of dissertation

Environmental and ecotoxicological aspects of cyanobacterial toxins - microcystins

Pavel BABICA

Dissertation thesis in Environmental Chemistry

Masaryk University, Faculty of Science, RECETOX - Research Centre for Environmental Chemistry and Ecotoxicology Brno, Czech Republic

Brno, 2006 Supervisor Assoc. Prof. Blahoslav Maršálek, Ph.D.

Bibliographic information Author:

Pavel Babica

Title of dissertation: Environmental and ecotoxicological aspects of cyanobacterial toxins - microcystins Title of dissertation (in Czech): Environmentální a ekotoxikologické aspekty sinicových toxinů - microcystinů Ph.D. study program:

Chemistry

Specialization:

Environmental Chemistry

Supervisor:

Assoc. Prof. Blahoslav Maršálek, Ph.D.

Year of defence:

2006

Keywords:

cyanobacteria, cyanotoxins, microcystins, analytical determination,

environmental

fate,

ecotoxicological

effects Keywords (in Czech):

sinice, cyanotoxiny, microcystiny, analytické stanovení, environmentální osud, ekotoxikologické efekty

© Pavel Babica, Masaryk University, 2006

Contents

Table of contents Table of contents ............................................................................................................. 5 Abstract ........................................................................................................................... 6 Abstrakt (Abstract in Czech) ........................................................................................... 7 List of original articles..................................................................................................... 8 The author’s contribution in the articles.......................................................................... 9 Abbreviations ................................................................................................................ 10 1. Introduction ...................................................................................................... 11 2. Aims of the present study ................................................................................. 15 3. Analyses of microcystins.................................................................................. 16 3.1 Background ...................................................................................................... 16 3.2 Extraction of microcystins from sediments ...................................................... 19 3.3 Analysis of microcystins by capillary electrochromatography ........................ 22 4. Environmental occurrence of microcystins ...................................................... 25 4.1 Background ...................................................................................................... 25 4.2 Environmental monitoring of dissolved microcystins in water reservoirs of the Czech Republic in 2004 .............................................................................. 27 4.3 Detection of microcystins in water bloom of Pseudanabaena limnetica ......... 30 4.4 First report on microcystin occurrence in Bulgaria and Ghana........................ 31 4.4.1 Detection of microcystins in water reservoirs in Bulgaria....................... 31 4.4.2 Analysis of microcystin in drinking water reservoirs in Ghana............... 32 4.5 Microcystin analyses in phototrophic biofilms and the toxicity assessment .... 34 5. Environmental fate of microcystins.................................................................. 37 5.1 Background ...................................................................................................... 37 5.2 Microcystins occurrence and dynamic in the sediments of Brno reservoir...... 39 5.3 Removal of microcystin by phototrophic biofilms........................................... 41 6. Effects of microcystins on aquatic organisms .................................................. 45 6.1 Background ...................................................................................................... 45 6.2 Effects of microcystins on photoautotrophic organisms - a review ................. 45 6.3 Effects of dissolved microcystins on planktonic photoautotrophs ................... 51 7. General discussion and summary ..................................................................... 55 8. Acknowledgements .......................................................................................... 58 References ..................................................................................................................... 59 Appendices I - XI

5

Abstract

Abstract Developments of cyanobacterial mass populations in freshwater, brackish and coastal marine waters have increased as a consequence of nutrient pollution of aquatic ecosystems within the last few decades. Thus, cyanobacterial toxins (cyanotoxins) have become an important group of toxic compounds in the environment. Microcystins belong to the most hazardous cyanotoxins with respect to their acute and chronic toxicity and common environmental occurrence. This work focused on development of analytical procedures and methods for detection of these contaminants, study of microcystin environmental distribution and fate, and investigation of microcystin effects on photoautotrophic organisms. Several analytical procedures and methods for determination of microcystins in sediments were optimised and compared in this study. The results revealed only partial efficiency of conventional extraction (strongly dependent on extraction solvent, sediment composition and microcystin structure), importance of hydrophilic interactions in adsorption of microcystins on sediments, suitability of HPLC and ELISA techniques for final detection of microcystins in the sediment extracts, as well as the presence of microcystins and dynamic of their concentrations in the natural sediments. Novel information on separation and detection of microcystins by capillary electrochromatography was also provided in the present work. Microcystins occur frequently in the environment, as was clearly confirmed in this study. Dissolved microcystins were detected in 85% of about 90 major water bodies in the Czech Republic in 2004 with median concentration 0.67 µg/L and maximal concentration up to 37 µg/L. First reports of microcystin occurrences in freshwaters of Bulgaria and Ghana illustrate worldwide distribution of these cyanobacterial toxins. It was also shown that microcystins were associated with blooms of cyanobacterial genera, which have not been considered as typical microcystin producers. However, no microcystins were detected in the samples of phototrophic biofilms dominated by benthic cyanobacteria and only weak toxicity was elicited by these biofilms in battery of in vitro bioassays. On the other hand, phototrophic biofilms were demonstrated as potent degraders of microcystins in model microcosms and previous contact of biofilm with cyanobacteria was identified as a factor selectively improving the rate of microcystin removal from water column. Available information showed that microcystins might affect not only animal and human health, but also various aquatic photoautotrophs. Nevertheless, the phytotoxicity of microcystins seems to be rather a side effect than a result of allelopathic activity. Correspondingly, growth of several planktonic photoautotrophs was affected only at high concentrations of microcystins in the experiments carried out in this study. The effects of microcystins were species-specific and congener-specific, but the general lack of growth inhibition at environmentally relevant concentrations did not support hypothesis on primarily allelopathic function of microcystins. 6

Abstrakt (Abstract in Czech)

