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First reported case of turtle deaths during a toxic Microcystis spp. bloom in Lake Oubeira, Algeria
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Ecotoxicology and Environmental Safety 71 (2008) 535–544 www.elsevier.com/locate/ecoenv
First reported case of turtle deaths during a toxic Microcystis spp. bloom in Lake Oubeira, Algeria Hichem Nasria,b, Soumaya El Herryb, Noureddine Bouaı¨ chab, a
Institute of Biology, University of El Taref, Algeria Laboratoire Sante´ Publique-Environnement, 5 Rue J.B. Cle´ment, Universite´ Paris-Sud 11, UFR de Pharmacie, 92296 Chaˆtenay-Malabry, France
b
Received 4 April 2007; received in revised form 7 December 2007; accepted 8 December 2007 Available online 30 January 2008
Abstract Microcystins analysis was conducted in field cyanobacterial bloom samples and dead terrapin tissues from Lake Oubeira (Algeria) with an aim of studying the cause of the mortality of the freshwater terrapin species Emys orbicularis and Mauremys leprosa during October 2005. The deaths of these two terrapin species were observed during a bloom of Microcystis spp. The total microcystin content per phytoplankton biomass evaluated with the methanol extraction-protein phosphatase methodology was 1.12 mg MCYST-LR equivalents/g dried bloom material. The analysis of this bloom extract by the LC/MS technique demonstrated the presence of three microcystin variants: microcystin-LR (MCYST-LR), microcystin-YR (MCYST-YR), and microcystin-RR (MCYST-RR). Microcystins were also detected in fresh carcasses of terrapin liver, viscera and muscle tissues using the GC/MS after Lemieux oxidation method and the PP2A inhibition assay. The high level of microcystins detected using the Lemieux oxidation-GC/MS method in the liver tissue (1192.8 mg MCYST-LR equivalent/g dw) and in the viscera tissue (37.19 mg MCYST-LR equivalent/g dw) of the species M. leprosa and E. orbicularis, respectively, and the liver crumbling observed after the necropsy examination of the fresh carcass of M. leprosa support the possibility that cyanobacterial microcystins contribute to the turtle mortalities. r 2007 Elsevier Inc. All rights reserved. Keywords: Freshwater terrapin; Cyanobacteria; Microcystis; Microcystins; Lake Oubeira; Algeria
1. Introduction In the present study, we report for the first time the possibility that the toxic Microcystis bloom in Lake Oubeira, Algeria, may contribute to freshwater turtle deaths. Since the first report of livestock poisoning associated with cyanobacterial blooms (Francis, 1878), numerous cases of animal deaths have been reported worldwide due to contact with cyanobacterial scums, indicating that this is a widespread phenomenon in Mediterranean, continental, and temperate climates in both hemispheres (Codd et al., 2005; Falconer, 2005). Lake Oubeira, one of the largest freshwater lake in Algeria, is an important natural reserve for migration birds and wildfowl species. It is also home to the two freshwater turtles Emys orbicularis (Linnaeus, 1758) and Mauremys Corresponding author. Fax: +33 1 46 83 57 32.
E-mail address: [email protected] (N. Bouaı¨ cha). 0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.12.009
leprosa (Schweigger, 1812). This lake provides considerable benefits for the region functioning as a drinking water source for many urban communities. However, during the past decades the lake has witnessed a steady increase in eutrophication, characteristic of a regular occurrence of toxic Microcystis blooms and high concentrations of microsystins (Nasri et al., 2004). Microsystins are cyclic peptide hepatotoxins causing liver damage by inhibition of serine/threonine protein phosphatase enzymes (Eriksson et al., 1990). The increase in bloom occurrence has led the authorities to cease using this lake as a source for the production of drinking water. However, it is still used for irrigation and aquaculture. Cyanotoxins are known to bioaccumulate in common aquatic vertebrates and invertebrates, including fish, mussels, and zooplankton (Eriksson et al., 1989; Kotak et al., 1996; Prepas et al., 1997; Watanabe et al., 1997; Williams et al., 1997a; Thostrup and Christoffersen, 1999; Magalha˜es et al., 2001; Sipia¨ et al., 2001; Mohamed et al., 2003; Chen and Xie, 2005; Chen
