Short Communication. First Occurrence of Cylindrospermopsin in Freshwater in France INTRODUCTION

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1 Short Communication First Occurrence of Cylindrospermopsin in Freshwater in France Luc Brient, 1 Marion Lengronne, 1 Myriam Bormans, 1 Jutta Fastner 2 1 Université de Rennes 1 - U.M.R., CNRS 6553 Ecobio - I.F.R. CAREN, France 2 Federal Environmental Agency, Germany Received 14 May 2008; revised 22 July 2008; accepted 9 August 2008 ABSTRACT: Eleven waterbodies in Western France dominated by cyanobacteria of the genera Aphanizomenon and Anabaena were analyzed in September 2006 for microcystins (MC) and cylindrospermopsin (CYN). CYN was detected for the first time in France in four of them in the presence of Aphanizomenon flos-aquae and in the presence of Anabaena planctonica in the other. The intracellular concentrations of CYN measured by LC-MS/MS ranged between 1.55 and 1.95 lg/l. The occurrence of CYN represents an additional health hazard to MC especially because Aphanizomenon flos-aquae is the third most common species in freshwaters in France. # 2008 Wiley Periodicals, Inc. Environ Toxicol 00: , Keywords: cylindrospermopsin; Aphanizomenon flos-aquae; Anabaena sp.; drinking and recreational waters INTRODUCTION Correspondence to: L. Brient; luc.brient@univ-rennes1.fr Published online in Wiley InterScience ( DOI /tox Massive proliferations of cyanobacteria are observed in many waterbodies on all continents as a consequence of increasing eutrophication (Whitton et al., 2000). Cyanobacteria are potent producers of different types of toxins such as neurotoxins, hepatotoxins, and dermatotoxins (Chorus and Bartram, 1999). Although the characterization of these toxins is well documented, only the group of microcystins (MC) is routinely analyzed in France during monitoring of recreational and drinking waters. In Brittany, the presence of toxic cyanobacteria in freshwater has been documented since 1995 (Vezie et al., 1997; Brient et al., 2008) with the presence of MC in nearly 65% of the sampled sites. Recently, other cyanotoxins have also been detected in France: anatoxin-a and homoanatoxin-a from benthic cyanobacteria have been found in association with dog poisoning (Gugger et al., 2005; Cadel-Six et al., 2007). In contrast, cylindrospermopsin (CYN) has so far not been detected in France. CYN has been identified for the first time in Western Europe only rather recently (Fastner et al., 2003; Manti et al., 2005), although it has been present in Australia for several decades (Byth, 1980) and in others parts of world (Padisak, 1997; Bouvy et al., 1999). In Germany, CYN has been detected as frequently as microcystin with maximal concentrations of 12 lg/l (Fastner et al., 2007; Rücker et al., 2007). An Australian study has shown that out of 47 waterbodies 14 had an average concentration of 3.4 lg/l of CYN (McGregor and Fabbro, 2000) Concentrations of more than 90 lg/l of CYN have been detected in treated water in Florida (Falconer and Humpage, 2006). Contrary to microcystins which are contained largely within the cell and of which only a very small proportion is extracellular, extracellular CYN can reach up to 90% of total CYN (Chiswell et al., 1999; Rücker et al., 2007). CYN is a water soluble stable alkaloid with hepatotoxic properties. The first intoxication of this toxin in humans goes back to 1979 on Palm Island, Queensland, Australia C 2008 Wiley Periodicals, Inc. 1

2 2 BRIENT ET AL. (Bourke et al., 1983), where it has been demonstrated to be produced by Cylindrospermopsis raciborskii. CYN causes irreversible inhibition of protein synthesis in humans while interacting with transfer RNA (Froscio et al., 2003; Kinear et al., 2008). It is genotoxic (Humpage et al., 2000) and probably carcinogenic (Falconer et al., 2006). The primary symptoms described are gastroenteritis, and pathological affects on the kidneys, heart, thymus, spleen, and intestine. The freshwater cyanobacterial species known to produce CYN are the following: Cylindrospermopsis raciborskii in Australia (e.g., Hawkins et al., 1985; Schaw et al., 1999), Hungary (Padisak, 1997), Brazil (Bouvy et al., 2000), New Zealand (Stirling and Quilliam, 2001) and Florida, USA (Chapman and Schelske, 1997); Aphanizomenon flos-aquae in Germany (Preubel et al., 2006); Aphanizomenon ovalisporum in Israel (Banker et al., 1997; Schaw et al., 1999); Anabaena bergii in Australia (Schembri et al., 2001); Anabaena lapponica in Finland (Spoof et al., 2006); Raphidiopsis curvata in China (Li et al., 2001); Umezakia natans in Japan (Harada et al., 1994); and Lyngbia wollei in Australia (Seifert et al., 2007). Among these species, Cylindrospermopsis raciborskii has been detected in a reservoir in France (Briand et al., 2002) but did not produce CYN. It is important to bear in mind the small number of taxonomists in France who can identify this species taking into account that it has several synonyms in particular that of Anabaena or Anabaenopsis raciborskii (Stuken et al., 2006). Among the other species in Europe producing CYN, recent studies highlighted Anabaena lapponica in Finland (Spoof et al., 2006) and Aphanizomenon flos-aquae in Germany (Preubel et al., 2005). The two genera Anabaena and Aphanizomenon are also regularly found in France and a recent report by AFSSA (2006) highlighted Aphanizomenon in 40% of samples and on 64% of the sites; Anabaena in 34% of and on 54% of the sites from a survey of 1700 samples collected over the years 2002 to These genera also occur regularly in Brittany and often reach high biomass (Brient et al., 2004). These results encouraged us to initiate a scoping study of the occurrence of CYN in waterbodies in Western France in the presence of the two genera Aphanizomenon and Anabaena. MATERIALS AND METHODS Study Sites and Sampling Procedures This preliminary investigation was carried out on 11 shallow lakes and ponds of Western France for the period from September 4th 12th, 2006 during which the phytoplankton community was dominated by the genera Aphanizomenon and Anabaena. Since 2004, phytoplankton analyses, identification, and counting have been carried out weekly between May and October and thus allow to trace the presence of these two genera. Lakes Ribou (Cholet), La Dathée (Vire), and Etang au Duc (Ploermel) are used for recreational activities and the production of drinking water. The other eight waterbodies are used for recreational activities only: Grand Lieu (Bouaye), Martigné Ferchaud, Marcillé Robert, Le Pertre, Apigné (Rennes), Vern sur Seiche, Boulet (Feins), and Chevreux. Their water volumes are all lower than 3 millions m 3 during the summer and their mean depth is less than 3 m. The samples were carried out using a 1 L tube extending over the top meter of the water column in the swimming area. Analytical Methods The fresh phytoplankton material was analyzed within 48 h of sampling. The counting of the cells was carried out with a Nageotte cell whose volume is 50 ll after concentration on a polycarbonate filter of 1 lm. Less than 200 ml of sample is filtered. The filter is washed with 1 ml of the original solution and aspirated with a pipette and injected in the Nageotte cell. This cell is divided into 40 bands and the number of bands observed is defined when at least 40 colony or filaments are counted (Brient et al., 2008). MC concentrations were determined with a HPLC with diode array detection (HPLC-DAD) and a variable-wavelength UV detector operating at 238 nm. Samples were harvested by filtration and filters were suspended in 1 ml of 85% methanol in water and centrifuged at g for 7 min. The separation was performed on a Microspher C18 reverse-phase column (3 lm) under isocratic conditions at a flow rate of 1 ml/min in a mobile phase of 10 mm ammonium acetate and acetonitrile (7.4:2.6) for 20 min. As MC- LR was the standard used, concentration was expressed as lg MC-LR/L and lg Eq MC-LR/L for other MC. For CYN analysis, a subsample of 200 ml was filtered on 1 lm glass fiber filter and dried to 408C. The determination of CYN was performed by LC-MS/MS as described in details previously (Fastner et al., 2007). This analysis corresponded to intracellular CYN only in this preliminary investigation. RESULTS CYN was found in six of the 11 waterbodies sampled. (Table I) Characteristic ion chromatograms from standard CYN and of a sample from Boulet are shown in Figure 1. Among the genera potentially producing CYN, the species identified in the corresponding toxic samples were the

