Planktonic and benthic cyanobacteria of European brackish waters: a perspective on estuaries and brackish seas

Abstract

Cyanobacteria are photosynthetic organisms found in aquatic and terrestrial biotopes and ecosystems throughout the world. Marine and freshwater cyanobacteria have been extensively studied due to the toxic hazards that some of them create and biotechnological interest. In contrast, the cyanobacteria of brackish waters have been less studied despite being able to form toxic blooms like their freshwater and marine counterparts. We review the occurrence, diversity and toxicity of cyanobacteria species in the brackish waters of Europe, mainly estuaries and brackish environments of the Atlantic Ocean and the Mediterranean and Baltic Seas. The dominant cyanobacteria belong mainly to the planktonic genera Nodularia, Aphanizomenon, Microcystis and Anabaena, but also the benthic forms of Anabaena and Phormidium. Most genera from brackish waters are reported to be hepatotoxin producers (microcystin and nodularin). However, anatoxin-a and other bioactive compounds (e.g. apoptogens) can also be found and are produced exclusively by benthic forms. Nodularin is the best-characterized brackish-water cyanotoxin, being primarily produced by N. spumigena. Data are presented on cyanotoxin production, accumulation, and potential food chain transfer, with a particular focus on nodularin, which induces multiple effects on food-chain dynamics. Microcystins and anatoxin-a are also considered. Potential risks to animals and humans from cyanotoxin exposure are discussed, although reports of animal poisoning by brackish-water toxic cyanobacteria are scarce and no cases of human intoxication are yet known.

Keywords:

• benthos
• brackish waters
• cyanobacteria
• cyanotoxin production
• Europe
• food-chain transfer
• intoxication incidents
• plankton
• toxins

Introduction

Cyanobacteria are widespread among different aquatic and terrestrial biotopes and ecosystems worldwide, including extreme biotopes like hot springs and Arctic and Antarctic lakes (Whitton & Potts, 2000; Seckbach, 2007). Aquatic cyanobacteria include both planktonic and benthic forms (Stal et al., 2003). Cyanobacteria are also known for producing a wide array of secondary metabolites, some being toxic to other organisms and causing poisonings of humans and animals (Carmichael, 1994). According to their effects on mammals, cyanotoxins may be divided into four major groups: (i) neurotoxins, (ii) hepatotoxins, (iii) cytotoxins and (iv) dermatoxins (irritant toxins) (Pearson et al., 2010). The first two can cause acute and sometimes lethal poisonings while cytotoxins are not lethal but show selective bioactivity and dermatoxins are linked mainly with inflammation of the skin (Dow & Swoboda, 2000). Either way, secreted metabolites can interact within the aquatic system and affect many organisms including phytoplankton and the benthic populations (Carmichael, 1994; Wase & Wright, 2008).

Most of the studies about cyanobacteria are limited to planktonic forms, which are the main bloom-producers and linked to toxicity (Golubic et al., 2010). But, the discovery of toxicity on benthic cyanobacteria, such as the production of common toxins (e.g. microcystin) and unknown compounds, is gradually attracting the attention of the scientific community (Golubic et al., 2010).

The first report about toxic planktonic cyanobacteria, by Francis (1878), was from a brackish-water environment. Since then, research about cyanobacteria has focused mainly on freshwater environments, particularly on monitoring and aquatic and public health implications. The growing interest on marine cyanobacteria, on the other hand, is mainly due to their potential as sources of new pharmacological compounds (Burja et al., 2001; Tan, 2007). The cyanobacteria of brackish waters (including estuaries), however, remain under-investigated, particularly in Europe, despite a number of significant contributions (Sivonen et al., 1989; Nehring, 1993; Lehtimäki et al., 1994, 1997, 2000; Willén & Mattson, 1997; Sivonen & Jones, 1999; Sipiä et al., 2001, 2002, 2006, 2007, 2008; Vasconcelos & Cerqueira, 2001; Laamanen et al., 2002; Rocha et al., 2002; Geiss et al., 2003; Hobson & Fallowfield, 2003; Lehtonen et al., 2003; Stal et al., 2003; Sobrino et al., 2004; Herfindal et al., 2005; Kankaanpää et al., 2005; Karjalainen et al., 2005, 2007; Karlsson et al., 2005; Peréz & Carrilo, 2005; Surakka et al., 2005; Marino et al., 2006; Mazur-Marzec et al., 2007, 2008; Paoli et al., 2007; Halinen et al., 2008; Ibelings & Havens, 2008; Jóźwiak et al., 2008; Lopes et al., 2010; Oftedal et al., 2010;). This lack of information for brackish-water cyanobacteria is especially significant in relation to the potential for production of new bioactive compounds. In addition, brackish waters are amongst the most biologically productive environments on earth and are important transition zones between different environments (McLusky, 1999). And finally, some brackish-water cyanobacteria are known to form toxic blooms or mats, which may constitute a threat to public and environmental health (Surakka et al., 2005).

The ability of cyanobacteria to form blooms and mats in fresh, brackish (estuarine) and marine waters has been described as playing an important role in the energy flow and in the dynamics of these environments (Telesh, 2004). In recent decades, global changes in climate and eutrophication have contributed to an increased frequency of cyanobacterial blooms, with enhanced risks to human health around the world (Carpenter et al., 1998; Smith et al., 1999). European brackish waters along the Atlantic Coast, from mid-Norway to southern Portugal, have been described as being enriched with organic matter (Kaebernick & Neilan, 2001; Davis & Koop, 2006; Jonasson et al., 2008), presenting favourable conditions for the occurrence of cyanobacteria blooms with all the hazards to animal and environmental health they bring. A careful examination of the evolutionary history of cyanobacteria, particularly the ability of cyanobacteria to thrive under environmental conditions with anthropogenic pressure and grow in abundance following mass extinction events, suggest that cyanobacteria may be particularly tolerant to global climate changes (Hallock, 2005), relative to other organisms.