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et al., 2005, 2007), which poses a risk to both animal and human heath if such aquatic animals are consumed (Carmichael and Falconer, 1993). The toxicity of microcystins is associated with irreversible binding and inhibition of these toxins to protein phosphatases 1 and 2A in the liver (Mackintosh et al., 1990, 1995; Yoshizawa et al., 1990), which has important implications for the quantitative determination of these toxins in food webs. Furthermore, the detection methods for microcystins, involved determination of the amount of toxins in methanol extracts of homogenized tissue via either a direct protein phosphatase (PPase) inhibition assay or an antibody assay, can underestimate the concentration of microcystins in the tissue sample. Nevertheless, a new analytical approach capable of detecting both free and covalently bound microcystin fractions has been described (Williams et al., 1997a, b; Kaya and Sano, 1999). This analytical methodology is based upon the chemically unique nature of the Adda [(2S, 3S, 8S, 9S)-3-amino-9methoxy-2,6,8-trimethyl-10-phenyl-4,6-dienoic acid] residue of the microcystins. It has been showed that Lemieux oxidation of pure microcystin-LR (MCYST-LR), or MCYST-LR present in methanol extracts of cyanobacteria blooms, gives 2-methyl-3-methoxy-4-phenylbutanoic acid in high yield (Sano et al., 1992). This last butanoic acid represents a unique marker for the presence of microcystins and, therefore, it can be readily isolated from Lumieux oxidation reactions and detected at low concentrations by GC/MS (Williams et al., 1997a, b; Kaya and Sano, 1999). In October 2005, 12 freshwater turtles were found dead in a small area (0.5 ha) of Lake Oubeira, Algeria. The present study is the first attempt to evaluate the contribution of cyanobacterial poisoning to the turtle deaths. The quantification of microcystins in animal samples was achieved using both the protein phosphatase assay and the Lemieux oxidation GC/MS method. The identification of the different microcystin variants in the cyanobacterial bloom sample was achieved by the LC/MS technique. 2. Materials and methods 2.1. Study site and characterisation of the natural diet of terrapin species Lake Oubeira (361530 N and 0081230 E, Fig. 1) located in north-eastern Algeria is the home to El Kala National Park (PNEK) and has an average elevation of 25 m above sea level. It is the first largest freshwater lake in Algeria, and has an estimated surface area of 2200 ha and a maximum depth of about 4 m. In 1984, the lake was included as a wetland under the Ramsar Convention (Ramsar Convention Official Website, 2007, www.ramsar.org). Lake Oubeira is an important natural reserve for migratory birds and wildfowl species. Two species of terrapin are also present in this lake, E. orbicularis (Linnaeus, 1758) and M. leprosa (Schweigger, 1812). The E. orbicularis is carnivorous and carrion feeder. Its diet consists of molluscs, watery insects, fish corpses even of frogs, tritons, or tadpoles (Lebboroni and Chelazzi, 1991; Kotenko, 2000). However, M. leprosa is an omnivorous species, with prevalence carnivore. Its natural diet is highly biased towards filamentous algae, insect larvae,
earthworms, molluscs, small amphibians and tadpoles, and various aquatic plants. It is also known to be particularly partial to carrion.
2.2. Sample collection In October 2005, cyanobacterial samples were hand collected on one site (Fig. 1) from off the northern shore of Lake Oubeira were scum formation was visible. The bloom samples were fixed in formalin (5% final volume) for cyanobacteria determination under light microscopy, or dried and stored at 20 1C for later cyanobacterial toxin analysis. Dominant species of cyanobacteria were determined using Koma´rek and Anagnostidis (1999) based on cell structure and dimension, and colony morphology, and mucilage characteristics and photographically documented using an Eclipse TE200 light microscope (Nikon, Paris, France). The cell diameter was determined for 50 cells (10 cells from five different colonies of each morphospecies of Microcystis). Because of avian influenza and banning access by the health authorities to the PNEK, and in particular Lake Oubeira, only 2 (out of the observed 12) turtles of each species were allowed to be used for the toxin analysis. The two terrapin species were identified according to Fritz (1992, 1996); Duguy and Baron (1998) and Kotenko (2000). The two fresh carcasses of each terrapin species E. orbicularis and M. leprosa were necropsied and the livers were weighed and examined externally for abnormalities. The terrapin tissues (viscera, liver, and meet) were immediately frozen at 20 1C and then freeze-dried for later microcystins analysis.
