faculty of resource science and technology shrimp, artemia ... assessment of cultured...shrimp,...

Faculty of Resource Science and Technology

Toxicity Assessment of Cultured Cyanobacteria Toxin by Using Bioassay (Brine

Shrimp, Artemia salina), Thin Layer and High Performance Liquid Chromatography

Lai Jia Rhou (21252)

Bachelor of Science with Honours

(Aquatic Resource Science and Management)

2011



Lai Jia Rhou

This project is submitted in partial fulfillment of the requirement for the degree of

Bachelor of Science with Honours (Aquatic Resource Science and Management)

Aquatic Resource Science and Management Programme

Department of Aquatic Science


Universiti Malaysia Sarawak

2011

I

Acknowledgement

First and the foremost, I would like to express my sincere gratitude to my supervisor, Dr.

Samsur Mohamad for the support in my final year project, for his patience, encouragement,

and measureless knowledge. His guidance helped me in all the time of project going on and

report writing. This project and report will not be completed without his assistance and

guidance.

Besides my supervisor, I would like to thank his Master student, Miss Jasmina Majit, for her

advices, comments, motivation, guidance, and questions. I am grateful to her for guiding me

the first glance of project. Without her assistance, I will not be able to complete my final year

project in time.

My sincere thanks also go to the lab assistant, En. Nazri bin Latib@Latip, for helping me to

prepare all of the glassware and equipments that I asked for, and also helping me to carry 40

kg of water samples during my sampling at Tarat.

Special appreciation to my fellow course mates, especially Miss Chen Wai Ling and Miss

Kathy Dang, for accompanying me to work overtime at the laboratory and discussions on how

to work out for our experiments that have similarities in our projects.

Last but not least, I would like to thank my family, for providing me financial support and

supporting me mentally and spiritually throughout my three years life in Unimas.

II

Declaration

The project entitled ‘Toxicity assessment of cultured cyanobacteria toxin by using bioassay

(Brine Shrimp, Artemia salina)’ was prepared by LAI JIA RHOU. There is no portion of the

work referred to this dissertation has been submitted in support of an application for another

degree of qualification of this or any other university of institution of higher learning. The

thesis presented was carried out under the supervision of Dr. Samsur Mohamad.

___________________________

LAI JIA RHOU

Aquatic Resource Science and Management Program

Department of Aquatic



III

Table of Contents

Acknowledgement ……………………………………………………………………………. I

Declaration …………………………………………………………………………………... II

Table of Contents ………………………………………………………………………..….. III

List of Abbreviations .............................................................................................................. VI

List of Tables ………………………...…………………………………………………….. VII

List of Figures ………………………………………………………………………..….... VIII

Abstract ….…………………………………………………………………………………… 1

Introduction …………………………………………………………………...……………… 2

Literature Review …………………………………………………………………………….. 4

Cyanobacteria ..............................….............................................................................. 4

Occurrence of toxic cyanobacteria ………...……………………………………….… 6

Cyanotoxins ……………………………………………………………...................... 8

Microcystins ...................................................................................................... 8

Neurotoxins ..............................…................................................................... 10

Cylindrospermopsin ........................................................................................ 11

Analysis of cyanotoxins .............................................................................................. 14

Physico-chemical methods for detection of the cyanotoxins .......................... 14

Biological methods for detection of the cyanotoxins ..............................….... 15

IV

Materials and Methods …………………………………………………………...……….… 17

Cyanobacteria Sampling ..............................…........................................................... 17

Culture establishment ………………………………………………………….......... 19

Determination of biomass using chlorophyll a analysis ............................................. 21

Sample preparation ..............................…....................................................... 21

Sample extraction ..............................….......................................................... 21

Spectrophotometer measurement ..............................….................................. 22

Calculation ...................................................................................................... 22

Determination of nutrients concentration ................................................................... 23

Analysis of phosphorus, PO43-

........................................................................ 23

Analysis of nitrogen-ammonia, NH3-N ...................................................…... 23

Analysis of nitrate, NO3--N ......................................................................…... 23

Analysis of nitrite, NO2--N .......................................................................…... 24

Toxin extraction of samples ........................................................................................ 24