This resilience is, with no doubt, linked to some unique features of cyanobacteria. Many coccoid and filamentous cyanobacterial species are able to fix nitrogen (N₂), allowing them to survive and prosper under a wide range of environmental conditions. This ability, in some cases, is conferred by the presence of N₂-fixing cells, the heterocysts. These specialized cells are essential for the good functioning of the oxygen-sensitive nitrogenase enzyme responsible for reducing nitrogen to ammonia, by providing a suitable anaerobic environment (Adams, 2000). Other strategies have been developed by some non-heterocystous cyanobacteria to fix N₂ under aerobic conditions, such as (i) temporal separation of N₂ fixation from photosynthesis, or (ii) combined spatial and temporal separation of both processes. Other non-heterocystous cyanobacteria fix N₂ but exclusively under anaerobic conditions (Bergman et al., 1997; Severin & Stal, 2008). Akinetes are another kind of specialized cell, which allow cyanobacteria to resist unfavourable conditions. Furthermore, cyanobacteria are known for their (i) capacity for buoyancy, due to the formation of gas vesicles, (ii) storage of phosphorus (P), (iii) grazing resistance, (iv) capability to grow at low irradiance and (v) tolerance to high pH and low carbon dioxide concentration (Engström-Öst et al., 2002).

The main goal of this work is to compile and discuss information about brackish-water cyanobacteria in Europe. We focus on the estuaries and brackish seas of three main areas, the Atlantic, the Mediterranean and the Baltic Sea, dealing with the occurrence and diversity of cyanobacteria and the factors that limit growth. Next, we discuss the production of cyanotoxins and other secondary metabolites, particularly the hepatotoxins nodularin (NOD) and microcystin (MC), which are the cyanotoxins most frequently found in European brackish-water and estuarine environments; NOD receives the most attention, due to its frequent occurrence in brackish waters and because it is only produced by the typical brackish-water species, Nodularia spumigena (Van Apeldoorn et al., 2007). Our final topic is the bioaccumulation of cyanobacterial toxins, since a few studies have suggested that cyanobacterial toxins bioaccumulate in brackish-water aquatic biota, possibly enhancing the risk of poisonings at the highest levels of the food chain (e.g. Sipiä et al., 2001).

Occurrence and diversity of cyanobacteria present in brackish waters

Background

In the last 50 years, the presence of cyanobacteria in brackish-water systems has been increasingly reported worldwide (Table 1), although remaining under-investigated (e.g. Finni et al., 2001; Verspagen et al., 2006). Recently, the occurrence of cyanobacteria in brackish water has come more into the spotlight, following the discovery by Miller et al. (2010) of a clear link between deaths of 21 marine otters and a thriving toxic bloom of Microcystis in a Californian estuary. This work demonstrated the trophic transfer of MC to marine invertebrates feeding in the contaminated freshwater plumes.

Table 1. Examples of cyanobacteria most commonly found in brackish-water environments worldwide (estuaries and brackish seas); ND: no data.

Species	Years	Location	Salinity*	Temperature (°C)	References
Anabaena lemmermannii	1985–1987	Baltic Sea	7.5	ND	Stal et al. (2003), Hajdu et al. (2007)
	1985–1997		6.3–6.8	19–25
Anabaena sp.	1996	Patos Lagoon, southern Brazil	ND	27.0	Yunes et al. (1996)
Aphanizomenon sp.	1987–1999	northern Baltic Sea	0–30	25	Stal et al. (2003), Cronberg & Annadotter (2006), Laamanen et al. (2002), Lehtimäki et al. (1994)
Aphanocapsa sp.	2003	Patos Lagoon, southern Brazil	ND	27	Yunes et al. (1996)
Aphanothece parallelliformis	1997	Baltic Sea	6.8–7.1	19–21	Hajdu et al. (2007)
Cyanobium bacillare	2003	ND	ND	ND	Cronberg & Annadotter (2006)
Merismopedia sp.	2003	Patos Lagoon, southern Brazil	ND	27	Yunes et al. (1996)
Microcystis ichthyoblabe	1990–2002	Lake Oued Mellah, Morocco	8.6–12.6	15–23.5	Sabour et al. (2002)
Microcystis aeruginosa	1960–2003	Estuary of river Odra, Baltic Sea; Bay estuary, USA	ND	ND	Cronberg & Annadotter (2006), Lehman et al. (2005), Finni et al. (2001)
Nodularia spumigena	1988–1999	Baltic Sea	5–20	19–21; 25	Stal et al. (2003), Cronberg & Annadotter (2006), Sivonen et al. (1989), Lehtimäki et al. (1994)
	1994–1997
Nostoc linckia	1978	ND	ND	ND	Cronberg & Annadotter (2006)
Planktothrix agardhii	1960	Estuary of river Odra, Baltic Sea	ND	ND	Finni et al. (2001)
Pseudanabaena limnetica	1994–1997	ND	ND	ND	Hajdu et al. (2007)
Snowella sp.	2003	Patos Lagoon, southern Brazil	ND	27	Yunes et al. (1996)
Synechococcus sp.	1987–2005	Baltic Sea	9	ND	Stal et al. (2003), Peréz & Carrilo (2005), Paoli et al. (2007), Uysal (2001)
		Adriatic Sea, Italy	3.0–37.2	10.6–27.2
		Black Sea	5–87	5.3–26.3
*Salinity was measured using the Practical Salinity Scale.

