the role of microcystins in heavy cyanobacterial bloom formation

Journal of Plankton Research Vol.20 no.4 pp.691-708, 1998

The role of microcystins in heavy cyanobacterial bloom formation

Bojan Sedmak and Gorazd KosiNational Institute of Biology, Department of Biology, Biotechnical Faculty,University of Ljubljana, Vecnapot 111, 1000 Ljubljana, Slovenia

Abstract The presence of high microcystin concentrations in cyanobacterial blooms additionallyaffects species diversity. Blooms with high toxin contents can reach higher cell densities, which is alsodemonstrated by microcystin cell contents. In vitro experiments show that microcystins influencephytoplankton proliferation. The action is strongly dependent on the phytoplankton species testedand light conditions. We propose that the environmental impact of different microcystins depends ontheir enzymatic inhibition activity and thus could not be measured merely on the basis of their toxicityto vertebrate species. Their role in heavy cyanobacterial bloom and scum formation is discussed, aswell as their impact on the massive proliferation of other species following toxic cyanobacterial bloomdegradation.

Introduction

The majority of authors point out the significance of cyanobacteria as a healthhazard to humans and agricultural livestock (e.g. Codd, 1994; Falconer et ai,1994). Nevertheless, we would like to emphasize the negative impact cyano-bacterial blooms have on the aquatic environment. Besides the health hazard tohigher vertebrates, they have a great influence on general environmental healthand must be considered as a warning of environmental degradation. Eutrophica-tion, together with blooms as the consequence, has a strong influence on all livingorganisms in water bodies (e.g. Harper, 1992). There are a great number ofmeasurable physicochemical and environmental factors that are responsible forphytoplankton distribution and species diversity, including bloom formation.These factors can affect all the organisms in a water body or only specific, moresusceptible ones, having regard to the conditions, amounts or concentrations.Variations in day length, insolation, temperature, wind mixing, rainfall, flushingand nutrient loading rates affect all kinds of water bodies, but are morepronounced in smaller ones. Physicochemical parameters vary more widely inponds and reservoirs than in deep natural lakes, and reflect more or less onlytemporary conditions. As blooms are frequent in smaller water bodies in Slovenia(Sedmak et al., 1994; Sedmak and Kosi, 1997), we focused our research mainly onphytoplankton species abundance and composition as an indicator of environ-mental status. In our opinion, phytoplankton species are the best reflection oflong-lasting physicochemical and biological factors, which are themselves func-tions of the climatic and hydrological regimes, since the majority of freshwaterphytoplankton organisms incorporate resistant benthic resting stages into theirlife history strategies. In this way, planktonic algae can compensate for noxiousexogenic short-term effects.

Despite the fact that cyanobacteria have lower maximal growth rates than themajority of other phytoplankton species, in certain conditions they are able tooutstrip all other species, resulting in cyanobacterial blooms. Their chances of

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RSedmak and G-Kosi

proliferation are better in low-light conditions (e.g. Oscillatoria rubescens), asdemonstrated by Mur et al. (1978). These authors unsuccessfully tried to demon-strate that conditions of low phosphate concentration or other nutrients could bethe factor that allowed cyanobacterial dominance. Two main factors can gener-ate low-light conditions: the depth of the water and its turbidity. The latter couldbe caused by materials suspended in the water, such as mud, or, more commonly,by phytoplankton. Light distribution in water is highly influenced by the mutualshading of phytoplankton. In this way, the turbidity of the water increases andfavours those organisms adapted to low light intensities. Light limitation alonedoes not elucidate the cyanobacterial bloom phenomenon. In a competitionexperiment, Mur et al. (1978) presumed that there must be an ecological factorwhich lowers the growth of algae more than the growth of cyanobacteria. Wewould like to rephrase this idea as: there must be an ecological factor whichaugments the growth of cyanobacteria more than the growth of algae.

It has long been known that the possibility of a cyanobacterial bloom beingtoxic is >50% (Olson, 1964). In our investigations, >80% of Microcystis bloomswere toxic (Sedmak et al., 1994; Sedmak and Kosi, 1997). Additionally, a long-term study showed that each year Microcystis aeruginosa was non-toxic at thebeginning of the growing season, and developed high toxicity during the firststrong biomass increase (Benndorf and Henning, 1989). Although there areseveral hypotheses elucidating the factors influencing cyanobacterial dominance(e.g. Shapiro, 1990), none of them deals directly with the influence of microcystinson bloom formation.