Brine shrimp bioassay ................................................................................................. 25

Toxin screening (Thin-layer Chromatography, TLC analysis) ................................... 26

High Performance Liquid Chromatography (HPLC) analysis .................................... 27

Results ..............................................................................................................................…... 29

Water quality ..............................…............................................................................. 29

V

Mass culture of Anabaena ....................................................................................…... 33

Bioassay analysis ..............................…...................................................................... 36

TLC analysis ........................................................................................................…... 40

HPLC analysis ......................................................................................................…... 41

Discussion ............................................................................................................................... 43

Conclusion .............................................................................................................................. 50

Future plans .......................................................................................................................….. 51

References .......................................................................................................................….... 52

Appendices .............................................................................................................................. 59

VI

List of Abbreviations

TLC Thin Layer Chromatography

HPLC High Performance Liquid Chrpmatography

VII

List of Tables

Table 1 Cyanobacterial Toxins and General Features ……………………………..... 12

Table 2 Composition of ASN3 Medium for the growth of isolated Anabaena sp. from

Pond 12 ........................................................................................................... 20

Table 3 Acute bioassay conditions for brine shrimp (A. salina) toxicity test ……….. 25

Table 4 Conditions of HPLC analysis …………………………………….………..... 28

Table 5 In-situ water quality parameter readings during first sampling of the sampling

ponds at IFRPC ……………........................................................................... 30

Table 6 In-situ water quality parameter readings during second sampling of the

sampling ponds at IFRPC ……………........................................................... 31

Table 7 Chlorophyll a concentrations (mg/L) analysis result of the sampling ponds at

IFRPC during second sampling ...................................................................... 30

Table 8 Nutrient concentration of water sampled from three different ponds during

second sampling at IFRPC .............................................................................. 32

Table 9 Retention factor (Rf) values from the extracts of samples from earth pond 1, 2

and 3 and cultured Anabaena sp. under TLC toxin screening ........................ 40

VIII

List of Figures

Figure 1 Systematic of cyanobacteria taxon according to Bold and Wynne …………... 5

Figure 2 Chemical structures of the cyanotoxin: the cyclic hepatotoxic peptides

microcystin (A) and nodularin (B), the hepatotoxic alkaloid

cylindrospermopsin (C) and the tricyclic neurotoxic alkaloid saxitoxin (D)

adopted by Kurmayer and Christiansen, 2009 ................................................ 13

Figure 3 Different color of algae blooms at the sampling ponds at Tarat. (a) Pond 1/first

sampling, (b) Pond 12/first sampling, (c) Pond 16/first sampling, (d) Earth

pond 1/second sampling, (e) Earth pond 2/second sampling, and (f) Earth pond

3/second sampling ........................................................................................... 18

Figure 4 Ten liters mass culture of isolated Anabaena sp. from Pond 12 at laboratory by

using ASN3 medium at day 4 in a 10 liter plastic carboy ..................................... 19

Figure 5 Position of distance traveled by the compound (x) and distance traveled by the

solvent front (y) for calculating of retention factor value on silica gel precoated

plates ............................................................................................................... 26

Figure 6 Micrograph of laboratory cultured Anabaena sp. isolated from Pond 12 during

first sampling at IFRPC taken by microscope model: Nikon Eclipse 80i,

magnification of 40X10 ................................................................................................... 33

Figure 7 Growth curve of cultured Anabaena sp. by using medium ASN3 within 12

days of culture period ...................................................................................... 35

Figure 8 Dose-response curve of brine shrimp (A. salina) bioassay by using extract from

cultured Anabaena sp. ..................................................................................... 36

Figure 9 Transformation of mortality percentages to probit, probit versus log10

concentration graph for brine shrimp (A. salina) bioassay ............................. 37

Figure 10 Morphological differences of brine shrimp (A. salina), before and after toxin

exposure of Anabaena sp. extracts (Figure 10 a-b, control; figure 10 c-d, toxin

concentration of 2 g/ml, figure e-f, toxin concentration of 50 g/ml) ............ 39

Figure 11 HPLC chromatogram for standard microcystin-LR and extracts of samples

from cultured Anabaena sp., earth pond 1, 2, and 3. HPLC: Waters column

C18, mobile phase methanol:0.05 M phosphate buffer (6:4), flow rate 1

ml/min, detection UV 238 nm ......................................................................... 42

1



Lai Jia Rhou

Aquatic Resource Science and Management



ABSTRACT

Cyanobacterial toxicity was both tested from the natural water samples and isolated cyanobacterial samples

collected from aquaculture ponds in Indigenous Fisheries Research and Production Center (IFRPC) Tarat,

Serian. Water quality parameters such as salinity, turbidity, dissolved oxygen, pH and temperature were taken.