Abstrakt (Abstract in Czech) V důsledku znečištění vodních ekosystémů živinami dochází v posledních desetiletích stále častěji k masovým rozvojům sinic (cyanobakterií) ve sladkých a brakických vodách pevnin a pobřežních moří. Sinicové toxiny (cyanotoxiny) se tak staly významnou skupinou toxických látek v prostředí. Microcystiny patří mezi nejnebezpečnější cyanotoxiny s ohledem na jejich vysokou akutní a chronickou toxicitu a častý výskyt v prostředí. Předkládaná dizertační práce byla zaměřena na vývoj a optimalizaci analytických metod pro stanovení těchto důležitých kontaminantů, studium jejich environmentální distribuce a osudu a na výzkum jejich účinků na fotoautotrofní organismy. V rámci práce bylo srovnáno a optimalizováno několik analytických postupů a metod pro stanovení microcystinů v sedimentech. Výsledky ukázaly pouze částečnou úspěšnost konvenčních extrakčních postupů (silně závisející na extrakčním rozpouštědle, složení sedimentu a struktuře microcystinů), význam hydrofilních interakcí pro adsorpci microcystinů na sedimenty, vhodnost použití HPLC a ELISA koncovky pro stanovení microcystinů v extraktech ze sedimentů, stejně jako přítomnost microcystinů a dynamiku jejich koncentrací v přírodních sedimentech. V práci byla rovněž nově popsána separace a stanovení microcystinů s využitím kapilární elektrochromatografie. Microcystiny jsou v prostředí velmi rozšířené sloučeniny, jak bylo potvrzeno výsledky předloženými v dizertaci. Rozpuštěné microcystiny byly v roce 2004 detekovány v 85% z asi 90 nejvýznamnějších vodních nádrží v České republice (mediánová koncentrace byla 0,67 µg/L a detekované maximum činilo 37 µg/L). První informace o výskytu microcystinů v sladkovodních nádržích v Bulharsku a Ghaně dokreslují globální rozměr problematiky toxických sinic. Ukázalo se rovněž, že microcystiny mohou být produkovány vodními květy s dominujícími druhy sinic, které dosud nebyly považovány za typické producenty microcystinů. Naproti tomu microcystiny nebyly detekovány ve vzorcích fototrofních biofilmů s dominujícími bentickými sinicemi a extrakty těchto biofilmů vykazovaly pouze slabé toxické efekty v sérii laboratorních in vitro experimentů. Na druhou stranu byly fototrofní biofilmy schopny účinně degradovat microcystiny v modelových mikrokosmech, a předcházející kontakt biofilmu se sinicemi selektivně zvyšoval rychlost odstraňování microcystinů z vodního sloupce. Podle dostupných informací microcystiny mohou poškozovat nejen živočichy, ale take fotoautotrofní organismy. Fytotoxicita microcystinů je však s nejvyšší pravděpodobností spíše vedlejším efektem nežli důsledkem cíleného alelopatického působení. Tomu odpovídají výsledky experimentů realizovaných v rámci této studie, podle nichž byl růst několika zástupců planktonních fotoautotrofů ovlivněn pouze vysokými koncentracemi microcystinů. Pozorované ovlivnění růstu bylo druhově specifické a závislé na struktuře microcystinů, nicméně absence efektů při působení environmentálně relevantních koncentrací microcystinů nepodporuje hypotézu o primární alelopatické funkci microcystinů. 7

Original articles

List of original articles The particular chapters of this thesis are based on the following articles, which are referred to by their Roman numerals in the text:

I Babica P., Kohoutek J., Bláha L., Adamovský O. & Maršálek B. (2006): Evaluation of extraction approaches linked with ELISA and HPLC for analyses of microcystin-LR, -RR and -YR in freshwater sediments with different organic material content. Analytical and Bioanalytical Chemistry 385(8):1545-1551 II Zeisbergerová M., Košťál V., Šrámková M., Babica P., Bláha L., Glatz Z. & Kahle V. (2006): Separation of microcystins on monolithic electrochromatography columns. Journal of Chromatography B 841:140-144 III Bláhová L., Babica P., Sukačová K., Geriš R., Feldmannová M., Maršálek B. & Bláha L.: Dissolved microcystins in the freshwaters of the Czech Republic - results of the national monitoring programme - 2004. Submitted to Water Research IV Maršálek B., Bláha L. & Babica P. (2003): Analyses of microcystins in the biomass of Pseudanabaena limnetica collected in Znojmo reservoir. Czech Phycology 3:195-197 V Pavlova V., Babica P., Todorova D., Bratanova Z. & Maršálek, B. (2006): Contamination of some reservoirs and lakes in Republic of Bulgaria by microcystins. Acta Hydrochimica et Hydrobiologica 34 (in press) VI Addico G., Hardege J., Komarek J., Babica P. & de Graft-Johnson K.A.A. (2006): Cyanobacteria species identified in the Weija and Kpong reservoirs, Ghana, and their implications for drinking water quality with respect to microcystin. African Journal of Marine Sciences 28(2):451-456 VII Bláha L., Sabater S., Babica P., Vilalta E. & Maršálek B. (2004): Geosmin occurrence in river cyanobacterial mats: is it causing a significant health hazard? Water Science and Technology 49(9):307-312 VIII Babica P., Bláha L. & Maršálek B. (2005): Removal of microcystins by phototrophic biofilms - a microcosm study. Environmental Science and Pollution Research 12(6):369-374 IX Babica P., Bláha L. & Maršálek B. (2006): Exploring the natural role of microcystins - a review of effects on photoautotrophic organisms. Journal of Phycology 42(1):9-20 X Babica P., Hilscherová K., Bártová K., Maršálek B. & Bláha L.: Effects of dissolved microcystins on growth of planktonic photoautotrophs. Phycologia (accepted for publication)

8

Original articles

The author’s contribution in the articles The author´s research described in this thesis and in the articles was carried out at the RECETOX - Research Centre for Environmental Chemistry and Ecotoxicology (Faculty of Science, Masaryk University) and at the Department of Experimental Phycology and Ecotoxicology (Institute of Botany, The Academy of Sciences of the Czech Republic).

