# Cyanobacterial Toxins: 

# Removal 

# during DrinkingWater

# Treatment, and 

# Human RiskAssessment

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 108(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 ofponds and lakes also present a 

### hazard to 

### wild and domestic 

### animals. 

### Providing the human pop

### ulation with 

### safe 

### drinkingwater is one 

### ofthe 

### most important issues in 

### public health and 

### will gainmore 

### importance in 

### the coming

### mil

### lennium. Reports 

### of toxic 

### blooms and poisonings 

### ofhumans 

### and cattle range 

### from the 

### first report 

### of a toxic 

### Nodularia bloom in 

### Lake

### Alexandrina, Australia, in 

### 1878 (1), to a 

### high incidence ofprimary 

### liver cancer 

### (PLC) in 

### China 

### attributed to 

### cyanobacterial toxin

### contaminated 

### 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. 

### Ofmuch 

### 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

### ofquestions 

### ranging 

### fromwhat 

### 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 MorphologyandTaxonomy 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, lacka

### nucleus, whereas in contrast to their closest 

### relatives, the 

### purple and the green 

### sulfur

### bacteria, 

### theyproduce oxygen (9). 

### According to 

### the current 

### taxonomy, 150 

### generawith 

### 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 

### maybe

### 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 gasvacuoles that allow them to 

### regulate their 

### position in the water 

### columnandgive thema

### distinct 

### ecologic advantage over

### other

### planktonic species. 

### BloomFormation Prevention ofbloom 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 eluci

### dated (12). 

### Although these factors can 

### be 

### considered 

### closely related to 

### bloom formation, 

### cell numbers and toxin 

### levels are 

### usually not closely

### related. Furthermore, fewgeneralizations 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, Aphanizomenonflos-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 113HITZFELD ETAL.

Table 1. Toxicityofcyanobacterial 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, Anabaenasp., M. viridis 50-300 M. aeruginosa, Aph. flos-aquae, M. viridis,

O. agardhii 250 0. agardhii, M. aeruginosa, Anabaenasp. 250 M. aeruginosa, Anabaenasp., 0. agardhii

> 1200 M. viridis

> 1200 M. viridis 50 N. spumigena 75 N. spumigena

> 2000 N. spumigena 200-250 Aph. flos-aquae, Anabaenaspp., Oscillatoria

sp., Aphanizomenonsp., Cylindrospermumsp. 20 Aph. flos-aquae 10 Aph. flos-aquae, A. circinalis, Cylindrospermopsis

raciborskii, Lyngbyawollei 2000 C. raciborskii, Umezakianatans, 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

