Questions à propos de l’éducation

R. J. R. Molica1,4*, E. J. Alécio-Oliveira2, P. V. V. C. Carvalho3, A. N. S. F. Costa3, M. C. C. Cunha1, G. L. Melo1, S. M. F. O. Azevedo4.

1

Instituto de Tecnologia de Pernambuco (ITEP), Av. Prof. Luis Freire 700, Recife-PE, 50.740-540, Brazil

2

Centro Federal de Educação Tecnológica de Pernambuco, (CEFET), Av. Prof. Luis Freire 500, Recife-PE, 50740-540, Brazil

3

Companhia Pernambucana de Sanamento – COMPESA, Gerência de Controle de Qualidade, Largo Dois Irmãos 1012, Dois Irmãos, Recife-PE, 52.071-440, Brazil

4

Instituto de Biofísica Carlos Chagas Filho, CCS, Blogo G, UFRJ, Ilha do Fundão, Rio de Janeiro, 21949-900, Brazil

*

Corresponding author: Instituto de Tecnologia de Pernambuco, Laboratório de Ecofisiologia de Microalgas, Av. Prof. Luis Freire 700, Cid. Universitária, Recife-PE, 50.740-540, Brasil. Fone: 55 81 3272-4264 Fax: 55 81 3272-4287 e.mail: [email protected]

ABSTRACT

The blooms of toxic cyanobacteria are very common in Brazilian waterbodies as a consequence of eutrophication process. This situation is more critical in the Northeast of Brazil, a region subject to recurrent periods of droughts and consequently with a greater number of surface reservoirs to store water. In this work we have investigated the presence of neurotoxins during a cyanobacterial bloom in Tapacurá reservoir, which is used to supply water to Recife city. It is a eutrophic system with frequent blooms of cyanobacteria. We also investigated the presence of neurotoxins in strains of Anabaena spiroides isolated from this reservoir. Samples were collected from March to May 2002 at the surface close to the dam. Cyanobacteria density was analyzed. The samples were assayed for toxicity by mouse bioassay, acetylcholinesterase inhibiting activity by colorimetric method and saxitoxins (paralytic shellfish toxins) by HPLC-FLD postcolumn derivatization method. The dominant species during the bloom were A. spiroides, Pseudoanabaena sp. Cylindrospermopsis raciborskii and Microcystis aeruginosa. The mouse bioassays have showed the presence of neurotoxins during A. spiroides and C. raciborskii dominance but an antiacetylcholinesterase activity only during A. spiroides dominance. The A. spiroides strains have also demonstrated posses an acetylcholinesterase inhibitor. HPLC-FLD chromatograms reveled the presence of saxitoxin, neosaxitoxin and dc-saxitoxin, probably produced by C. raciborskii. The presence of saxitoxins and anatoxin-a(s)-like in a drinking water supply alerts the local authorities for the possibility of the presence of these high toxic compound in potable water.

KEYWORDS: Anabaena spiroides, Cylindrospermopsis raciborskii, acetylcholinesterase inhibitor, anatoxin-a(s), saxitoxin, cyanobacterial bloom, Tapacurá reservoir.

INTRODUCTION

Eutrophication favors occurrence of cyanobacterial blooms and many of which are toxic (Bartram et al., 1999). Animal poisonings by toxic cyanobacteria are extensively recorded in the literature (Carmichael, 1992). The most serious event involving human being took place in Caruaru city, Northeast Brazil, when 76 people died during hemodialysis treatment (Jochimsen et al., 1998; Carmichael et al., 2001). The toxic compounds produced by cyanobacteria can be grouped in two classes according to the targets of their toxic actions as hepatotoxins and neurotoxins (Sivonen, 1996). The first one includes microcystins and nodularins, cyclic peptides that inhibit protein phosphatases type 1 (PP1) and type 2 (PP2A) and cylindrospermopsin, an alkaloid suppressor of protein synthesis (Sivonen and Jones, 1999). Neurotoxins include anatoxin-a, a postsynaptic cholinergic nicotinic agonist, anatoxina-a(s), an inhibitor of acetylcholinesterase activity and PSP toxins (saxitoxins), sodium channel blockers in the nerves (Sivonen and Jones, 1999).