Paper I

Pavel Babica participated in the experimental design, performed part of the final HPLC-DAD and ELISA analyses, evaluated data and interpreted the results. He wrote parts of the article and finalized the entire manuscript.

Paper II

Pavel Babica prepared the extracts of cyanobacterial bloom, purified microcystins and performed HPLC analyses. He participated in the revision and correction of the manuscript.

Paper III

Pavel Babica participated in the design and organization of the surveillance and sampling, performed part of ELISA analysis, contributed to the data evaluation, the manuscript revision and correction.

Paper IV

Pavel Babica performed the analysis of microcystins using HPLC-DAD and evaluated data, contributed to the revision and correction of the manuscript.

Paper V

Pavel Babica performed analyses of the extracts using HPLC-DAD, evaluated and interpreted data, revised and corrected the manuscript.

Paper VI

Pavel Babica performed the final HPLC-DAD analysis of microcystins, contributed to the revision and correction of the manuscript.

Paper VII

Pavel Babica analysed the microcystin content in the biofilm matrices, participated in the optimisation of acetylcholinesterase inhibition assay and measured inhibitory activity of the biofilm extracts. He contributed to the revision and correction of the manuscript.

Paper VIII

Pavel Babica contributed to the experimental design, realized the experiments and analysed microcystins using HPLC-DAD. He evaluated data, interpreted the results and wrote the article.

Paper IX

Pavel Babica summarized the current knowledge on the discussed issue and wrote the article.

Paper X

Pavel Babica designed and carried out the majority of the experiments, contributed to the data evaluation and the interpretation of the results, wrote the article.

9

Abbreviations

Abbreviations BF CEC d.m. DAD DNA DW ELISA GPx GSH GST HPLC LOD LOQ MC MES MMPB MS ODS POD PPBA PPIA ROS SOD SPE TFA TRIS UV WHO

10

biofilm capillary electrochromatography dry matter diode array detector deoxyribonucleic acid dry weight enzyme-linked immunosorbent assay glutathione peroxidase glutathione glutathione S-transferase high performance liquid chromatography limit of detection limit of quantitation microcystin 4-morpholinoethanesulfonic acid monohydrate 2-methyl-3-methoxy-4-phenylbutyric acid mass spectrometry octadecyl silica peroxidase protein phosphatase binding assay protein phosphatase inhibition assay reactive oxygen species superoxide dismutase solid phase extraction trifluoroacetic acid tris(hydroxymethyl) aminomethane ultraviolet World Health Organization

Introduction

1.

Introduction

Chemical pollution is a serious problem of the modern era. Thousands of different chemical compounds are released into the environment because of various human activities. Many of these substances exhibit toxic effects in low concentrations or doses, may occur at environmental levels hazardous for living organisms, and thus might represent human health as well as ecological risk. Besides toxic chemicals, nutrient pollution has also become an important issue in the last few decades all over the world. The increased input of nutrients into surface waters (i.e., anthropogenic eutrophication) is considered to be the main factor responsible for massive proliferations of cyanobacteria in freshwater, brackish and coastal marine ecosystems. Cyanobacteria, an ancient group of photosynthesising prokaryotic organisms, are integral and natural parts of many planktonic or benthic communities, and as such cause no significant problem (Whitton & Potts 1999). However, high levels of nutrients (especially phosphorus and nitrogen) lead, together with other factors, to accelerated growth of cyanobacteria, which often form so-called blooms, scums or mats (Bartram et al. 1999; Mur et al. 1999). Cyanobacterial blooms formed by planktonic species or mats of benthic cyanobacteria have severe impacts on ecosystem functioning, e.g., disturbances of relationships among organisms, changes of biodiversity, light conditions, pH values or oxygen levels (Briand et al. 2003). Occurrence of cyanobacterial mass populations can create a significant water quality problem, particularly as many cyanobacterial species are capable of synthesising a wide range of odours, bioactive compounds or potent toxins (Carmichael 1992). It has been estimated that approximately 60% of cyanobacterial blooms produce one or more of the known and well-recognized cyanobacterial toxins (Sivonen & Jones 1999). Production of cyanobacterial toxins (cyanotoxins) is human and animal health hazard, which can result in risk of illness and mortality at environmentally relevant concentrations (Codd et al. 2005a). Thus, in addition to pollutants released by and produced within different industrial and agricultural processes, cyanotoxins represent an important group of chemical contaminants from the viewpoints of ecotoxicology, toxicology and environmental chemistry. Cyanotoxins are a diverse group of chemical substances with different toxicological properties. From hundreds of bioactive compounds of cyanobacterial origin (Carmichael 2001; Rao et al. 2002), several cyanotoxins have been recognized as priority hazard to human and animal health. These cyanotoxins comprise three types of neurotoxic alkaloids (anatoxin-a, anatoxin-a(S), saxitoxins), cyclic peptides, which are chiefly hepatotoxic (microcystins and nodularins), hepatotoxic and cytotoxic alkaloid cylindrospermopsin, and dermatotoxic compounds from marine cyanobacteria (aplysiatoxins and lyngbyatoxins).