### onlyin

### Nodularia (17-21). Toxin 

### synthesis. Although the environ

### mental 

### conditions underwhich

### cyanobacteria produce toxins remain 

### largely unknown, the
way 

### these toxins are 

### synthesized is 

### becoming clearer. Their small size, 

### cyclic structure, 

### and
content 

### ofunusual 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 

### theirexpression 

### under certain 

### environmental conditions. With the emergence 

### ofa 

### molecular 

### genetics-based tax

### onomy 

### of

### cyanobacteria together with 

### the 

### development of

### polymerase chain reaction 

### primers 

### and DNA

### probes specific for toxic strains 

### ofcyanobacteria, 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 

### ofthese 

### cyclic pep

### tides in rat 

### liver (28-30). As a 

### result 

### of

### this, toxicity 

### ofmicrocystins 

### 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-D

### MeAsp3-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

### andunique to 

### cyanobacterial toxins: 

### (2S, 3S, 8S, 

### 9S)-3- 

### amino-9-methoxy-2,6,8-trimethyl- 10- 

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

### pentapeptide with the 

### general structure 

### cyclo- (D-MeAspl

### -L-arginine2-Adda3-D

### glutamate4-Mdhb5). Mdhb is 

### 2-(methylamino)-2-dehydrobutyric acid. The most

common 

### structuralvariants 

### occur in

### positions 2 

### and 4, resulting in 

### substitutions of the L-amino 

### acids, and 

### demethylation 

### ofamino 

### acids at 

### positions 3 

### and/or 7. 

### The current 

### nomenclature names 

### themost 

### common struc

### tural 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 

### reportedto

### be nontoxic in 

### the 

### standardmouse 

### bioassay, with LD50 

### (median lethal dose) values 

### of> 

### 1,200 

### pg/kgbodyweight 

### (bw). 

### Cylindrospermopsin Cylindrospermopsin (Figure 1) is a 

### struc

### turallydistinct toxin 

### that has been 

### found in 

### tropical and 

### subtropical waters ofAustralia, where it causes 

### problems in water 

### supplies (35). 

### This alkaloid cyto- and 

### hepatotoxin is 

### produced mainlyby 

### Cylindrospermopsis raci

### borskii but also 

### byAph. ovalisporum and 

### Umezakianatans.


### Neurotoxins
The neurotoxins 

### (Figure 2) 

### described in 

### cyanobacteria can 

### be classified into 

### three distinct 

### groups: 

### a) anatoxin-a 

### and homoana

### toxin-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-aquaeand other 

### Anabaena spp., 

### Planktothrixsp. 

### (Oscillatoria sp.), 

### Aphanizomenonsp., 

### Cylindrospermumsp. 

### 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). Anatoxin

### a(s) is 

### the 

### only naturally occurring 

### organo

### phosphate and 

### has been isolated 

### from A. 

### flos-aquae andA. 

### 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 

### theyare 

### responsible 

### for par

### alytic 

### shellfish poisoning 

### after consumption 

### ofcontaminated 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 

### Lyngbyawollei (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 

### offarm animals drinking scums 

### ofcyanobacterially contami

### nated

### ponds or 

### poisonings 

### ofdogs 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 

### formicrocystin toxicity; itwas 

### shown to accu

### mulate 20-70% 

### of a 

### radioactively 

### labeled toxin 

### dose 

### (intravenous) (48-54). 

### Studies in mice 

### and pigs 

### exposed to 

### extracts 

### ofa toxic M. 

### aeruginosa bloom 

### demonstrated dose

### dependent toxicity 

### (55,56). Increasedmortal

### ity, 

### liver weight, and 

### plasma 

### alanine

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



### aminotransferase levels were 

### associated 

### with loss 

### ofbodyweight. 

### Neither 

### other organ 

### systems nor 

### lactate 

### dehydrogenase levels were 

### affected. Deathofthe organism

### through intra

### hepatic

### hemorrhage and shockis 

### rapid, occurring 

### within about 3 

### hr in 

### the case 

### ofmice. 

### Pathologic and 

### ultrastructural features commonly 

### observed in 

### the liver are 

### centrilobular hepatic necrosis, 

### destruction 

### of sinusoidal endothelium, 

### disruption ofbile 

### canalicular 

### function, 

### intrahepatic hemorrhage, loss of 

### microvilli 

### and bleb formation in 

### hepatocytes, andhepatocytenecrosis 

### (29,57-61). 

### InhibitionofProtein

### Phosphatases 1 

### and 2A
The 

### toxicityofmicrocystins 

### and

### nodularins is 

### due to 

### inhibition 

### ofthe 

### catalytic subunit of protein 

### phosphatases 1 

### and 2A 

### (PP1, PP2A) 

### (62-64). In 

### the case 

### ofmicrocystins it 

### has been 

### suggested that 

### covalent 

### binding to 

### cys

### teine-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 ofhepatocytes (28,67-71). It is not clear, 

### however, if

### the covalent bindingofthe 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 

### TPA

### independent pathway (75). 

### Cyanobacterial
extracts 

### or 

### microcystin-LR in 

### drinkingwater 

### induce skin tumors in 

### rats 

### andmice 

### 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 

### ofmicrocystin-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 

### ofinitiators 

### (80,81). 

### Both 

### microcystin-LR and 

### nodularin induce 

### the expression 

### oftumor 

### necrosis 

### factor-cc and 

### early response 

### genes 

### (c-jun, jun B, 

### jun D, 

### c-fos, fos B, 

### fra-1) in 

### rat 

### liver 

### andhepatocytes (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 

### ofcyanobacterial extract or 

### microcystin-LRin

### 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 ofmicrocystin, nodularin, andcylindrospermopsin. 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 5HITZFELD ETAL.

Table 2. Toxic cyanobacteria episodes.