3 FIRST OCCURRENCE OF CYN IN FRESHWATER IN FRANCE 3 TABLE I. Distribution of dominant species and production of CYN from waterbodies in western France Sites Ribou La Dathée Vern/Seiche Boulet Marcillé Robert Le Pertre CYN (lg/l) Traces a Traces a Dominant species Planktothrix agardhii Woronichinia Anabaena planctonica Anabaena planctonica Planktothrix agardhii Aphanizomenon gracile Second most dominant Aphanizomenon Microcystis Aphanocapsa sp. Planktothrix Anabaena Oscillatoria sp. species flos-aquae aeruginosa agardhii spiroides Third most dominant Aphanizomenon Aphanizomenon Aphanizomenon Oscillatoria sp. Aphanizomenon species Issatchenkoi flos-aquae flos-aquae flos-aquae Total cyanobacteria 404, ,000 48,000 84, , ,800 (cell/ml) Dominant species 266, ,000 17,000 38, , ,800 (cell/ml) Second most dominant 85, ,000 16,000 21, ,000 60,000 species (cell/ml) Third most dominant species (cell/ml) 22,400 82, ,000 16,000 a Traces: \limit of quantification (0.01 lg/l). following: Aphanizomenon flos-aquae, Aphanizomenon gracile, Aphanizomenon issatchenkoi (called now Cuspidothrix issatchenkoi), Anabaena planctonica, and Anabaena spiroides. Aphanizomenon flos-aquae was present in four of them as the dominant species in Ribou with a biomass of 85,600 cells/ml and La Dathée with a biomass of 82,000 cells/ml. For two other sites Aphanizomenon flos-aquae was present but nondominant, with 5000 cells/ml in Vern/Seiche and 16,000 cells/ml in Marcillé Robert. The pond at Le Pertre indicated traces of CYN in the presence of Aphanizomenon gracile, whereas there was neither Aphanizomenon flosaquae in the September sample nor in any of the samples covering the period May to October. The site of Boulet Fig. 1. Reconstructed ion chromatograms of a cylindrospermopsin standard (retention time 4.50 min) and a sample from Lake Boulet (Feins) with the MRM transitions of m/z 416 > 194 and m/z 416 > 176 characteristic for cylindrospermopsin. Chromatographic conditions were as in Materials and Methods.

4 4 BRIENT ET AL. indicated the existence of CYN in the presence of Anabaena planctonica (38,000 cells/ml), although there were no other species of Aphanizomenon or Anabaena. The three sampling sites at Apigné and Chevreux were characterized by the absence of Aphanizomenon flos-aquae and Anabaena planctonica but by the presence of the species Aphanizomenon aphanizomenoides, Aphanizomenon issatchenkoi, and Aphanizomenon gracile without detection of CYN. The remaining two sites at Martigné Ferchaud and Etang au Duc indicated the presence of Aphanizomenon flos-aquae without CYN. The 11 waterbodies studied for intracellular CYN show the presence of this cyanotoxin with concentrations varying from traces to 1.95 lg/l for biomasses of potential producers from 16,000 cells/ml to 85,000 cells/ml. It is also interesting to note that the two hepatotoxic types of cyanotoxins, CYN and MC, were present at the same time in four waterbodies: Le Pertre, Marcillé Robert, Ribou, and La Dathée (Table II). Three other waterbodies Grand Lieu, Apigné, and Martigné-Ferchaud contained cyanobacteria producing MC but not CYN. DISCUSSION In this preliminary study, we highlighted the possible role of Aphanizomenon flos-aquae and Anabaena planctonica, as CYN producers on the waterbodies Ribou and Dathée, Vern/Seiche, and Boulet. This hypothesis is in agreement with recent publications in Europe where the two species were reported in the presence of CYN (Preubel et al., 2006; Spoof et al., 2006). The toxin concentrations found in this study are also similar to those reported for Cylindrospermopsis raciborskii, namely 1 lg/l of CYN with 20,000 cells/ml (Mc Gregor and Fabbro, 2000). The presence of traces of CYN in Le Pertre may be explained by the presence of Aphanizomenon gracile as this species is a suspected CYN producer in Germany (Rücker et al., 2007). This investigation highlights that in France waterbodies used for human activities should be monitored for cylindrospermopsins, as well as for the routinely monitored MC, in the presence of species of Aphanizomenon and Anabaena genera. This is of special importance as Aphanizomenon flosaquae is the third