11

Chapter 1

Also lipopolysaccharides, which are integral parts of cell walls of all cyanobacteria, cause irritant and pyrogenic effects (Chorus 2001a; Codd et al. 2005a). The cyclic peptide toxins of the microcystin and nodularin family are probably the most frequently observed cyanotoxins in the environment (Sivonen & Jones 1999). Microcystins are monocyclic heptapeptides with the characteristic feature, unusual β-amino acid Adda (3amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4E, 6E-dienoic acid). General structure of microcystins is a cyclo (- D-alanine1 - L-X2 - D-erythro-ß-methylaspartic acid3 - L-Y4 Adda5 - D-glutamate6 - N-methyldehydroalanine7). The molecular weight of microcystins varies in the range of 909 to 1115 (Duy et al. 2000). The main structural variations in microcystins are observed in the L-amino acid residues X and Y, which are indicated by two-letter suffix in the name. For example, microcystin-LR contains leucine (L) and arginine (R) in these positions (Fig. 1). Nevertheless, modifications of the other parts of molecule exist and more than 70 structural variants (congeners) of microcystins have been identified so far (Codd et al. 2005a). However, different structural variants are not equally prevalent in the environment. For instance, microcystin-RR, -YR and -LR can be found very frequently in environmental samples (de Figueiredo et al. 2004), whereas others are detected only rarely and usually in trace amounts (Lawton & Edwards 2001). Microcystins are synthesised by ancient biochemical pathways involving unique mixed polyketide synthases and nonribosomal peptide synthetases (Nishizawa et al. 2000; Tillett et al. 2001). However, in spite of intensive research of these cyanotoxins, the question on their natural physiological or ecological role has not been clearly answered (Kaebernick & Neilan 2001). D-glutamate

N-methyldehydroalanine

6

CH3

COOH Adda

5

H3C

OCH3

CH3

O

O NH

CH3

D-alanine

O

NH

CH3

CH2

CH3

H N

HN H N

CH3 CH3

COOH O

L-leucine

4

HN HN

1

O

O L-arginine

O

N

HN

7

D-erythro-ß-methylaspartic acid

2

3

NH2

Fig. 1: Structure of microcystin-LR. Adda is (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10phenyldeca-4E, 6E-dienoic acid.

12

Introduction

The main mode of action of microcystins is binding to the catalytic units of protein phosphatases 1 and 2A and inhibition of their function (MacKintosh et al. 1990). Moreover, ATP-synthase (Mikhailov et al. 2003) and aldehyde dehydrogenase (Chen et al. 2006a) have been identified or proposed as further cellular targets of microcystins. The exposure to microcystins affects intracellular regulatory processes and signal transduction. It can lead into hyperphosphorylation of structural proteins and cell necrosis, mitochondrial permeability transition, mitochondrial membrane potential alteration, formation of reactive oxygen species, induction of oxidative stress, oxidative damage of DNA, modulation of apoptosis, changes in cell proliferation and cytokinesis (Falconer 2006). However, the exact and complete mechanism of microcystin toxicity at the cellular level remains to be elucidated. Microcystins primarily act as hepatotoxins in mammals, since hepatocytes highly express organic anion transport proteins, which are responsible for active cellular uptake of microcystins. However, organic anion transporters are present also in other organs than liver, e.g., in the gastrointestinal tract, the kidney or the brain (Hagenbuch & Meier 2003). In fact, microcystins might affect multiple organs and tissues. They have been showed to induce renal damage (Nobre et al. 1998, 1999; Beasley et al. 2000; Milutinovic et al. 2002, 2003) or neurological symptoms (Azevedo et al. 2002; Ito et al. 2002; Maidana et al. 2006). Microcystins affected erythrocytes (Sicinska et al. 2006) or immune cells (Hernandez et al. 2000; Yea et al. 2001; Chen et al. 2004b; Lankoff et al. 2004a, 2004b; Teneva et al. 2005). There is increasing evidence on tumour promotion properties of microcystins from laboratory experiments (Falconer & Buckley 1989; Falconer 1991; Nishiwaki-Matsushima et al. 1992; Ohta et al. 1993; Ito et al. 1997; Sekijima et al. 1999; Humpage et al. 2000; Dietrich & Hoeger 2005). Mass developments of microcystin-producing cyanobacteria caused many incidents of animal poisonings and deaths (Duy et al. 2000; Briand et al. 2003). There are also several examples of negative human health outcomes associated with the occurrence of microcystin-producing cyanobacteria in recreation water bodies, drinking water reservoirs or in reservoirs supplying haemodialysis units (Kuiper-Goodman et al. 1999; Codd et al. 2005a). Potential health risk might result from the consumption of contaminated food (due to bioaccumulation of microcystins in fish tissue or in vegetables irrigated with microcystincontaminated water) or the consumption of contaminated food supplements made from cyanobacteria (Dietrich & Hoeger 2005). Besides acute poisonings, the chronic exposure of humans to low concentrations of microcystins in drinking water can lead to promotion of liver or colorectal cancer (Yu 1995; Zhou et al. 2002). Microcystin-producing mass populations of cyanobacteria therefore represent a serious global issue of public health. Special attention given to microcystins is reflected by World Health Organization (WHO) guideline value set for concentration of microcystin-LR in drinking water (1 µg/L) (WHO 1998). Consequently, limits for microcystin concentrations have been implemented in legislations of many countries as well, and numerous studies have addressed risk assessment and management of these cyanotoxins (Codd et al. 2005b; Dietrich & Hoeger 2005; Chorus 13

Chapter 1

2005). Furthermore, microcystins affect wildlife organisms and might have negative impact on aquatic ecosystems (Wiegand & Pflugmacher 2005; Zurawell et al. 2005). Because of ecological and human health risks related to microcystins, the research of these cyanobacterial toxins is highly required. Therefore, the present dissertation focuses on microcystins to fill in several gaps of knowledge regarding their analyses, environmental occurrence, fate and effects on selected groups of aquatic organisms.

14

Aims of the present study

2.