Location Year Case Organism Toxin Reference

LakeAlexandrina, Australia 1878 Livestockstupor Nodulariaspumigena Nodularina (1) OhioRiver, U.S. 1931 Humans: gastroenteritis Unspecified cyanobacteria Unknown (86) Harare, Zimbabwe 1966 Humans: gastroenteritis Microcystisaeruginosa Unknown (87) Alpine Lakes, Switzerland 1974-1994 Cattledeaths, liverdamage Oscillatoria limosa, 0. tenuis, Phormidium PPI-toxinb (151) sp., Tychonemasp., Pseudoanabaena catenata, P. autumnalea.o. Armidale, Australia 1983 Humans: liverdamage Microcystisaeruginosa 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, Microcystisaeruginosa Rudyard Reservoir, U.K. 1989 Humans: recruits, pneumonia, diarrhea Microcystisaeruginosa Microcystin-LR (154) Darling River, Australia 1990-1991 Livestockdeaths Anabaenacircinalis Saxitoxin (42) 1000-kmbloom Neosaxitoxin Loch lnsh, Scotland, U.K. 1992 Dogdeath Oscillatoriasp. Anatoxin-a (155) Itaparica Dam, Brazil 1993 Humans: 88 deaths, gastroenteritis Anabaenasp., Microcystissp. Unknown (89) Loch Leven, Scotland, U.K. 1994 Fishdeaths Anabaenaflos-aquae Microcystin (44) Nandong District, Jiangsu Province, 1994-1995 Humans: PLC 100 of 105toxins act M. aeruginosa, Planktothrixagardhii, Microcystins (156) Nanhui/Shanghai, Fusui, China withHBsAg andaflatoxin Anabaenasp., 0. tenuis, Lyngbyasp. (2-4) Caruaru, Brazil 1996 Hemodialysis patients: 60 deaths Aphanizomenonsp., Oscillatoriasp. Microcystins (5,6)

Abbreviations: HBsAg, hepatitis Bantigen; PLC, primarylivercancer; PPI, proteinphosphatase inhibition. &Suspected. bProteinphosphatase-inhibiting activity.