2.3. LC/MS analysis of the cyanobacterial bloom extract The LC/MS technique was carried out on an Ion Trap LC/MS system (Bruker, France) and a Hewlett Packard (France) HP-1100 Series system equipped with a binary solvent pump coupled to an analytical work station. The bloom material was dried (50 1C, 1 h) and then extracted three times (overnight at 4 1C) with 100 mL/g of 75% (v/v) aqueous methanol. Extracts were centrifuged (2000g, 10 min.) and supernatants pooled and diluted to a 20% methanol final concentration. The total microcystin content per phytoplankton biomass was then evaluated by the PP2A inhibition assay as described below. The 20% (v/v) aqueous methanol extract was then preconcentrated over a Bakerbond SPE cartridge (Baker, The Netherlands) according to the method described by Maatouk et al. (2002). The toxin fraction after solid phase extraction was then evaporated to dryness under nitrogen and dissolved in 1 mL of 50% (v/v) aqueous methanol. This last fraction was then chromatographed on an Inertsil ODS (150 mm 2 mm i.d., 5 mm) stainless steel column with a guard column LiChrosorb RP-18 (35 mm 4.6 mm, 5 mm) both from Chrompack (Les Ulis, France) using acetonitrile (solvent A) and water with 0.5% formic acid (solvent B) as mobile phase. The gradient program selected for LC/MS was set: 0 min 20% A, 10 min 40% A, 15 min 60% A, 20 min 80% A, and 25 min 100% A, flow-rate 0.3 mL/min. The ES/MS interface in positive mode operated at 350 1C gas temperature, 13 L/min drying gas flow, 40,000 Pa nebulizer gas pressure and 4000 V capillary voltage. Fullscan LC/MS chromatograms were obtained by scanning from m/z 300–1100 with a scan time of 0.75 s.
2.4. Methanol extraction of terrapin tissues and protein phosphatase analysis for free microcystins One portion of the frozen dried tissues of each terrapin was extracted at room temperature for 12 h in methanol: 5, 10, and 20 mL/g for liver, viscera, and muscle, respectively. The different extracts were filtered (GF/C, Millipore, France) and the residues were again re-extracted two times with fresh methanol in the same conditions stated above. The combined methanol extracts were evaporated to dryness in vacuo on a rotary evaporator. The resultant brown oils were dissolved in methanol and kept at 20 1C until analysis. The different tissue extracts of each terrapin were then assayed for the ability to inhibit the catalytic subunit of
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Fig. 1. Map showing the location of the study area. The star indicates the sampling site of cyanobacterial bloom samples and the carcasses of the two terrapin species Emys orbicularis and Mauremys leprosa.
PP2A phosphatase, and the equivalent unit quantity of MCYST-LR was determined according to Bouaı¨ cha et al. (2002).
2.5. Lemieux oxidation of terrapin tissues A quantity of 100 mg of each dried terrapin tissues (viscera, liver, and muscle) was subjected to exhaustive Lemieux oxidation (Williams et al., 1997a, b; Ott and Carmichael, 2006). After 3 h of oxidation reaction (Ott and Carmichael, 2006), the erythro-2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) was converted to its methyl ester using 12% trifluoroborate in methanol according to Kaya and Sano (1999).
2.6. Preparation of the methyl ester of MMPB The methyl ester of the MMPB used as a standard was prepared as follows: cyanobacterial cells from a bloom of Microcystis spp. were collected as a thick scum from the water surface in the lake Oubeira (Algeria). The bloom material was dried (50 1C, 1 h) and then extracted three times (overnight at 4 1C) with 100 mL/g of 75% (v/v) aqueous
methanol. Extracts were centrifuged (2000g, 10 min) and supernatants pooled and diluted to a 20% methanol final concentration. The 20% (v/v) aqueous methanol extract was then preconcentrated over a Bakerbond SPE cartridge (Baker, The Netherlands) according to the method described by Maatouk et al. (2002). The toxin fraction after solid phase extraction was then evaporated to dryness under nitrogen and dissolved in 0.4 mL of 1 M K2CO3, and was reacted with 10 mg of sodium metaperiodate and 0.4 mL of 0.024 M potassium permanganate as described in Kaya and Sano (1999). After 3 h of oxidation reaction (Ott and Carmichael, 2006), the MMPB was extracted with 10 ml of n-hexane and then converted to its methyl ester using 14% trifluoroborate in methanol as described in Kaya et al. (1985). The methyl ester of MMPB was then purified using high performance liquid chromatography on a HP1090 LC system (Agilent Technologies, France) coupled to a diode array detector, and a Partisil silica (150 4.6 mm i.d., 5 mm) column using n-hexane with 20% ethyl acetate as mobile phase, flow-rate 1 mL/min and detection absorbance at 254 nm. The purified methyl ester of MMPB was then analyzed by capillary GC/MS as described below, and its mass spectrum was compared to that reported by Kaya and Sano (1999).
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2.7. GC/MS analysis and quantification of the methyl ester of MMPB from oxidized terrapin tissues
triplicate using the m/z 190.10, 131.00, and 75.05 calibration ions at retention time of 14.73 min.
