Cyanobacterial Toxins: Removal during Drinking Water Treatment, and

Human Risk Assessment

Bettina C. Hitzfeld, Stefan J. Hoger, and Daniel R. Dietrich

Environmental Toxicology, University of Konstanz, Konstanz, Germany

Cyanobacteria (blue-green algae) produce toxins that may present a hazard for drinking water safety.

These toxins (microcystins, nodularins, saxitoxins, anatoxin-a, anatoxin-a(s), cylindrospermopsin) are

structurally diverse and their effects range from liver damage, including liver cancer, to

neurotoxicity. The occurrence of cyanobacteria and their toxins in water bodies used for the

production of drinking water poses a technical challenge for water utility managers. With respect to

their removal in water treatment procedures, of the more than 60 microcystin congeners,

microcystin-LR (L, L-leucine; R, L-arginine) is the best studied cyanobacterial toxin, whereas

information for the other toxins is largely lacking. In response to the growing concern about

nonlethal acute and chronic effects of microcystins, the World Health Organization has recently set

a new provisional guideline value for microcystin-LR of 1.0 pg/L drinking water. This will lead to

further efforts by water suppliers to develop effective treatment procedures to remove these

toxins. Of the water treatment procedures discussed in this review, chlorination, possibly micro-

/ultrafiltration, but especially ozonation are the most effective in destroying cyanobacteria and in

removing microcystins. However, these treatments may not be sufficient during bloom situations or

when a high organic load is present, and toxin levels should therefore be monitored during the

water treatment process. In order to perform an adequate human risk assessment of microcystin

exposure via drinking water, the issue of water treatment byproducts will have to be addressed in

the future. Key words: cyanobacteria, ozone, risk assessment, toxin, water treatment. - Environ

Health Perspect 1 08(suppl 1 :113-122 (2000).

http.//ehpnetl.niehs.nih.gov/docs/2000/suppl-1/1 13-122hitzfeld/abstract.html

Toxic blue-green algae in water used as

drinking water or for recreational purposes

poses a hazard to humans but has long been

neglected or at most been treated on a local

level. Scums of blue-green algae or cyanobacteria accumulating along the shores of ponds

and lakes also present a hazard to wild and

domestic animals. Providing the human population with safe drinking water is one of the

most important issues in public health and

will gain more importance in the coming millennium. Reports of toxic blooms and poisonings of humans and cattle range from the

first report of a toxic Nodularia bloom in

Lake Alexandrina, Australia, in 1878 (1), to a

high incidence of primary liver cancer (PLC)

in China attributed to cyanobacterial toxincontaminated drinking water (2-4), to the

recent tragic deaths of 60 dialysis patients in

Caruaru, Brazil, in 1996 due to the presence

of cyanobacterial toxins in the water supply

used in a hemodialysis unit (5,6). The presence of cyanobacterial toxins in drinking

water supplies poses a serious problem to

water treatment facilities, since not all technical procedures are able to effectively remove

these toxins to below acceptable levels.

Despite this, it is highly unlikely that lethal

poisonings would occur following consumption of drinking water contaminated with

cyanobacterial toxins. Of much higher concern are low-level chronic exposures, since the

risks associated with long-term exposure have

not been adequately described. Drinking

water suppliers are nevertheless confronted

with a variety of questions ranging from what

levels actually occur in the drinking water

sources to the current state of knowledge

about acute and chronic effects and effective

water treatment technologies in removing

toxins (7). This review addresses these issues.

Cyanobacteria

Morphology and Taxonomy

Cyanobacteria are an ancient group of

organisms whose habitats range from hot

springs to temporarily frozen ponds in

Antarctica (8). They occur both in freshwater

and in marine environments. Cyanobacteria,

like eubacteria, lack a nucleus, whereas in contrast to their closest relatives, the purple and

the green sulfur bacteria, they produce oxygen

(9). According to the current taxonomy, 150

genera with about 2,000 species, at least 40 of

which are known to be toxicogenic, have been

identified (10). Cyanobacteria grow as single

cells, as single cells in colonies, or as single

cells in filaments, whereas some filamentous

genera contain special nitrogen-fixing heterocysts. Cells growing in colonies may be packed

in a mucilaginous sheath like Microcystis sp.

or, in the case of filamentous species, grow as

floating mats or as free-floating strands. Many

cyanobacterial species possess gas vacuoles that

allow them to regulate their position in the

water column and give them a distinct ecologic

advantage over other planktonic species.

Bloom Formation

Prevention of bloom formation is the most

efficient method for avoiding cyanobacterial

toxin contamination of drinking water.

Unfortunately the factors leading to

cyanobacterial bloom development (cell

numbers > 106/L), whether of toxic or nontoxic species, have not been satisfactorily

identified. Factors such as nitrogen, phosphorus, temperature, light, micronutrients

(iron, molybdenum), pH and alkalinity,

buoyancy, hydrologic and meteorologic

conditions, and the morphology of the

impoundment have all been implicated [for a

discussion see Chorus and Bartram (11)].

More importantly, factors influencing toxin

production have not been conclusively elucidated (12). Although these factors can be

considered closely related to bloom formation, cell numbers and toxin levels are usually

not closely related. Furthermore, few generalizations can be made from the few laboratory

studies that have been conducted to date

(7,13-16).

Cyanobacterial Toxins

Cyanobacteria produce a variety of toxins,

subsequently called cyanotoxins, that are

classified functionally into hepato-, neuro-,

and cytotoxins. Additionally, cyanobacteria

produce lipopolysaccharides (LPS) as well as

secondary metabolites that are potentially

pharmacologically useful. The former are

responsible for the irritant nature of

cyanobacterial material. Defined by their

chemical structure, cyanotoxins fall into

three groups: cyclic peptides (the hepatotoxins microcystins and nodularin), alkaloids

(the neurotoxins anatoxin and saxitoxins),

and LPS. The species most often implicated

with toxicity are Microcystis aeruginosa,

Planktothrix (= Oscillatoria) rubescens,

Aphanizomenon flos-aquae, Anabaena flosaquae, Planktothrix agardhii, and Lyngbia

spp. (Table 1).

Address correspondence to B.C. Hitzfeld, PO Box

X918, D-78457 Konstanz, Germany. Telephone: 49

7531 88 4105. Fax: 49 7531 88 3170. E-mail:

bettina.hitzfeld@uni-konstanz.de

This work was partly supported by the Swiss

Federal Office of Public Health, grant FE 316-98-0715.