Cyanobacterial blooms are very frequent events in drinking water supplies in Brazil (Yunes et al., 1996; Souza et al., 1998; Bouvy et al., 2000; Huszar et al., 2000) and the cyanotoxins registered to occur in Brazilian waterbodies until today are microcystins (Azevedo et al., 1994; Yunes et al., 1996; Matthiensen et al., 2000), saxitoxins (Lagos et al., 1999; Molica et al., 2002) and anatoxin-a(s)-like (Monserat et al., 2001). Cylindrospermopsin was only detected in a carbon from a hemodialysis clinic in Caruaru (Carmichael et al., 2001) and anatoxin-a has never been reported.

In this report we present results of presence of saxitoxins and anatoxin-a(s)-like during a cyanobacterial bloom in Tapacurá reservoir. This eutrophic waterbody is located in Pernambuco state - Northeast of Brazil - and it is used to supply water to Recife city (Braga, 2001; Bouvy et al., 2003). The local Water Company has been realizing a water-monitoring program in Tapacurá reservoir since 2001 including cyanobacteria cells count, toxicity measurements and some limnological parameters. We also investigated the presence of neurotoxins in cultures of Anabaena spiroides isolated from this reservoir.

MATERIALS AND METHODS

Sampling site, analytical procedures and nutrients

Sampling was carried out weekly from 19 March to 10 April and from 8 May to 30 May at the surface close to the dam of Tapacurá reservoir. The interruption on sampling between 10 April and 8 May was due a technical problem. The conductivity and pH were recorded using specific electrodes. Water samples for nutrients (NH4-N, NO3-N, NO2-N and total phosphorus) determinations were transported under refrigeration to the laboratory. Analysis were performed according to APHA (1992).

Cyanobacteria identification and counts

Cyanobacteria species were identified by light microscopy and cells counts were performed using a Sedgwick-Rafter Cell.

Isolation and culture

Non-axenic cultures of A. spiroides were initiated from a bloom sample by transferring successively single trichomes with a Pasteur pipette to drops of culture media and at last to a culture tube containing approximately 5 ml sterile ASM-1 medium. The three strains (ITEP-024, ITEP-025 and ITEP-026) were maintained under 26oC + 2oC with a photon flux density of 40 µmol photon flux m-2 s-1 (Biospherical Instruments QSL-100) from cool-white fluorescent tubes on a 12:12 light:dark cycle. The strains were cultivated in 2 L Erlenmeyers flasks containing 1.5 L ASM-1 medium (Gorham et al., 1964), under 80 µmol photon flux m-2 s-1 with a 12:12 h light:dark cycle and aeration. Cultures were harvested at the late exponential phase by centrifugation. The cell material was lyophilized and stored at -18oC.

The measurements of A. spiroides were made taking 50 vegetative cells, heterocytes, akinets, spirals wide and distance between coils of each strain and natural population.

Toxins extraction and mouse bioassays

The toxicity of bloom samples and A. spiroides strains were tested by i.p. injection in at least three Swiss Webster mice weighting 15-22 g.

Bloom samples were collected in 5 liters plastic bottles and vacuum-filtered through 47 mm fiberglass filters and the volumes were recorded. The toxins were extracted disrupting cells by adding 4 mL of 0,9% NaCl pH 4.0 (adjusted with HCl 0.1 M) to the filters and submitted them to freeze-thawed process. The saline solution was separated from the filters by centrifugation (8,014 g, 10 min). The doses injected in three mice varied from 150 to 520 mg Kg-1 (mg of dry weight of seston per Kg of mice body weight). The dry weights of bloom material (seston) were determined by filtering know volumes through tared 25 mm fiberglass filters, which were then dried for 24 h at 100oC and weighted.

Lyophilized culture material was resuspended in 0.9% NaCl pH 4.0 (adjusted with HCl 0.1 M) and sonicated (High Intensity Ultrasonic Processor - 50W Model) during 1 min at low temperature. Two or three doses in the range of 14 to 156 mg Kg-1 were injected i.p. into mice.

Animals were observed for poisoning symptoms during the first 2 hours and deaths were recorded until the first 24 hours. Control animals were injected with 1 mL of 0.9% NaCl (pH 4.0).