In Europe, research on brackish-water cyanobacteria seems undervalued. Most work has focused on the largest brackish-water system, the Baltic Sea. This is a unique biotope, which comprises different environments very variable in terms of morphology, size and physical and chemical composition. Some of these distinct environments are highly contaminated, like other brackish-water systems around the world (Telesh, 2004; Segal et al., 2006; Paoli et al., 2007). The phytoplankton of the Baltic is dominated during summer by filamentous, bloom-forming cyanobacteria, in particular Nodularia, Anabaena and Aphanizomenon (Willén & Mattsson, 1997; Laamanen et al., 2002; Karjalainen et al., 2007; Palinska & Surosz, 2008). Two distinct genotypes of Nodularia have been detected in the Baltic Sea through 16 S rRNA gene sequence analysis and total genome techniques. One genotype resembles the non-toxic N. sphaerocarpa whereas the other fits the description of N. baltica and the toxic N. spumigena (Lehtimäki et al., 2000).

Various Anabaena species, which comprise heterocystous filamentous, planktonic and benthic forms, have been reported in European brackish waters, especially in the Baltic (Willén & Mattsson, 1997; Halinen et al., 2008). Aphanizomenon species possess diverse morphologies. The main morphotype reported in the Baltic Sea is a colonial form, with a cellular ultrastructure that differs from the only known colony-forming species, A. flos-aquae. Nevertheless, molecular data retrieved from the 16 S rDNA sequence, SSU rDNA and PC-IGS analysis place this particular population of Aphanizomenon close to A. flos-aquae, suggesting that only one species occurs in brackish waters (Laamanen et al., 2002; Stal et al., 2003). Features shared by Anabaena, Aphanizomenon and Nodularia genera are their diazotrophic metabolism (capability to fix N₂) and the presence of gas vesicles, which favours their occurrence and proliferation in eutrophic waters (Laamanen et al., 2002; Stal et al., 2003; Palinska & Surosz, 2008).

The occurrence of Phormidium, Nostoc, Cyanothece and Calothrix in Baltic waters has been reported (Herfindal et al., 2005; Surakka et al., 2005). Interestingly, although the filamentous Prochlorothrix is known to be a genus sensitive to high salinity, the uncommon species P. hollandica has been found in the Baltic Sea, in an environment with salinity up to 12 psu (Burger-Wiersma et al., 1989; Geiss et al., 2003). In Sweden and in the Netherlands particularly, the most commonly reported cyanobacteria have been Microcystis aeruginosa, Aphanizomenon flos-aquae, Oscillatoria spp., Planktothrix spp. and Anabaena spp. (Willén & Mattsson, 1997; Rocha et al., 2002; Verspagen et al., 2006). Filamentous forms of Pseudanabaena and the unicellular Cyanodictyon, Cyanonephron, Aphanothece, Aphanocapsa and Cyanocatena have also been recorded in brackish-water environments of the Baltic Sea, but less frequently than the genera already mentioned (Lehtimäki et al., 2000; Finni et al., 2001; Rocha et al., 2002; Stal et al., 2003; Surakka et al., 2005). Due to the significance of cyanobacteria overall in the dynamics of Baltic Sea communities, several comprehensive revisions have been published in recent years (e.g. Hällfors, 2004).

Other important brackish-water systems in Europe are the Atlantic estuaries, such as those of the Guadiana, Minho and Tagus rivers. In these brackish waters, the most dominant cyanobacteria reported have been Oscillatoria spp., Microsytis aeruginosa, Planktothrix spp., Anabaena spp., as well as some unidentified picocyanobacteria (Rocha et al., 2002; Lehman et al., 2005; Verspagen et al., 2006; Domingues & Galvão, 2007; Domingues et al., 2007). In brackish waters of NW Spain, less frequent cyanobacteria such as Aphanocapsa conferta, Gomphosphaeria salina, Gloeocapsa deusta, Gloeocapsa granosa, Aphanothece stagnina, Gloeothece palea, Xenococcus pyriformis, Phormidium papyraceum, Spirulina labyrinthiformis, Symploca funicularis, Hassalia bouteillei, Tolypothrix tenuis, Nodularia spumigena and Rivularia nitida have also been reported (Calvo & Rbara, 2003). In the Ebro estuary (Spain), as in Adriatic brackish-water environments and the Black Sea, the coccoid picocyanobacteria Synechococcus spp. and the Cyanobium spp. dominate the phytoplankton (Uysal, 2001; Peréz & Carrilo, 2005 ; Paoli et al., 2007). Regarding benthic cyanobacteria, the most frequently reported in European brackish waters are mainly Anabaena, Calothrix, Cyanothece, Nostoc, Oscillatoria and Phormidium (Edwards et al., 1992; Mez et al., 1997; Vasconcelos & Cerqueira, 2001; Herfindal et al., 2005; Surakka et al., 2005; Oftedal et al., 2010).

It is evident, therefore, that a wide diversity of cyanobacteria species can be found in brackishwater systems in Europe. In the particular case of estuaries, it is known that environmental heterogeneity produces a high biodiversity of benthic and planktonic microbial communities. Work in our laboratory (unpublished information) has shown, by phylogenetic analysis of 16 S rRNA, that the benthic estuarine cyanobacteria of Portuguese estuaries group with their marine and freshwater counterparts, and with cyanobacteria from geographically distant and ecologically dissimilar habitats, suggesting an allochthonous origin of the species occurring in these ecosystems.