Anabaena sp. was isolated and mass cultured by using medium ASN3. The cells were harvested at their late

exponential phase of growth. Crude extracts were obtained from natural samples and Anabaena sp. by using 95

% methanol. Brine shrimp (Artemia salina) bioassay was carried out on Anabaena strain by using eight

concentrations (2, 5, 10, 20, 30, 40, 50, 100 g/ml) in filtered seawater. Endpoint of the experiment was

mortality of brine shrimp. LC50 value obtained was approximately 8.32 g/ml and lethality at 30 g/ml. Thin-

layer chromatography (TLC) and high performance liquid chromatography (HPLC) were used for further

examination of microcystin by using standard of microcystin-LR. Microcystin were not detected in all of the

samples, however, other toxins such as neurotoxin are expected to be available based on the mortality of the

brine shrimps, retention factor values in TLC and overlay of peaks at 5.0 minutes retention times shown in

HPLC chromatogram.

Key words: cyanotoxins, culture, bioassay, TLC, HPLC

ABSTRAK

Toksin sianobakteria diuji dari sampel air kolam akuakultur dan sel yang diisolasi diperoleh dari Pusat

Penyelidikan Perikanan dan Pengeluaran, Tarat, Serian. Parameter yang diambil untuk kajian kualiti air

merangkumi saliniti, kekeruhan, oksigen terlarut, pH dan suhu. Anabaena sp. diisolasi dari air kolam dan

dikultur dengan menggunakan medium ASN3. Sel-sel diekstrak pada akhir fasa eksponen. Sample air kolam dan

Anabaena sp. diekstrak dengan mengunakan 95 % metanol. Pengujian bioesei dengan menggunakan Artemia

salina dijalankan dengan menggunakan lapan konsentrasi (2, 5, 10, 20, 30, 40, 50, 100 g/ml) ekstrak

Anabaena di dalam air masin yang sudah ditapiskan. Keputusan eksperimen ini adalah berdasarkan kematian

A. salina. Nilai LC50 adalah pada 8.32 g/ml dan aras kematian pada 30 g/ml. Kromatografi lapisan nipis dan

kromatografi cecair prestasi tinggi digunakan bagi pemeriksaan microsytin dengan menggunakan standard

microsytin-LR. Microcystin tidak dikesankan dalam semua sampel, tetapi toksin lain misalnya neurotoksin

dijangka terdapat dalam sampel yang dikaji berdasarkan kematian A. salina dalam eksperimen bioesei, indek

faktor yang dicapai dari kromatografi lapisan nipis dan puncak bertindih pada masa rentensi minit 5.0 yang

ditunjukkan di kromatogram kromatografi cecair prestasi tinggi.

Kata kunci: sianotoksin, pengkulturan, bioesei, TLC, HPLC

2

1.0 Introduction

Cyanotoxin refer to toxins that are produced by many genera of cyanobacteria and are

generally known as hepatotoxins and neurotoxins (Chorus & Bartram, 1999). Hepatotoxins

are generally portrayed by cyclic peptides (microcystins, MCs and nodularins) and alkaloids

(cylindrospermopsins), and neurotoxins by alkaloids (anatoxina-a, anatoxina-a (s) and

saxitoxins (STX).