Acetylcholinesterase assay

All the chemicals products were of analytical grade purity. Eel acetylcholinesterase (AChE), propionylthiocoline iodide, 5,5’-dithiobis- (2-nitro) benzoic acid (DTNB) and eserine were purchased from the Sigma Co. (St. Louis, MO, USA).

The centrifuged saline aqueous extracts of bloom samples were used for the assay. The lyophilized culture material of the three strains (25 mg) were extracted in ethanol:1M acetic acid (20:80) solution for 4 hours and centrifuged as described by Henriksen et al. (1997).

The in vitro inhibition of acetylcholinesterase (AChE) was determined by Ellman method (Dietz et al., 1973) using propionylthiocoline iodide (10 mM) as a substrate. The capacity of inhibition of eel AChE by extracts was determined by incubation of enzyme in an end point assay. The strains extracts were diluted 1:10 in saline solution and bloom extracts tested without dilution. All activity assays were made in triplicate.

Eel AChE solution (100 µL) and extracts (10 µL) were incubated 2 min at 37ºC. Then 290 µL of PBS and 1200 µL of DTNB (0.423 M) were added and incubated for 5 min. Substrate (400 µL) were added and incubate more 3 min at 37ºC. The reaction was stopped with 80 µL of eserine (2.4 mM) and absorbance read at 410 nm (Hitachi U- 2000). The positive control (PBS) and blank reaction were assayed during the period of the test. For negative control eserine (80 µL) was added first and PBS instead extract solution. The final absorbance at 410 nm was utilized as inhibition measurement.

PSP toxins analysis

The saline aqueous extracts of bloom samples and lyophilized culture material utilized for mice bioassay were analyzed for the presence of the three classes of saxitoxins (C toxins, gonyautoxins and saxitoxin) separately according to Oshima (1995). The Shimadzu HPLC system used a silica-base reversed phase column (125 x 4.0 mm, 5µm; Lichrospher 100 RP-8e) and separations were carried out under specifics mobile phases for each group of saxitoxins. Samples were filtered through 0.22 µm Millex (Millipore) prior injection. Post column oxidation also followed the method of Oshima (1995) excepted for the temperature of the oven (Shimadzu CTO-10Avp) that was 80o C. Fluorescent saxitoxins derivatives were detected using a Shimadzu RF- 10Axl fluorometric detector with excitation at 330 nm and emission at 390 nm. Toxins were identified and quantified by comparison of retention times and integrated areas with standards, respectively. Standards (Reference Material) of saxitoxins (saxitoxin, neosaxitoxin, GTX1/4 and GTX2/3) were purchased from Institute for Marine Bioscience – National Research Council of Canada (Halifax, Canada). A mixture of saxitoxin, neosaxitoxin and dc-saxitoxin of unknown concentration and C1/C2 toxins were kindly provided by Dr. Nestor Lagos of Chile University (Chile).

RESULTS

Environmental parameters and nutrients

The pH was predominantly alkaline and as conductivity showed a tendency to

decrease, but progressively rises up from mid-April to the end of the survey (Fig.1). Nitrite and ammonium were always below the detection limit (d.l.) of the method (25 and 40 µg L-1, respectively) except on 24 May when ammonium concentration was 40 µg L-1 and 15 May when nitrite concentration was 150 µg L-1. Nitrate (d.l. 50 µg L-1) was not detected during the entire period and total phosphorus varied in the range of 20 to 228

µg L-1 (Fig. 1).

Figure 1: Environmental parameters in Tapacurá reservoir from 19 March to 30 May 2002. (A) pH; (B) Conductivity and (C) = Total phosphorus.

0 50 100 150 200 250 P -P O4 3 - (m g L -1) 7 7,4 7,8 8,2 8,6 9 p H 420 430 440 450 460 470 19/0 3 27/0 3 04/0 4 12/0 4 20/0 4 28/0 4 06/0 5 14/0 5 22/0 5 30/0 5 µ S . c m -1