Bloom and mat-forming brackish-water cyanobacteria and limiting factors

Despite the diversity of cyanobacteria found in brackish-water in general, it seems that only a few species can bloom and cause potential environmental and human health problems. Blooms and mats of toxic cyanobacteria in brackish-water have been reported throughout Europe, particularly in Finland, Sweden, Poland and Portugal (Sivonen & Jones, 1999; Vasconcelos & Cerqueira, 2001), but most data refer to bloom species and the factors triggering their proliferation, rather than to mat-forming species. (We use ‘mat’ in a general sense for mass growths on solid substrata such as rocks, sediments and corals.)

The success of bloom-forming cyanobacteria has been mainly attributed to the presence of specific morphological or physiological characteristics such as (i) buoyancy, (ii) ability to grow under low irradiance, (iii) the presence of differentiated cells, for reproduction or dormancy, (iv) the ability to fix atmospheric nitrogen (N₂) and presence of heterocysts, and (v) the ability to store phosphorus (P) (Lehtimäki et al., 1994; Kaebernick & Neilan, 2001). The ability to fix N₂, however, is not always correlated with bloom formation. For example, Microcystis can be a successful bloom former but does not fix N₂. In addition, N₂ fixation is not always correlated with heterocystous cyanobacteria: several reports showed nitrogenase activity in some filamentous and coccoid non-heterocystous cyanobacteria under anaerobic or aerobic conditions (Stal & Krumbein, 1985; Bergman et al., 1997).

Bloom development has been shown to be associated with particular sets of physical, chemical and biotic factors. Features such as the physical stability of the water column, warm waters and high incidence of photosynthetically active radiation (PAR) (Paerl, 1988), the input of inorganic N and/or P, adequate availability of trace metals, organic matter loading, high pH, and the presence of suitable sediments for storing akinetes, have all been described as important factors contributing to cyanobacterium bloom formation (Davis & Koop, 2006; Marino et al., 2006). Contrarily, turbulence has been pointed out as a factor affecting bloom development (Thomas & Gibson, 1990). Turbulent environments have more deeply mixed waters, which can limit light availability to cyanobacteria (Marino et al., 2006), and microscale turbulence can have adverse impacts on N₂ fixation by non-heterocystous species, thus affecting their growth (Paerl et al., 1995). In contrast, turbulence does not inhibit N₂ fixation by heterocystous planktonic cyanobacteria (Howarth et al., 1995).

In brackish-water and estuarine habitats, the development of nuisance blooms of cyanobacteria has been shown to be mainly dependent on salinity. Blooms usually occur in oligohaline or oligohalobous conditions, with salinities of 0–15 psu, such as are encountered in the Baltic Sea (Marino et al., 2006). Moreover, the vertical stratification pattern common in several brackish-water and estuarine habitats of the northern hemisphere contributes to hypoxia or anoxia of bottom waters, which can also promote successful bloom formation (Davis & Koop, 2006).

Biotic interactions, such as cyanobacterial–bacterial symbioses, cyanobacterial–micrograzer (rotifers and protists) symbioses, and the absence or reduced activity of macrograzers (e.g. crustacean zooplankton and fish), may also affect bloom development (Paerl, 1988). In brackish-water systems, selective zooplankton grazing has been suggested to play an active role controlling the density of toxic and non-toxic heterocystous cyanobacteria, by preferentially selecting cyanobacterial cells with high content of N₂ (Chan et al., 2006). This can be particularly important in the Baltic Sea where cyanobacterial blooms are mainly constituted by the N₂-fixing filamentous cyanobacterial species, Nodularia spumigena, Aphanizomenon flos-aquae and Anabaena species, as previously mentioned.

Little is known about the physical, chemical and biotic factors triggering the proliferation of benthic mats and their ability to produce toxins. Recently, it was shown in a New Zealand estuary that water temperature and flow can influence benthic cyanobacterium proliferation (Heath et al., 2010). The success of mat-building cyanobacteria might be due to two main factors: (i) N₂ fixation, which is another advantageous characteristic of cyanobacteria in mat environments, commonly described as nutrient-poor and nitrogen-depleted; and (ii) their ability to adapt to the wide environmental fluctuations experienced by mats (Badger et al., 2006; Severin et al., 2010). Up to now, no work on these aspects has been published for European brackish waters.

Cyanotoxin production in brackish waters

Introduction

Bloom and mat-forming cyanobacteria in fresh, brackish and marine waters produce a broad variety of toxins. Cyanotoxins production has been already documented for more than 40 cyanobacteria genera including Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Microcystis, Nostoc, Nodularia, Planktothrix, Phormidium and Oscillatoria. More than 600 cyanobacterial secondary metabolites (SMs) have been isolated and characterized from freshwater, brackish-water and marine cyanobacteria. They comprise peptides, macrolides and glycosides with a broad spectrum of bioactivity, ranging from antifungal, antibacterial, antiviral and cytotoxic to antineoplastic activity (Lehtimäki et al., 2000; Welker & Von Döhren, 2006). The first report of toxicity induced by cyanobacterial metabolites was published in the late nineteenth century (Francis, 1878) but only in 1950 was lethality induced by cyanotoxins demonstrated in the laboratory (Carmichael, 1994).

Cyanotoxins and other SMs have been responsible for acute and chronic poisonings of cattle, pets and wildlife as well as humans. According to their chemical nature, cyanotoxins can be divided into three groups: cyclic peptides (microcystins [MC] and nodularins [NOD]), alkaloids (cylindropermopsins [CYN], aplysiatoxins, lyngbyatoxin-a, anatoxin-a, anatoxin–a(s), saxitoxins [STX]) and lipopolysaccharides (LPS). Cyanotoxins have also been classified as hepatotoxins, neurotoxins and dermatotoxins (irritant toxins), according to their symptoms in intoxicated mammals (Pearson et al., 2010). Some authors consider yet another group of cyanotoxins, the cytotoxins, which are not highly lethal to animals but exhibit more selective bioactivity (Keil et al., 2002; Welker & Von Döhren, 2006).