in vivo

### data have to 

### be 

### viewed in 

### the 

### light of 

### observations in

### China, where consumption of 

### microcystin-contaminated drinkingwater 

### has been 

### associatedwith a

### high incidence 

### ofPLC 

### (2-4) (see next 

### section).



### Human

### HealdthEffects Evidence ofhuman poisonings 

### bycyanobacterial toxins 

### ranges 

### from health effects 

### after recreational exposure to 

### poisonings 

### following consumption 

### of

### contaminated

### drinkingwater 

### (Table 2). Acute 

### andsubchronic exposures. 

### The earliest case 

### ofgastroenteritis 

### from 

### cyanobacteriawas 

### reported in 

### 1931 in towns 

### along the Ohio River, 

### where low

### rainfall had causedthe 

### development ofa 

### 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 car

### bon, permanganate, 

### ammonia, and dechlorination) 

### all 

### proved to 

### be ineffective in 

### reducing taste, 

### odor, or toxin content 

### ofthe 

### drinking water. A 

### natural 

### Microcystis

### bloom in a water reservoir in 

### Harare, 

### Zimbabwe, caused gastroenteritis in 

### children each year 

### when the bloomwas 

### decaying (87). A

### partic

### ularly extensive 

### and toxic 

### (microcystin-YM [Y, L-tyrosine; M, 

### L-methionine]) (31) M. 

### aeruginosa bloom in 

### Malpas Dam, near 

### Armidale, Australia, was 

### treatedwith copper 

### sulfate in 

### 1981 after 

### complaints of

### bad taste in 

### the

### drinkingwater

### were 

### received (88). The 

### plant treating 

### thewater 

### usedprechlorination, alum 

### flocculation, sedimentation, rapid sand 

### filtration, postchlorination, and 

### fluoridation. The 

### effect ofthis 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 

### ofthe liver enzyme

### y-glutamyltransferase was 

### elevated in 

### that part 

### of the population using 

### the

### Malpas Damwater

### during 

### the bloom 

### and after the bloomwas 

### 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 well

### documented case 

### occurred in a 

### hemodialysis
center in 

### Caruaru in 

### 1996 

### (5,6). The water 

### used in 

### the 

### dialysis unit, 

### taken 

### fromTabocas Reservoir, was 

### normally sedimented with 

### alum, 

### filtered, and 

### chlorinated in 

### the munici

### pal water treatment 

### plant prior to 

### being supplied 

### by truck to 

### the clinic. At 

### the dialysis unit, 

### thewaterwas 

### further

### purifiedbypassing 

### 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 

### chlo

### rinated 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 thewater 

### received from the 

### trucks had been 

### visibly turbid. In 

### February 1996, the majority (85%) 

### of the 

### hemodialysis patients 

### developeda toxic 

### illness ofvarying severity, with a

### wide range 

### ofneu

### rologic 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

### fromcomplications 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 

### ofan 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 

### ofmicrocystin 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 con

### geners 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 ofPLC in 

### regions of China where pond and ditch water are 

### used as 

### drinkingwater supplies. In Haimen 

### and Qidong (Jiangsu Province), 

### pond and 

### ditchwater used as drinkingwater showedaveragemicrocystin concentrations of 160 

### pg/mL (60% 

### of the samples analyzed

were 

### positive), whereas microcystins could not be 

### detected in well water (2-4). PLC

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

# 1116CYANOBACTERIAL 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/ditchwater, 

### respectively (2). It has been 

### calculated that humans living in areas with a 

### reported high PLC 

### incidence

consume 

### 0.19 

### pg microcystin per 

### dayduring the 4 

### summer 

### months from June to 

### September over 

### their 40- to 

### 50-year 

### life span 

### (4). Coexposure to 

### the potent 

### liver carcino

### gen 

### aflatoxin B1 or to 

### hepatitis B 

### virus may 

### result in 

### the 

### high incidence 

### of PLC in 

### this region 

### (4). 

## EfficacyofWaterTreatment Procedures

Water treatment measures 

### should 

### always be just one 

### option 

### after other 

### techniques such as 

### selection 

### ofintake 

### depth, 

### offtake bybank filtration, 

### and/or use 

### ofbarriers to restrict scum 

### movement 

### have been used. When 

### evaluating water treatment

### procedures for the removal 

### of 

### cyanobacterial toxins, one is 

### facedwith

### problems 

### regarding soluble and 

### suspended substances. 

### Cyanotoxins are

### producedwithin 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 

### drinkingwater. Most 

### studies, especially earlyones, 

### had to 

### rely on 

### relatively crude measurements 

### of acute 

### toxicity, since more 

### specific analytical 

### methods were 

### not 

### available. The 

### cyclic hep

### tapeptides have been the focus ofmost 

### of these 

### studies, but some 

### studies on 

### removal 

### of saxitoxins 

### andanatoxin-a

### also exist. 

### Coagulaton/Flocculation, Dissolved AirFlotation, 

### and

### ActivatedCarbon 

### Adsorption Coagulation or 

### flocculation involves 

### the aggregation 

### ofsmaller

### particles into 

### largerpar

### ticles using

### chemicals suchas 

### ferric chloride or 

### aluminum 

### sulfate. 

### Coagulation can

### be an 

### efficient 

### method for 

### eliminating cyanobacterial cells fromwater, 

### whereas soluble cyanotoxins

are not 