Reported cases relating infection of cyanotoxin of uninhabited and household animals, and

humans have been published globally (Jochimsen et al., 1998; Chorus & Bartram, 1999;

Azevedo et al., 2002). The adverse effects associated with the occurrence of cyanobacterial

water blooms have been recorded. The effects include depletion of dissolved oxygen that

leads to deaths of fish (Jacobsen, 1994), bloom that produce scums and unpleasant odour that

bring to destruction of recreational use of water bodies, (Pilotto, et al., 1997), and toxic

compounds production that bring health effects to human (Falconer, 1998). The health

significance of toxic cyanobacteria can be seen in many reports on injuries and deaths of

animals all over the world (Lawton & Codd, 1991). Thus, cyanotoxin represent a risk to

aquatic organisms and human health, especially when consumed in drinking water, through

the food or skin contact.

A lot of researches have been done on determining and assessing microcystins. On the

contrary, information and methods of detecting other known toxins such as anatoxins and

cylindrospermopsins are limited due to little work has been done on. Aside from saxitoxins

3

which can be found generally in marine environment, several methods have been established

in detecting saxitoxins. Nevertheless, detection methods are insufficient in freshwater

samples. Moreover, it is challenging in selecting an ideal technique to assess the toxicity level

of a bloom where some species produce multiple classes of toxins. For instance, Anabaena sp.

produces microcystin, saxitoxin and also anatoxin (Lawton & Edward, 2008).

Several biological method involving bioassay have been established for assessing

cyanobacterial toxicity. For example, mouse bioassay is one of the methods that have been

single used for the detection of all kinds of cyanotoxin. Nonetheless, considerable research

efforts have been made to find suitable alternative methods and brine shrimp bioassay is one

of them (Marsalek & Blaha, 2000).

The cyanobacterial samples were collected from three aquaculture ponds in Indigenous

Fisheries Research and Production Center (IFRPC) Tarat, Serian. This project is important in

providing the information regarding the occurrence of potential toxin producer which can

bring hazard to public and introduce data concerning cyanotoxin and their levels as the

information regarding cyanobacterial blooms especially in tropical countries are limited. The

purposes of this study are to isolate the potential toxic cyanobacteria species (Anabaena sp.)

from IFRPC and to establish the culture of isolated cyanobacteria, and to assess the toxicity of

cyanotoxin of cultured cyanobacteria by using bioassay (Artemia salina).

4

2.0 Literature Review

2.1 Cyanobacteria

Cyanobacteria are an ancient group of simple microorganisms with characteristics in common

with bacteria and algae. They resemble bacteria in the prokaryotic cellular organization and

similar to algae in oxygen-evolving photosynthesis. Cyanobacteria are said to have been the

first oxygen releasing organisms on our planet, two and a half billion years ago. As such, they

created the atmosphere that further allowed the development of life. This group of micro-

organisms comprises unicellular to multicellular prokaryotes that possess chlorophyll a and

perform oxygenic photosynthesis associated with photosystems I and II (Castenholz et al.,

2001).

Cyanobacteria were classified in the Monera Kingdom under Phylum of Cyanophyta (Bold &

Wyyne, 1985) and can be divided into 5 Orders, namely Chroococcales, Oscillatoriaceae,

Stigonematales, Nostocales and Chaemasiphonales (Figure 1).

The basic morphology comprises unicellular, colonial and multicellular filamentous forms.

For example, unicellular and isopolar (Chroococcales), unicellular and heteropolar

(Chamaesiphonales), pseudoparenchymatous (Pleurocapsales), multicellular, trichal, and

heterocysts not present (Oscillatoriales), multicellular, trichal, with branch, and heterocysts

present (Stigonematales), multicellular, trichal, and heterocysts present (Nostocales).

5

Figure 1: Systematic of cyanobacteria taxon according to Bold and Wynne (1985)

Kingdom: Monera

Division: Cyanophyta

Order: Chroococcales

Family: Chroococcaceae

Order: Chaemasiphonales

Family: Pleurocapsaceae

Dermocarpaceae

Chaemasiphonaceae

Order: Oscillatoriales

Family: Oscillatoriaceae

Borziaceae

Stigonematoceae

Order: Nostoles

Family: Nostoceceae

Rivulariaceae

Scytonematoceae

Order: Stigonematales

Family: Chlorogloeosacceae

Capsosiraceae

6

2.2 Occurrence of toxic cyanobacteria

Genera Anabaena, Microcystis and Planktothrix are among the freshwater cyanobacteria.