In European brackish waters, most reports of toxin production have involved planktonic cyanobacteria but publications about toxicity by benthic cyanobacteria are increasing. Hepatotoxins are the main toxins found so far, being produced by planktonic Nodularia spumigena, Microcystis sp., Nostoc sp., Planktothrix sp., Phormidium sp. and some Anabaena species (Vasconcelos & Cerqueira, 2001; Rocha et al., 2002; Sobrino et al., 2004; Karlsson et al., 2005; Halinen et al., 2007).

Hepatotoxins: microcystin and nodularin

Hepatotoxins are the most widespread toxins occurring in brackish waters worldwide and comprise the microcystins and nodularins. Hepatotoxins target the liver via an adenosine triphosphate-dependent transporter (Carmichael, 1994). Once in the liver cells, hepatotoxins inhibit the eukaryotic protein serine/threonine phosphatases 1 and 2 A (PP1 and PP2A), resulting in excessive phosphorylation of cytoskeletal filaments (Carmichael, 1994; Kaebernick & Neilan, 2001). About 70 different structural variants of MCs and a few NODs are known, varying in potency from highly toxic to non-toxic, depending on the specific chemical structure. MCs are monocyclic heptapeptides containing both D- and L-amino acids together with N-methyldehydroalanine and a unique ß-amino acid side-group, 3-amino-9-methoxy-2-6, 8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA) (Botes et al., 1985; Carmichael et al., 1988). MCs are mainly produced by Microcystis, Planktothrix, Phormidium, Oscillatoria, Nostoc and Anabaena genera, with Microcystis being the first described MCs-producer.

MC-producing Microcystis blooms have been reported in European brackish waters (Rocha et al., 2002; Karlsson et al., 2005; Verspagen et al., 2006) and a bloom of M. aeruginosa has been recorded from the Minho River estuary (Portugal) that contained MC-LR (MC with leucine and arginine) and another variant of MC that is as yet uncharacterized (Vasconcelos & Cerqueira, 2001). In the Guadiana estuary (Portugal) the presence of MCs has also been reported, produced either by Microcystis or Oscillatoria species (Sobrino et al., 2004). MCs had already been reported from the north-east of Europe, in the brackish waters of Baltic Sea, where they were produced by planktonic and benthic Anabaena species (Halinen et al., 2007, 2008). Outside Europe, microcystins have been detected mainly in North America (e.g. San Francisco bay estuary), South America (e.g. Patos lagoon, Brazil) and Australia (e.g. Swan river estuary) (Matthiensen et al., 2000; Orr et al., 2004; Lehman et al., 2005).

Another well-known hepatotoxin is NOD, which is commonly produced by the brackish-water cyanobacterium Nodularia spumigena (Sivonen et al., 1989; Kaebernick & Neilan, 2001; Keil et al., 2002). Toxic blooms of N. spumigena are frequent during the summer months in the Baltic Sea and have also been reported on the German North Sea coast (Carmichael et al., 1988; Sivonen et al., 1989; Galat et al., 1990; Nehring, 1993; Jones et al., 1994; Heresztyn & Nicholson, 1997). NOD has been found outside Europe in brackish waters from Australia, New Zealand and Tasmania (Van Apeldoorn et al., 2007).

NOD is a cyclic pentapeptide with a structure similar to MC, consisting of ADDA, D-glutamic acid (D-Glu), N-methyldehydrobutyrine (MeDhb), D-erythro-β-methylaspartic acid (D-MeAsp) and L-arginine (L-Arg) (Rinehart et al., 1988; Nehring, 1993). NOD can produce toxic effects similar to MC and it has been reported to have harmful effects on numerous organisms within the ecosystem, including invertebrates and fish (Karjalainen et al., 2007). We consider NOD production further in a later section.

Neurotoxins – Anatoxin-a

Another important group of cyanotoxins are the neurotoxins, which affect the nervous system causing death by respiratory arrest. Neurotoxins are usually grouped into three distinct families: (i) anatoxin-a and homoanatoxin-a, a very potent analogue of anatoxin-a, (ii) anatoxin-a(S) and (iii) saxitoxins (STX), also known as paralytic shellfish poisons (PSPs) (Sivonen & Jones, 1999).

Anatoxin-a synthesis has been documented for Aphanizomenon, Anabaena, Oscillatoria and Phormidium in Polish brackish waters (Sivonen & Jones, 1999; Mazur-Marzec et al., 2008). Outside Europe, anatoxin-a has been described until now in the largest estuary in the United States, Chesapeake Bay (Tango & Butler, 2008 ).

Anatoxin-a, a low molecular weight alkaloid (MW = 165), is a secondary amine, 2-acetyl-9-azabicyclo (4-2-1) non-2-ene that mimics the neurotransmitter acetylcholine, which binds to the nicotinic acetylcholine receptor. Since this neurotoxin cannot be degraded by the enzyme acetylcholinesterase, the muscles are over-stimulated, paralysing and leading to death (Carmichael, 1994). The other neurotoxins, homoanatoxin-a, anatoxin-a (S), and STX have not yet been reported in brackish waters of Europe or elsewhere.

Further cyanotoxins

Dermatoxins comprise, among others, lyngbyatoxins, aplysiatoxins and their variants and are produced by a large group of species (e.g. Lyngbya, Oscillatoria and Schizothrix). They can cause dermatitis upon direct contact between the skin and cyanobacterial filaments. These toxins have been isolated chiefly from planktonic and benthic marine cyanobacteria but up till now, no reports have been published about dermatoxins from brackish-water habitats in Europe or elsewhere (Smith et al., 2008).

