Notes
HILTON, J. 1990. Greigite and the magnetic properties of sediments. Limnol. Oceanogr. 35: 509-520.
JAKSIC,M., G. W. GRIME,J. HENDERSON,
ANDF. WATT.
199 1. Quantitative PIXE analysis using a scanning proton microbeam. Nucl. Instr. Meth. 54:
49 l-498.
JOHANSS~N,S. A. E., AND J. L. CAMPBELL. 1988.
PIXE-A novel technique for elemental analysis.
Wiley.
Momrr,
K. D. 1988. Trace metal dynamics in a
seasonally anoxic lake. Ph.D. thesis, Lancaster
Univ. 175 p.
-,
W. DAVISON, AND J. HAMILTON-TAYLOR. 1988.
Trace metal dynamics in a seasonally anoxic lake.
Environ. Geol. Water Sci. 11: 107-l 14.
1777
MYERS, C. R., AND K. NEALSON. 1988. Microbial
reduction of manganese oxides: Interactions with
iron and sulfur. Geochim. Cosmochim. Acta 52:
2727-2732.
STUMM,W., ANDJ. J. MORGAN. 198 1. Aquatic chemistry, 2nd ed. Wiley-Interscience.
TIPPING, E., C. WOOF, AND D. COOKE. 198 1. Iron
oxide from a seasonally anoxic lake. Geochim.
Cosmochim. Acta 45: 1411-1419.
Submitted: 6 December 1991
Accepted: 25 June 1992
Revised: 23 July 1992
Limnol. Oceanogr., 37(S), 1992, 1777-1783
0 1992, by the American Society of Limnology and Oceanography, Inc.
Toxic compounds isolated from Microcystis PCC7806 that are
more active against Daphnia than two microcystins
Abstract-Microcystis strain PCC7806 was toxic
to Daphnia pulicaria. The toxicity of a crude water extract from lyophilized cells of Microcystis
was determined in order to characterize the toxic
compound. The LC,, ofthe crude extract was 47.4
~1 ml- I. The crude extract was fractionated by
solid-phase extraction and tested for toxicity. The
microcystins in the different fractions were analyzed by HPLC. The crude extract contained 22
pg ml-’ microcystin-LR and 12.3 rg ml-’ 3-desmethylmicrocystin-LR,
the C- 18 eluate contained 16.3 and 9.1 kg ml-l of these compounds,
respectively. Neither compound was detected in
the C-18 cartridge-passed crude extract. Nevertheless the C-18 cartridge-passed fraction was
toxic to Daphnia. Hence I conclude that the two
microcystins in the crude water extract are not
the compounds in Microcystis PCC7806 which
are toxic to Daphnia.
Microcystis spp. are the most common
bloom-forming
cyanobacteria in lake ecosystems. The toxicity of Microcystis blooms
to warm-blooded animals has been reported
worldwide
(Carmichael
1988). In recent
years, however, many Microcystis strains
Acknowledgments
I thank W. Lampert for critical comments on the
manuscript, and W. R. DeMott, W. W. Carmichael,
and an anonymous reviewer for comments and critiques. N. Zehrbach and I. Griineberg provided linguistic improvements.
have been reported to be toxic to Daphnia
(Lampert 198 1a; Pefialoza et al. 199 1; Jungmann et al. 199 1). Daphnia avoids ingesting
such Microcystis strains which can be detected by measuring the filtering rate of
Daphnia. Daphnids whose only food source
is Microcystis cells die more rapidly than
starved animals (Lampert 198 1a), indicating that Microcystis cells contain a compound (compounds) toxic to Daphnia. In
contrast DeBernardi et al. (1980) reported
that they sampled Microcystis sp. which exhibited no toxicity to Daphnia but provided
a suitable food source for the animals. Comparison of these results show that not every
Microcystis strain is toxic to daphnids. The
toxicity of a Microcystis strain and its ability
to reduce the grazing pressure of the zooplankton have been suggested to be an algal
defense that favors formation of a bloom
(Lampert 198 1a, b). It is therefore ‘important to characterize the toxic compound in
more detail.
The microcystins
(cyanoginosine) compounds in Microcystis, which are cyclic hepatotoxic heptapeptides with a known amino-acid sequence (Botes et al. 1985) are
toxic to many vertebrates. They have been
isolated from various Microcystis strains
(Carmichael 1988; Codd et al. 1989). They
1778
Notes
are toxic to many warm-blooded
animals
by oral and intraperitoneal
application
(Carmichael 1988). Animals that died by
these agents show a swollen and damaged
liver (Runnegar and Falconer 198 1). However, not all isolated Microcystis strains are
toxic to warm-blooded animals (Codd et al.