Capillary GC/MS analysis was carried out on a HP-5890 (HewlettPackard) gas chromatograph linked to HP-5973 Mass Selective Detector. A HP-1 capillary column (0.32 mm 30 m) was used with helium as carrier gas (flow-rate: 1.2 mL/min; splitless). The program rate for the analysis was 80 1C (1 min) to 250 1C at 6 1C/min. The other conditions were as follows: source 190 1C, injector temperature 250 1C, detector temperature 250 1C, and interface temperature 280 1C. Calibration curve was established for the standard, methyl ester of the MMPB, with a retention time of 14.73 min. Curve was developed in triplicate on a SIM (Singal Ion Monitoring) mode for the three strong fragment ions with m/z 190.10, 131.00, and 75.05 of the methyl ester of the MMPB standard. The concentration of the standard injected ranged from 0.1 to 50 ng in 1.0 mL of n-hexane. Oxidized tissue extracts of terrapins were then analyzed in
3. Results 3.1. Characterisation of dominant cyanobacteria and the dead terrapin species Based on the microscopic analyses, the cyanobacteria communities were dominated by the genus Microcystis. Nine morphotypes of Microcystis (Fig. 2) showing diverse variations in individual cell sizes, colony morphology and size, and mucilage characteristics were observed in the cyanobacterial bloom sample collected in October 2005 in
Fig. 2. Different morphotypes of the genus Microcystis observed in the cyanobacterial bloom sample collected in Lake Oubeira in October 2005.
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Table 1 Morphological characteristics of Microcystis colonies (morphospecies) observed in Lake Oubeira (October 2005) Morphospecies
Diameter of cells (mm)
Shape of colonies
Cell density
Mucilaginous margin
A
5.1 (4–6)
Densely
Distinct, not very wide margin around cells
B C
4.83 (4–6) 5.7 (4.5–6)
More or less spherical, with one distinct hole Spherical Shaped arc
Distinct, not very wide margin around cells Distinct, not very wide margin around cells
D E F
5 (4.5–6) 3.26 (2.5–3.5) 4 (3–4.5)
Shaped stick Irregular, later becoming rounded or elongated Two subcolonies with distinct holes
G
4.5 (4–5)
H
5.4 (5–6)
I
4.5 (4–5)
Elongated and irregular in outline, spheroidal lobate Irregular in outline and composed of small subcolonies Elongate colonies
Densely Very densely and irregular agglomerated Densely Densely aggregated in all the mucilage surface Very densely and irregular agglomerated cells Densely and irregular agglomerated cells Densely packed cells Densely and irregular agglomerated cells
Distinct, with delimited smooth margin Indistinct, diffuse, slightly and irregularly enveloping cells there is no wide mucilaginous envelope Distinct, not very wide margin around cells
Distinct, not very wide margin around cells Colourless, very thick and distinctly exceeding the outline of cell cluster by more than 20 mm Distinct, not very wide margin around cells
Diameter of cells: median (typical range).
peripheral fringes of the lake or it covered the entire lake, and in this case the mortality of the terrapin could probably reach several tens of individuals. Necropsy revealed liver crumbling in M. leprosa, but not E. orbicularis. The fresh weights of recuperated livers are 2.1070.07 and 16.0570.35 g, respectively. 3.2. Microcystins quantification and variants identification in the cyanobacterial bloom extract
Fig. 3. Surface bloom of Microcystis spp. in Lake Oubeira (Algeria) associated with freshwater turtle deaths.
Lake Oubeira (Table 1). According to Koma´rek and Anagnostidis (1999), morphospecies A, F, G, and I can be assigned as Microcystis aeruginosa, morphospecies E as M. panniformis, and morphospecies I as M. botrys. However, morphospecies B, C, and D are likely young stages of known species and, therefore, could not be unequivocally determined to species level. Twelve dead terrapins (eight individuals of the species M. leprosa and four individuals of the species E. orbicularis) were observed in a small area (0.5 ha) where a scum of Microcystis spp. bloom was observed on only one accessible shore of Lake Oubeira (Fig. 3). Due to the large surface area of the lake (2200 ha) and the difficulty of access to all the places, sampling did not covered large parts of this lake. Therefore, we cannot confirm if the bloom is limited to the
The total microcystin content per phytoplankton biomass was estimated to 1.12 mg MCYST-LR equivalents/g dried bloom material. The identification of microcystin variants in this extract was carried out by LC/MS analysis. Positive-ion mass spectra are shown in Fig. 4. In the mass range of m/z 300–1100 Da numerous peaks were detected, of which m/z 995.7, 1038.5, and 1045.5 [M+H]+ ions assigned these as MCYST-LR, microcystin-RR (MCYSTRR), and microcystin-YR (MCYST-YR), respectively. 3.3. Microcystin concentrations in terrapin tissues Table 2 summarizes the total levels of MCYST-LR equivalents in terrapin liver, viscera and muscle tissues as determined using the GC/MS after Lemieux oxidation as well as the levels of methanol extractable MCYST-LR equivalents as determined using the Protein phosphatase inhibition (PP2A) assay. The PP2A inhibition assay analysis of methanol extracts of the fresh carcasses of the two terrapin species tissues showed very low levels of microcystins. In contrast, the Lemieux oxidation method showed a high concentration of microcystins. Of the total amount of MCYST-LR equivalents calculated in the two terrapin tissues, up to 99% was detected by the Lemieux
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Fig. 4. LC/MS spectra of the crude extract of Microcystis spp. bloom sample (October 2005) exhibit parent ions [M+H]+ of MCYST-LR (m/z: 995.7), MCYST-YR (m/z: 1045.5), and MCYST-RR (m/z: 1038.5).