The cytotoxins have been less well studied and comprise mainly bioactive secondary metabolites with antialgal, antimycotic, antibacterial or moderate antitumour activity. Cytotoxins can also be highly toxic to mammals and include the potent toxin cylindrospermopsin, which has not yet been reported in brackish waters in Europe (Dow & Swoboda, 2000).

The endotoxins, which are lipopolysaccharides (LPS), are hydrophilic heteropolysaccharides with a lipid component called ‘lipid A’, which have been implicated in human illnesses (Dow & Swoboda, 2000). In contrast to exotoxins, the mechanism of action of endotoxin LPS requires the active response of host cells, rather than killing them or inhibiting cellular functions (Rietschel et al., 1994). Endotoxins have been isolated from a range of cyanobacteria belonging to Synechococcus, Microcystis, Aphanizomenon, Anabaena and Phormidium (Annadotter et al., 2005), but none have yet been analysed and reported from brackish-water cyanobacteria in Europe (Lippy & Erb, 1976; Herbert et al., 1992; Anderson et al., 1996). However, there is some controversy about whether LPS should be considered toxins or not, since LPS are key constituents of the outer membranes of Gram-negative prokaryotes, which include the cyanobacteria. For example, Stewart et al. (2006) stated that the cyanobacterial LPS by themselves may not be enough to justify the toxicity cited in some reports and further research should be done.

A new neurotoxic amino acid, β-N-methylamino-L-alanine (BMAA), has recently been described and it seems that it can be produced by most terrestrial, freshwater, brackish-water and marine cyanobacteria, as well as by cyanobacterial symbionts (Cox et al., 2005; Jonasson et al., 2008). Acute neurotoxic effects related with BMAA (in vivo) were observed in 1968 (Vega et al., 1968), and recent studies revealed hippocampus injury after BMAA administration, suggesting a possible association of BMAA with neurological diseases (Buenz & Howe, 2007).

In contrast to their planktonic counterparts, benthic cyanobacteria have been traditionally regarded as innocuous. Nevertheless, recently, benthic cyanobacteria species of the genera Anabaena, Calothrix, Cyanothece, Nostoc, Oscillatoria and Phormidium, have been shown to produce toxins such as MCs, anatoxin and BMAA, and other cytotoxic and apoptosis-inducing compounds in several European estuarine systems, such as in France, Norway, Finland, Switzerland, Poland, Scotland and Portugal (Edwards et al., 1992; Mez et al., 1997; Vasconcelos & Cerqueira, 2001; Aboal & Puig, 2005; Herfindal et al., 2005; Surakka et al., 2005; Jonasson et al., 2008; Oftedal et al., 2010). Furthermore, the presence of anabaenopeptins A and D in a benthic brackish-water Nostoc isolate from an Atlantic estuary (Minho River, Portugal) has been reported by Lopes and co-workers (unpublished information). Moreover, Lopes et al. (2010) and Lopes & Vasconcelos (2011) have shown that benthic cyanobacteria from Atlantic estuaries (e.g. Synechococcus, Cyanobium, Phormidium, Microcoleus genera) are able to produce other bioactive compounds, which have not yet been characterized. A novel compound, nostocyclopeptide, has been found to be produced by a Nostoc isolate from the Baltic Sea (Jokela et al., 2010). This potent cyclic peptide is an inhibitor of microcystin-induced apoptosis in hepatocytes, an activity not previously associated with cyanobacterial natural compounds. Herfindal et al. (2005) and Surakka et al. (2005) also showed that benthic strains of Anabaena, Phormidium and Nostoc genera could cause cytotoxicity and apoptosis in mammalian cell lines by uncommon compounds, again not yet characterized. In addition, Lopes et al. (2011) have shown cytotoxicity against L929 cells and antiviral activity by estuarine cyanobacteria. These results suggest that a great diversity of bioactive compounds is produced by brackish-water cyanobacteria in Europe and elsewhere.

Comprehensive reviews of cyanotoxins in aquatic systems have been published in the last few years (e.g. Van Apeldoorn et al., 2007; Pearson et al., 2010) and so the present review will focus on the one toxin most commonly found and primarily studied in brackish-water and estuarine environments, NOD.

Nodularin – production and limiting factors

NOD is the best-known toxin produced by brackishwater cyanobacteria, specifically Nodularia spumigena. In Europe, toxic N. spumigena blooms have frequently been reported in the Baltic Sea and because of that NOD is considered to be one of the most abundant naturally occurring compounds in this brackish-water environment (Pearson et al., 2010). Production of NOD in situ and in laboratory conditions has been well studied. Nevertheless, available data on the influence of environmental parameters on NOD production in the field are rather scarce (Sivonen et al., 1989; Lehtimäki et al., 2000; Stal et al., 2003; Mazur-Marzec et al., 2007).

During N. spumigena blooms in the Baltic Sea, NOD concentrations range between 3.96 and 25.85 mg NOD l⁻¹, whilst in cultures of N. spumigena they are usually lower (Sivonen et al., 1989; Lehtonen et al., 2003; Mazur-Marzec et al., 2007; Jóźwiak et al., 2008). NOD concentration was shown to increase with temperature and irradiance, factors that also favour the growth and N₂ fixation by Nodularia (Lehtimäki et al., 1994). Determining the environmental conditions most favourable for NOD production has not been straightforward. Conditions of 20°C, 5 psu and 600 µg l⁻¹ phosphate were documented as the most favourable for NOD synthesis by Lehtimäki et al. (1994), while Lehtimaki et al. (1997) indicated increased NOD production at high P (5500 µg l⁻¹), 25–28°C, an irradiance of 155 µmol m⁻² s⁻¹ and 5–10 psu. In contrast, Hobson & Fallowfield (2003) recorded the highest NOD production by N. spumigena at the lowest temperature used (10°C), irradiances of 30–80 µmol m⁻² s⁻¹ and a salinity of 13 psu. Furthermore, Hobson & Fallowfield (2003) showed that temperature combined with irradiance had the greatest influence on both the intracellular and extracellular NOD content. Surprisingly, the highest values of total NOD were equally recorded at combinations of 30 µmol m⁻² s⁻¹ and 30°C and 80 µmol m⁻² s⁻¹ and 20°C. Besides, the influence of salinity and temperature on the total toxin concentration was shown to be strain-dependent (Hobson & Fallowfield, 2003). Significant influence of silicate on NOD production has also been reported, but nothing is known about the mechanism of this effect (Repka et al., 2004). The various parameters affecting NOD production are summarized in Table 2. The use of different strains by different groups may explain some of the contradictions in these results.