Cyanobacteria are most probably the first organisms in the Earth's history, withfossil records dated 3.3-3.5 billion years ago, and therefore it is unlikely thatmicrocystins play the role of a defensive substance. We propose that microcystinsact as growth regulators, helping cyanobacteria to produce as many offspring aspossible, giving a better opportunity for successful adaptation. Our goal is toemphasize the effects of microcystins on the phytoplankton community duringhepatotoxic blooms.

Method

Research sites

We analysed data from seven fish ponds and three reservoirs over a 3 year period(Tables I and II). The locations were grouped according to outflow operativityand hepatotoxin contents. Some data from Lake Bled (a natural lake) are alsodiscussed.

Field sampling

Qualitative samples were taken in late summer and autumn, during cyano-bacterial blooms, with a 25 mesh net as a vertical profile and analysed for phyto-plankton species community composition. The surface bloom was avoided inorder to achieve a representative sample of the community. Samples werepreserved in 5% formaldehyde. With the use of an inverted microscope, they

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Microcystins and bloom formation

Table L Description of locations with relatively stable conditions during cyanobacterial blooms,grouped according to cumulative hepatotoxin contents of the bloom

Locations without outflowSampling Description Year Chi a Phytoplankton Trophic categorysite ("gl-1) species no.a (OECD values)

Blooms with high microcystin contents (ZMC > 10 ug I"1)BOC Gravel pit 19% 67HLP Gravel pit/fish pond 1994 169HGD Fishpond 1994 249SAV Reservoir/fishpond 1994 91

Blooms with low microcystin contents (SMC < 10 ug I"1)BOR Gravel pit/ 1994 31

fish pond 19% 11LUT Fishpond 1994 56

19% 40PODC Gravel pit/fish pond 1994 51POD Gravel pit/fish pond 19% 57

BOC, Boreci; BOR, Borovci: BUK, Lake Bukovnik; HGD, Hotinja Village (fish pond); HLP, HotinjaVillage (gravel pit); KOS, Koseze; LED, Ledava; LUT, Lutverci; POD, Podgrad; SAV, Savci.'Figures in parentheses represent phytoplankton taxa other than cyanobacteria.•"Because of low rainfall, the outflow was inoperative.Intermediate value.

Table II. Description of locations with operational outflows resulting in unstable conditions duringcyanobacterial blooms with subsequent higher phytoplankton diversity

6(2)6(2)5(2)8(5)

21 (16)24 (19)18(13)20(15)12(8)19(14)

HypertrophicHypertrophicHypertrophicHypertrophic

Hypertrophic

Hypertrophic

HypertrophicHypertrophic

Locations with outflowSamplingsite

BUKKOSKOSLEDSAV

Description

ReservoirClay pit/fish pondClay pit/fish pondReservoirReservoir

Year

1996199419%19951996

Chi a(MgH)

1149203130

Phytoplanktonspecies no.°

15 (12)18(16)28(25)24(22)22 (17)

Trophic category(OECD values)

EutrophicEutrophicEutrophicHypertrophicHypertrophic

"Figures in parentheses represent phytoplankton taxa other than cyanobacteria.

were analysed for phytoplankton species composition and abundance. Thespecies were identified according to Komarek (1958,1991), Starmach (1966,1980)and Hindak (1978).

Toxin analysis

Bloom samples were purified, concentrated and extracted according to themethod of Harada et al. (1988), as described elsewhere (Sedmak and Kosi,1997). The toxic fractions were separated using HPLC. All equipment wasobtained from Waters, Millipore Division, and consisted of a Waters 600 Multi-solvent Delivery System, a Waters 616 Pump, a Waters 996 Photodiode ArrayDetector and a Waters Fraction Collector (Milford, MA) equipped with ananalytical Hibar Pre-Packed RT 125-4 (Merck) LiChrospher 100 RP-18 (5 um)

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B^edmak and G.Kosi

column. The equipment was fed through an NEC Image 466 ES computer runby a Millenium 2010 Chromatography Manager (Millipore). The amounts oftoxins were estimated by comparison of the peak area at 238 nm of the testsample after separation with methanol:0.05 M phosphate buffer (58:42, pH 3.0)with those of standard samples (MC-LR, ICN Biomedicals Inc.; MC-LR, MC-RR and MC-YR, Calbiochem]. Microcystin isolation was performed under thepreviously described conditions using a preparative Spherisorb S10 ODS2column (Phase Separation Inc. USA) with a flow rate of 10 ml min"1.