We also thank the Zurich Water Works, Zurich,

Switzerland, for logistical support.

Received 20 July 1999; accepted 1 September

1999.

Environmental Health Perspectives * Vol 1 08, Supplement 1 * March 2000 113
HITZFELD ET AL.

Table 1. Toxicity of cyanobacterial toxins.

Toxin

Microcystin-LR

Microcystin-LA

Microcystin-YR

Microcystin-RR

lD-Asp3]microcystin-LR

[D-Asp3]microcystin-RR

[Dha7]microcystin-LR

[(6Z)-Adda]microcystin-LR

[(6Z)-Addalmicrocystin-RR

Nodularin

[LD-Aspl]nodularin

l)6Z)-Adda3)nodularin

Anatoxin-a

Anatoxin-a(s)

Saxitoxin

Cylindrospermopsin

LD50 (pg/kg,

ip, mouse) Organism

50 M. aeruginosa, Aph. flos-aquae, M. viridis

50 M. aeruginosa, M. viridis

70 M. aeruginosa, M. viridis

600 M. aeruginosa, Anabaena sp., M. viridis

50-300 M. aeruginosa, Aph. flos-aquae, M. viridis,

O. agardhii

250 0. agardhii, M. aeruginosa, Anabaena sp.

250 M. aeruginosa, Anabaena sp., 0. agardhii

> 1200 M. viridis

> 1200 M. viridis

50 N. spumigena

75 N. spumigena

> 2000 N. spumigena

200-250 Aph. flos-aquae, Anabaena spp., Oscillatoria

sp., Aphanizomenon sp., Cylindrospermum sp.

20 Aph. flos-aquae

10 Aph. flos-aquae, A. circinalis, Cylindrospermopsis

raciborskii, Lyngbya wollei

2000 C. raciborskii, Umezakia natans, Aph. ovalisporum

Cyclic Peptides

Microcystins and nodularins are the most

widespread cyanotoxins. They can be found

in cyanobacterial blooms ranging from freshwater bodies to oceans. Microcystins have

been described from the genera Microcystis,

Anabaena, Planktothrix, Nostoc, and

Anabaenopsis, whereas nodularin has been

found only in Nodularia (17-21).

Toxin synthesis. Although the environmental conditions under which cyanobacteria

produce toxins remain largely unknown, the

way these toxins are synthesized is becoming

clearer. Their small size, cyclic structure, and

content of unusual amino acids indicate that

these peptides are synthesized nonribosomally

rather than on ribosomes (22,23). The

enzymes involved in nonribosomal peptide

synthesis, peptide synthetases, have highly

conserved structures. The genes coding for

these peptide synthetases are modular, each

module containing information for a single

peptide synthetase unit. Using two conserved

sequence motifs of the adenylate-forming

domain of peptide synthetases to search for

homologous sequences in toxic and nontoxic

strains of M. aeruginosa, it was found that

only the toxic strain contains peptide synthesis gene sequences (23). The ability of a

cyanobacterial strain to produce toxins may

thus depend primarily on the possession of

these genes and on their expression under certain environmental conditions. With the

emergence of a molecular genetics-based taxonomy of cyanobacteria together with the

development of polymerase chain reaction

primers and DNA probes specific for toxic

strains of cyanobacteria, these toxin-producing strains may be identified more rapidly in

the future (24-26).

Structure and uptake. These cyclic

peptides (Figure 1) are rather small molecules

with a molecular weight ranging from

Reference

(31,125)

( 138)

(31)

(139-141)

(142, 143)

(19,139)

(139,144)

(143)

(143)

(145)

(146)

(146)

(145,147)

(148)

(42,149)

(150)

800-1,000 Da (17,27). Most congeners are

hydrophilic and generally not able to penetrate vertebrate cell membranes and therefore

require uptake via an adenosine triphosphate

(ATP)-dependent transporter. One thus far

unidentified multispecific organic anion

transporter (or bile acid transporter) has been

described as the carrier of these cyclic peptides in rat liver (28-30). As a result of this,

toxicity of microcystins and nodularins is

restricted to organs expressing the organic

anion transporter on their cell membranes,

such as the liver. The structure of the heptapeptide microcystin was first identified in

1982 from an isolate of M. aeruginosa.

Meanwhile, about 60 congeners with the

general structure cyclo-(D-alaninel-X2-DMeAsp3-Z4-Adda5-D-glutamate6-Mdha7)

have been characterized (Table 1) (17,27,

31-33). X and Z are two variable L-amino

acids, D-MeAsp is D-erythro-f-methylaspartic

acid, and Mdha is N-methyldehydroalanine.

Adda is an unusual amino acid and unique to

cyanobacterial toxins: (2S, 3S, 8S, 9S)-3-

amino-9-methoxy-2,6,8-trimethyl- 1 0-

phenyldeca-4,6-dienoic acid. Nodularin is a

pentapeptide with the general structure

cyclo- (D-MeAspl -L-arginine2-Adda3-Dglutamate4-Mdhb5). Mdhb is 2-(methylamino)-2-dehydrobutyric acid. The most

common structural variants occur in positions

2 and 4, resulting in substitutions of the

L-amino acids, and demethylation of amino

acids at positions 3 and/or 7. The current

nomenclature names the most common structural variation, i.e. microcystin-LR (L,

L-leucine; R, L-arginine) or microcystin-LW

(W, L-tryptophane) (34). 6Z-stereoisomers of

Adda have been reported for nodularin and

microcystin (Table 1). [(6Z)-Adda5]nodularin, [(6Z)-Adda5]microcystin-LR, and

[(6Z)-Adda5]microcystin-RR have all been

reported to be nontoxic in the standard mouse

bioassay, with LD50 (median lethal dose)

values of > 1,200 pg/kg body weight (bw).

Cylindrospermopsin

Cylindrospermopsin (Figure 1) is a structurally distinct toxin that has been found in

tropical and subtropical waters of Australia,

where it causes problems in water supplies

(35). This alkaloid cyto- and hepatotoxin is

produced mainly by Cylindrospermopsis raciborskii but also by Aph. ovalisporum and

Umezakia natans.

Neurotoxins

The neurotoxins (Figure 2) described in

cyanobacteria can be classified into three distinct groups: a) anatoxin-a and homoanatoxin-a; b) anatoxin-a(s), which is structurally

not related to anatoxin; and c) saxitoxins or

paralytic shellfish poisons (PSPs). Anatoxin-a

has been described in A. flos-aquae and other

Anabaena spp., Planktothrix sp. (Oscillatoria

sp.), Aphanizomenon sp., Cylindrospermum sp.

and in small amounts even in Microcystis sp.