Cyanobacteria belong to the genus Anabaena comprises filamentous, heterocyst-forming

cyanobacteria that may have gas vacuoles (Rippka et al., 2001). Microcystis strains are

unicellular colony-forming cyanobacteria with gas vesicles (Herdman et al., 2001). Genus

Planktothrix are filamentous, able to move by gliding and have abundant gas vacuoles

(Castenholz et al., 2001b). Three of these genera are potential in producing microcystins in

freshwater environments (Sivonen & Jones, 1999). Three types of neurotoxins: an alkaloid

(anatoxin-a), an organophosphate (anatoxin-a(S)) and carbamate toxins (saxitoxins) are the

potential toxin produced by Anabaena strains (Sivonen, 2000). However, anatoxin-a can be

found in a few strains of Microcystis and benthic Oscillatoria (Sivonen, 2000). While,

homoanatoxin-a, an anatoxin-a derivative, is produced by some Oscillatoria/Planktothrix

strains (Sivonen, 2000).

According to Sivonen and Jones, (1999), toxic cyanobacteria can be found worldwide in

inland and coastal water environments. Microcystis spp., Cylindrospermopsis raciborskii,

Planktothrix (syn. Oscillatoria) rubescens, Synechococcus spp., Planktothrix (syn.

Oscillatoria) agardhii, Gloeotrichia spp., Anabaena spp., Lyngbya spp., Aphanizomenon spp.,

Nostoc spp., some Oscillatoria spp., Schizothrix spp. and Synechocystis spp. are among the

most common toxic cyanobacteria in freshwater. Toxicity may be available in the similar

species and genera. More of the toxic species are likely to be discovered as more research has

been carried out in more regions worldwide. Hence, it is possible to presume a toxic potential

in a cyanobacterial bloom.

7

Microcystins and neurotoxins are the most widespread cyanobacterial toxins. Neurotoxin and

microcystin may be contained in some species simultaneously. Microcystis, are almost always

toxic (Carmichael, 1995), but non-toxic strains do occur. Generally, toxicity is not a trait

specific for certain species; rather, most species comprise toxic and nontoxic strains. For

microcystins, it has been shown that toxicity of a strain depends on whether or not it contains

the gene for microcystin production (Dittmann et al., 1996) and that field populations are a

mixture of both genotypes with and without this gene (Kurmayer et al., 2002). Experience

with cyanobacterial cultures also shows that microcystin production is a fairly constant trait of

a given strain or genotype, only somewhat modified by environmental conditions (Chorus,

2001).

Worldwide, about 60% of cyanobacterial samples investigated contain toxins. The toxicity of

a single bloom may, however, change in both time and space. Demonstrations of toxicity of

the cyanobacterial population in a given water body does not necessarily imply an

environmental or human hazard as long as the cells remain thinly dispersed. Mass

developments and especially surface scums pose the risks.

8

2.3 Cyanotoxins

Cyanotoxins fall into three broad groups of chemical structure: cyclic peptides, alkaloids and

lipopolysaccharides (LPS) (Chorus & Bartram, 1999). The specific toxic substances within

these broad groups that have been identified to date from different genera of cyanobacteria,

together with their primary target organs in humans (Table 1).

2.3.1 Microcystins

Microcystins are the most frequently occurring and widespread of the cyanotoxins. They are

cyclic heptapeptides containing a specific amino acid (ADDA) side chain which, to date, has

been found only in microcystins and nodularin. About 70 structural analogues of microcystin

have been identified (Rinehart et al., 1994; Sivonen & Jones, 1999). They vary with respect to

methyl groups and two amino acids within the ring. This has consequences for the tertiary

structure of the molecule and results in pronounced differences in toxicity as well as in

hydrophobic/hydrophilic properties. Microcystins block protein phosphatases 1 and 2a which

are important molecular switches in all eukaryotic cells with an irreversible covalent bond

(MacKintosh et al., 1990).

The chief pathway for microcystins entry into cells is the bile acid carrier, which is found in

liver cells and, to a lesser extent, in intestinal epithelia (Falconer, 1993). For vertebrates, a

lethal dose of microcystin causes death by liver necrosis within hours up to a few days.