Aims of the present study

Microcystins are widespread distributed and highly toxic compounds of cyanobacterial origin. They have been recognized to be hazardous for living organisms including humans, and represent important contaminants of aquatic ecosystems. The primary goal of this study was to contribute to ongoing microcystin research, namely to: 1) Optimisation of analytical procedures for detecting microcystins in sediments (Paper I, Chapter 3.2) 2) Development of new analytical method for microcystins based on capillary electrochromatography (Paper II, Chapter 3.3) 3) Investigation of microcystin occurrence in the environment: - Environmental monitoring of dissolved microcystins in the Czech Republic (Paper III, Chapter 4.2) and their detection in the selected water bloom (Paper IV, Chapter 4.3) - Detection of microcystins in Bulgaria (Paper V, Chapter 4.4.1) and Ghana (Paper VI, Chapter 4.4.2) - Analyses of microcystins in the matrices of phototrophic biofilms and evaluation of their toxicity (Paper VII, Chapter 4.5) 4) Investigation of microcystin fate in sediments (Paper I, Chapter 5.2) and their biodegradation by phototrophic biofilms (Paper VIII, Chapter 5.3) 5) Investigation of microcystin effects on photoautotrophic organisms and discussion of the natural role of these cyanotoxins (Paper IX and X, Chapter 6.2 and 6.3)

15

Chapter 3

3.

Analyses of microcystins 3.1

Background

A number of analytical techniques are currently used for detecting and identifying of this class of toxins in a wide range of sample matrices. Extraction of microcystins from the sample matrix is a basic step of any analytical procedure and determines the results regardless of the final analytical method employed. Solvent extraction of microcystins from cyanobacterial biomass is relatively well optimised and usually performed with aqueous methanol under ultrasonication (Fastner et al. 1998; Barco et al. 2005). Alternative extraction methods were developed, such as microwave oven extraction, water bath boiling (Metcalf & Codd 2000), pressurized liquid extraction (Aranda-Rodriguez et al. 2005), or supercritical fluid extraction (Pyo et al. 2004b). Many studies have described extraction of microcystin from animal tissues (Williams et al. 1997b; Amorim & Vasconcelos 1999; Cazenave et al. 2005; Karlsson et al. 2005b) and plant materials (McElhiney et al. 2001; Chen et al. 2004a; Pflugmacher 2004; Mitrovic et al. 2005; Yin et al. 2005). However, there are still a limited number of available studies critically comparing the efficiencies of individual extraction procedures, subsequent cleanup, pre-concentration steps and final method of detection (Ernst et al. 2005; Karlsson et al. 2005b; Moreno et al. 2005). To date, less attention has been paid to analysis of microcystins (or closely related nodularins) in sediments. Extraction with methanol (Kankaanpaa et al. 2001, 2005) or 5% acetic acid (Ihle et al. 2005) was employed for this purpose. However, only poor recoveries of microcystins from sediment were observed after convential solvent extraction and different approach for analysis of microcystins in sediments using MMPB method was therefore suggested (Tsuji et al. 2001). Sample clean-up and microcystin pre-concentration is usually performed by solid phase extraction (SPE) on C18 (octadecyl silica, ODS) cartridges (Lawton et al. 1994) or extraction disks (Tsuchiya & Watanabe 1997). However, other sorbents are also suitable for SPE of microcystins, e.g., styrene-divinylbenzen copolymers (Hormazabal et al. 2000; Kondo et al. 2000; Rapala et al. 2002; Aranda-Rodriguez et al. 2003) or highly selective immunoaffinity sorbents (Kondo et al. 2000, 2002; Tsutsumi et al. 2000a; Lawrence & Menard 2001; Aranda-Rodriguez et al. 2005). Application of molecularly imprinted polymers (Chianella et al. 2003) or recombinant antibody fragments (McElhiney et al. 2002) in SPE of microcystins has been recently described. Analytical methods for microcystins can be divided into three principle groups: physicochemical (instrumental) methods, biochemical and immunochemical assays, and whole organism bioassay.

16

Analyses of microcystins

Instrumental techniques are the only analytical methods that separate microcystin congeners and allow their individual identification (Meriluoto 1997). Routine analyses of microcystins are usually performed by reversed phase high performance liquid chromatography (HPLC) on C18 (ODS) columns (McElhiney & Lawton 2005). Diode array detector (DAD) is probably most widely used detection technique, because microcystins have characteristic absorption profiles between 200 - 300 nm (Meriluoto 1997). More accurate identification of microcystins can be achieved using HPLC coupled with mass spectrometry (MS) detection. The main ionisation techniques described for MS analyses of microcystins are the frit-fast atom bombardment (Kondo et al. 1995), electrospray ionisation (Poon et al. 1993), and matrix-assisted laser desorption ionisationtime of flight (MALDI-TOF) (Erhard et al. 1997; Welker et al. 2002). MS techniques have become more accessible and represent sensitive, reliable and promising methods for microcystin analysis not only for monitoring but also for research purposes, because it facilitates characterisation and quantification of biotransformation or (bio)degradation products of microcystins (Liu et al. 2003; Karlsson et al. 2005b). HPLC methods for microcystin analyses have been also described in combination with electrochemical detector (Meriluoto et al. 1998), fluorescence detection of microcystinderivates with fluorogenic substrate (Shimizu et al. 1995), or chemiluminiscence detection of derivatised microcystins (Murata et al. 1995). Very sensitive screening method employing thin layer chromatography with visual detection was developed (Pelander et al. 2000). Electromigration techniques represent other methods of choice, which are complementary to liquid chromatography. Capillary zone electrophoresis has been optimised for microcystin analyses using UV detection (Boland et al. 1993; Bateman et al. 1995; Siren et al. 1999; Aguete et al. 2001; Gago-Martinez et al. 2003a, 2003b; Vasas et al. 2004, 2006), MS detection (Bateman et al. 1995) or fluorescence detection of microcystin derivatives (Wright 1989; Li et al. 1999). Furthermore, there has been reported also application of capillary electrophoresis in the modes of micellar electrokinetic capillary chromatography (Bouaicha et al. 1996; Onyewuenyi & Hawkins 1996; Siren et al. 1999; Aguete et al. 2001; Gago-Martinez et al. 2003b; Vasas et al. 2004, 2006) and capillary isotachophoresis (Aguete et al. 2001) in combination with the UV or MS detection of microcystins. Alternative instrumental method for microcystins analysis is based on the oxidation of their molecules, following extraction and detection of the 2-methyl-3-methoxy-4phenylbutyric acid (MMPB), which is formed from the Adda residue. The oxidation of the toxins can be achieved using permanganate and periodate (Sano et al. 1992) or by treatment with ozone (Harada 1996). MMPB can be detected after its esterification by gas chromatography coupled with MS detector (Kaya & Sano 1999) or flame ionisation detector (Sano et al. 1992). HPLC has been employed for MMPB analysis in combination with either MS detector (Ott & Carmichael 2006) or fluorescence detector after labelling of MMPB by fluorescent reagent (Sano et al. 1992). The MMPB method can be time-consuming and it is 17