Cyanobacteria species composition during the bloom

In general, a bloom is considered as a drastic development of one or two species of microalgae. We observed a cyanobacterial bloom with a succession of different species in Tapacurá reservoir. It was recorded from mid-March to the end of May and during this period the average number of cyanobacterial cells was about 140,000 cells mL-1 (Fig 2). The monitoring program realized at Tapacurá reservoir just managed cyanobacteria species, thus the data about other phytoplankton group are not available. On 19 March A. spiroides was the dominant species representing 72% (approximately 1.4 x 105 cells mL-1) of total cyanobacteria and their number gradually declined until 3 April when Pseudoanabaena sp. become the dominant species. Cylindrospermopsis raciborskii started to increase at end of March and dominate until 10 April. The samples between 10 April and 8 May could not be collected but C. raciborskii dominance continued to be registered from 8 to 15 May. The last part of the bloom was dominated by Microcystis aeruginosa constituting 66% to 82% (3.7 and 2.7 x 104 cells mL-1, respectively) of cyanobacterial cells. Other cyanobacterial species were observed during the bloom but their numbers were not expressive (Fig. 2). Raphidiopsis-like was included in the enumeration of C. raciborskii because the difficult to separate them as recommended by McGregor and Fabbro (2000).

Figure 2: Cyanobacteria composition and concentration (cell number mL-1) during a bloom in Tapacurá reservoir. Anabaena spiroides, Anabaena sp., Synechocystis sp., Planktothrix sp., Merismopedia sp., Microcystis aeruginosa,

Gomphosphaeria sp., Pseudoanabaena sp. and Cylindrospermopsis raciborskii.

Characterization of isolated strains

Three clones of A. spiroides were isolated and cultivated. Under the culture conditions the organism formed solitary trichomes, coiled, with many aerotopes, spirals 32-62 µm wide and distance between the coils 10-50 µm. In general all the taxonomic characteristics of strains and natural population agreed well with those described for A. spiroides. The only exception was the strain ITEP-026, which spirals wide and distance between coils were smaller (Table I).

1 9 /M a r 2 7 /M a r 0 3 /A p r 1 0 /A p r 0 8 /M a y 1 5 /M a y 2 4 /M a y 3 0 /M a y 0 50000 100000 150000 200000 C e ll n u m b e r m L -1

Toxicity of bloom samples and isolated strains

Mouse bioassay demonstrated a neurotoxic response of bloom material from 19 March to 24 May except the sample collected on 03 April that was not toxic. The time of death varied from 3 to 12 min. Anatoxin-a(s)-like anticholinesterase symptoms, including salivation and limbs fasciculation, were only observed for sample collected on 27 March (Table II).

The acetylcholinesterase assay reveled an AChE inhibitor activity in samples collected on 19 and 27 March with 100 and 88% of inhibition, respectively. This period coincided with A. spiroides dominance. All other samples did not show AChE inhibition (Table II).

The three isolated strains were toxic in mouse bioassay and the observed symptoms were typical for that caused by anatoxin-a(s). The AChE enzymatic assay reveled a 100% of inhibition for strains extracts (Table II).

HPLC-FLD analysis

HPLC-FLD chromatograms reveled the presence of saxitoxins variants only in sample collected on 8 May. The peaks had retention times identical to those of the standards (Fig. 3). The variants identified were saxitoxin (Stx), neosaxitoxin (NeoStx) and dc-saxitoxin (dc-Stx). The concentrations calculated by comparison of peak areas with those of the standards were 52 and 51 ng L-1 for Stx and NeoStx, respectively.

Table 1. Morphological features of three strains and natural population of A. spiroides from Tapacurá reservoir. Cell lenght (µm) Cell width (µm) Heterocyst length (µm) Heterocyst width (µm) Akinetes length (µm) Akinetes width (µm) Spirals width (µm) a Between coils (µm) N.P 4.87 ± 0.70 5.47 ± 0.50 6.18 ± 0.7 6.55 ± 0.7 10.05 ± 1.0 10.01 ± 1.0 42.90 ± 7.10 32.60 ± 11.80 ITEP-024 5.30 ± 0.52 5.95 ± 0.57 7.22 ± 1.0 7.09 ± 0.8 9.04 ± 1.3 9.24 ± 1.0 42.80 ± 6.26 30.20 ± 11.02 ITEP-025 5.28 ± 0.50 6.04 ± 0.50 7.88 ± 0.6 7.80 ± 0.7 N.D N.D 39.85 ± 4.70 24.35 ± 8.59 ITEP-026 5.03 ± 0.40 5.60 ± 0.50 7.21 ± 0.6 7.33 ± 0.6 N.D N.D 14.30 ± 3.71 32.00 ± 3.81 b - 6 – 8 - 6 - 8 17 - 21 10 - 14 20 - 45 20 - 45 A. spiroides c - 6.5 – 8 - 6 - 7 13 - 18 8 - 9 30 - 40 30 - 48 NP – natural population ND – not determined a