1989). Ecological aspects must also be considered during investigations
into microcystins. Toxicity to higher animals cannot
be of evolutionary
adaptive value to Microcystis; nevertheless only a few publications deal with the response of microcystinLR in lake ecosystems (Pefialoza et al. 199 1;
DeMott et al. 1991). The present study reports experiments to clarify whether microcystin-LR or 3-desmethylmicrocystin-LR
is
the compound extracted from Microcystis
PCC7806 which is toxic to Daphnia puli-
caria.
Axenic Microcystis PCC7806 was originally supplied by R. Rippka (Pasteur Culture Collection) and checked for sterility by
plating on DEV-agar (Merck No. 1147).
Stock cultures were cultivated in sterile 300ml Erlenmeyer flasks filled with 100 ml of
autoclaved cyanobacteria medium (Cbm)
(Jungmann et al. 1991) and continuously
illuminated (50 PEinst s-l m-2) in a culture
room at 25 + 1°C. New cultures were established from an inoculum every 60th day.
Microcystis PCC7806 was precultured in
sterile 300-ml glass tubes filled with 250 ml
of autoclaved Cbm and a lo-ml inoculum
from a stock culture. The tubes were bubbled from the bottom (300 ml min-l) with
CO,-enriched sterile air (-0.2% C02, vol/
vol) and continuously illuminated with fluorescent lamps (65 PEinst s-l m-2) at
25+ 1°C. Microcystis PCC7806 was mass
cultured in sterile 5-liter glass tubes (4, 7.3
cm; 150 cm long) with a precultured 1O-l 4d-old culture as inoculum. The tubes were
bubbled from the bottom (500 ml min-‘)
with CO,-enriched sterile air and continuously illuminated with fluorescent tubes (100
PEinst s-l me2) at 25& 1°C.
Microcystis cells were harvested in the declining logarithmic growth phase by means
of a continuous-flow-through
centrifuge
(Heraeus Labofuge 15000, 10,000 rpm,
room temperature). The resulting pellet was
resuspended in 400 ml of Cbm and centri-
fuged once more (19,000 x g, 4°C 15 min)
in 300-ml polyethylene tubes. The supernatants were decanted, the pellets shockfrozen in fluid nitrogen and after lyophilization the powder was stored in glass vessels
at - 18°C until needed. Particulate organic
C (POC) of lyophilized Microcystis was determined (Krambeck et al. 198 1). D. pulicaria was cultured as described earlier
(Jungmann et al. 1991). Survival time of
starved daphnids
(exposed to 0.45-pm
membrane-filtered lake water) placed in 1.2liter glass bottles (1 5-20 animals bottle-l)
was compared with results of starved
daphnids placed in 2-ml Eppendorf caps
(polyethylene) to test the effect of container
size. Live animals were counted every 24 h.
No statistically
significant difference was
found between these two treatments for 72
h (data not shown); hence container size did
not affect survival in the absence of food.
Toxicity experiments were carried out in
2-ml Eppendorf caps.
Lyophilized
Microcystis cells (500 mg)
were diluted in a glass bottle containing 50
ml of double-deionized
water (NANOpure,
- 18 MQ cm-l conductivity)
and sonified
at room temperature (15 min). For extraction the pH was adjusted to 4 with 2 N HCl
and the bottle was placed on a magnetic
stirrer (150 + 25 rpm) overnight (12 h). The
suspension was distributed to 30-ml polycarbonate tubes and centrifuged (18,000 x
g, 4”C, 30 min). The supernatants were
pooled, filtered (0.8 pm, celluloseacetate
membrane), and the pH adjusted to 7.5 with
1 N NaOH. To calculate the LCso (Weber
1972) I distributed different amounts of the
crude extract to 2-ml Eppendorf caps. A
single 4-d-old Daphnid was transferred into
each cap and the volume was filled to 2 ml
with membrane-filtered
(0.45 pm) lake water.
For further differentiation,
the crude Microcystis extract was fractionated by solidphase extraction. A Bond Elut C-18 polyethylene cartridge (Analytichem
Int., 500
mg of sorbents) was activated with 10 ml
of methanol and washed with 10 ml of double-deionized water. Half of the total volume of the crude extract was tested for toxicity to Daphnia and the other half was
passed through the C- 18 cartridge and also
1779
Notes
tested for Daphnia toxicity.