Table 2 Comparison of the Lemieux oxidation-GC/MS method and the protein phosphatase (PP2A) inhibition assay for the analysis of microcystins in viscera, liver, and muscle tissues of fresh carcasses of two terrapin species Emys orbicularis and Mauremys leprosa collected in Lake Oubeira (Algeria) in October 2005 Terrapin tissues
Viscera Liver Muscle Total
PP2A assays (mg MCYST-LR equivalent/g dw)
Lemieux oxidation-GC/MS (mg MCYST-LR equivalent equivalent/g dw)
Emys orbicularis
Mauremys leprosa
Emys orbicularis
Mauremys leprosa
0.006 (1.95%) 0.300 (97.7%) 0.001 (0.35%) 0.307
0.02 (6.77%) 0.27 (87%) 0.02 (6.23%) 0.31
37.19 (52.8%) 23.84 (33.8%) 9.40 (13.4%) 70.43
90.25 (6.98%) 1192.80 (92.2%) 10.13 (0.82%) 1293.18
Each value represents an average amount of microcystins in the organs of two individuals. The value in the brackets represents the percentage of microcystins in each terrapin tissue compared to the total level of microcystins.
oxidation method. The total amount of MCYST-LR equivalents detected by the PP2A assay in the viscera, liver and muscle tissues of the species E. orbicularis (0.307 mg/g dw) was similar than that detected in the species M. leprosa (0.31 mg/g dw). Up to 97.7% and 87% of these total amounts were accumulated in the liver tissues of E. orbicularis and M. leprosa, respectively. However, the total amount of microcystins detected by Lemieux oxidation was higher in the species M. leprosa (1293.2 mg/g dw)
than that detected in the species E. orbicularis (70.4 mg/g dw), with up to 92.2% of this total amount. In contrast, the total amount of microcystins E. orbicularis was distributed in the viscera (52.8%), then the liver (33.8%), and muscle (13.4%) tissues. In the viscera tissue of the fresh carcass of the species E. orbicularis, the concentration of MCYST-LR equivalents detected using the GC/MS after Lemieux oxidation was 6200 times than that detected using the PP2A assay, and representing 52.8% and 1.95% of total
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microcystin levels, respectively. However, in the viscera tissue of the fresh carcass of the species M. leprosa, although the concentration of MCYST-LR equivalents detected using the GC/MS after Lemieux oxidation was approximately 4500 times than that detected using the PP2A assay, these concentrations represent similar percentage of total microcystin levels (Table 2) when calculated using the two analytical methods. 4. Discussion Animal mortalities due to cyanobacterial toxins can occur by two routes: through consumption of cyanobacterial cells from the water, or indirectly through consumption of other animals that have themselves fed on cyanobacteria and accumulated cyanotoxins. Consequently, there is considerable potential for toxic effects to be magnified in aquatic food chains. The massive development of cyanobacteria is common in warm countries and in eutrophic reservoirs. Within the North-African basin, in Egypt (Abdel-Rahman et al., 1993; Brittain et al., 2000; Mohamed et al., 2003) and in Morocco (Oudra et al., 2001, 2002; Sabour et al., 2002) microcystins have been detected in reservoirs during the warmest months particularly in summer and in autumn. In Algeria, the presence of toxic cyanobacterial blooms dominated by the genus Microcystis was first established in Lake Oubeira in 2000–2001 (Nasri et al., 2004). The results of 2001 study showed that microcystin concentrations in the lake water ranged between 3 and 29,163 mg MCYST-LR equivalents per liter with a peak in August 2001. In October 2005, the death of two species of freshwater turtle was observed in this lake, during a Microcystis spp. bloom (Fig. 3). As shown in Fig. 2 and in Table 1, morphospecies A, F, H, and I, morphospecies E, and morphospecies H can assign as M. aeruginosa, M. panniformis, and M. botrys, respectively. However, the three other morphospecies B, C, and D observed in the bloom samples could not be unequivocally determined to species level and thus were grouped as Microcystis spp. In previous studies, Nasri et al. (2004) reported that eight morphospecies were observed in a bloom of Microcystis in Lake Oubeira in which only four morphospecies: M. aeruginosa, M. ichthyoblabe, M. wesenbergii, and M. parasitica have been clearly distinguished. It is inferred that terrapins died of cyanobacterial hepatotoxicoses due to the ingestion of microcystins. Indeed, during this period microcystins were detected in cyanobacterial sample from Lake Oubeira and in the liver, viscera, and muscle tissues of dead terrapins. The total microcystin content per phytoplankton biomass evaluated with the methanol extraction-PPase methodology was 1.12 mg MCYST-LR equivalents/g dried bloom material. The analysis of this bloom extract by the LC/MS technique demonstrated the presence of three microcystin variants (Fig. 4): MCYST-LR, MCYST-YR, and MCYST-RR. However, it is possible that the turtles have already been weakened by other anthropogenic