Evidence for the permeability of other cell membranes to microcystins is controversial. It is

possible that hydrophobic structural analogues can penetrate into some cell types even

without the bile acid carrier. In addition, Fitzgeorge et al. (1994) published evidence for

disruption of nasal tissues by the common hydrophilic analogue microcystin-LR. While

9

toxicity by oral uptake is generally at least an order of magnitude lower than toxicity by

intraperitoneal (i.p.) injection, intranasal application in these experiments was as toxic as i.p.

injection, and membrane damage by microcystin enhanced the toxicity of anatoxin-a. This

uptake route may be relevant for water sports activities that lead to inhalation of spray and

droplets, such as waterskiing.

Microcystins are found in most populations of Microcystis spp. and in strains of some species

of Anabaena. High microcystin content has also been observed in Planktothrix (syn.

Oscillatoria) agardhii and P. rubescens (Fastner et al., 1999). P. agardhii, however, never

forms scums, and where it occurs P. rubescens does not usually form scums during the

recreational water use season, thus reducing the hazard to swimmers.

Fitzgeorge et al. (1994) demonstrated that microcystin toxicity is cumulative: a single oral

dose resulted in no increase in liver weight (which is a measure of liver damage), whereas the

same dose applied daily over seven days caused an increase in liver weight of 84% and thus

had the same effect as a single oral dose 16 times as large. This may be explained by the

irreversible covalent bond between microcystin and the protein phosphatases and subsequent

substantial damage to cell structure (Falconer, 1993). Healing of the liver probably requires

growth of new liver cells.

As a result of the lack of apparent symptoms at moderate exposure, exposure is likely to be

continued by people uninformed of the risk, for instance, for consecutive days of a holiday or

a hot spell, which will increase the risk of cumulative liver damage. There are two aspects of

chronic microcystin damage to the liver: progressive active liver injury (Falconer et al., 1988)

and the potential for promotion of tumour growth. Tumour-promoting activity of microcystins

10

is well documented, although microcystins alone have not been demonstrated to be

carcinogenic. Promotion of mouse skin tumours has been shown after initiation by topical

exposure to a carcinogen (dimethylbenzanthracene) followed by ingestion of a Microcystis

aeruginosa extract (Falconer & Buckley, 1989; Falconer & Humpage, 1996). In rat liver

studies, the appearance of pre-neoplastic liver foci and nodules was promoted by pure

microcystin-LR in a protocol involving one i.p. dose of diethylnitrosamine and i.p. doses of

microcystin-LR over several weeks (Matsushima et al., 1992).

2.3.2 Neurotoxins

Irrespective of somewhat different modes of action, all three neurotoxins (Table 1) have the

potential to be lethal by causing suffocation: anatoxin-a, a(s) through cramps, and saxitoxins

through paralysis. However, no human deaths from exposure to neurotoxins associated with

recreational use of water are known. Anatoxin-a(s) is the only known naturally occurring

organophosphate cholinesterase inhibitor and causes strong salivation (the‘s’ in its name

stands for salivation), cramps, tremor, diarrhoea, vomiting and an extremely rapid death

(within minutes). Saxitoxins and anatoxin-a(s) are among the most neurotoxic substances

known. However, evidence is accumulating that in lakes and rivers they do not occur as

frequently as microcystins. Furthermore, concentrations even of these highly toxic substances

in scums will scarcely reach levels acutely neurotoxic to a human ingesting a mouthful. In

contrast, neurotoxicity may be experienced by livestock that drink many litres of

contaminated water and pets—especially dogs—that gather scum material in their fur and

ingest it through grooming with the tongue. After ingestion of a sub-lethal dose of these

neurotoxins, recovery appears to be complete, and no chronic effects have been observed to

11

date. For these reasons, the neurotoxins are a hazard to be aware of when using waters

populated with cyanobacteria for recreation.