(33,36,37). Anatoxin-a exerts its neurotoxic

effect by mimicking acetylcholine with an

LD50 of 200-250 pg/kg bw (36). Anatoxina(s) is the only naturally occurring organophosphate and has been isolated from A.

flos-aquae and A. lemmermannii (38). It is a

highly toxic compound with an LD50 of 20

pg/kg bw (mouse ip) (39). Saxitoxins are

better known from marine dinoflagellates

(red tide) where they are responsible for paralytic shellfish poisoning after consumption

of contaminated shellfish. But saxitoxins, a

group of carbamate alkaloid neurotoxins,

have also been detected in relevant amounts

in freshwater cyanobacteria such as Aph. fibsaquae, A. circinalis, C. raciborskii, and

Lyngbya wollei (40-42).

Microcystins

Animal Toxicity

Since the first description in 1878 of a

Nodularia spumigena bloom in Lake

Alexandrina, Australia (1), numerous cases of

animal poisonings have been reported (Table

2). Most commonly, deaths of farm animals

drinking scums of cyanobacterially contaminated ponds or poisonings of dogs swimming

in cyanobacterial scum have been described

(7). Fish kills have been reported in conjunction with cyanobacterial blooms and have

often resulted in significant economic losses

(43-47). The liver is the major target organ

for microcystin toxicity; it was shown to accumulate 20-70% of a radioactively labeled

toxin dose (intravenous) (48-54). Studies in

mice and pigs exposed to extracts of a toxic

M. aeruginosa bloom demonstrated dosedependent toxicity (55,56). Increased mortality, liver weight, and plasma alanine

114 Environmental Health Perspectives * Vol 108, Supplement 1 * March 2000
CYANOBACTERIAL TOXINS IN DRINKING WATER TREATMENT

aminotransferase levels were associated with

loss of body weight. Neither other organ systems nor lactate dehydrogenase levels were

affected. Death of the organism through intrahepatic hemorrhage and shock is rapid, occurring within about 3 hr in the case of mice.

Pathologic and ultrastructural features commonly observed in the liver are centrilobular

hepatic necrosis, destruction of sinusoidal

endothelium, disruption of bile canalicular

function, intrahepatic hemorrhage, loss of

microvilli and bleb formation in hepatocytes,

and hepatocyte necrosis (29,57-61).

Inhibition ofProtein Phosphatases 1

and 2A

The toxicity of microcystins and nodularins is

due to inhibition of the catalytic subunit of

protein phosphatases 1 and 2A (PP 1, PP2A)

(62-64). In the case of microcystins it has

been suggested that covalent binding to cysteine-273 and cysteine-266 on PPI and

PP2A, respectively, is responsible for this

effect (65,66). PP1 and PP2A dephosphorylate phosphoseryl or phosphothreonyl

proteins and their inhibition leads to hyperphosphorylation of cytoskeletal proteins

resulting in the deformation of hepatocytes

(28,67-71). It is not clear, however, if the

covalent binding of the toxin to PP1 or PP2A

is in fact responsible for the inactivation,

since inactivation precedes covalent modification and nodularin does not bind covalently

(72). Furthermore, it has been suggested that

the Adda side chain and possibly the planar

ring portion of the peptide are responsible

for both recognizing and inhibiting protein

phosphatases (73,74).

Glu Mdha

CO2H

Adda

OCH3

CH3

Ala

CH3

0 HO2C

Microcystin-LR

Arg

NH=i

Leu

Masp

Tumor Promotion

The cyanobacterial cyclic peptides possess

tumor-promoting activity (TPA) by a TPAindependent pathway (75). Cyanobacterial

extracts or microcystin-LR in drinking water

induce skin tumors in rats and mice after initiation with 7,12-dimethylbenz[a]anthrazene

(76,77). Glutathione-S-transferase placental

form positive foci were detected in livers of

rats after ip injection of microcystin-LR or

nodularin and initiation with diethylnitrosamine (78-80). It has been speculated

that these toxins may be liver carcinogens,

since they induce foci or small neoplastic

nodules without the use of initiators (80,81).

Both microcystin-LR and nodularin induce

the expression of tumor necrosis factor-cc and

early response genes (c-jun, jun B, jun D,

c-fos, fos B, fra- 1) in rat liver and hepatocytes

(80,82). In addition, mutations in the K-ras

codon 12 in the RSa cell line (83) and DNA

fragmentations have been reported after ip

injection of cyanobacterial extract or microcystin-LR in mice (84,85). These in vitro and

H

o N

9 \C-cH3

8

5 4

Anatoxin-a

ADDA

Nodularin

-NH

CH3 CH3

H02C

NH'

Arg |

NH=C

NH2

OH

-03SO - 0

Cylindrospermopsin N NH HN NH

Me

NH O

Figure 1. Structure of microcystin, nodularin, and cylindrospermopsin.

R = H; saxitoxin dihydrochloride

R = OH; neosaxitoxin dihydrochloride

/S~~~ \ / ~CH3 HN3 5 N

N\

>0

+H2N CH

0

Anatoxin-a(s)

Figure 2. Structure of anatoxin-a, saxitoxins, and

anatoxin-a(s).

Environmental Health Perspectives * Vol 108, Supplement 1 * March 2000 11 5
HITZFELD ET AL.

Table 2. Toxic cyanobacteria episodes.

Location Year Case Organism Toxin Reference

Lake Alexandrina, Australia 1878 Livestock stupor Nodularia spumigena Nodularina (1)

Ohio River, U.S. 1931 Humans: gastroenteritis Unspecified cyanobacteria Unknown (86)

Harare, Zimbabwe 1966 Humans: gastroenteritis Microcystis aeruginosa Unknown (87)

Alpine Lakes, Switzerland 1974-1994 Cattle deaths, liver damage Oscillatoria limosa, 0. tenuis, Phormidium PPI-toxinb (151)

sp., Tychonema sp., Pseudoanabaena

catenata, P. autumnale a.o.