Organisms and growth conditions

For in vitro experiments, non-axenic phytoplankton species isolated in ourlaboratory were grown in continuous culture. Twelve hour illumination wasprovided by Osram L 18 W/72 Biolux white fluorescent lamps together withSylvania Gro-Lux F 18 W/GRO-T8 lamps. The incident illumination wasmeasured outside the vessels with a Delta-T Logger (Delta-T Devices Ltd, UK),equipped with a QS Quantum sensor.

Growth experiments were carried out on MultiDish 24 (Nunc, Denmark) cellculture plates. They were performed at least in triplicate, each in 2 ml of BG11media (Steiner et al, 1971). The starting inoculum depended on the maximumcell count that could be reached by the specific species, in the range from 1 X 10s

to 5 X 10s cells I"1, and on the chlorophyll a content. The experiments werecarried out at three light intensities: in the dark, and at 4 and 40 u£ nr2 s"1 at aconstant temperature of 20°C. The chronic influence of three different micro-cystin-RR concentrations of 1 X 10"7,5 X 10"7 and 1 X 10"* M on phytoplanktonin culture was tested. The microcystin tested was isolated in the laboratory andits characteristics compared with a standard. Microcystin-RR was chosen as themost frequent toxin in our waters, with a share up to >88% of total microcystincontents (Sedmak and Kosi, 1997). The growth was determined by cell countingin a Burker-Tiirk haemocytometer (Brand) under a microscope.

The permeability of cyanobacterial cells to microcystin-RR was tested in anacute experiment. Microcystis aeruginosa Kuetz. cells, a non-toxic unicellularstrain, were concentrated by centrifugation to 107 cells ml"1 and exposed to thetoxin at a final concentration of 5.5 X 10~5 M MC-RR for 1 h at 0°C to preventcell division. The cells were washed twice with a 10-fold excess of cold BG11 toremove the toxin from the medium. A cell growth experiment was then carriedout and proliferation determined as described previously.

Analytical methods

For chlorophyll a determination, the Vollenweider (1974) method was used. Twotechniques of cell harvesting were used: by centrifugation at 13 000 r.p.m. andby filtering through glass microfibre Whatman GF/C filters (Whatman Ltd,Maidstone, UK) and extracting with hot methanol.

694



Results

Phytoplankton diversity

The most striking similarity in all the water bodies studied was the sometimesextreme reduction in species diversity, especially in blooms with higher micro-cystin contents with a rise of cyanobacterial species. In summer and autumn,there are well over 30 different phytoplankton species (data not presented) inindividual water bodies, but during cyanobacterial blooms their count decreasessignificantly (Tables I and II).

A correlation was found between the total microcystin content and the numberof phytoplankton species present in the blooms. Plotting the amount of micro-cystins against species diversity gave an exponential relationship. The data werefitted by the exponential function:

y = A X exp(-fc X x) + Residue

The results in Figure 1 are arranged into two groups, where 10 ug microcystinsh1 seems to be the boundary level. Above this concentration, the species diver-sity count falls to a minimal level.

Not only microcystin contents, but also bloom density, were negatively corre-lated with species diversity (Figure 2). The values for the locations with an oper-ating outflow were dispersed, indicating unstable conditions during the bloom,deriving from the hydraulic wash-out of cyanobacteria. In stable conditions, thebloom could actually exert a much stronger influence on various species throughthe resulting toxin release, and through the creation of adverse light conditionsin the water body. The influence of toxic cyanobacterial blooms on species diver-sity in stable conditions was clearly divided into two separate groups described

XS

Iver

slty

(N

° of

ies

dpe

c

(0

30-,

25-

20-

15-

10 -

5 -

0 -

0 # Fit statisticsSS = 260.8936

# df= 12m r = 0.807477

; .