Distance between coils

b Komárková.-Legnerová & Eloranta (1992) c Sant’Anna & Azevedo (2000)

Table 2. Toxicity and achetylcholinesterase inhibition assay of bloom samples and A. spiroides strains. Date Injected Dose

(mg Kg-1) Toxicity by mouse bioassay Acetylcholinesterase assay - % inhibition Symptoms 19 March 519.4 + 100 Convulsions and respiratory arrest

27 March 495.2 + 88 Salivation, tremors, convulsions, respiratory arrest and limbs fasciculation

03 April 276.4 - 0 No observed symptoms

10 April 319.3 + 0 Trembling head, convulsions, jumping and respiratory arrest

08 May 493.0 + 0 Trembling head, convulsions, jumping and respiratory arrest

15 May 149.1 + 0 Trembling head, convulsions, jumping and respiratory arrest

24 May 444.4 + 0 Trembling head, convulsions, jumping and respiratory arrest

30 May 253.0 - 0 No observed symptoms

ITEP-024 148.4 + 100 Salivation, tremors, convulsions, respiratory arrest and limbs fasciculation

ITEP-025 150.4 + 100 Salivation, tremors, convulsions, respiratory arrest and limbs fasciculation

Figure 3: HPLC-FLD chromatograms of (A) 8-May bloom sample, (B) standards (reference material) and (C) mixture of standards of unknown concentrations.

It was not possible to calculate dc-Stx concentration because its concentration on mixture standards was unknown. When analyzed replacing the oxidizing reagent by water the peaks changed in the same manner as with the respective standards toxins. NeoStx increased several fold while Stx and dc-Stx almost disappeared (data not shown). No matched peaks were detected under the conditions for Gtx and C toxins (data not shown). Moreover, no suspected peaks of these groups of saxitoxins variants were observed in the chromatograms. The three A. spiroides strains were also analyzed for the presence of saxitoxins but no suspicious peaks were observed.

Minutes

A

B

DISCUSSION

The Northeast Brazil is subjected for periods of drought especially in years of El Niño phenomenon. Man-made reservoirs, most designed for multiple purposes, including drinking water sources, are very common in this region as a consequence of this meteorological characteristic (Bouvy et al., 1999). Many of these waterbodies receive a high level of nutrients from human activities – mainly industry and urban effluents – creating favorable conditions for cyanobacterial blooms (Bouvy et al., 2000). Since the hemodialysis accident in Caruaru city (Azevedo et al., 2002) more attention has been given for the presence of cyanobacteria in drinking water supplies in Pernambuco state. Tapacurá reservoir supplies water for about 1.35 million inhabitants in Recife city and as cyanobacterial blooms are recurrent in this environment (Nascimento et al., 2000; Ferreira, 2002) the local Water Company realizes a monitoring program due to the potential of human health consequences.

Bouvy et al. (2003) through a study during two years (May 1998 – May 2000) showed that Tapacurá reservoir is a eutrophic environment. These authors observed concentrations as high as 124 µg L-1 chlorophyll-a, 200 µg L-1 of particulate phosphorus and 2 mg L-1 of ammonium. In our study the concentration of total phosphorus was also high and ranged from 20 to 228 µg L-1 (Fig.1C). On the other hand, the level of ammonium was nearly always below the detection limit. The lack of dissolved nitrogen show a nitrogen limitation. According to Sas (1989) concentrations lower than 100 µg L-1 would be considered limiting. The dominant species during the bloom were the nitrogen-fixing A. spiroides and C. raciborskii. The exception was Pseudoanabaena sp. which dominance was during a short period of time. Low concentration of ammonium was detected in 24 May coinciding with M. aeruginosa non-nitrogen fixing species dominance.