Armidale, Australia 1983 Humans: liver damage Microcystis aeruginosa Unknown (88)

Palm Island, Australia 1983 Humans: hepatoenteritis Cylindrospermopsis raciborskii Cylindrospermopsin (152)

Richmond Lake, U.S. 1988 Livestock, birds, dogs, fish Anabaena flos-aquae, Aphanizomenon Anatoxin-a(s) (153)

flos-aquae, Microcystis aeruginosa

Rudyard Reservoir, U.K. 1989 Humans: recruits, pneumonia, diarrhea Microcystis aeruginosa Microcystin-LR (154)

Darling River, Australia 1990-1991 Livestock deaths Anabaena circinalis Saxitoxin (42)

1000-km bloom Neosaxitoxin

Loch lnsh, Scotland, U.K. 1992 Dog death Oscillatoria sp. Anatoxin-a (155)

Itaparica Dam, Brazil 1993 Humans: 88 deaths, gastroenteritis Anabaena sp., Microcystis sp. Unknown (89)

Loch Leven, Scotland, U.K. 1994 Fish deaths Anabaena flos-aquae Microcystin (44)

Nandong District, Jiangsu Province, 1994-1995 Humans: PLC 100 of 105toxins act M. aeruginosa, Planktothrix agardhii, Microcystins (156)

Nanhui/Shanghai, Fusui, China with HBsAg and aflatoxin Anabaena sp., 0. tenuis, Lyngbya sp. (2-4)

Caruaru, Brazil 1996 Hemodialysis patients: 60 deaths Aphanizomenon sp., Oscillatoria sp. Microcystins (5,6)

Abbreviations: HBsAg, hepatitis B antigen; PLC, primary liver cancer; PPI, protein phosphatase inhibition. &Suspected. bProtein phosphatase-inhibiting activity.

in vivo data have to be viewed in the light of

observations in China, where consumption of

microcystin-contaminated drinking water has

been associated with a high incidence of PLC

(2-4) (see next section).

Human Healdth Effects

Evidence of human poisonings by cyanobacterial toxins ranges from health effects after

recreational exposure to poisonings following

consumption of contaminated drinking water

(Table 2).

Acute and subchronic exposures. The

earliest case of gastroenteritis from cyanobacteria was reported in 1931 in towns along the

Ohio River, where low rainfall had caused the

development of a large cyanobacterial bloom

(86). The water treatment procedures

employed over months to combat this bloom

(prechlorination, sedimentation, filtration,

chlorination, copper sulfate to lyse the

cyanobacterial cells, aeration, activated carbon, permanganate, ammonia, and dechlorination) all proved to be ineffective in

reducing taste, odor, or toxin content of the

drinking water. A natural Microcystis bloom

in a water reservoir in Harare, Zimbabwe,

caused gastroenteritis in children each year

when the bloom was decaying (87). A particularly extensive and toxic (microcystin-YM

[Y, L-tyrosine; M, L-methionine]) (31) M.

aeruginosa bloom in Malpas Dam, near

Armidale, Australia, was treated with copper

sulfate in 1981 after complaints of bad taste

in the drinking water were received (88). The

plant treating the water used prechlorination,

alum flocculation, sedimentation, rapid sand

filtration, postchlorination, and fluoridation.

The effect of this toxic bloom event was then

monitored in a retrospective epidemiologic

study of liver function in the population

consuming the water. It was found that the

serum level of the liver enzyme y-glutamyltransferase was elevated in that part of the

population using the Malpas Dam water during the bloom and after the bloom was lysed

with copper sulfate. The two most lethal poisonings attributed to cyanobacteria in drinking water occurred in Brazil. A massive

Anabaena and Microcystis bloom in Itaparica

Dam was responsible for 2,000 gastroenteritis

cases resulting in 88 deaths, mostly children

(89). A very tragic though relatively welldocumented case occurred in a hemodialysis

center in Caruaru in 1996 (5,6). The water

used in the dialysis unit, taken from Tabocas

Reservoir, was normally sedimented with

alum, filtered, and chlorinated in the municipal water treatment plant prior to being supplied by truck to the clinic. At the dialysis

unit, the water was further purified by passing

through sand, charcoal, and an ion-exchange

resin, and finally by micropore filtration.

During the 1996 summer drought, the dialysis center received water from the municipal

plant that was only treated with alum, but

not filtered or chlorinated. There are conflicting reports as to whether the water was chlorinated in the trucks prior to delivery to the

clinic. Furthermore, the charcoal, sand, and

micropore filters at the clinic had not been

changed in the 3 months prior to this

episode, even though the water received from

the trucks had been visibly turbid. In

February 1996, the majority (85%) of the

hemodialysis patients developed a toxic illness

of varying severity, with a wide range of neurologic symptoms as well as acute liver injury.

Up to 23 patients died in the first 2 weeks of

this episode with either neurologic symptoms

or from liver failure. About 37 more patients

died in the following 5 weeks either directly

from hepatotoxic effects or from complications

such as sepsis, gastrointestinal bleeding, or

cardiovascular effects. The patients displayed

cholestatic jaundice with high bilirubin and

alkaline phosphatase concentrations, and

increases in hepatic enzymes (aspartate and

alanine aminotransferase). Liver pathology

showed the presence of an acute novel toxic

hepatitis similar to that seen in animals

exposed to microcystins (6). Histopathology

showed panlobular hepatocyte necrosis,

together with cell-plate disruption and apoptosis. However, in contrast to animal models

of microcystin intoxication, no intrahepatic

hemorrhage could be observed. After initial

uncertainties as to the causative agents of

these fatal intoxications, microcystin concentrations were determined in serum and in

liver tissue as well as in the water filtration

columns. The latter contained intact and

fragmented cyanobacterial cells as well as

microcystin-LR. Levels in serum ranged from

1 to 10 ng/mL; concentrations in the liver

were as high as 0.6 mg/kg tissue. Toxin congeners were reported to be microcystin-YR,

microcystin-LR, and microcystin-AR (A,

L-alanine). At the time of the outbreak,

cyanobacterial counts had not been made in

the reservoir, but in March 1996, it was

found that the most common cyanobacterial

genera present were Aphanizomenon,

Oscillatoria, and Spirulina.

Chronic exposure. When considering the

chronic effects of long-term exposure to

microcystins in drinking water, one has to

take into account the high incidences of PLC

in regions of China where pond and ditch

water are used as drinking water supplies. In

Haimen and Qidong (Jiangsu Province),

pond and ditch water used as drinking water

showed average microcystin concentrations of

160 pg/mL (60% of the samples analyzed

were positive), whereas microcystins could

not be detected in well water (2-4). PLC

1116 Environmental Health Perspectives * Vol 108, Supplement 1 * March 2000
CYANOBACTERIAL TOXINS IN DRINKING WATER TREATMENT

incidences of 4.28 per 100,000 and 100.13

per 100,000 were observed in humans using

well water and pond/ditch water, respectively

(2). It has been calculated that humans living

in areas with a reported high PLC incidence

consume 0.19 pg microcystin per day during

the 4 summer months from June to

September over their 40- to 50-year life span

(4). Coexposure to the potent liver carcinogen aflatoxin B1 or to hepatitis B virus may

result in the high incidence of PLC in this

region (4).