Table 2. Ranges of environmental parameters affecting NOD production by Nodularia in laboratory conditions.

Environmental parameter	Values	Nodularin intracellular concentration (µg mg^−1dry weight)	References
Salinity (PSU)	5–10; 13	4.0–8.2; 3.6	Lehtimäki et al. (1994, 1997, 2000)
Temperature (°C)	25–28; 10	6–12; 3.6
Irradiance (µmol m^−2 s^−1)	105–155; 80	8–9; 3.6
Phosphate (µg l^−1)	600; 200–5500	11–15	Hobson & Fallowfield (2003)
Silicate (µM)	8.5	∼6.5*	Repka et al. (2004)
* mg toxin (g. dry weight)^−1.

Table 3. NOD concentrations in different plant, vertebrate and invertebrate species in brackish-water systems. Analysis done by HPLC except where indicated. ND: no data.

Organism	Organ	Toxin content (µg kg^−1dry weight)	Location	References
Algae
Fucus vesiculosus	whole	45.1^1	Baltic Sea	Pflugmacher et al. (2007)
	receptacle	14.1^1
Cladophora fracta	whole	42.0
Molluscs
Mytilus edulis	ND	100–13800	Sweden	Strogyloudi et al. (2006)
Mussels	ND	1490 ± 60^3	western Gulf of Finland, Baltic Sea	Sipiä et al. (2002)
	whole	139	Gulf of Gdansk, southern Baltic Sea	Kankaanpää et al. (2007), Mazur-Marzec et al. (2007)
Dreissena polymorpha	whole	135	western Gulf of Finland, Baltic Sea	Sipiä et al. (2002)
Macoma balthica	whole	100 ± 10 to 130 ± 10
Crustaceans
Neomysis integer	whole	770 ± 60	Baltic Sea	Karjalainen et al. (2005)
Fish
Gasterosteus aculeatus	viscera	2.8–700	northern Baltic Sea	Sipiä et al. (2007)
Clupea harengus	liver, stomach	0–90	northern Baltic Sea	Sipiä et al. (2007)
Platichthys flesus	liver	489; 400^3 410 ± 12 137 ± 7 to 399 ± 5	western Gulf of Finland, Baltic Sea	Sipiä et al. (2002)
	muscle	5 and 100^2 18 and 23^3	western Gulf of Finland and Gulf of Bothnia, Baltic Sea	Sipiä et al. (2001)
	whole	390 20–2230^3	northern Baltic Sea	Kankaanpää et al. (2005)
Rutilus rutilus	liver	20–900^2 17–440^3	western Gulf of Finland, Baltic Sea	Sipiä et al. (2006)
Salmo salar	liver	10	northern Baltic Sea	Sipiä et al. (2002)
Esox lucius	whole	410 ± 20	Baltic Sea	Karjalainen et al. (2005)
Gadus morhua	liver	53–56^3,4	western Gulf of Finland and the Gulf of Bothnia, Baltic Sea	Sipiä et al. (2001)
Aquatic birds
Somateria mollissima	liver	∼60–200^2 7.2 and 11.8 (mean^3)	western Gulf of Finland, Baltic Sea	Sipiä et al. (2006), Sipiä et al. (2002)
	muscle	20^2, 0.7 (mean^3)
	liver	0.1–5.8^2; 3–180^3	Gulf of Finland, northern Baltic Sea
^1µg kg^−1 fresh weight.
^2LC-MS.
^3ELISA.
^4Phosphatase assay.

Accumulation and transfer of toxins along the brackish-water trophic chain

One of the most important aspects of cyanotoxins is that they accumulate and transfer in the food chain. This is a phenomenon of major concern for public health because the high temperatures used in traditional food preparation methods do not destroy these toxins (Morais et al., 2008).

Nodularin accumulation and transfer

NOD accumulation is higher in mussels, clams and some fish species than in other organisms of other trophic levels (Table 3). In a work by Karjalainen et al. (2005), blue mussels presented the highest amount of accumulated NOD, up to 13 800 µg kg⁻¹dry weight, while mysid shrimps accumulated more NOD than zebra mussels or the muddy sediment bivalve Macoma balthica. In fish, NOD accumulation can range from 0.1–2230 µg kg⁻¹dry weight, depending on the organs analysed and the technique used. In contrast, primary producers such as Cladophora fracta and Fucus vesiculosus accumulate the lowest amounts of the toxin (Sipiä et al., 2001, 2002, 2007; Karjalainen et al., 2005; Strogyloudi et al., 2006; Mazur-Marzec et al., 2007; Pflugmacher et al., 2007). Despite the low NOD concentrations reported for the lower trophic levels, it has been suggested that zooplankton may act as a toxin vector, increasing its range (Karjalainen et al., 2005). Nevertheless, the high degree of depuration and/or detoxication processes across trophic levels observed in both field and experimental studies could explain the tolerance of some zooplankton species to Nodularia (Karjalainen et al., 2007). Indeed, data accumulated over recent years suggest that NOD is transformed and degraded to less toxic compounds at each trophic level (Strogyloudi et al., 2006; Karjalainen et al., 2007; Mazur-Marzec et al., 2007; Pflugmacher et al., 2007; Sipiä et al., 2007).