Subsamples
were stored at 4°C until HPLC analysis (not
longer than 24 h). The C-l 8 cartridge was
eluted with 10 ml of methanol and the eluate
was dried in a stream of nitrogen at room
temperature. The dried C- 18 eluate was diluted stepwise in phosphate buffer, pH 8 (5
ml per step, placed for 10 s in a sonification
bath and pooled in a glass flask) until the
original volume of the C- 18 cartridge-passed
crude extract was reached (25 ml). The diluted C-18 eluate was tested for Daphnia
toxicity and analyzed by HPLC with a Beckman System Gold module (programmable
solvent module 126, diode array detection
module 168). The column was a Pep-S
C2C18 (4 x 250 mm) with a precolumn
from Pharmacia (Freiburg).
For HPLC separation of the different
crude extract fractions, the mobile phase was
an isocratic mixture of A (64%): 25 mM
CH3COONH4 in H,O-NANOpure;
B (36%):
25 mM CH,COONH,
in 80% CH,CN and
a flow of 1 ml min-l. Microcystis PCC7806
contains two closely related hepatotoxins
(Birk et al. 1989; Martin 199 1); the separation method for microcystin-LR
and
3-desmethylmicrocystin-LR
is described in
more detail by Martin (199 1). Authentic
microcystin-LR
was purchased from Medor
(Biochemicals, Hersching). The UV spectrum (190-4 10 nm) of authentic microcystin-LR was compared with the UV spectra
of the microcystin-LR
and 3-desmethylmicrocystin-LR
corresponding
peaks in the
tested fractions and regression coefficients
were calculated (Beckman software 5.0). All
chemicals of analytical grade and for HPLC,
which were of gradient grade, were from
Merck. Acetonitrile was from Baker (GroBGerau).
Toxicity experiments of the different fractions ended after 48 h; daphnids were counted every 24 h. The C-l 8 solid-phase extraction described above was also carried
out with lake water (0.45-pm membrane filtered), which was sampled and tested for
Daphnia toxicity to show that the used cartridges contained no compounds toxic to
daphnids. The pH and conductivity
of the
water of dead animals were determined. If
pH (7.5 +0.5) or conductivity (450& 150 PS
cm-2) were out of the ranges mentioned,
7.5
Probit
1
90
: 60
%
i-30
2.5 /’
0.3
I
1.3
‘0
2.3
Log (concn)
Fig. 1. Relative survival times (right axis) of Da&niu pulicuria exposed to different amounts of crude
Microcystis PCC7806 extract (7.8-, 15.6-, 3 1.3-, 62.5,
125-, and 250 ~1 ml-‘, on a logarithmic x-axis) as a
PROBIT-transformed plot (left axis, Weber 1972).
the experiment was discarded. All experiments were carried out three times with 5 1
daphnids per treatment. The statistically
significant difference (P < 0.05) between the
treatments was determined by x2-test.
The experiments were conducted to clarify whether microcystin-LR
and 3-desmethylmicrocystin-LR
are the compounds
in Microcystis PCC7806 that are toxic to
Daphnia. POC was measured to compare
experiments with other natural and laboratory data. Lyophilized Microcystis (1 mg)
contained 0.528 mg of C. The crude extract
was prepared from material equivalent to
264 mg of C. Figure 1 shows the relative
survival times of daphnids exposed to different amounts (7.8, 15.6, 31.3, 62.5, 125,
and 250 ~1 ml-l) of crude Microcystis extract as a PROBIT-transformed
plot (Weber
1972). Survival times of treatments with
lower amounts of the crude extract (7.8 and
15.6 ~1 ml-l) showed no statistically significant difference to the control (starved)
animals. Survival times of treatments with
higher amounts (> 3 1.3 ~1 ml- l) of the crude
extract showed Daphnia toxicity. These animals died significantly faster than starved
animals.
With these data a LCsO of 47.4 (+8.6,
- 6.5) ~1ml- 1was calculated and an amount
of 125 ~1 ml- 1 was set for further toxicity
experiments
with purified extracts. The
Daphnia toxicity of the crude extract was
compared (Table 1) with the toxicity of C- 18
cartridge-passed crude extract and C-l 8
Notes
1780
Table 1. Comparison of microcystin-LR concentration in different fractions of the Microcystis PCC7806
crude extract and their toxicity against Daphnia. MCYST-LR-microcystin-LR;
ND-not detected; in parentheses- the 3-desmethylmicrocystin-LR concentrations.