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toxicants and this increases their susceptibility to cyanotoxin poisoning. Since microcystins covalently bind to the protein phosphatases (PP1 and PP2A), conventional methods such as organic extraction only extract the unbounded portions of the toxin, which will lead to a false determination of total toxin concentration. Thus, in this study, a comparison of microcystin concentrations as determined by the Lemieux oxidation-GC/MS methodology and the methanol extraction-PPase methodology has been used to look for evidence of covalently bound forms of microcystins in the viscera, liver, and muscle tissues of two died species of freshwater turtles, M. leprosa and E. orbicularis. Free microcystins was detected in the liver, viscera and in the muscle tissues of the fresh carcasses of the two terrapin species, and the highest concentrations were located in the liver tissues (Table 2). Free microcystins in the visceral tissues of E. orbicularis was present in concentration 11 times the concentration in the muscle tissue (Table 2). However, free microcystins in the visceral tissues of M. leprosa was present in concentration approximately similar to that in muscle tissue (Table 2). In contrast, when we determine both free and bound microcystin concentrations, the results obtained for these two species (Table 2) show that greater than 99% of the total level of microcystins was not detected by the methanol extraction-PPase methodology. In fact, microcystins are known to interact with PP1 and PP2A by a two-step mechanism involving initial rapid reversible binding and inactivation of PPase activity followed by slower formation of an irreversible covalent linkage between toxin and enzyme (Mackintosh et al., 1995; Runnegar et al., 1995). Williams et al. (1997b) demonstrated with Atlantic salmon, which had received an i.p. injection of MCYST-LR, that the PPase inhibition assay is compromised as more than 60% of the total microcystin burden is bound covalently and irreversibly in the liver tissue. In the same work, authors showed that the Lemieux oxidation-GC/MS method detected 10,000-fold greater microcystin concentrations in Cypress Island Dungeness crab larvae than did the methanol extraction-PPase method. In the present study, the total level of microcystins detected by the Lemieux oxidation technique in the tissue samples of the terrapin species M. leprosa was approximately 4000-fold greater than that detected by the PP2A assay. However, in the species E. orbicularis was approximately 230-fold greater. The difference in the total level of microcystins observed between these two species can be explained by their natural diet. The species E. orbicularis is carnivorous and carrion feeder. Its diet consists of molluscs, watery insects, fish corpses even of frogs, tritons, or tadpoles. Thus, this terrapin species can accumulate microcystins particularly by feeding on aquatic organisms that contained bound covalently microcystins. In fact, as shown in Table 2, the highest concentration of toxin detected by the Lemieux oxidation-GC/MS method, indicating of free and covalently bound microcystins, in the terrapin species
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E. orbicularis was observed in the viscera, with 52.8% of the total toxin levels, and was approximately 6200 times the concentration detected by the methanol extraction-PPase methodology (1.95% of the total toxin levels). However, M. leprosa is an omnivorous species, with prevalence carnivore. Its natural diet is highly biased towards filamentous algae, insect larvae, earthworms, molluscs, small amphibians and tadpoles, and various aquatic plants. Thus, this terrapin species can accumulate microcystins particularly by direct uptake of free cyanobacterial cell toxins from the water. Although the toxin concentration detected in the viscera of the species M. leprosa by the Lemieux oxidation-GC/MS method (90.25 mg MCYST-LR equivalents/g dw) was higher than that detected by the methanol extraction-PPase methodology (0.02 mg MCYSTLR equivalents/g dw), these concentrations represent similar percentage of the total toxin levels with the two analytical methods (6.77% and 6.98%, respectively). As shown in Table 2, up to 92.2% of the total microcystin levels were accumulated in the liver tissue of the species M. leprosa. The indication of hepatotoxicoses, consistent with microcystins poisoning, was provided both by the accumulation of