NOD transfer among pelagic organisms is quite slow, although negative effects on the feeding and growth rates of fish larvae have been described (Karjalainen et al., 2005, 2007). This may be due to the high energy cost associated with NOD detoxification, leading to a decrease in the growth rate of fish larvae and increased vulnerability to predation (Karjalainen et al., 2007). In contrast, benthic organisms may exhibit faster NOD transfer. Juvenile flounders can show high concentrations of NOD even if they are not in direct contact with Nodularia (Karjalainen et al., 2007). Moreover, benthic filter-feeding organisms seem to be more efficient in uptake of NOD than the pelagic ones (Engström-Öst, 2002; Engström-Öst et al., 2002; Karjalainen et al., 2005).

From a human health perspective, mussels are the organisms that pose highest health risks upon consumption (Sipiä et al., 2008), since they can accumulate NOD up to 13 800 µg kg⁻¹dry weight. In the absence of mammalian oral toxicity data, the Tolerable Daily Intake of MC–LR (40 ng kg⁻¹ body weight) is provisionally assumed for NOD.

Microcystin accumulation and transfer

Regarding MC in brackish-water environments, available data are very limited. Published reports mention that some fish species, such as Tilapia rendalli, cannot avoid MC-producing cyanobacteria but they can reduce grazing, suggesting that this may be used as a defence mechanism against the transfer of MC via seston (De Magalhaes et al., 2001). De Figueiredo et al. (2004) showed that microcystins could accumulate in mussels, crayfish and fish growing in estuarine waters of Europe used for human consumption.

Intoxication incidents and ecological impacts

Intoxication events caused by brackish-water cyanobacteria are scarce compared with those induced by freshwater cyanobacteria. No human intoxications have been reported after direct ingestion of brackish-water cyanobacteria in Europe. The first animal poisoning due to N. spumigena in European ecosystems occurred in the 1960s in Germany, affecting ducks (van Apeldoorn et al., 2007). Later, in 1975, in the Danish Baltic Sea coast, a Nodularia bloom intoxicated 30 dogs, 20 of which died. From 1982 to 1984, poisoning incidents of dogs and cattle by N. spumigena were also reported along the Swedish, German and Finnish coasts (Duy et al., 2000, Lehtimäki et al., 2000; cited in van Apeldoorn et al., 2007).

Adverse effects on aquatic biota caused by brackish-water cyanobacteria of Europe producing NOD, MC and anatoxin-a have been observed (Reinikainen et al., 2002). The egg production of copepods declined when they fed on cyanobacteria and ingested NOD, leading to a decrease in population growth. Estuarine calanoid copepods were also affected by high concentrations of anatoxin-a and MC, and with MC-LR, reduced survival was observed at lower concentrations than those known to affect other meso-zooplankton.

Another, indirect, effect is via organisms such as the mysid shrimps, which can be tolerant to brackish-water cyanobacteria and therefore remain able to reduce the feeding and growth of fish larvae in the presence of cyanotoxins (Karjalainen et al. 2005, 2007); such organisms can alter the ecosystem composition by positive selection. Cyanobacterial filaments and colonies can also pose hydromechanical problems to planktivores, by clogging the feeding appendages and affecting prey detection (Karjalainen et al., 2007; Ibelings & Havens, 2008).

Closing remarks

Although there are few studies of brackish-water cyanobacteria in Europe and those that exist often focus on Nodularia species, other cyanobacteria such as Anabaena, Aphanizomenon, Microcystis, Oscillatoria, Planktothrix and Phormidium are increasingly being investigated and some of them reported as hepatotoxin producers. Furthermore, the existing data are biased towards planktonic bloom-formers, rather than benthic mat-forming cyanobacteria.

Among the toxins produced by European brackish-water cyanobacteria, hepatotoxins are the most widespread and NOD the best described. Thus, the production of NOD in situ and in laboratory conditions has been well studied and is known to be affected by different environmental factors, such as light, water temperature, salinity, phosphate and silicate. However, available data on the influence of environmental parameters on NOD production in the field are rather scarce. This cyanotoxin is commonly responsible for inducing multiple effects on food chains of European brackish waters. At lower trophic levels, plants and macroalgae accumulate low amounts of NOD compared with mussels, clams and some fish species, suggesting that the zooplankton can act as a vector of the toxin.

Other cyanotoxins such as MC, anatoxin-a, BMAA and other cytotoxic compounds are present in brackish waters of European countries and confirmed to be synthesized either by planktonic or by benthic species. However, the current literature about these toxins, in particular their production, transfer along the food chain and factors triggering their production, is sparse, though it is known that MC can accumulate in mussels, crayfish, and fish growing in estuarine waters and used for human consumption.

Even though few intoxication episodes (mostly linked to animal poisonings) have been reported in brackish waters of European countries, it is imperative to improve the study of cyanobacteria from these systems, not only because of the effects cyanobacteria can have on aquatic ecosystems, but also because of the potential biotechnological interest of the bioactive compounds they synthesize.

Acknowledgements

This review was funded by a PhD grant to VL (SFRH/BD/32846/2006) from the Foundation for Science and Technology (FCT) and to VV (SFRH/BSAB/988/2010). We also acknowledge FCT for partial funding of this research (PTDC/MAR/102258/2008) and the Interreg project ATLANTOX.