MCYST-LR (pg ml-l)
Tested vol. (~1 ml-l)
MCYST-LR (pg test vol.-l)
Survivors (24 h, %)
Survivors (48 h, %)
Crude extract
C- 18 cartridge-passed
extract
C- 18 eluate
22 (12.3)
62.5
1.4 (0.8)
68
0
ND
125
ND
58
0
16.3 (9.1)
125
2.0 (1.1)
94
78
eluate (methanol, dried and diluted in phosphate buffer, pH 8). The relative survival
times of daphnids exposed to the crude extract or to the C-l 8 cartridge-passed crude
extract showed no significant difference.
Both treatments are significantly different
from control (starved) animals. The relative
survival times with C-l 8 eluate (dried and
diluted in phosphate buffer, pH 8) are significantly different from the two other treatments but not from the control treatment.
The chromatograms
of quantitative
HPLC analysis of microcystin-LR
are presented in Fig. 2. The HPLC chromatogram
of authentic microcystin-LR
(monitored at
238 nm) is shown in Fig. 2A. Retention time
under the conditions
used is 5.48 min.
Quantitative
analysis of the HPLC chromatogram ofthe crude Microcystis PCC7806
extract (Fig. 2B) resulted in a microcystinLR concentration of 22 pg ml-‘. The tested
volume of the crude extract contains 1.4 pg
of microcystin-LR.
The crude extract also
contained 12.3 pg ml-l of 3-desmethylmicrocystin-LR (i.e. 0.8 pg tested vol-‘). The
HPLC chromatogram
of the C-l 8 eluate
(dried and diluted in phosphate buffer, pH
8, Fig. 2C) showed a microcystin-LR
and a
3-desmethylmicrocystin-LR
peak with concentrations of 16.3 and 9.1 pg ml-l respectively. The volume for testing Daphnia toxicity (125 ~1 ml-l) contained 2 and 1.1 pg
of these two compounds respectively. The
correlation
coefficient
of the DAD-UV
spectra of the microcystin-LR
corresponding peaks (Fig. 2A-C) for the analysis data
is >0.99 (Beckman software 5.0). This correlation coefficient verifies that the compounds of the corresponding peaks in the
different fractions are equal. No microcystin-LR and 3-desmethylmicrocystin-LR
were detected in the C-l 8 cartridge-passed
crude extract (Fig. 2D).
HPLC analysis data were compared with
the survival times of D. pdicaria exposed
to the different fractions of the crude extract
in Table 1. The crude extract was toxic to
Daphnia at this concentration (62.5 ~1 ml-’ ;
containing 1.4 and 0.8 pg microcystin-LR/
3-desmethylmicrocystin-LR).
The fraction
containing
microcystin-LR
and 3-desmethylmicrocystin-LR
(C- 18 eluate, dried
and diluted in phosphate buffer, pH 8)
showed no Daphnia toxicity at this concentration (125 ~1 ml-l; containing 2 pg microcystin-LR/ 1.1 pg 3-desmethylmicrocystin-LR). However, the fraction in which
these compounds were not detected (C- 18
cartridge-passed crude extract) was toxic to
daphnids.
Microcystin-LR
is the compound frequently isolated from Microcystis spp. and
its toxicity to warm-blooded animals is well
described (Runnegar and Falconer 198 1;
Honkanen et al. 1990). The role of this cyclic heptapeptide in the lake ecosystem is
Fig. 2. HPLC analysis of different fractions of the Microcystis extract. A. Chromatogram presents authentic
microcystin-LR (MCYST-LR). B. Chromatogram of the crude extract of Microcystis PCC7806 shows a microcystin-LR peak and a closely related toxin reported as 3-desmethylmicrocystin-LR (3-dm-MCYST-LR). C. The
C- 18 eluate, diluted in phosphate buffer, contains both toxins. D. In the C- 18 cartridge-passed crude extract the
toxins were not detected.
0
v
0
0
0
0
-h
aii
C
I
1
1
1
Absorbance
I
u
I
1
Absorbance
MCYST-LR
0
.
N
1782
not clear. Earlier experiments show the toxicity of Microcystis strain PCC7806 to
Daphnia and the possibility of separating
the toxic factor from freeze-thawed cells with
water (Jungmann et al. 199 1). Codd et al.