• \

• \

\

0 10 20 30 40 50 60

Microcystins (ug/l bloom)

Ctll

num

btr

4x10*-

3x10*-

2x105-

1x105-

0 -

s0

controlMC-RR 5x10"7 M

/

/

i < i i i

2 4 6 8 10

Days

Fig. L (A) The relationship between microcystin concentration and species diversity for all locations.(B) The relationship between microcystin concentration and species diversity in small water bodieswith stable conditions during the bloom. The dashed vertical line indicates the 10 |ig microcystin I*1

bloom boundary.

695


RSedmak and G-Kosi

40-.

s* 30.o

%

ie

20-

COS 10-

Q Locations with outflow0 Locations without outflow < 10 (ig/l MCY• Locations without outflow > 10 |ig/l MCYO Intermediate value

0 - _

107" 10*

Bloom density (cells/1)

10'

Fig. 2. The influence of bloom density on species diversity. The linear regression curves were calcu-lated only for the locations without outflow, separately for groups with lower IMC and higher XMCcontents.

by linear regression curves with good correlation coefficients. We divided thetoxic blooms into two categories: blooms with low toxicity (<10 \ig total micro-cystins I"1) and blooms with high toxicity (>10 ug total microcystins I"1), assuggested by the results presented in Figure IB.

Regression statistics

• locationscSSd.f.5 2

rr2

= <10 ug MC I"1 bloom= 0.003893= 3= 0.001298= 0.966158= 0.933461

• locationscSSdf5 2

rr2

= >10 ug MC I"1 bloom= 0.371687= 2= 0.185843= 0.676206= 0.457254

(sum of squares)(degrees of freedom)

(corr. data versus mo

The results from Figure 2 indicate that very high bloom densities (>1.8 x 108 cellsI"1) were associated with cumulative microcystin concentrations of >10 jig I"1

bloom.

Description of species

Cyanophyta. The most frequent bloom-forming genus in smaller water bodies isMicrocystis, with two species: M.aeruginosa and M.wesenbergii Kom. (Tables IIIand IV). Other bloom-forming cyanobacteria are O.rubescens DC (Sedmakand Kosi, 1991) and two (in our case) non-toxic filamentous representatives:

696



Anabaena flos-aquae (Lyngb.) Breb. and Aphanizomenon flos-aquae (Sedmakand Kosi, 1997).

Other species present. In Lake Bled, only two green algae were found. Threeother species were abundant in this period: Dinobryon sociale Eh. (Chrysophyta),a diatom belonging to the genus Stephanodiscus (Bacillariophyta) and Crypto-monas ovata Eh. (Pyrrhophyta). The non-toxic Aphanizomenon bloom in Ledavareservoir had little effect on species diversity. The appearance of the Microcystisbloom in Radehova reservoir decreased the number of species present, with onlyone alga reaching a higher abundancy: Melosira varians Ag. (Bacillariophyta). Aheavy toxic bloom of the genus Microcystis in the Savci reservoir provoked adrastic decrease in diversity. In 1994, there were only eight different species, threeof them belonging to the cyanobacteria. Koseze is a smaller water body withblooms of Microcystis in both of the years 1994 and 1995. In 1994, the bloomcontained only very small amounts of toxins (Sedmak and Kosi, 1997). Thevariety was affected, but there were two other species abundant: Uroglena amer-icana Calk. (Chrysophyta) and Peridinium cinctum (O.F. Muller) Eh. (Pyrrho-phyta). In the bloom of 1995, which was more toxic, but very weak in terms ofbiomass, the diversity was high, with several species other than cyanobacterialones abundant. Among the gravel pits, the greatest diversity was found in theweak bloom at Borovci. The sample from Lutverci contained only one diatomspecies, M.granulata, with a small count. Both toxic Microcystis blooms inPodgrad had a strong negative effect on diversity. In the bloom in 1994, an abun-dance of Ceratium hirudinella (O.F. Muller) Schrank (Pyrrhophyta) wasobserved. In 1994, the effect on diversity was stronger, with only one species,M.granulata (Bacillariophyta), being abundant besides cyanobacteria.

We inspected two locations, Podgrad and Savci, after 23 days in the phase ofbloom degradation. The cyanobacterial scum disappeared, although the speciescomposition remained similar. The overall species diversity increased in Podgradfrom 12 to 16 and in Savci from 8 to 11. In both locations, in the period of exten-sive blooming there were no Bacillariophyta species that reappeared with thedecline of cyanobacteria. An increase in Chlorophyta was especially evident inPodgrad.