Efficacy of Water Treatment

Procedures

Water treatment measures should always be

just one option after other techniques such as

selection of intake depth, offtake by bank filtration, and/or use of barriers to restrict scum

movement have been used. When evaluating

water treatment procedures for the removal of

cyanobacterial toxins, one is faced with problems regarding soluble and suspended substances. Cyanotoxins are produced within the

cyanobacterial cells and thus toxin removal

involves measures to destroy or avoid the

cells. The cyanotoxins also are all water soluble, thus remediation measures involve chemical procedures reducing the toxicity or

completely removing the toxins from the

drinking water.

Most studies, especially early ones, had to

rely on relatively crude measurements of

acute toxicity, since more specific analytical

methods were not available. The cyclic heptapeptides have been the focus of most of

these studies, but some studies on removal of

saxitoxins and anatoxin-a also exist.

Coagulaton/Flocculation, Dissolved

Air Flotation, and Activated Carbon

Adsorption

Coagulation or flocculation involves the

aggregation of smaller particles into larger particles using chemicals such as ferric chloride or

aluminum sulfate. Coagulation can be an efficient method for eliminating cyanobacterial

cells from water, whereas soluble cyanotoxins

are not very efficiently removed by this

method (90,91). The efficiency of cyanobacterial removal is dependent on an optimization of chemical doses and coagulation pH

(92). Coagulation may cause additional problems such as lysing of cyanobacterial cells leading to release of toxins (90). When employing

dissolved air flotation (DAF), it is important

to consider that different cyanobacterial

species behave differently depending on their

physical properties: in a Belgian DAF plant

Microcystis was removed by 40-80%,

Anabaena by 90-100%, but Planktothrix only

by 30% (93). Because conventional water

treatment usually involves a combination of

these methods, most of the research has

focused on the effect of coagulation/flocculation in combination with other measures. In

one of the earliest studies, toxins isolated from

algal material were subjected to a) activated

carbon filtration; b) prechlorination, flocculation with FeCI3, sedimentation, sand filtration, and activated carbon filtration; and

c) lime pretreatment, flocculation with FeCl3,

chlorination, and activated carbon filtration

(94). The toxicity of these samples was then

tested in the mouse bioassay. Chlorination,

flocculation, or sand filtration were unable to

destroy the toxins; only the last step, powdered activated carbon (PAC) at a ratio of

1:10 to 1:100 (toxin:activated carbon),

removed a toxin concentration of 3 pg/mL to

below toxic levels. Studies using 50 mg

lyophilized cyanobacteria also show that conventional flocculation, filtration, and chlorination are not efficient in destroying the toxins:

high performance liquid chromatography

(HPLC) analysis shows a toxin reduction of

up to only 34% (95,96). Only the inclusion

of a treatment step with activated carbon

resulted in 100% removal of the toxins from

water. On the basis of the results of these laboratory studies, Lahti and Hiisvirta (97) conducted pilot-scale experiments to study the

feasibility of predicting the behavior of

cyanobacterial toxins in water treatment practice. Both fresh and freeze-dried cyanobacteria

were subjected to the following processes: a)

flocculation with A12(S04) plus sedimentation

plus filtration, and b) PAC plus flocculation

with A12(S04) plus sedimentation plus filtration. Toxicity was measured using the mouse

bioassay and HPLC. As was to be expected

from the laboratory scale studies, only the

inclusion of PAC significantly reduced toxicity. Activated carbon is, however, not always a

very efficient method. A study aimed at the

removal of cyanobacterial cells with rapid sand

filtration and activated carbon found a reduction of cyanobacteria of only 42% (98). More

detailed studies with activated carbon show

that both PAC as well as granular-activated

carbon (GAC) effectively and quickly (contact

times of 30 min are sufficient) eliminate cyanotoxins from water (99-101). In the case of

PAC, dosing is an important parameter (10

pg/L toxin: > 200 mg/PAC/L), whereas when

using GAC, the choice of the carbon source is

important (coal, wood > peat, coconut),

probably due to the different pore sizes relative to the size of the microcystin molecule

(102). A major concern when using activated

carbon in water treatment plants is the formation of a biofilm, which can significantly

impair the ability of the filter to adsorb toxins; biodegradation by the biofilm does not

seem to occur (99,103,104). Furthermore,

below concentrations of 0.15 pg microcystinLR/L, very little microcystin will be removed

by activated carbon in the presence of a

biofilm or natural organic matter (103). This

finding has consequences for the risk

assessment of a chronic exposure to low

microcystin concentrations.

Rapid Filtration and Slow Sand

Filtraion

The performance of rapid filtration, a method

usually employed after coagulation to remove

the floc, does not effectively remove cyanobacterial cells (98,105). Conventional water treatment requires regular backwashing of the

filters, but if this washing process is performed

inadequately, lysis of cyanobacterial cells on

the filters can lead to release of toxins into

the water (11,106). Furthermore, sand filtration alone does not lead to substantial reduction of toxicity (99), and blocking caused by

overloading should be avoided (11).

Chlorinatinon

In general, chlorination is not an effective

process in destroying cyanotoxins (94-96,

107). The efficiency of chlorination seems to

depend largely on the chloride compounds

and the concentration used. Aqueous chlorine

and calcium hypochlorite at . 1 mg/L remove

more than 95% of microcystins or nodularin,

while sodium hypochlorite at the same dose or

chloramine achieve 40-80% removal at most

(91,108,109). A chlorine residual of at least

0.5 mg/L should be present after 30 min contact time in order to destroy cyclic peptides

completely (110). It should be noted, however, that even when acute toxicity, as measured by the mouse bioassay, was removed by

this process, progressive liver damage could

still be detected in the animals. This subacute

toxicity may be due to incomplete toxin

removal or to the formation of chlorination

byproducts, which have been implicated in

toxicity (111). Anatoxin-a or saxitoxins could

neither be destroyed with chlorine doses

exceeding a 30-min chlorine demand nor

by changes in pH (107,108). Cylindrospermopsin, on the other hand, was effectively

oxidized by 4 mg/L chlorine at pH 7.2-7.4

(toxin concentration 20-24 pg/L) (108).