microcystins in the liver tissues and by the crumbling of the liver observed after the necropsy examination of the fresh carcass of this species. In consequence, the liver weight of the species M. leprosa (2.1070.07 g) was 8 times lower when compared to that of E. orbicularis (16.0570.35 g). In fact, toxicity of microcystins is mediated through the active transport of these toxins into hepatocytes by the bile acid organic anion transport system, followed by inhibition of eukaryotic serine/threonine protein phosphatases 1 and 2A (Eriksson et al., 1990). This inhibition leads to hyperphosphorylation of proteins associated with the cytoskeleton in hepatocytes (Toivola et al., 1997). Therefore, the rapid loss of the sinusoidal architecture and attachment to one another leads to the accumulation of blood in the liver, and death most often results from hemorrhagic shock. Indeed, acute poisoning, leading to death from massive hepatic hemorrhage, has been reported to occur in both animals and humans (Beasley et al., 1989; Jochimsen et al., 1998; Pouria et al., 1998). Even though the liver is the target organ for microcystins, these two species and particularly the species E. orbicularis were found to accumulate toxins in the viscera and muscle tissues also. Mohamed et al. (2003) reported that tilapia fish accumulated microcystins in the liver, kidney and muscle tissues. Furthermore, several studies have been reported that the liver is the major target organ for microcystins toxicities, it was shown to accumulate 20–70% of a radioactively labeled toxin dose (i.v.) in mice (Brooks and Codd, 1987; Falconer et al., 1986). 5. Conclusions A contribution of the cyanobacterial toxins to the deaths of the freshwater terrapin species E. orbicularis
and M. leprosa in Lake Oubeira (Algeria) during October 2005 is suggested by: (a) the presence of high concentrations of microcystins in the Microcystis spp. bloom sample occurred during the deaths of terrapins; (b) the presence of high concentrations of the cyanobacterial microcystins in terrapin viscera and liver contents; and (c) observations of the liver crumbling after the necropsy of the fresh carcass of the species M. leprosa. Intoxication with cyanobacterial microcystins could occur either by direct toxicoses from consuming contaminated water or indirectly by the food chain. Although the most usual route of human intoxication is drinking contaminated water, the accumulation of toxins in the food chain may increase the number of intoxications and heighten the long-term effects, including the risk of hepatocancer. Therefore, the presence of these hepatotoxins in Lake Oubeira led us to suggest that monitoring programs of cyanobacteria and their toxins should be implemented. Acknowledgments We acknowledge the critical comments of the referees and the help of Dr S. Ghouti (The language Department, University of Paris-Sud 11, UFR Pharmacy) for critically reading of the manuscript. We acknowledge the help of Dr. R. Zenki (Veterinary Doctor) for the necropsy of dead terrapins. This study was supported in France by grant from the Ministe`re de l’Enseignement Supe´rieur de la Recherche et de la Technologie (EA3542) and in Algeria by the National Agency for the Development of Research in Health (A.N.D.R.S. 03050004124); The National Observatory of Environment and Durable Development (O.N.E.D.D., Code 222), and a fellowship to Hichem Nasri from the Ministry for Higher Education and Scientific Research (M.E.R.S.), Algeria. We confirm that no studies involving humans or experimental animals were conducted in this work. References Abdel-Rahman, S., El-Ayouty, Y.M., Kamael, H.A., 1993. Characterization of heptapeptide toxins extracted from Microcystis aeruginosa (Egyptian isolate)—comparison with some synthesized analogs. Int. J. Pept. Protein Res. 41, 1–7. Beasley, V.R., Dahlem, A.M., Cook, W.O., Valentine, W.M., Lovell, R.A., Hooser, S.B., Harada, K.I., Suzuki, M., Carmichael, W.W., 1989. Diagnostic and clinically important aspects of cyanobacterial (blue-green algae) toxicoses. J. Vet. Diagn. Invest. 1, 359–365. Brittain, S., Mohamed, Z.A., Wang, J., Lehmann, V.K.B., Carmichael, W.W., Rinehart, K.L., El-Sharouny, H.M., 2000. Isolation and characterization of microcystins from a River Nile strain of Oscillatoria tenuis Agardh ex Gomont. Toxicon 38, 1759–1771. Brooks, W.P., Codd, G.A., 1987. Distribution of Microcystis aeruginosa peptide toxin and interactions with hepatic microsomes in mice. Pharm. Toxicol. 60, 187–191. Bouaı¨ cha, N., Maatouk, I., Vincent, G., Levi, Y., 2002. A colorimetric and fluorometric microplate assay for the detection of microcystin-LR in drinking water without preconcentration. Food Chem. Toxicol. 40, 1677–1683.
