, which is within a similar range of MC concen-
tration used in our study, 5 and 50 mg L1. Besides the
specific toxic effects of cyanobacterial compounds, cyano-
bacterial cells affect daphnids also by malnutrition and
impairment of feeding. The availability of green algae as
alternative food source always increased survival in those
studies.
Survival of daphnids in E50 treatment strongly
decreased whereas that in E5 only slightly declined from
the third week to the end of exposure (Fig. 2B). In F1
daphnids, cyanobacterial crude extract caused both death

Fig. 8. Body length of exposed mother Daphnia magna and their offspring (mean value  SD of n ¼ 30 for mother and 100 for offspring at the start) during the
first week of incubation. Asterisks indicate significant differences by Kruskal–Wallis test (***, P < 0.001). The symbol # indicates significant difference by Kruskal–
Wallis test (P < 0.05) in F1 between increasing concentration within either toxin or crude extract treatment (A–C; B–D). Abbreviation as in Fig. 1.
1252 T.S. Dao et al. / Toxicon 55 (2010) 1244–1254
Fig. 9. Maturation of exposed mother Daphnia magna and their offspring
(mean value  SD of n ranging between 28 and 97), asterisks indicate
significant difference by Kruskal–Wallis test (***, P < 0.001). n as indicated in
the columns. Abbreviation as in Fig. 1.
and malformation occurring from the third clutch of
mother daphnids. In total, E50 treatment induced higher
mortality for mother daphnids but lower malformed
neonates and decomposed clutches than E5. Apparently,
the lower crude extract concentration weakened the
mother daphnids to the expense of their offspring, whereas
the higher directly affected the mother daphids survival.
The M50 treatment not only affected survival of the mother
daphnids, but in addition the offspring suffered malfor-
mation and abortion.
Although dissolved toxins were less harmful than cell-
bound toxins to daphnids, exposure to dissolved toxins as
in our study provides a more evenly contact to the animals.
On the other hand, our results may extrapolate the toxicity
of dissolved cyanobacterial toxins to the survival of daph-
nids in nature because the animals are almost unable to
filter big colonies or long filaments of cyanobacteria in
blooms.
Unexpectedly, our study revealed that daphnids in E5
incubation grew faster and significantly bigger than the
control animals during the exposure. Possibly, the toxin
concentration of 5 mg L1 did not inhibit the animals’
growth, as also evidenced by Lürling and Van der Grinten
(2003), but beside toxic metabolites there might be nutri-
tional compounds in the crude extract contributing to
growth enhancement of the daphnids. However, MC
concentration of 50 mg L1 was sufficiently potent to
suppress the growth of daphnids by the last weeks of
exposure. Hence, despite growth stimulation at the
beginning of the treatment by cyanobacterial toxins it was
limited afterwards. The inhibition of ingestion process and
activity of digestive enzyme trypsin in daphnids (Rohrlack
et al., 2003; Czarnecki et al., 2006) resulted in starvation
which induced adverse effects on the animals’ life traits
including the decrease of growth. It could be inferred that
in our study the D. magna might withstand the toxin at first
due to the constant Scenedesmus availability as food source.
However, the animals were impaired by the toxin after-
wards. Still the mechanisms by which daphnids in M50
grew faster than those in control within the first two weeks
of experiment remained unknown.
Contrasting to other studies, where treatment with
toxic Microcystis delayed the age to first reproduction in D.
pulex (Hietala et al., 1995), we found earlier maturation and
start of reproduction in treated D. magna. Ebert (1992)
found that maturity of D. magna is positively related to
their body length and maturation is initiated above
a threshold size. In our study exposed daphnids grew faster
during the first weeks of incubation (Fig. 3). Therefore, the
exposed animals matured and reproduced sooner than the
control according to the hypothesis of threshold size of
D. magna.
Our results are in agreement with previous investiga-
tions evidencing a reduction or completely inhibition of
daphnid reproduction due to cyanobacterial compounds
(Lürling, 2003; Lürling and Van der Grinten, 2003;
Gustafsson et al., 2005; Trubetskova and Haney, 2006).
The M5, E5 and E50 adults had a reproducing reduction of
around 10% of total neonates whereas the fecundity of M50
had a severe decrease of more than 60% of total offspring by
the end of incubation. Gustafsson et al. (2005) showed that
offspring number of daphnids in a mixed food treatment
(Microcystis and Scenedesmus, ratio of 5/95 biomass) were
lower than those of control in every clutch. In our study
MC-LR at 50 mg L1 depressed the reproduction during
lifetime of the animals, whereas the other treatments had
some fluctuation in the beginning, before fecundity
decreased. However, the total numbers of neonates of all
exposures were lower than that of their controls.
The decrease of offspring happened via two possibili-
ties: (1) 10–83.3% of exposed mother aborted their eggs/
embryos (Table 1) and the abortion happened repeatedly in
several females; (2) in addition to dead neonates in brood
chamber or malformed neonates, incomplete development
of the antennae and un-rejected tail spine were observed in
many offspring, which usually died some hours after birth.
Abortion of D. magna were observed when they were
treated with toxic M. aeruginosa (Gustafsson et al., 2005) or
fungicides such as prochloraz, fenarimol and pyrifenox
(Hassold and Backhaus, 2009). In addition, malformations
of neonates were described after fungicide application
(Hassold and Backhaus, 2009). Our study traced back
malformations of D. magna offspring to be caused by cya-
nobacterial toxins.