Light

Microcystins are very stable under natural sunlight (112), whereas ultraviolet (UV) light

around the absorption maxima of microcystinLR and microcystin-RR rapidly decomposed

the toxins (113). A photocatalytic process

using a TiO2 catalyst and UV radiation also

quickly decomposed microcystin-LR, -YR, and

.YA with half-lives of < 5 min (114). The efficiency of this process was largely dependent on

the organic load of the water (114).

Membrane Processes

Microfiltration (MF) and ultrafiltration (UF)

are technologies that have emerged in recent

Environmental Health Perspectives * Vol 108, Supplement 1 * March 2000 11 7
HITZFELD ET AL.

years and have therefore not been thoroughly

investigated as to their efficiency in removing

cyanobacterial cells or toxins (7). One study

showed that both UF and MF can be very

efficient (> 98%) in removing whole cells of

toxic M. aeruginosa (115). An important

point when considering filtration is the lysis

of cells. In the case of the above-cited study,

some damage to cells could be observed, but

toxin was not detected in the filtrate. UF was

also effective in reducing microcystin and

nodularin levels in the filtrate. This may be

expected from a membrane with a very

low-molecular-weight cut-off pore size

(nanofiltration membrane) (101,116).

Ozonation

In Europe and North America, ozonation has

been used primarily for disinfection purposes

or to remove color and/or odor (117). Ozone

was initially used at the beginning of the water

treatment train mainly to inactivate viruses

and bacteria. In recent years, though, many

water treatment plants have included a twostage ozonation treatment, either with preand interozonation, inter- and postozonation,

or with pre- and postozonation.

In water, two pathways for the oxidation

of organic pollutants by ozone have been

described (117,118): direct attack by molecular ozone via cycloaddition or electrophilic reaction, and indirect attack by

free radicals (primarily OH) formed by the

decomposition of ozone. The mechanism

involving cycloaddition in water usually

results in the formation of aldehydes, carboxylic acids, ketones, and/or carbon dioxide. The electrophilic attack by molecular

ozone probably occurs on atoms carrying

negative charge such as N, P, 0, or nucleophilic C. An indirect attack by free radicals

generally occurs via one of three pathways:

hydrogen abstraction, electron transfer, or

radical addition.

Microcystins and nodularin. Ozone is one

of the most powerful oxidizing agents and its

potential to destroy cyanobacterial toxins has

been investigated in the last 10 years (Table 3).

In one of the earliest studies looking at the

effect of ozone on cyanotoxins, researchers at

the British Foundation for Water Research

ozonated microcystin-LR purified from M.

aeruginosa and assessed toxicity using a mouse

bioassay (90,119). After ozonation, the toxicity of the cyanotoxin is reduced, which could

be shown by a prolongation of mouse survival

time, but the results cannot be quantified

since the authors omitted to detail ozone or

toxin concentrations. HPLC and fast atom

bombardment-mass spectrometry analysis also

show a reduction in the microcystin peak after

a 2-sec ozonation. Interestingly, several new

peaks appeared but were not tested separately

for toxicity. This very fast destruction of

microcystin was corroborated in Australian

studies quantifying the effect of ozone on different microcystin-LR concentrations

(91,108,120) and in our own work (121).

These studies showed that up to 800 pg/L

microcystin-LR can be oxidized to below the

HPLC detection limit by < 0.2 mg/L ozone

within seconds to minutes. The reaction of

ozone with nodularin also occurs very rapidly:

when reacting 88 pg/L nodularin with 0.05

mg/03, there was zero toxin recovery after 15

sec (120). With these studies it was also

demonstrated that the removal of microcystins

is proportional to the ozone dose when the

microcystin concentration is below the ozone

demand (91). Complete removal of microcystin is achieved and an ozone residual is

detected when the ozone demand of the water

has been met.

Cyanobacterial extracts and cells and

organic load. Obviously, a more realistic way

to test the efficiency of ozonation would be to

use either cyanobacterial extracts or whole

cells (Table 4). Oxidation reactions of ozone

with cyanobacterial toxins are always in competition with other organic compounds in the

water. As a result, naturally occurring organic

matter is one of the most important factors to

consider in terms of toxin dynamics. In a

study designed to model continuous operation, ozone doses from 1 to 10 mg/L were

tested over 5-10 min for their ability to

degrade 10 pg/L microcystin added to different water sources (101). Two milligrams per

liter ozone added to raw water leads to a 60%

removal of microcystin, whereas the same

dose added to treated water removes toxins by

98%. A similar ozone demand was measured

in a study; 500 pg/L microcystin-LR was oxidized with 0.2 mg/L 03 over 4 min in

organic-free water (122). The author calculated an ozone demand of 0.6 mg/L with

almost complete microcystin removal.

However, only 50% of the same microcystin

concentration was removed when filtered

Seine River (France) water was oxidized with

0.5 mg/L 03 over 10 min. This led to a

much higher ozone demand of 1.6 mg/L.

Our own results show that cyanobacterial

extracts (M aeruginosa or P. rubescens) containing 50-100 ,ug/L microcystin-LR-equivalents need to be oxidized with at least 1.0 mg

03/L to effectively destroy the toxins present,

whereas ozone residuals were undetectable

after 10 min (106). These results show that

ozone consumption by natural organic matter

still occurs at the preozonation stage. During

postozonation, 1 mg/L 03 removed 38% of

the microcystin, whereas > 2 mg/L 03

removed toxin related toxicity below the limit

of detection. The importance of organic load

and ozone concentration was also demonstrated in Australian studies: cyanobacterial

extracts containing 135-220 pg/L microcystin-LR required 1.0 mg/L ozone over 5

min for complete toxin destruction

(108,120). After this treatment, the ozone

residual was zero, reflecting the higher

organic load and resultant high ozone

demand. The critical importance of ozone

dose, especially with respect to the organic

load of the water, was also shown in several

Finnish studies (95-97). Fresh and freezedried natural bloom material (M aeruginosa,

M. wesenbergii, M. viridis) from a Finnish

lake (LD50 60-75 mg/kg bw, mouse ip) as

Table 3. Effect of ozone on destruction of cyanobacterial toxins in the presence or absence of organic matter.