In vitro experiments

All algae tested exhibited an enhanced growth in the presence of microcystin inthe early stage and a resulting growth suppression afterwards, strongly dependenton light intensity. A 10-fold increase in microcystin concentration did not resultin a proportional effect. On the contrary, the growth of M.aeruginosa wasenhanced with no inhibitory action even in the presence of higher microcystinconcentrations. In the absence of light, when the cells did not proliferate, wedetected no effects of microcystin on phytoplankton in culture.

Cyanobacteria. (i) Microcystis aeruginosa Kuetz. (non-toxic unicellular strain)—chronic experiment. The influence of microcystin-RR on this most widely known

697


B-SedmaV and G-Kosi

2x107 -,

1.8x107

1.6x107 \

1.4x10' \

Ic

1x10'

S 8x10*:

6x10* i

4x10*-

2x10*-i

0 -

2x10'

1.8x107

1.6x10T

1.4x10T

5 1.2X107

g 1x107

% 8x10*

6x10*

4x10*

2x10*

0 J

B B Control• MC-RR 5x10"7 M

8 10

Days

12 14 16 18

Fig. 3. The growth of M.aeruginosa at 40 \xE nr2 s"1 illumination at two microcystin concentrations:(A) XQr1 M and (B) 5 X 10"7 M microcystin-RR. The arrow indicates the onset of growth stimulation(mean ± SE).

hepatotoxin producer depends strongly on light conditions and toxin concentra-tions. A higher toxin concentration shortened the response time and augmentedcell division (Figure 3).

The stimulation of cell division was evident at higher (5 X 10~7 M) microcystin-RR concentrations, with a pronounced effect at lower light intensities (Figure 4).

(ii) Microcystis aeruginosa Kuetz. (non-toxic unicellular strain)—acute experi-ment. Cyanobacterial cells were exposed to microcystin-RR at low temperatureto prevent cell division. After 1 h incubation, the toxin was removed and thecyanobacteria grown at 40 uE nr2 s"1 illumination. Cell proliferation wasenhanced, indicating the permeability of the membrane to microcystin (Figure 5).

(iii) Chroococcus minutus (Kuetz.) Nag. The influence of microcystin-RR onthis cyanobacterium was strongly light dependent. Higher light intensities (40 uE

698



1x10' -,

9x10*-

8x10*:

7x10* ̂

I 6x10*:|c% 4x10*-O

3x10*:

2x10*:

1x10*:

0 -

• Control• MC-RR 10"7 M

1x107 -,

9x10*:

8x10* •:

7x10* \

6x10*:

5x10* \

4x10* '-

3x10*-

2x10*-

1x10* '-

B

0 2 4 6 8 10 12 14 16 18 20

Days

Fig. 4. The growth of M.aeruginosa at 4 uE nr2 s~' illumination at two microcystin concentrations:(A) 10~7 M and (B) 5 x 10"7 M microcystin-RR. The arrow indicates the onset of growth stimulation.

m~2 s"1) caused growth suppression, with fluctuations in cell count suggesting celldegradation. The onset of cell growth stimulation was positively correlated withmicrocystin concentration (Figure 6). At a defined illumination and toxin concen-tration, stimulation of cell proliferation could be achieved (Figure 7A).

Algae

Coelastrum microporum Nag., Monoraphidium contortwn (Thur.) Kom.-Legn.and Cryptomonas erosa Eh. The growth of C.microporum under low-lightconditions was strongly affected, resulting in inhibition of cell proliferation(Figure 8).

The green alga M.contortwn, also growing successfully at low light intensities,

699


B£edmak and G-Kosi

o

oU

2x10'

1.5x10'

1x107

5x10*

g ControlDMC-RR5.5x10"5M

0 2 4 6 8 10 12

Days

Fig. 5. Growth stimulation of M.aeruginosa after 1 h acute exposure to 5.5 X 10"5 M microcystin-RR

showed enhanced proliferation in the presence of microcystin-RR (Figure 9). Thesame results were obtained with S.quadricauda (Chlorophyta) (data notpresented). In contrast, the initial stimulation of C.erosa (Cryptophyta) resultedin cell loss at the same light and temperature conditions (Figure 10).

Phytoplankton species behave heterogeneously to a microcystin environmentunder different light conditions. Unlike other phytoplankton species, cyano-bacteria respond to a higher toxin concentration by shortening the responsetime required for enhanced proliferation, indicating cell permeability tomicrocystin-RR.