(1989) reported that a water extract of lyophilized Microcystis cells contains large
amounts of microcystin-LR
which may be
the compound toxic to Daphnia. For toxicity experiments and for further purification steps of the compound in Microcystis
that is toxic to Daphnia, it was necessary to
determine the LCSO [47.4 (+8.6, -6.5) ~1
ml-l] of the crude extract from lyophilized
cells. With the determination of POC of lyto
ophilized Microcystis cells-necessary
harvest the LCSO amount (0.3 mg of C)-a
comparison of laboratory and natural data
became possible.
Lampert (198 lb) showed that the compound in Microcystis that was toxic to
Daphnia must be an endotoxin. Therefore
the animals must ingest the cells to become
toxified. Testing extracts from Microcystis
cells for toxicity to daphnids can be problematic because the mechanism of toxin uptake by the animals (not the molecular
mechanism for toxicity) is unclear. One can
expect that the effective dosage applied from
extracts is higher than in natural environments where whole cells are ingested.
Therefore the uptake mechanism of toxincontaining cells can be more effective in nature. Lampert (198 1a,b) and Jungmann et
al. (199 1) showed that a concentration of 1
mg C liter-’
of Microcystis cells is high
enough to kill daphnids within 4 d. These
results correspond to Microcystis quantities
found in nature (Benndorf and Henning
1989) and to theoretical considerations,
suggesting that the toxin must be effective
at low cell densities in order to allow bloom
formation (Lampert 198 1b).
The calculated LCSo for the crude extract
seems to be comparable. My results suggest
that the C- 18 cartridge-passed crude extract
contains a compound or compounds extracted from Microcystis PCC7806, exhibiting a higher toxicity against D. pulicaria
than microcystin-LR
or 3-desmethylmicrocystin-LR
because both compounds
were detected in the nontoxic fraction. A
higher toxicity of other compounds con-
firms the results of Kiviranta et al. (1991).
They detected toxicity against Artemia saZina in different nonmicrocystin-containing
fractions extracted from various cyanobacteria strains. Pefialoza et al. (199 1) reported
a Microcystis bloom toxic to daphnids and
tried to isolate the toxic compound. After
different purification steps they determined
a molecular weight for the toxic compound
close to the molecular weight of microcystin-LR, implying that a microcystin-XY
was
the purified compound that is toxic to
daphnids. They also reported that toxicity
of the different fractions was lost after boiling ( 1OOOC).However, microcystins are heatstable to > 160°C (Weckesser and Martin
1990). The temperature instability
of the
isolated compounds is another indication
that microcystin-LR
is not the compound
toxic to Daphnia.
DeMott et al. (199 1) incubated D. pulicaria with different amounts of purified microcystin-LR and calculated a LCSO of 2 1.4
pg ml-l. If one considers this quantity to
be 0.2% dry weight of Microcystis cells
(DeMott et al. 199 l), an extract from 10.7
mgdrywt ml-l would be needed to reach this
concentration,
meaning that 107 mg,,, ti
ml-l of Microcystis material would be required-more
than 1,OOO-fold higher than
the amount of Benndorfand Henning (1989)
observed in a natural bloom of Microcystis
aeruginosa. At this concentration microcystin-LR seems to be toxic to daphnids.
However, this dosage is not comparable with
laboratory or natural observations, as 2 1.4
hg ml-l can be more than the total DOC in
eutrophic lakes (Wetzel 1983).
Until now only one specific cell type is
known (the liver cells of vertebrates) to have
a molecular uptake mechanism for microcystins (Weckesser and Martin 1990). Other
experiments carried out to clarify the molecular mechanism of toxicity deal with extracts of cells from plants or mammals containing protein phosphatases 1 and 2A
(MacKintosh et al. 1990). Zooplankton may
have these ubiquitous enzymes also, but until now no cellular molecular uptake mechanism has been established, which could be
the reason for the low response of different
zooplankton to purified toxins (DeMott et
al. 199 1). Further purification
steps of the
Notes
1783
JUNGMANN,D., M. HENNING,AND F. J~~T~NER.199 1.
Are the same compounds in Microcystis responsible for toxicity to Daphnia and inhibition of its
filtering rate? Int. Rev. Gesamten Hydrobiol. 76:
47-56.
KIVIRANTA, J., K. SIVONEN,AND S. I. NIEMELA. 199 1.
Detection of toxicity of cyanobacteria by Artemia
salina biotest. Environ. Toxicol. Water Qual. 6:
423-436.
Dirk Jungmann
KRAMBECK, H.-J., W. LAMPERT, AND H. BREDE. 198 1.