Chapter 3

unable to distinguish between different microcystin congeners. However, it was shown useful in detecting the toxins in complex matrices, such as sediments (Tsuji et al. 2001) or animal tissue (Williams et al. 1997a, 1997b; Ott & Carmichael 2006). The method provides information on total microcystin content in the sample, including products of microcystin biotransformation as well as strongly adsorbed or covalently bound molecules of toxin. More recently, conventional analytical pyrolysis followed with gas chromatography coupled with MS detection was also employed for microcystin analysis (Camean et al. 2005). Biochemically, microcystins are potent inhibitors of serine/threonine protein phosphatase enzymes. Protein phosphatase inhibition assay (PPIA) is therefore based on measuring of the inhibitory effect of microcystins on the release of phosphate from phosphorylated substrates in the presence of protein phosphatase enzyme. The reaction can be quantified using 32P-labelled substrates (MacKintosh et al. 1990; Xu et al. 2000), chromogenic (An & Carmichael 1994; Heresztyn & Nicholson 2001) and fluorogenic substrates (Fontal et al. 1999; Bouaicha et al. 2002), or using bioluminiscence (Isobe et al. 1995). PPIA represents sufficiently sensitive method for microcystin detection in water below the WHO guideline level (1 µg/L) without the need for sample pre-concentration, but suffers from generally poor robustness. Biosensors based on the immobilized protein phosphatases have been also developed for microcystin detection (Campas et al. 2005; Szydlowska et al. 2006). Another approach, so called protein phosphatase binding assay (PPBA), involves competition between the unknown sample and radiolabelled microcystin for binding to the catalytic subunit of protein phosphatase. The PPBA arrangement using immobilized enzymes and microcystin labelled with fluorescent probe has been recently reported (Kleivdal et al. 2004) and biosensor based on similar principle has been described as well (Sadik & Yan 2004). PPBA is a highly sensitive and relatively simple method more robust than PPIA, and non-radioactive fluorescent format could be promising technique for rapid screening analyses of microcystins. Yang et al. (1999) suggested a biosensor using surface plasmon resonance method to examine interaction between microcystin and protein phosphatase (Yang et al. 1999). Immunochemical analyses can be also used for microcystin detection. Different formats involving antibodies isolated against microcystins have been developed. Immunoassays for microcystins include Enzyme-Linked ImmunoSorbent Assays (ELISAs) (Lin & Chu 1994; Saito et al. 1994; McDermott et al. 1995; Tsutsumi et al. 2000b; Zeck et al. 2001a; Lindner et al. 2004), an immuno-protein phosphatase inhibition assay (Metcalf et al. 2001), immunofluorescent assay (Lei et al. 2004), immunochromatographic techniques (Kim et al. 2003; Pyo et al. 2004a, 2005b, 2006) or nonseparation electrochemical enzyme immunoassay (Zhang et al., in press). Immunoassays are rapid, easy, sensitive and selective methods capable of detecting microcystins within the WHO guideline levels without sample pre-concentration. These assays have been developed using both polyclonal (An & Carmichael 1994; Liu et al. 1996; Metcalf et al. 2000) and monoclonal antibodies (Nagata et al. 1995; Fischer et al. 2001; Zeck et al. 2001a, 2001b), or recombinant antibody 18

Analyses of microcystins

fragments (McElhiney et al. 2000, 2002). Currently, the ELISA arrangement is probably the most widespread immunochemical method and several diagnostic ELISA kits are now commercially available (McElhiney & Lawton 2005). The ELISA technique has been recently integrated into a microchip (Pyo et al. 2005a). Also various biosensors employing antibodies (Kim et al. 2003; Campas & Marty, in press) have been constructed and represent promising technologies especially for routine monitoring of waters. A novel, perspective techniques involve specific artificial receptors (molecularly imprinted polymers), which have been recently designed for microcystin-LR (Chianella et al. 2002). They have been employed not only for SPE sample clean-up, but also for construction of sensitive and inexpensive competitive assays or biosensors for microcystin analysis (Chianella et al. 2003; Osten et al. 2005). The whole organism bioassays can be used for determination of the presence of microcystins in a sample. The mouse bioassay has been most frequently employed for this purpose, but also several invertebrate bioassays (Kiviranta et al. 1991b; Tarczynska et al. 2001; Marsalek & Blaha 2004), plant bioassays (Kos et al. 1995; Romanowska-Duda & Tarczynska 2002; Gehringer et al. 2003) and in vitro methods (Fastner et al. 1995) have been described for microcystin detection. However, these bioassays suffer from low sensitivity, selectivity (McElhiney & Lawton 2005) and provide information on biological effect and sample toxicity rather than on amounts of specific cyanotoxins.