2.3.3 Cylindrospermopsin

Cylindrospermopsin can be found in Cylindrospermopsis raciborskii, the compound of

Cylindrospermopsin is an alkaloid (Ohtani et al., 1992). This type of cyanotoxin causes

obstruction in protein synthesis, it thus collapses the kidney and liver. Besides, extraction of

the toxin also hurts intestine, lungs and adrenals. The adverse effects will only visible after

many days of exposure, therefore cause-effect relationship is hard to establish. As an

example, victims in Australia recovered from intake of cylindrospermopsin through drinking

water by skillful hospital staffs (Falconer, 1996). C. raciborskii can be found in tropic and

sub-tropic countries; however the appearance had been detected in the north such as Vienna

(Roschitz, 1996). Northeastern Germany reported an abundant community of C. raciborskii, it

seems to affect temperate regions (Padisák, 1997).

12

Table 1: Cyanobacterial Toxins and General Features

Cyanobacteria Toxin(s) Structure Primary target organ in

mammals

Microcystis aeruginosa Microcystin Cyclic

peptide Liver

Microcystis-type-c Peptide Liver

2 Microcystin-like-

toxins Peptides Liver

Microcystin-like Peptide Liver

Aphanizomenon flos-

aquae Aphantoxins Alkaloids

Neosaxitoxin Alkaloids Nerve axons

Saxitoxin Alkaloids Nerve axons

Anabaena flos-aquae Anatoxin-a Alkaloids Nerve synapse

Anatoxin-b

Nerve synapse

Anatoxin-c

Nerve synapse

Anatoxin-c

Nerve synapse

Schizothrix calcicola Aplysiatoxins Alkyl

phenols Skin

Lyngbya gracilis Debromoaplysiatoxin Alkyl

phenols Skin, gastrointestinal tract

L. majuscule Debromoaplysiatoxin Alkyl

phenols Skin, gastrointestinal tract

Lygbyatoxin

Skin, gastrointestinal tract

Oscillatoria nigroviridis Aplysiatoxin Alkyl

phenols Skin

Calothrix crustacean Aplysiatoxin Alkyl

phenols Skin

Nostoc muscorum Aplysiatoxin Alkyl

phenols Skin

S. muscorum Aplysiatoxin Alkyl

phenols Skin

Nodularia Nodularin Peptides Liver

Cylindrospermopsis Cylindrospermopsins Alkaloids Liver

All Lipopolysaccharides Alkaloids Potential irritant: affect any

(LPS) exposed tissue

(Sources: Chorus & Bartram, 1999)

13

Figure 2: Chemical structures of the cyanotoxin: the cyclic hepatotoxic peptides microcystin (A) and nodularin

(B), the hepatotoxic alkaloid cylindrospermopsin (C) and the tricyclic neurotoxic alkaloid saxitoxin (D) adopted

by Kurmayer and Christiansen, 2009

14

2.4 Analysis of cyanotoxins

2.4.1 Physico-chemical methods for detection of the cyanotoxins

Toxicity testing of cyanobacteria by using chemical methods is chosen based on the chemical

structure of the respective compound. A few of considerable methodological application have

been established in detecting and quantifying cyanotoxin. One of the most commonly used

methods recently is high performance liquid chromatography (HPLC).

While reversed phase HPLC (RP-HPLC) is among the most commonly used technique in

peptide microcystins detection. Ultra violet (UV) detector is used because of the typical

absorption spectra of microcystins. A few of RP-HPLC modified conditions were published

(Harada et al., 1988; 1991; Namikoshi et al., 1992). The most broadly used as mobile phase

comprised from various combinations of acetonitrile with other solvents (Meriluoto &

Eriksson, 1988; Sivonen et al., 1990; Vasconcelos et al., 1993; Luukainen et al., 1993; 1994;

Lawton et al., 1994).

Apart from UV detection, microcystins can be detected with other systems linked with RP-

HPLC which have been successfully applied. Poon et al. (1993) first reported the adoption of

electrospray ionization mass spectrometry for microcystin identification. Application of

electrochemical detector following detection techniques by derivatization with fluorogenic

(Shimizu et al., 1995) or chemiluminescent reagents (Murata et al., 1995) were also reported.

Thin layer chromatography (TLC) has been acknowledged as one of the analytical techniques

used for the detection of microcystins (Harada et al., 1988; Lanaras & Cook, 1994). Though,

TLC method is usually limited for semi-preparative purposes or check of the sample purity.

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