### very 

### efficiently removed 

### by this method (90,91). The 

### efficiency of

### cyanobac

### terial removal is 

### dependent on an 

### optimization 

### of chemical doses and 

### coagulation pH (92). 

### Coagulation may cause 

### additional 

### problems 

### suchas 

### lysingof

### cyanobacterial cells leading to release 

### oftoxins 

### (90). When

### employing dissolved air 

### flotation 

### (DAF), it is 

### important to 

### consider that 

### different 

### cyanobacterial species behave 

### differentlydepending 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 ofcoagulation/flocculation in 

### combination with other measures. In



### one 

### ofthe 

### 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 

### ofthese samples was 

### then 

### tested in 

### the mouse 

### bioassay. Chlorination, 

### flocculation, or 

### sand 

### filtration were 

### unable to 

### destroy the toxins; 

### only the 

### last step, pow

### dered activated 

### carbon (PAC) at a ratio 

### of 1:10 to 

### 1:100 

### (toxin:activated carbon), removed a toxin 

### concentration 

### of3 

### pg/mL to 

### below toxic 

### levels. Studies using 50 mg 

### lyophilized cyanobacteria also 

### show that conventional 

### flocculation, filtration, andchlorination 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 

### thebasis ofthe 

### results 

### ofthese laboratory

### studies, Lahti and Hiisvirta (97) con

### ducted 

### 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) 

### flocculationwith

### A12(S04) plus sedimentation 

### plus 

### filtration, and b) PAC 

### plus flocculation with

### A12(S04) plus sedimentation plus 

### filtration. 

### Toxicitywas 

### 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. 

### Activatedcarbon is, 

### however, not 

### always a very 

### efficient method. A 

### study aimed at 

### the removal of

### cyanobacterial cellswith

### rapidsand 

### filtration and activated carbon found a 

### reduction

### ofcyanobacteriaofonly42% (98). More 

### detailed studies with 

### activated carbon 

### show that both PAC as 

### well as 

### granular-activated carbon (GAC) 

### effectivelyand

### quickly (contact times 

### of30 min are

### sufficient) eliminate cyanotoxins 

### fromwater 

### (99-101). In 

### the case 

### of PAC, 

### dosing is an 

### important parameter 

### (10 

### pg/L toxin: > 

### 200 

### mg/PAC/L), 

### whereaswhen using

### GAC, the choice ofthe 

### 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 concernwhen 

### using activated 

### carbon inwater treatment

### plants is 

### the formation 

### of a 

### biofilm, which can 

### significantly impair the 

### ability of the 

### filter to 

### adsorb tox

### ins; 

### biodegradation by the 

### biofilm does not

seem to occur 

### (99,103,104). Furthermore, below concentrations 

### of0.15 

### 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. 

### RapidFiltrationandSlowSand 

### Filtraion
The 

### performance ofrapid filtration, amethod 

### usually employed 

### after coagulation to remove 

### the

### floc, does not

### effectivelyremove 

### cyanobacterial cells 

### (98,105). Conventionalwatertreatment requires 

### regular 

### backwashing of the 

### filters, but ifthiswashingprocess is

### performed inadequately, lysis ofcyanobacterial cells on 

### the filters can 

### lead to 

### release of toxins into 

### thewater 

### (11,106). 

### Furthermore, sandfiltration 

### alone does not 

### lead to 

### substantial 

### reduction 

### oftoxicity (99), 

### and blocking causedby 

### overloadingshouldbe avoided (11). 

### Chlorinatinon

In 

### general, chlorination is not an 

### effective
process in 

### destroying cyanotoxins 

### (94-96, 107). 

### The 

### efficiency ofchlorination 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% 

### ofmicrocystins or 

### nodularin, while 

### sodiumhypochloriteat 

### the samedose or 

### chloramine achieve 40-80% removal at most 

### (91,108,109). A 

### chlorine residual ofat least 0.5 

### mg/L 

### shouldbe 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 mea

### sured

### bythe 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 ofchlorination 

### 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

### stableundernatural sun

### light (112), 

### whereas 

### ultraviolet (UV) light around the

### absorption maxima

### ofmicrocystinLR 

### and microcystin-RR 

### rapidly

### decomposed the toxins 

### (113). A 

### photocatalytic process using a 

### TiO2 

### catalyst and UV 

### radiation also 

### quickly

### decomposedmicrocystin-LR, 

### -YR, and 

### .YAwith

### half-lives 

### of< 

### 5 min 

### (114). The 

### efficiency

### ofthisprocesswas 

### largelydependent on 

### theorganic 

### loadofthewater 

### (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 7HITZFELD ETAL.



### 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 

### ofcells. In 

### the case 

### ofthe 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 

### andNorthAmerica, ozonation 

### has been 

### used

### primarily for disinfection purposes
or to remove 