3.2

Extraction of microcystins from sediments

Although analytical procedures have been relatively well optimised for microcystin analysis in bloom, water or animal tissue samples (see Chapter 3.1), analysis of microcystin in sediments and soils represents insufficiently resolved problem. Microcystin sorption and degradation in sediments and soils were shown to dramatically influence their environmental fate (Rapala et al. 1994; Miller et al. 2001; Miller & Fallowfield 2001; Holst et al. 2003; Chen et al., in press). More detailed investigation of biodegradation, sorption and mobility of microcystins in sediments or soils is highly needed, because these processes affect the distribution of microcystin in the environment and consequently the exposure of living organisms and humans. Moreover, study of the microcystin production by cyanobacterial inoculum overwintering in sediments might contribute to the explanation of their natural ecological or physiological function (Ihle et al. 2005). However, this research has been limited due to lack of efficient and accessible analytical procedures for microcystin determination in these matrices. The extraction of microcystins from sediments seems to be a crucial step of the whole analytical procedure. There has been a lack of critical comparison of various extraction and analytical methods, particularly with regard to various aspects of sediment composition such

19

Chapter 3

as organic carbon content, which is one of the main factors that affects the sorption of compounds to natural sediments or soils (Hance 1988). Thus, the objective of the Paper I was to evaluate the extraction efficiencies of three different solvents (5% acetic acid, 5% acetic acid in 0.1%TFA-methanol, 5% acetic acid in 0.2%TFA-methanol) in several sediments with different contents of organic carbon (0.5%, 1.25%, 5.35%) spiked with three common structural variants of microcystins (microcystinLR, -RR, -YR) at environmentally feasible levels. Two-step extraction, ultrasonication and subsequent sample clean-up by SPE were employed. The analytical detection was performed using both instrumental (HPLC-DAD) and immunochemical (ELISA) techniques. The total microcystin extraction efficiencies for various sediments analysed with HPLC and ELISA are summarized in Fig. 2. Despite the extraction of microcystins from sediments using 5% acetic acid was previously described (Ihle et al. 2005), this solvent appeared to be the least efficient, regardless of the sediment used. Methanol with 5% acetic acid and 0.10.2% TFA was shown to be more efficient solvent extracting about 24.8-50.1% (HPLC) of externally added microcystins. Similarly to these results, Tsuji et al. (2001) reported that 5% acetic acid in 0.1% TFA-methanol extracted microcystin from sediment more efficiently than methanol acidified with TFA (Tsuji et al. 2001). Moreover, Chen et al. (2006) have recently confirmed that conventional extractions with 5% acetic acid, methanol or methanol0.1%TFA were not effective for microcystin extraction from sediments or soils, whereas better recoveries were obtained with 60% EDTA (0.1M)-40% aqueous methanol (70%) or 0.1M EDTA-0.1M Na4P2O7 (Chen et al. 2006b). 100 I - sediment 0.5% OC

II - sediment 1.25% OC

III - sediment 5.35% OC

% recovery

80

HPLC

60

40

ELISA 20

0 A

B

C

A

B

C

A

B

C

extraction solvent

Fig. 2: Recovery of total microcystins externally added into the three model sediments using different extraction solvents (A: 5% acetic acid, B: 5% acetic acid in 0.1% TFA-methanol, C: 5% acetic acid in 0.2% TFA-methanol). Two different analytical methods were used (ELISA and HPLC). OC - organic carbon. Bars represent means of duplicate experiments with standard deviations.

20

Analyses of microcystins

The recoveries of microcystins did not clearly correlate with the contents of organic carbon (Fig. 2) and the other sediment components were probably responsible for microcystin sorption. Correspondingly, several studies have reported predominant role of clays in microcystins adsorption (Morris et al. 2000; Miller et al. 2001; Chen et al., in press). It has been suggested, that hydrophilic interactions between ionizable groups (carboxylic- or amino-) of microcystins and clay particles (Tsuji et al. 2001) or even microcystin chelatation with the metal ions in clays (Chen et al., in press) take part in the adsorption mechanism. It is supported also by our results, where the extraction efficiencies of different structural variants of microcystins increased with their hydrophobicity (microcystin-RR < YR < -LR; Fig. 3), which indicates that the hydrophilic interactions are responsible for the adsorption. Similarly, other studies have also reported generally lower extractability of more hydrophilic microcystins from sediments or soils (Tsuji et al. 2001; Chen et al. 2006b). 100

I - sediment 0.5% OC

II - sediment 1.25% OC

III - sediment 5.35% OC MC-RR

% recovery

80 60

MC-YR 40 20

MC-LR

0 A

B

C

A

B

C

A

B

C

extraction solvent

Fig. 3: Recovery of different structural variants of microcystin (MC) externally added into three sediments and extracted with different solvents (A: 5% acetic acid, B: 5% acetic acid in 0.1% TFAmethanol, C: 5% acetic acid in 0.2% TFA-methanol). Results of HPLC analyses. OC - organic carbon. Bars represent means of duplicate experiments with standard deviations.