Discussion

From the field data, it is evident that small water bodies with stable conditionsbetter reflect all the noxious effects of toxic cyanobacterial blooms on the phyto-plankton community in comparison to those with unstable conditions. Differentmicrocystins exhibit similar effects on mammalian cells, in vitro and in vivo (e.g.Runnegar et al., 1993), or even on imaowsb a n d p i a n t s (MacKintosh et al., i§90).We presume that thmy might a i s o exert a similar influence on phytoplanktonorganisms at their cellular level. We therefore plotted cumulative values of themicrocystins present in a bloom againsi §pe§i§§ diversity. Water bodies withhigher total microcystin valugs In the bleeifl (5»10 H| MC H) have a higher nega-tive impact on species diversity, and such a bloom fea"§&§§ higher cyanobacterialcell densities (Figures 1 and 2). The calculated results of ffllef oeystin contents percyanobacterial cell also confirm the last statement (Tables III and IV).

In vitro results demonstrate that microcystin-RR, particularly in low-lightconditions, can act heterogeneously on phytoplankton organisms. The effects canrange from augmentation of cell proliferation, as in the case of M.aeruginosa and

700



1.5x107 -,

1.2x10'

9x10§

6x10*-

3x10*-

0 -I

1.5x107 -,

1.2x10' -

9x10*:

% 6x10*-O

3x10* -

• ControlQ MC-RR10"' M

0 2 4 6 8 10 12 14 16

Days

B • Control• MC-RR 5x10"' M

\

,mT\0 2 4 6 8 10 12 14 16

Days

Fig. 6. The growth of C.minutus at 40 nE in"2 s~' illumination at two microcystin concentrations:(A) 10-7 M and (B) 5 x 10-' M microcystin-RR.

M.contortum (Figures 4 and 9), through inhibition, in the case of C.microporum(Figure 8), or even result in cell loss, e.g. Cerosa (Figure 10). It seems that theseeffects become evident only in specific light conditions, presumably depending onthe energetic light requirements of the species. These microcystin effects ongrowth seem to be minor, but added together could represent an ecologicalfactor, promoting heavy bloom or scum formation. In order to act on cell prolif-eration, microcystins must penetrate the cell or at least the cell membrane. Micro-cystis aeruginosa cells are permeable to microcystin-RR, as demonstrated in theacute experiment (Figure 5). Cells exposed to microcystin-RR proliferate fasterthan controls. That means, in favourable conditions when the bloom is partiallyformed and therefore turbidity rises, the microcystin producers proliferate fastersince the microcystin release in this phase is minimal (Watanabe et al, 1992). On

701


RSedmak and GJCosi

Table UL Toxic cyanobacterial blooms grouped according to total amount of hepatotoxins fromlocations without stable conditions

Locations with outflowSampling Year Dominant speciessite

Abundance(cells 1-')

I M C(Mgl-1)

SMC cell-(Pg)

Blooms with high microcystin contents (IMC > 10 ug I"1)BOC 1996 M.wesenbergii 50% 1.9 X 108

M.aeruginosa 50%HLP 1994 M.aeruginosa

4.7 X 10s

HGD 1994 M.aeruginosa 95%6.6 X 10s

SAV» 1994 M.aeruginosa 92%2.2 x 10s

Blooms with low microcystin contents (IMC < 10 ug I"1)

19.38 0.102

51.45

24.15

11.76

0.11

0.037

0.054

BOR

BOR

LUT

LUT

PODb

POD

1994

1996

1994

1996

1994

1996

M.aeruginosa 90%M.wesenbergii 10%M.aeruginosa

M.wesenbergii 75%M.aeruginosa 25%M.aeruginosa 70%M.wesenbergii 30%M.wesenbergii 75%M.aeruginosa 25%M.wesenbergii 80%M.aeruginosa 20%

7.8 X 10'

1.5 x 10'

1.5 x 108

1.1 x 10s

1.4 x 108

1.6 x 10s

1.88

0.04

2.26

5.63

2.43

1.28

0.024

0.003

0.014

0.051

0.017

0.008

'Because of low rainfall, the outflow was inoperative,intermediate value.