### color and/or odor (117). Ozone



### was 

### initiallyusedat 

### the

### beginningofthewater 

### treatment 

### train 

### mainly to 

### inactivate viruses 

### and bacteria. In recent 

### years, 

### though, many water treatment 

### plants have 

### included a two

### stage ozonation 

### treatment, 

### either with pre

### and interozonation, inter- 

### and postozonation,

or

### withpre- 

### andpostozonation. In

### water, two 

### pathways for 

### the oxidation 

### of organic 

### pollutants 

### by ozone 

### have 

### been 

### described 

### (117,118): direct attack 

### by mole

### cular ozone via 

### cycloaddition or 

### elec

### trophilic 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, car

### boxylic 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 

### nucle

### ophilic C. An 

### indirect attack

### byfree 

### radicals 

### generally occurs via one 

### of three 

### pathways: hydrogen abstraction, electron 

### transfer, or 

### radical addition. 

### Microcystins 

### and

### nodularin. Ozone is one ofthe most 

### powerful oxidizing agents 

### and its 

### potential to 

### destroycyanobacterial toxins 

### has been

### investigated in

### thelast 10

### years 

### (Table 3). In one 

### ofthe 

### earliest studies 

### looking at 

### the 

### effect ofozone on 

### cyanotoxins, 

### researchers at 

### the British Foundation 

### for Water 

### Research ozonated 

### microcystin-LR 

### purified from M. 

### aeruginosaand 

### assessed 

### toxicityusing a

### mouse 

### bioassay (90,119). After 

### ozonation, the 

### toxicity 

### ofthe cyanotoxin is reduced, which could be shown 

### bya

### prolongation ofmouse 

### 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 

### showa

### reduction in 

### themicrocystin

### peakafter

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 ofozone 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, therewas zero toxin recovery

### after 15

sec 

### (120). With these studies it was 

### also demonstrated that 

### the removal 

### ofmicrocystins 

### is 

### proportional to 

### the ozone 

### dose when 

### the microcystin 

### concentration is 

### below the ozone 

### demand (91). 

### Complete removal 

### ofmicro

### cystin is 

### achieved and an ozone 

### residual is 

### detectedwhen 

### the ozone 

### demandofthewater 

### hasbeenmet. 

### Cyanobacterial extracts and cells and 

### organic load. 

### Obviously, amore realistic 

### way to test 

### the

### efficiencyofozonation

### wouldbe to

use 

### either 

### cyanobacterial extracts or 

### whole 

### cells 

### (Table 4). Oxidation reactions 

### ofozone 

### with 

### cyanobacterial toxins are 

### always in com

### petitionwith 

### other organic 

### compounds in 

### the water. As a 

### result, naturallyoccurring organic matter is one 

### ofthemost 

### 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 rawwater 

### leads to a 

### 60% 

### removal 

### ofmicrocystin, 

### whereas the same 

### dose added to 

### treatedwater removes toxins

### by 98%. A 

### similar ozone 

### demand was 

### measured in a study; 500 

### pg/Lmicrocystin-LRwas oxi

### dized with 0.2 

### mg/L 03 over 

### 4 min in 

### organic-free water 

### (122). The author calcu

### lated an ozone 

### demand of 0.6 mg/L 

### with 

### almost 

### complete microcystin 

### removal. However, 

### only 50% 

### ofthe same microcystin 

### concentration was 

### removed when filtered Seine River 

### (France) water was 

### oxidizedwith 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/Lmicrocystin-LR-equiva

### lents need to 

### be oxidizedwith at least 1.0 mg 

### 03/L to 

### effectivelydestroythe toxins 

### present, 

### whereas ozone 

### residuals were 

### undetectable 

### after 10 min 

### (106). 

### These 

### results show that

ozone 

### consumption 

### bynatural organicmatter 

### still occurs at 

### the preozonation stage. During postozonation, 1 

### mg/L 

### 03 

### removed 38% 

### of the microcystin, 

### whereas > 2 

### mg/L 

### 03 removed toxin

### relatedtoxicity

### belowthe limit ofdetection. The importance 

### oforganic 

### load and ozone 

### concentration was 

### also demonstrated in 

### Australian 

### studies: cyanobacterial extracts 

### containing 135-220 pg/L micro

### cystin-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, especiallywith respect to 

### the organic 

### load ofthe 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 ondestruction ofcyanobacterial toxins inthe presence orabsence of organicmatter. Microcystin-LR Ozonedose Duration OM Destruction Ozonedemand 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, notdetermined; OM, organic matter.

Table4. Effectofozone ondestructionofcyanobacterial toxinsfromcells inthepresenceorabsence oforganicmatter. 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 x106 2.5 12 100 29 ND (120) 1 x 104 0.8 10 60 ND 0.01 (107) 1x i05a 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, notdetermined. 5In rawwater; all otherexperiments inpure orfilteredwater.