Microcystin-LR Ozone dose Duration OM Destruction Ozone demand Ozone residual

(pg/L) (mg/L) (min) present (%) (mg/L) (mg/L) Reference

21 1.2 5 - 73 ND 0.13 (101)

9 1.0 5 + 50 ND 0 (101)

500 0.2 4 - 99 0.6 (122)

500 0.5 10 + 50 1.6 (122)

< 200 1.0 5 - 100 ND 0 (108,120)

15 1.0-1.5 30 + 50 ND ND (95-97)

50 1.0-1.5 30 + 90 ND ND (95-97)

10 0.5-1.5 9 - 90-100 ND 0.4-1.2 (121)

50-100 0.5-1.5 9 + 0-100 ND 0.1-0.6 (121)

ND, not determined; OM, organic matter.

Table 4. Effect of ozone on destruction of cyanobacterial toxins from cells in the presence or absence of organic matter.

Microcystis Ozone Ozone Ozone

aeruginosa dose Duration Destruction demand residual

(cells/mL) (mg/L) (min) (%) )mg) (mg/L) Reference

1.63 x 106 3.7 5 36 ND 0 (120)

2.05 x 106 2.5 12 100 29 ND (120)

1 x 104 0.8 10 60 ND 0.01 (107)

1 xi05a 1.3 10 65 ND 0 (107)

1 x 105 1.0-1.5 9 50-100 ND 0.25-1.4 (106)

5 x 105 1.0-1.5 9 30-75 ND 0.4-0.8 (106)

ND, not determined. 5In raw water; all other experiments in pure or filtered water.

118 Environmental Health Perspectives * Vol 108, Supplement 1 * March 2000
CYANOBACTERIAL TOXINS IN DRINKING WATER TREATMENT

well as a laboratory culture of P. agardhii

(NIVA-CYA 126; LD50 190 mg/kg bw,

mouse ip) were used. In a pilot plant setup, 35

mg/L fresh (50 jig/L toxin) or 24 mg/L (15

,ug/L toxin) freeze-dried cyanobacteria were

subjected to preozonation at a dose of 1.0-1.5

mg 03/L. This treatment resulted in a reduction of toxicity by 50% (freeze-dried) and 90%

(fresh). As can be seen, toxin reduction from

fresh cyanobacteria was better than from

freeze-dried material. This may be explained

by the improved coagulation caused by preozonation. Preozonation has been widely used

to assist coagulation (117). The major problem associated with this method is the danger

of cell lysis and toxin release. A second postozonation step, using an ozone concentration

high enough to oxidize the remaining organic

matter and toxin, would then be essential. The

experiment with freeze-dried material corresponds to a situation where the bloom disintegrates and cells lyse due to chemical treatment

or as a result of natural causes. Preozonation

with 0.5-1.0 mg 03/L in that case is not the

most effective treatment. Postozonation is a

preferred method, since more of the oxidation

capacity could be used on toxins instead of on

other organic material. A common problem

with these early studies is, however, that toxicity was determined by the mouse bioassay

(detection in the microgram range) or by

HPLC (detection in the nanogram-microgram

range), two relatively insensitive assays (123).

Ozonation of intact cells during preozonation steps poses the risk of cyanobacterial

lysis and increased ozone demand. If cyanobacteria are not monitored at the water intake

level and thus enter the water treatment

process, the treatment plant may not be prepared to meet the increased ozone demand.

This leads to either an increase in the soluble

toxin concentration in the water and/or to

incomplete degradation of the cyanotoxins.

Simulating bloom situations, studies were performed with M. aeruginosa concentrations

from 1 x 104 to 2 x 106 cells/mL (107,120)

(Table 4). Depending on cell number and

organic load of the spiked water, an ozone

demand between 2 and 3 mg/L (over 5 min)

and 29 mg/L (over 12 min) was calculated.

This ozone demand is relatively high considering that water treatment plants regularly

employ a concentration of 0.5 mg/L at preozonation and 1.0 mg/L at postozonation

stages, respectively. This can also be seen in

our own study where ozonation with 1.0

mg/L 03 did not completely destroy the toxins present in 1 x 105 cells/mL (106) (Table

4). Furthermore, when a culture of 1.63 x 106

M aeruginosa cells/mL was ozonated with a

maximum of 3.7 mg 03/L, only 36% of total

toxin was removed after 5 min (120).

Anatoxin-a, anatoxin-a(s), and saxitoxins.

The efficiency of oxidation with ozone with

respect to anatoxin-a, anatoxin-a(s), or the

saxitoxins (PSPs) has not been well characterized (95,97). Using raw and filtered waters,

the British Foundation for Water Research

determined that anatoxin-a is more resistant

to removal by ozone than microcystin-LR

(107). The maximal ozone dose applied (4.5

mg/L) in raw water reduces the anatoxin-a

concentration from 2.4 pg/L to 0.6 pg/L,

whereas no ozone residual could be detected.

In filtered water, without competition from

natural organic material, 2.2 mg/L 03

destroys an anatoxin-a concentration to

below the limit of detection (0.3 jig/L). But

again, no ozone residual could be detected.

The PSPs require even higher 03 doses.

Ozonation over 15 min at 4.2 mg/min was

necessary to reduce the neurotoxicity of an

A. circinalis extract to near the lethal threshold

concentration (120). After 30 min ozonation,

the mice survived the doses. There are indications that other PSP toxins, such as GTX2,

dcGTX2, dcGTX3, C1, and C2, may also be

effectively oxidized by ozone (110). These

studies stress the need for more detailed and

quantifiable studies regarding the efficiency of

ozone in destroying the neurotoxins.

pH. A very important parameter in the oxidation efficiency of ozone is pH. At pH values

> 7.5, toxins can still be detected in the samples. This is due to the lower oxidation potential of ozone under alkaline conditions (1.24V)

compared to acidic conditions (2.07V).

Ozonation byproducts. In contrast to

chlorination byproducts, the issue of ozonation

byproducts has not been properly addressed.

One has to keep in mind though, that the

amount of ozone applied is always less than

what would be required to oxidize all the

organic material to CO2 and H20, especially

in water high with organic content. One can

therefore expect semioxidation products to

form (124). Such oxidation products were

found by HPLC when cyanobacteria were preozonated. Their toxicity was, however, not

investigated (97). Our work points to ozonation products that still exhibit phosphatase

inhibitory activity, but their structure has not

yet been determined (121). An indication of

the effect of ozone on microcystins stems from

chemical characterization studies, since ozonolysis has been widely employed for structural

characterization of organic compounds by

cleavage of carbon-carbon double bonds

(125,126). In the case of microcystins and

nodularin, ozonolysis has been applied in the

determination of the absolute configurations of

the Adda moiety (126). It has been described

that the double bond between C-6 and C-7 of

the Adda side chain is easily cleaved by ozone

to give 3-methoxy-2-methyl-4-phenylbutyric

acid. In order to realistically assess the consequences of ozonation on cyanobacterial

toxins, the ozonation byproducts have to be

identified and their toxicity tested not only in

acute tests but also in subacute tests such as

the phosphatase inhibition assay as well as in

chronic situations.