Table IV. Bloom composition and total hepatotoxin contents in locations subjected to wash-out

Locations with outflowSamplingsite

BUKKOSKOSLEDSAV

Year

19961994199619951996

Dominant species

M.wesenbergiiM.aeruginosaM.aeruginosa 90%Aph.flos-aquaeM.aeruginosa 70%M.wesenbergii 30%

Abundance(cells I"1)

3.1 x 10'1.4 x 10s

5.5 x 10'7.8 X 108

8.3 x 10'

IMC(ug I'1)

00.722.900

3.78

IMC cell-'(Pg)

00.0050.0530

0.046

the other hand, when microcystin-producing clones dominate the bloom, toxinrelease could affect other species, leading to the disappearance of susceptibleones.

On the basis of our results, we propose the following theory of bloomformation. Eutrophic waters favour the growth of different phytoplanktonspecies that contribute to water turbidity. At the beginning, different cyano-bacterial clones are evenly dispersed in the water body. Since the optimal lightintensities for toxin production are up to 40 uE nr2 s"1, where the maximum

702


Microcystins and bloom fonnation

1.5x107 ,

1.2x10' -I

^ 6x10* -IO

3x10* J

I ControlO MC-RR10'7 M

0 2 4 6 8 10 12 14 16

Days

E

O

1.5x10' -,

1.2x107

9x10*

6x10'

3x10*

B• ControlD MC-RR 5x10 7 M

0 2 4 6 8 10 12 14 16

Fig. 7. The growth of C.minutus at 4 uE m~2 s-' illumination at two microcystin concentrations:(A) 10-7 M and (B) 5 x 10"7 M microcystin-RR.

toxicity and maximum ratio of toxin to protein are achieved (Utkilen and Gjolme,1992), hepatotoxin producers are in a favourable position, due to water turbidity.The buoyant cyanobacteria additionally generate even poorer light conditions,allowing themselves to remain in a position to produce more toxins that arereleased during the senescence of cyanobacteria (Watanabe et al, 1992; Lahti etal, 1997). In combination with low light intensity, they can exclude the major partof susceptible species from the bloom. In such a situation, microcystin productionis a self-enhancing mechanism augmenting cyanobacterial cell division, gener-ating denser blooms than the non-producing cyanobacterial strains. We presumethat there is no environmental factor which converts an organism that does notproduce toxins into a toxin producer, but that toxin producers proliferate fasterand thus are able to capture the major share in the bloom.

703


RSedmak and CKosi

10'-,

g

10'

8x10*

5 6x10*

c^ 4x10*O

2x10*

Fig. 8. Growth of C.microporwn at 4 \i£. m~2 s~' illumination at two microcystin concentrations:(A) 10-7 M and (B) 5 X 10"7 M microcystin-RR.

Microcystins dissolved in water exert a chronic influence on other phyto-plankton species. Although the concentrations are usually low, we cannot neglectthe microenvironment. This means the organisms that are in close proximity tothe toxin releaser may be subjected to essentially higher microcystin concentra-tions, influencing their growth (Figures 6, 7 and 8), which in unfavourable lightconditions may even lead to cell disintegration of certain species (Figure 10).

We have several field results confirming our hypothesis on the influence ofmicrocystins on species diversity. In Lake Bled, O.rubescens is a permanentinhabitant together with sporadic blooms of An.flos-aquae and Aph.flos-aquae(Vrhovsek et al, 1982, 1984; Sedmak and Kosi, 1991). During the mixed toxicbloom of O.rubescens and An.flos-aquae in November 1994, we found anextremely low number of planktonic species; only nine phytoplankton species

704



3x107 -

,_ 2x107 -e

.a

c

0 2 4 6 8 10 12 14 16

1x10'-

0 J

Fig. 9. Growth stimulation of M.contortum; 1O"7 M microcystin-RR, illumination 4 jiE nr2 s~'.