The most common analytical methods were compared for detection of microcystins in sediments in the present work. Good correlations between the results from HPLC and ELISA were revealed not only for spiked sediment (Fig. 2), but also for 34 samples of natural sediments (Fig. 4). However, only weak correlation between ELISA and HPLCDAD results was observed in the samples of natural sediments with concentrations below 0.1 µg/g d.m. It might be caused by greater variability and lower robustness of ELISA assays as well as by the presence of many impurities, which can interfere with HPLC analysis, especially at low concentrations of microcystins. Nevertheless, it can be

21

Chapter 3

concluded, that both ELISA and HPLC methods are suitable for detecting of microcystin in sediments or soils. In summary, several factors affecting microcystin analyses in sediments were revealed in this part of the study. The extraction efficiency depends strongly on the employed solvent, the microcystin structure and the sediment composition. Because the total organic carbon content was not evaluated as the critical parameter influencing microcystin extraction, and hydrophilic microcystins were less extractable, it seems that hydrophilic interactions of microcystins with inorganic particles (probably clays) substantially affect their sorption onto sediments. 0.5

HPLC (µg/g d.m.)

0.4 0.3

all values

y = 1.0601x + 0.0206 R2 = 0.9118

0.2

LOD]

0.827

1.253

0.94

1.01

Standard deviation [>LOD]

0.74

1.5

1.08

1.16

1

LOD - limit of detection (8 µg/L); 4 Median, mean and standard deviation for the subset of samples with concentrations >LOD. 3

In this study, the substantial part of samples contained microcystins during the whole summer season. The occurrence of microcystins was higher in August or September than in July, because the portion of samples with microcystins increased from 58% to approximately 80% during the season. However, the extreme concentrations of microcystins (up to 19 µg/L) were found in July and the median values were comparable with those observed in September. More detailed investigation of seasonal trends was performed in selected reservoirs (N=31). The majority of the localities had the peak in microcystin concentrations during the later months of the season, 11 sites in August (i.e., 36% of localities) or 13 sites (42%) in September. It could result from the increase of total microcystin concentrations (cell-bound and dissolved) during the season, which is often observed in the environment (Chorus 2001b). In the present study, the seasonal increase of dissolved microcystins could be attributed to seasonal shift towards dominance of 28

Environmental occurrence of microcystins

Microcystis sp. (see below), which is potent microcystin producer. On the other hand, this increasing trend is not general, because seven localities (22%) in the present study had maximal microcystin levels in July. Correspondingly, peaks of microcystin concentrations during early summer were previously reported by other papers (Pawlik-Skowronska et al. 2004; Briand et al. 2005). It is evident that high concentrations of dissolved microcystins can occur under certain conditions also at the beginning of the season and this observation should be considered in monitoring studies as well as in water treatment practices.

microcystin concentration (µg/L)

37 36 4 3 2 1 0 jul

aug

sept

Fig. 8: Concentrations of dissolved microcystins in the Czech Republic during the 2004 summer months (July, August, September) in all analysed samples (black, N=204) and the subset of samples from drinking water reservoirs (empty, N=27). Data represent median (central square point), 25-75 percentiles (boxes), 95 percentiles (whiskers), outliers (circles) and extremes (asterisks).

Evaluation of relationships between phytoplankton dominants and dissolved microcystins showed that green algae and diatoms were the most prevalent phytoplankton groups in the samples with microcystin concentrations 1000 µg/L) (Casanova et al. 1999). Also macroalga C. fracta was shown quite resistant to microcystin-LR exposure, because concentrations up to 10 000 µg/L had no significant effects on growth or peroxidase activity, in spite of observed bioaccumulation of toxin by this macroalga (Mitrovic et al. 2005). Floating (pleustonic) water plants from Lemnaceae family are rather insensitive to microcystins as well, with exception of Spirodella oligorrhiza. Although studies with L. minor (Weiss et al. 2000; Mitrovic et al. 2005), Lemna gibba (LeBlanc et al. 2005) and Wolffia arrhiza (Mitrovic et al. 2005) showed significant growth inhibition, malformations, necrosis, chlorosis, inhibition of electron transport rate, elevation of peroxidase, reduction of total carotenoids and chlorophyll a/b content, these effects were observed only at high levels of microcystins (above 3000 - 15 000 µg/L). However, experiments with another pleustonic plant S. oligorrhiza showed significant growth reduction, depletion of chlorophyll a/b and inhibition of acid phosphatase activity at low concentrations (≥ 10 µg/L) of microcystin-LR (Romanowska-Duda & Tarczynska 2002). Increase of RNAse activity was observed at the concentration corresponding to the growth IC50 (200 µg/L) (Romanowska-Duda & Tarczynska 2002). Taken together, available experimental studies on microcystin effects in aquatic plants provided highly variable concentration-dependent and species-specific results. Based on the number of available papers, less attention has been paid to microalgae and cyanobacteria than to macrophytes. Table 6 compiles the results of studies with microcystins and this group of photoautotrophic organisms published so far. The observed effects of microcystins on planktonic photoautotrophs ranged from growth inhibition (Christoffersen 1996; Sedmak & Kosi 1998; Kearns & Hunter 2000; Singh et al. 2001; Hu et al. 2004, 2005) to growth stimulation (Sedmak & Kosi 1998; Ou et al. 2005). Toxin uptake (Singh et al. 2001), biodegradation (Ou et al. 2005) as well as microcystin effects on GST activities and inductions of oxidative stress (Pietsch et al. 2001; Vardi et al. 2002; Hu et al. 2005; Ou et al. 2005) were documented in microalgae or cyanobacteria. Microcystins were also shown to affect photosynthesis-related processes (Escoubas et al. 1995; Singh et al. 2001; Hu et al. 2004), cell motilities (Kearns & Hunter 2001), cell and chloroplast 47

Chapter 6

volumes, cell aggregation and pigment contents (Sedmak & Elersek 2005). Documented effects of microcystins on other important enzymatic activities include nitrogenase (Singh et al. 2001), nitrate reductase (Hu et al. 2004) and protein kinases (Vardi et al. 2002). However, only a few studies with planktonic algae demonstrated negative effects of dissolved microcystins within environmentally relevant concentrations (