Environmental Health Perspectives * Vol 108, Supplement 1 * March 2000 118CYANOBACTERIAL 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

### pilotplant 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

### oftoxicity

### 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 bypreozonation. Preozonation 

### has 

### been

### widely used to assist 

### coagulation (117). 

### The major 

### problem 

### associatedwith this method is 

### the 

### danger of

### cell lysis and toxin 

### release. A

### second posto

### zonation 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 corre

### sponds to a 

### situation

### where thebloom

### disintegrates 

### and cells lyse due to 

### chemical treatment

or 

### as a 

### result ofnatural 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 

### ofthe oxidation capacity

### could be 

### used on toxins 

### instead ofon 

### other organic 

### material. A common 

### problem with 

### these 

### early

### studies is, 

### however, that toxic

### itywas 

### determined 

### by the mouse 

### bioassay (detection in 

### the microgram 

### range) or 

### by HPLC 

### (detection in

### thenanogram-microgram 

### range), two 

### relativelyinsensitive

### assays 

### (123). Ozonation 

### of intact 

### cells 

### during preozonation 

### stepsposes 

### the

### riskof

### cyanobacterial lysis and increased ozone 

### demand. Ifcyano

### bacteria are not

### monitored at 

### thewater 

### intake level and thus enter 

### the water treatment 

### process, 

### the treatment 

### plant maynot 

### be pre

### pared 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 ofthe cyanotoxins. 

### Simulatingbloomsituations, 

### studieswere

### per

### formed 

### 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 

### relativelyhighconsidering 

### 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 

### completelydestroy the toxins 

### present in 1 

### x 

### 105 cells/mL 

### (106) 

### (Table 4). 

### Furthermore, whena

### culture of 

### 1.63 x 

### 106
M 

### aeruginosa cells/mL was 

### ozonated with a maximum

### of3.7 mg 

### 03/L, only 36% oftotal toxinwas 

### removed

### after 5 min 

### (120). Anatoxin-a, 

### anatoxin-a(s), andsaxitoxins. 

### The 

### efficiency ofoxidation 

### with ozone 

### with respect to anatoxin-a, 

### anatoxin-a(s), or 

### the saxitoxins 

### (PSPs) 

### has not 

### beenwell 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. circinalisextract tonearthelethal 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