According to Reynolds et al. (2002) A. spiroides can be classified in a H1 assemblage – a separation of group H - that includes heterocytic cyanobacteria. C. raciborskii was previously grouped in H, but a new one (Sn) was created for this

species due to its low light requirements rather than its N2 fixing capacity (Reynolds 1997). However, Ferreira (2002) reported a variation from 0 to 77% in the percentage of thricomes of C. raciborskii carrying heterocytes in Tapacurá reservoir indicating that the capacity of C. raciborskii to fix N2 could not be disregarded. Huszar et al. (2000) observed a codominance of C. phillippinensis and A. spiroides during a strong N-limitation in Juturnaíba reservoir. These authors observed that 30% of C. phillippinensis carried heterocytes, indicating that at least part of the population was fixing nitrogen. We did not count the number of heterocytes of C. raciborskii but according to the data of those authors, it is reasonable to suppose that even though C. raciborskii is included in Sn assemblage the lack or low concentration of dissolved inorganic nitrogen observed in Tapacurá reservoir during our study would not be a limitation factor for its development. Moreover, C. raciborskii has been reported as a species with high affinity for ammonium, using this nutrient in such concentrations in which the other heterocytic species already need to fix N (Padisák, 1997). Huszar et al. (2000) pointed out Cylindrospermopsis as genus with alternative physiological adaptations between S (Oscillatoriales – non-N2-fixing) and H (heterocytic cyanobacteria) assemblages.

Neurotoxic cyanobacterial blooms have already been observed in Tapacurá reservoir but the toxins identified were saxitoxins detected during C. raciborskii dominance (Nascimento et al., 2000). The 19 March sample showed typical symptoms of neurointoxication during mouse bioassay but no clinical signs of anatoxin-a(s) toxicosis were observed (Tab II). These results suggests that saxitoxins or even anatoxin-a could be the mainly toxins produced. However, anatoxin-a(s)-like anticholinesterase symptoms were observed in sample collected on 27 March. Henriksen et al. (1997) reported that mouse bioassays developed with high doses generally could mask the characteristic sings of anatoxin-a(s)-like anticholinesterase and only doses around LD50 would allow the visualization of typical symptoms. In fact, the acetylcholinesterase assay revealed an inhibition in samples collected on 19 and 27 March of 100 and 88% (Tab II), respectively.

The percentages of inhibition were significantly different (t = 19.34; p < 0.00004), suggesting that AChE inhibitor was in a higher concentration in 19 March sample. The period in which was observed an AChE inhibition coincided with a dominance of A. spiroides (Tab II and Fig 2). Interesting to note that the presence of this species was not reported during a two years survey (May 1998 – May 2000) conducted in Tapacurá reservoir. In that time the dominant species of cyanobacteria were C. raciborskii and Raphidiopsis cf. mediterranea (Ferreira, 2002). The appearance and dominance of A. spiroides probably are related to a specific limnological and nutrients conditions not present during that period. However, the environmental parameters analyzed in this study are not enough to explain the change in the environment that favored A. spiroides. Since March 2002, A. spiroides neurotoxic blooms have been observed in Tapacurá reservoir (data not showed).

The production of anatoxin-a(s) was only described for species of Anabaena genus including A. flos-aquae (Mahmood and Carmichael, 1986) and A. lemmermanii (Henriksen et al., 1997; Onodera et al., 1997). Monserrat et al. (2001) reported an AChE inhibition by an aqueous extract from A. spiroides bloom collected from an ornamental lake in South of Brazil. In order to confirm whether A. spiroides observed in Tapacurá reservoir synthesize anatoxin-a(s)-like anticholinesterase three strains were isolated and cultivated. The mouse and anticholinesterase bioassay reveled that the three strains are AChE inhibitor producer (Tab. II). It has been recently verified by LC/MS that strain ITEP-024 produces anatoxin-a(s) (Dr. Wayne Carmichael, Wright State University, personal communication, 2003). The others strains and bloom material will also be analyzed to confirm the presence of this toxin. Nevertheless, the mouse and anticholinesterase bioassay more the LC/MS analysis suggest that the toxin present in Tapacurá reservoir during A. spiroides dominance and the toxin produced by the other two A. spiroides strains is anatoxin-a(s).

During the Pseudoanabaena sp. dominance no toxicity was observed by the mouse bioassay (Fig. 2; Tab II). It is reasonable, since there are no reports about toxins production for this genus. From 10 April to 24 May the mouse bioassay