Risk Assessment

The health risk posed by exposure to cyanotoxins is difficult to quantify, since the actual

exposure and resulting effects have still not

been conclusively determined, especially for

the human situation. The most likely route for

human exposure is the oral route via drinking

water (127,128), recreational use of lakes and

rivers (129), or consumption of algal health

food tablets (130). The dermal route may

play a role during the recreational use of water

bodies (swimming, canoeing, etc.) (127,128).

Due to the growing concern about health

effects of cyanotoxins, especially via drinking

water, the World Health Organization

(WHO) has adopted a provisional guideline

value for microcystin-LR of 1.0 jig/L in 1998

(131). This guideline value is based on a tolerable daily intake (TDI) value derived from

two animal studies (56,132). The first study is

a 13-week mouse oral study that determined a

no-observable adverse effect level (NOAEL) of

40 jg/kg bw per day based on serum enzyme

levels and liver histopathology (132).

Applying a total uncertainty factor of 1,000

(10 for intra- and interspecies variability,

respectively, and 10 for limitations in the

database, especially lack of data on chronic

toxicity and carcinogenicity), a provisional

TDI of 0.04 pg/kg bw per day has been

derived for microcystin-LR. This TDI was

supported by a 44-day pig oral study that

determined a lowest observable adverse effect

level (LOAEL) of 100 jig microcystin-LR

equivalents/kg bw per day (56). In this study,

the cyanobacterial material fed to the pigs

contained several microcystin congeners, but

only microcystin-YR was tentatively identified. To this LOAEL a total uncertainty factor

of 1,500 was applied (10 for intraspecies variability, 3 for interspecies variability, 5 for

extrapolating from a LOAEL to a NOAEL,

and 10 for the less-than-lifetime exposure).

This resulted in a provisional TDI of 0.067

pg/kg bw per day. WHO used the lower of

these two values for establishing the provisional guideline value. This value is calculated

by applying the TDI (0.04 pg/kg bw) to a

typical daily water intake in liters (L = 2 liters)

by an individual of a given body weight (bw =

60 kg) and a proportion (P = 0.8) of the total

daily intake to the intake by drinking water:

Guideline value - TDIxbwxP

L

The resulting value of 0.96 pg/L was

rounded to 1.0 uig/L and should be applied to

cyanobacterial cell-bound and extracellular

Environmental Health Perspectives * Vol 108, Supplement 1 * March 2000 11 9
HITZFELD ET AL.

microcystins. This provisional guideline value

is applicable only for microcystin-LR, since

the database for other microcystin congeners

or even other cyanotoxins such as the saxitoxins is too small to derive a TDI. Health

Canada is applying an uncertainty factor of

3,000 to the NOAEL of 40 pg/kg bw per day

from the 13-week mouse study by adding a

factor of 3 for evidence of tumor promotion

and weak evidence of a potential for carcinogenicity in humans (133). They thus derive a

TDI of 0.013 pg/kg bw per day and conclude

that the consumption of 1.5 L drinking water

containing < 0.5 pg microcystin-LR/L by a

60-kg person would not exceed this TDI.

This discussion of the WHO guideline value

opens many questions for operators of water

treatment plants. Because the guideline value

is really only valid for microcystin-LR, in situations where it is not the most dominant

congener or not even present, the evaluation

of quantitative measurements with respect to

the guideline may be problematic. This is

true for HPLC analysis as well as for the

mouse bioassay and the protein phosphatase

inhibition assay (11). Results should thus

always be reported with these points in mind

and should, if possible, be reported for microcystin-LR concentration equivalents or toxicity equivalents. The next question that

obviously arises is which water treatment procedures are adequate to reduce cyanotoxin

levels to at least below the WHO guideline

value of 1.0 pg/L?

Assessment of water treatment procedures

has shown that most methods would result in

a reduction of cyanobacterial toxins concentrations to below acutely toxic levels as well as

below the new WHO guideline value of

1 pg/L drinking water. A completely different

situation may arise, however, during a bloom

and when water treatment procedures such as

chlorination and/or activated carbon are not

used together. Even when using ozonation, the

specific situation during a bloom has to be

observed. Parameters such as organic load of

the water have to be determined and toxin

levels during the treatment steps have to be

monitored. Until issues such as ozonolysis

byproducts have been resolved, even a very

efficient method such as ozonation has to be

treated with caution. These byproducts, which

may especially be formed when an insufficient

ozone dose has been used, have been detected

in several studies, but neither their structure

nor their toxicity has been determined.

Binding to and inhibition of protein phosphatases is considered a key mechanism by

which microcystins and nodularins exhibit

their toxicity. It is therefore critical to know

which structural modification to the toxin

molecule changes the affinity to and inhibition

of the phosphatase. So far, neither the Adda

residue alone (74,134) nor linear nodularin- or

microcystin-precursor peptides (135) bind

protein phosphatase or bind with high IC50

values of 0.5-1.0 mM and show no toxicity in

the mouse bioassay. However, it has also been

shown that substitution of the Adda side chain

with an L-Cys residue still leads to interaction

of the toxin with the hydrophobic groove of

the catalytic subunit of the phosphatase (135).

The mechanism of tumor promotion by

microcystins and nodularins as well as the

quantitative relationships have not been satisfactorily elucidated. It is therefore not clear if

the inhibition of protein phosphatase constitutes the only or major pathways for toxicity or

tumor promotion. Our work has shown that

in hepatocytes, microcystin binds to proteins

other than the phosphatases (136,137). A

chronic exposure to cyanobacterial toxins

and/or to the ozonolysis byproducts should

therefore be avoided. The situation for the saxitoxins, anatoxin-a, anatoxin-a(s), and cylindrospermopsin is even less clear. A broader

scientific background on which risk assessment

and management steps are based should be

developed. This can lead to sound processbased risk assessment and to the development

of effective procedures for water treatment

strategies aimed at specific situations.

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122 Environmental Health Perspectives * Vol 108, Supplement 1 * March 2000