Gustafsson and Hansson (2004) found that offspring of
toxic Microcystis exposed mother D. magna survived better
if fed with Microcystis than offspring of mothers fed with
Scenedesmus. However, our study displayed a controversial
result that more than 97% of offspring of control mothers
survived the first week in toxic or non-toxic medium. In
contrast, offspring of toxin treated mothers suffered from
mortalities even if reared in non-toxic medium. Addition-
ally, abortion of daphnids, decomposed eggs and embryos,
and dead neonates were observed after toxin treatment. It
was commonly observed that egg proteins of exposed
mother daphnids coagulated and eggs broke or decom-
posed inside the brood chamber. Hence, there must be
adverse effects of MC already on the egg/embryos devel-
opment in brood chamber. Wiegand et al. (2009) proved
that MC was not only absorbed by the gut cells but also
occurred in developing eggs in the brood chamber of adult
D. magna even within 48 h exposure to the toxin. Possibly,
the neonates of MC exposed mother daphnids suffered
from toxic stress and energy cost for detoxification before
 T.S. Dao et al. / Toxicon 55 (2010) 1244–1254
they were released. As a consequence they became more
vulnerable and their survival was decreased even if they
were raised in non-toxic medium.
In exposure scenarios with cyanobacterial cells, the
amount of toxin that reaches physiological targets in
daphnids may be lower than the total available cell-bound
toxin of the cyanobacteria because of decreased food
filtering and impaired digestion (Rohrlack et al., 2001;
Czarnecki et al., 2006). Concentrations of cell-bound MC
used for pre-trial exposure to daphnids in the study of
Gustafsson and Hansson (2004) were increased from
1.2 mg L1 at the first week to 4.8 mg L1 at the fourth week
of incubation. Differently, daphnids in our experiments
were constantly exposed to 5 and 50 mg L1 starting at
neonate stadium. Possibly, slowly increasing toxin
concentration from 1 mg L1 to 4.8 mg L1 induced maternal
effects (Gustafsson and Hansson, 2004; Gustafsson et al.,
2005), whereas a starting concentration of 5 or
50 mg L1 MC from new born age might be already too high.
In addition, out of the three clones of daphnids tested by
Gustafsson and Hansson (2004), only two developed
tolerance against toxic Microcystis. Therefore, different
toxin concentrations at start of exposure and clone
susceptibilities to toxin could be the root of the different
results.
Contrasting to Gustafsson et al. (2005), where offspring
of previously exposed D. magna to toxic Microcystis
increased their fitness, attributed to genetic inheritance,
our study revealed significant and concentration depen-
dent decrease of body length (Fig. 8) and increased dura-
tion to maturity (Fig. 9) of F1 of exposed mother daphnids.
The reduction of fitness in our case might be caused by
damage of offspring before hatching as mentioned above.
A good evidence for that is that the offspring developed
slower and suffered from mortalities even if raised in non-
toxic medium.
Despite exposed mother D. magna reached maturity
earlier than the control, the offspring of exposed animals
significantly delayed the maturation compared to their
mother, due to slower growth so that they later reached the
threshold size of maturation according to the hypothesis of
Ebert (1992). The postponement of maturation raises an
additional vulnerability of sustainable population in the
field, e.g. by predation.
In summary, chronically exposed to MC-LR or cyano-
bacterial crude extract the D. magna clone used in our study
strongly decreased survivorship from generation to
generation without inheritance of toxin tolerance. In
addition, development of the exposed animals was stimu-
lated at first but inhibited afterwards. However, the
explanation for the growth stimulation remains unknown.
Besides, maturation and time to first reproduction was
reached sooner in exposed mother daphnids, but total
fecundity was decreased. Egg/embryo and neonate devel-
opment in brood chamber of D. magna was strongly influ-
enced by cyanobacterial toxin inducing abortion or
malformation of neonates. The offspring of previously
exposed D. magna were not only repressed in development
and delayed in maturation compared to their mothers; they
also suffered mortalities, even if raised in non-toxic
medium.
1253
In consequence, population and community of this
investigated D. magna would decrease or become rare if
continuously affected by cyanobacterial toxins in nature.
Maternal effects of daphnids could be induced with cya-
nobacterial toxin concentration of around 1 mg L1
(Gustafsson et al., 2005) but might not be at 5–50 times
higher concentration at the neonate stadium exposure.
Study on chronic effects of dissolved cyanobacterial toxins
on different clones of D. magna should be implemented as
daphnid clones have different susceptibilities to the toxins.
Further investigations are in need for understanding the
mechanisms of abortion and malformation in daphnids
caused by cyanobacterial toxins.
Acknowledgement
The authors would like to thank Dr. Jorge Nimptsch,
Institute of Freshwater Ecology and Inland Fisheries (IGB),
Berlin, Germany, for providing biomass of toxic Microcystis
aeruginosa culture and preparing cyanobacterial crude
extract for this study. Rafael Ortiz, IGB, is acknowledged for
discussion on data treatment. Thanh Son Dao’s financial
support by The Swiss Agency for Development and Coop-
eration Project is gratefully acknowledged.
Conflict of interest
None.
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