most dominant species after Planktothrix agardhii and Microcystis sp. in France (AFSSA, 2006). The maximum concentration of CYN allowed in France for drinking water is 0.3 lg/l (AFSSA, 2006). Threshold values relate to the intracellular fraction of CYN but should also considered extracellular dissolved fraction. Indeed, several recent studies have demonstrated the predominance of dissolved CYN of around 80% of total CYN in the presence of Cylindrospermopsis raciborskii, Aphanizomenon ovalisporum, and Aphanizomenon sp. (Schaw et al., 1999; Rücker et al., 2007). This study has exclusively considered TABLE II. Distribution of CYN and MC from waterbodies Water Bodies Le Pertre Marcillé Robert Ribou La Dathée CYN (lg/l) Traces Traces MC LR (lg/l) 0.72 \0.2 \ Total equi MC-LR (lg/l) intracellular CYN and thus most probably underestimated the concentration of total CYN in these shallow lakes. It is worth noting that the treatment of drinking water does not satisfactorily remove CYN (Falconer and Humpage, 2006). In the absence of a global toxicity method which would identify each type of toxins, cyanobacteria monitoring in France should include testing for MC and for CYN. However, to reduce costs, testing for CYN could be preferentially conducted in the presence of Anabaena and Aphanizomenon genera. Indeed, the determination of the species themselves of these genera is not easy (Komarek and Kovacik, 1989). Aphanizomenon flos-aquae is relatively simple to identify by the shape of its colony but this is not the case for the Anabaena genus and other species of Aphanizomenon by the shape of their solitary filaments (Hindàk, 2000; Stüken et al., 2006). Moreover, the distinction between Anabaena and Aphanizomenon is also criticized because the equivalent phenotypes for certain species justify the existence of only one genus. CYN is an alkaloid which is difficult to detect when it is enzymatically-bound within the cells of animals or plants (Duy et al., 2000). Very few studies exist on the impacts of CYN on primary producers and their transfer through the foodweb (White et al., 2005) and as a protein synthesis-inhibitor on plants (Metcalf et al., 2004; Kinear et al., 2007). The inhibiting effects of CYN on the metabolism of Sinapsis mustard seedlings have been demonstrated with 50% reduction in growth (Vasas et al., 2002) and of bioaccumulation in an aquatic macrophyte having inhibiting effects (White et al., 2005). The relative impact of CYN on different aquatic organisms is not known although for MC the ingestion of toxic cyanobacterial cells was found more toxic than when they were exposed to the soluble form (Lance et al., 2006). CYN and MC are two hepatotoxins with different synthesis pathways whose combined effect are unknown (Falconer, 2005). Moreover unlike MC which kill mice relatively quickly within 1 2 h after a single dose, CYN toxic effect on hepatocytes is delayed and progressive, causing death usually within h in mice (Hawinks et al., 1985; Runnegar et al., 1994). The presence of CYN in waterbodies reinforce the need to ban copper sulfate in reservoirs used for drinking water as the inhibition of degradation of soluble CYN has been reported (Smith et al., 2008). Considering that CYN is abundant in the extracellular fraction and that it can persist in the water for weeks without degradation in some settings

5 FIRST OCCURRENCE OF CYN IN FRESHWATER IN FRANCE 5 (Wormer et al., 2008), the monitoring of CYN must be regarded as being highly recommended. The presence of two hepatotoxins MC and CYN at the same time in waterbodies (Table II) confirms the need for the management of water used for drinking or recreational purposes with monitoring measures base on cells numbers (Griffiths et al., 2003) and MC concentrations, not exclusively. In conclusion, it is necessary to better understand the mechanisms of CYN excretion and the possible influences of environmental conditions for its production. The results of this investigation also show that CYN and MC can be present together in waterbodies demonstrating the need for carrying out studies on the combined effects of these hepatotoxins at the ecological level and on human health. 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