### destroyingtheneurotoxins. 

### pH. A

### veryimportantparameter in

### theoxi

### dation

### efficiencyofozone is

### pH. At 

### pH

### values

> 

### 7.5, toxins can 

### still be detected in 

### the sam

### ples. This is 

### due to 

### the lower 

### oxidation poten

### tial 

### ofozone

### under

### alkalineconditions (1.24V) 

### comparedto

### acidic

### conditions 

### (2.07V). Ozonation 

### byproducts. In 

### contrast to 

### chlorination

### byproducts, the issue

### ofozonation 

### byproducts has not 

### been properlyaddressed. 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 

### highwith organic content. One can 

### therefore expect 

### semioxidation 

### products to 

### form 

### (124). Such oxidation 

### products were 

### found

### byHPLC

### when

### cyanobacteriawere

### pre

### ozonated. Their toxicitywas, 

### 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 ofozone 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 

### determinationoftheabsolute

### configurations of theAdda moiety 

### (126). It 

### has been 

### described that the

### double bondbetween C-6 

### and C-7 of theAdda side chain is 

### easilycleaved

### by ozone to 

### give 

### 3-methoxy-2-methyl-4-phenylbutyric acid. In 

### order to 

### realistically assess 

### the con

### sequences 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 

### chronicsituations. 

## Risk

## Assessment
The health 

### riskposed by exposure to cyanotoxins is 

### difficult to 

### quantify, since 

### the actual exposure 

### and resulting effects have still not 

### been conclusively determined, especially for thehuman situation. 

### The most 

### likelyroute 

### for human exposure is 

### the oral route via 

### drinking water 

### (127,128), recreational use 

### of

### lakes and rivers (129), or consumption 

### ofalgal health 

### food tablets (130). 

### The dermal route 

### may 

### playa

### role

### duringthe recreational use 

### ofwater 

### bodies 

### (swimming, canoeing, etc.) 

### (127,128). Due to 

### the growing concern 

### about health 

### effects ofcyanotoxins, 

### especiallyvia 

### 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

### guidelinevalue is

### basedona

### toler

### able 

### daily intake (TDI) 

### value 

### derived from two 

### animal 

### studies (56,132). 

### The first studyis

a 

### 13-weekmouse 

### oral 

### studythat

### determined a 

### no-observable adverse

### effect level (NOAEL) 

### of 40 

### jg/kg bwper 

### daybased 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 

### ofdata 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/kgbwper

### 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 vari

### ability, 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 provi

### sional 

### guideline value. This value is 

### calculated 

### by applying the TDI 

### (0.04 

### pg/kg bw) to a 

### typical 

### dailywater 

### intake in

### liters 

### (L = 2 

### liters) byan 

### individual ofa

### given

### bodyweight (bw = 60 

### kg) and a

### proportion 

### (P= 

### 0.8) ofthe total 

### dailyintake to 

### the intake

### bydrinkingwater:



### Guidelinevalue

- 

### TDIxbwxP

L



### The 

### resulting value of 0.96 

### pg/L was 

### rounded to 1.0

### uig/Land

### shouldbe

### applied to 

### cyanobacterial cell-bound and extracellular

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



### microcystins. 

### This provisional guideline

### value is applicable 

### only 

### for microcystin-LR, 

### since 

### the 

### database for other microcystin congeners
oreven 

### other 

### cyanotoxins 

### suchas 

### the saxitoxins is too 

### small to derive a TDI. Health 

### Canada is 

### applying an uncertainty 

### factor of 3,000 to 

### theNOAEL 

### of40 

### pg/kgbwper

### day from the 13-week mouse 

### study by adding a 

### factor of 3 

### for evidence of tumor promotion 

### andweak 

### evidence ofa

### potential 

### for carcino

### genicityin

### humans (133). 

### Theythus derive a 

### TDI 

### of0.013 

### pg/kgbwper

### dayandconclude that 

### the consumption of 1.5 L

### drinkingwater 

### containing < 

### 0.5 pg microcystin-LR/L 

### by a 

### 60-kg person 

### would not 

### exceed this TDI. 

### This 

### discussion ofthe 

### WHO guideline value
opens many questions 

### for operators 

### ofwater 

### treatment 

### plants. Because 

### the 

### guideline value is 

### reallyonlyvalid 

### for microcystin-LR, in 

### situations 

### where it is not 

### the most 

### dominant
congener or 

### not even 

### present, 

### the evaluation ofquantitative 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 

### reportedwith these points in 

### mind 

### and

### should, if

### possible, be

### reported formicro

### cystin-LR concentration 

### equivalents or toxic

### ity 

### equivalents. The next 

### question 

### that 

### obviouslyarises is

### whichwater treatment

### procedures are 

### adequate to 

### reduce cyanotoxin 

### levels to at 

### least below the 

### WHO 

### guideline value of1.0 

### pg/L? Assessment 

### ofwater 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/Ldrinkingwater. A

### completelydifferent situation 

### may

### arise, 

### however, during a 

### bloom andwhenwater treatment 

### procedures such as 

### chlorination 

### and/or activated carbon are not 

### used

### together. Even

### whenusingozonation, 

### 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 treatedwith 

### caution. 

### These 

### byproducts, which may

### especiallybe 

### formedwhen 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 ofprotein 

### phos

### phatases is 

### considered a keymechanism 

### by 

### which microcystins and 

### nodularins exhibit their toxicity. It is 

### therefore critical to 

### know which structural 

### modification to 

### the toxin 

### molecule 

### changes the

### affinityto 

### and inhibition of

### the 

### phosphatase. So 

### far, neither theAdda residue alone (74,134) norlinearnodularin- or microcystin-precursor peptides (135) bind 

### protein phosphatase or 

### bindwith 

### high 

### IC50 values 

### of0.5-1.0 mM 

### andshowno 

### toxicity in 

### the mouse 

### bioassay. However, it

### has also been 

### shown that substitution ofthe

### Addaside chain 

### with an L-Cys 

### residue still leads to interaction 

### of

### the toxin

### with 

### the 

### hydrophobic groove 

### of the catalytic subunit ofthe

### phosphatase (135). 

### The mechanism 

### of tumor 

### promotion 

### by microcystins 

### and 

### nodularins as 

### well as 

### the quantitative 

### relationships have not 

### been satis

### factorilyelucidated. It is 

### therefore not 

### clear if the inhibition ofprotein 

### phosphatase consti

### tutes 

### the

### onlyor

### major

### pathways fortoxicityor 

### tumor promotion. Our 

### workhas 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 

### thereforebe

### avoided. The situation

### forthe sax

### itoxins, anatoxin-a, 

### anatoxin-a(s), 

### and 

### cylindrospermopsin is even 

### less clear. A

### broader 

### scientific

### background on

### whichriskassessment 

### and management 

### steps are 

### based should be 

### developed. This can 

### lead to 

### sound process

### based risk assessment 

### and to 

### the 

### development ofeffective 

### procedures for water treatment 

### strategies

### aimedat

### specificsituations.

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