Messung germger Mengen von partikullrem KohMax-Planck-Institut
fur Limnologie
lenstoff in natiirliche Gewisser. GIT Fachz. Lab.
Abteilung Okophysiologie
25: 1009-1012.
August-Thienemann
Str. 2
LAMPERT, W. 198 1a. Inhibitory and toxic effects of
blue-green algae on Daphnia. Int. Rev. Gesamten
D-2320 Plan, Germany
Hydrobiol. 66: 285-296.
-.
198 1b. Toxicity of blue-green Microcystis
References
aeruginosa: Effective defence mechanisms against
grazing pressure by Daphnia. Int. Ver. Theor. AnBENNDORF,J., AND M. HENNING. 1989. Daphnia and
gew. Limnol. Verh. 21: 1436-1440.
toxic blooms of Microcystis aeruginosa in Bautzen
MACKINTOSH, C., K. A. BEAT-HE, S. KLUMP, P. COHEN,
reservoir (GDR). Int. Rev. Gesamten Hydrobiol.
AND G. A. CODD. 1990. Cyanobacterial micro74: 233-248.
cystin-LR is a potent and specific inhibitor of proBIRK, I. M., AND OTHERS. 1989. Nontoxic and toxic
tein phosphatases 1 and 2A from both mammals
oligopeptides with D-amino acids and unusual resand higher plants. FEBS (Fed. Eur. Biol. Sot.) Letidues in Microcystis aeruginosa PCC7806. Arch.
ters 264: 187-l 92.
Microbial. 151: 4 1l-4 15.
BOTES,D. P., AND OTHERS. 1985. Structural studies MARTIN, C. 1991. Toxische und nicht-toxische Peptide aus Cyanobakterien der Gattung Microcystis.
on cyanoginosin-LR, -YR, -YA, and -YM, peptide
Ph.D. thesis, Albert-Ludwigs-Univ., Freiburg.
toxins from Microcystis aeruginosa. J. Chem. Sot.
PEIULOZA, R. M., M. ROJAS,I. VILA, AND F. ZAMPerkin Trans. 1: 2747-2749.
BRANO. 199 1. Toxicity of a soluble peptide from
CARMICHAEL,W. W. 1988. Toxins of freshwater alMicrocystis sp. to zooplankton and fish. Freshgae, p. 12 1-147. In A. T. Tu [ed.], Handbook of
water Biol. 24: 233-240.
natural toxins. V. 3. Dekker.
CODD, G. A., AND OTHERS. 1989. Production, detec- RUNNEGAR,M. T., AND I. R. FALCONER. 198 1. Isolation, characterization and pathology of the toxin
tion, and quantification of cyanobacterial toxins.
from the blue-green alga Microcystis aeruginosa,
Toxicol. Assess. 4: 499-5 11.
p. 325-342. In W. W. Carmichael [ed.], The water
DEBERNARDI,R., G. GIUSSANI,ANDE. LASSOPEDRETTI.
environment. Plenum.
1980. The significance of blue-green algae as food
for filterfeeding zooplankton: Experimental stud- WEBER, E. 1972. Probitanalyse, p. 579. In E. Weber
[ed.], GrundriB der biologischen Statistik. Gustav
ies on Daphnia spp. fed Microcystis aeruginosa.
Fischer.
Int. Ver. Theor. Angew. Limnol. Verh. 21: 477WECKESSER,
J., AND C. MARTIN. 1990. Toxine aus
483.
Cyanobakterien im Wasser: Microcystin und verDEMOTT, R. W., Q. ZHANG,AND W. W. CARMICHAEL.
wandte Peptide. Forum Microbial. 7-8: 364-369.
199 1. Effects of toxic cyanobacteria and purified
toxins on the survival and feeding of a copepod WETZEL, R. G. 1983. Organic carbon cycling and
detritus, p. 667-669. In R. G. Wetzel [ed.], Limand three species of Daphnia. Limnol. Oceanogr.
nology, 2nd ed. Saunders.
36: 1346-l 357.
HONKANEN,R. E., AND OTHERS. 1990. CharacterizaSubmitted: 27 December 1991
tion of microcystin-LR, a potent inhibitor of type- 1
Accepted: 26 May 1992
and type-2 protein phosphatases. J. Biol. Chem.
265: 19,401-l 9,404.
Revised: 23 July 1992
crude water extract or other extraction nrocedures of Microcystis PCC7806 should lead
to identification
of the compound or compounds in Microcystis spp. that exhibit a
higher toxicity against Daphnia than the
well-known microcystins.