5x10*-

4x10*-

Z 3 x 1 °*"IO 2x10*-

1x10' -

0 - 0 2 4 S 8 10 12Days

Fig. 10. Cell loss of C.erosa due to the presence of microcystin. Light conditions: 4 jtE nr2 s"1

were present, three of them belonging to the cyanobacteria. Because of theecophysical characteristics of the stratified lake, we can compare the effects ofcyanobacterial blooms in this type of lake to smaller water bodies. The actual areain which the phytoplankton multiply is relatively narrow, comprising the meta-limnion-hypolimnion layer. This means that the eventual toxin release from thegrowth of Oscillatoria is also restricted to this area. Diatoms that are usuallypresent all the year around in the lake, such as Asterionella formosa Hass. andFragilaria crotonensis Kitt. (Vrhovsek et al., 1984, 1985; several unpublished

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results), disappeared completely during this bloom which contained very highmicrocystin-YR levels (Sedmak and Kosi, 1997). Two other diatom species werefound in the samples: Stephanodiscus sp. in higher amounts and Cyclotella sp.,demonstrating the availability of silicon. Asterionella formosa together withF.crotonesis are described by several authors as very common and abundantoligotrophic species present in association with different cyanobacteria(Reynolds, 1980; Rosen, 1981; Bucka, 1987; Zhang and Prepas, 1996). However,there are no data describing the presence of these two species in association withhepatotoxic blooms.

In the Ledava reservoir, there was no bloom in 1994; nevertheless, in 1995 weobserved a non-toxic Aph.ftos-aquae bloom which did not markedly influence thediversity of species composition. The Savci reservoir was in both years the site ofheavy Microcystis blooms. Owing to the low rainfall in 1994, the reservoir wasexposed to a mixed Microcystis bloom with high concentrations of microcystin-RR, resulting in extremely low species composition (Table II). We noticed thecomplete disappearance of Chrysophyta and Bacillariophyta species. In 1995,because of the abundant rainfall and drainage through surface outflow of the lesstoxic M.wesenbergii bloom, the phytoplankton community was less subject tonoxious effects. The result was an essentially higher species diversity. The gravelpit in Borovci was the site of a weakly toxic Microcystis bloom with a moreheterogeneous composition, in spite of poor light conditions caused by waterturbidity arising from occasional gravel excavations. We noted the completeabsence of Chrysophyta and Euglenophyta taxa and several Bacillariophytaspecies. Both the Lutverci and Podgrad fish ponds were dominated by similartoxic mixed cyanobacterial blooms. In Podgrad, which is the main centre of a localfishing community and therefore subjected to additional anthropogenic pollution,we noted the disappearance of Chrysophyta and Bacillariophyta. Similarly, onlyone diatom species was found in Lutverci. Two locations from the village ofHotinja were added to our comparison and their detailed data will be discussedelsewhere. In these two locations, we observed M.aeruginosa practically as amonoculture, with an extremely low count of other species (Table I). These fieldresults show how entire higher taxonomic groups can temporarily be cleared fromthe environment of a toxic cyanobacterial bloom, with several species beinginhibited or excluded.

Chlorophyta have a significant share in the phytoplankton species compositionof all the studied locations, with the exception of Lake Bled. Scenedesmus quadri-cauda is present in all highly eutrophic water bodies. Generally, the generaScenedesmus and Staurastrum are frequently associated with cyanobacterialblooms (Reynolds, 1980; Rosen, 1981; Bucka, 1985; Lewis, 1986; Jensen et ai,1994; Komarkova and Hejzlar, 1996). Inspection of the two locations after bloomcollapse revealed a rise in phytoplankton diversity with an increase in the abun-dancy of Chlorophyta species. It is well known that after the decline of cyano-bacterial blooms, green algae became dominant (e.g. Lin, 1972). We ascribe theintensified development of green algae after toxic cyanobacterial blooms to theimprovement of light conditions in the presence of microcystins, which canaugment their proliferation. The growth rates in culture of certain species of

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Scenedesmus can be doubled by adding water extracted from the end of a summerbloom caused by M.aeruginosa (Hartman, 1960), which we presume containedmicrocystins.

After bloom decomposition, large amounts of microcystins are dissolved inwater, with a simultaneous decrease in water turbidity. Toxins act as growthstimulators for other species like S.quadricauda commonly present in cyano-bacterial blooms and appearing in large numbers specifically after hepatotoxicblooms (Bucka, 1989).

Acknowledgements

The authors wish to thank Andrej Blejec for his suggestions on statistical analy-sis, K.Stanic and I.Dragan for valuable assistance, and Professor C.S.Reynolds fora fruitful discussion in Vigo. This research was supported by Ministry of Scienceand Technology grant number L4-7403. Additional support was provided by theMinistry of Agriculture and Forestry.

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Received on September 2,1997; accepted on November 21,1997

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