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Ecotoxicology and Environmental Safety 71 (2008) 535–544 www.elsevier.com/locate/ecoenv
First reported case of turtle deaths during a toxic Microcystis spp. bloom in Lake Oubeira, Algeria Hichem Nasria,b, Soumaya El Herryb, Noureddine Bouaı¨ chab, a
Institute of Biology, University of El Taref, Algeria Laboratoire Sante´ Publique-Environnement, 5 Rue J.B. Cle´ment, Universite´ Paris-Sud 11, UFR de Pharmacie, 92296 Chaˆtenay-Malabry, France
b
Received 4 April 2007; received in revised form 7 December 2007; accepted 8 December 2007 Available online 30 January 2008
Abstract Microcystins analysis was conducted in field cyanobacterial bloom samples and dead terrapin tissues from Lake Oubeira (Algeria) with an aim of studying the cause of the mortality of the freshwater terrapin species Emys orbicularis and Mauremys leprosa during October 2005. The deaths of these two terrapin species were observed during a bloom of Microcystis spp. The total microcystin content per phytoplankton biomass evaluated with the methanol extraction-protein phosphatase methodology was 1.12 mg MCYST-LR equivalents/g dried bloom material. The analysis of this bloom extract by the LC/MS technique demonstrated the presence of three microcystin variants: microcystin-LR (MCYST-LR), microcystin-YR (MCYST-YR), and microcystin-RR (MCYST-RR). Microcystins were also detected in fresh carcasses of terrapin liver, viscera and muscle tissues using the GC/MS after Lemieux oxidation method and the PP2A inhibition assay. The high level of microcystins detected using the Lemieux oxidation-GC/MS method in the liver tissue (1192.8 mg MCYST-LR equivalent/g dw) and in the viscera tissue (37.19 mg MCYST-LR equivalent/g dw) of the species M. leprosa and E. orbicularis, respectively, and the liver crumbling observed after the necropsy examination of the fresh carcass of M. leprosa support the possibility that cyanobacterial microcystins contribute to the turtle mortalities. r 2007 Elsevier Inc. All rights reserved. Keywords: Freshwater terrapin; Cyanobacteria; Microcystis; Microcystins; Lake Oubeira; Algeria
1. Introduction In the present study, we report for the first time the possibility that the toxic Microcystis bloom in Lake Oubeira, Algeria, may contribute to freshwater turtle deaths. Since the first report of livestock poisoning associated with cyanobacterial blooms (Francis, 1878), numerous cases of animal deaths have been reported worldwide due to contact with cyanobacterial scums, indicating that this is a widespread phenomenon in Mediterranean, continental, and temperate climates in both hemispheres (Codd et al., 2005; Falconer, 2005). Lake Oubeira, one of the largest freshwater lake in Algeria, is an important natural reserve for migration birds and wildfowl species. It is also home to the two freshwater turtles Emys orbicularis (Linnaeus, 1758) and Mauremys Corresponding author. Fax: +33 1 46 83 57 32.
E-mail address: [email protected] (N. Bouaı¨ cha). 0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.12.009
leprosa (Schweigger, 1812). This lake provides considerable benefits for the region functioning as a drinking water source for many urban communities. However, during the past decades the lake has witnessed a steady increase in eutrophication, characteristic of a regular occurrence of toxic Microcystis blooms and high concentrations of microsystins (Nasri et al., 2004). Microsystins are cyclic peptide hepatotoxins causing liver damage by inhibition of serine/threonine protein phosphatase enzymes (Eriksson et al., 1990). The increase in bloom occurrence has led the authorities to cease using this lake as a source for the production of drinking water. However, it is still used for irrigation and aquaculture. Cyanotoxins are known to bioaccumulate in common aquatic vertebrates and invertebrates, including fish, mussels, and zooplankton (Eriksson et al., 1989; Kotak et al., 1996; Prepas et al., 1997; Watanabe et al., 1997; Williams et al., 1997a; Thostrup and Christoffersen, 1999; Magalha˜es et al., 2001; Sipia¨ et al., 2001; Mohamed et al., 2003; Chen and Xie, 2005; Chen
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et al., 2005, 2007), which poses a risk to both animal and human heath if such aquatic animals are consumed (Carmichael and Falconer, 1993). The toxicity of microcystins is associated with irreversible binding and inhibition of these toxins to protein phosphatases 1 and 2A in the liver (Mackintosh et al., 1990, 1995; Yoshizawa et al., 1990), which has important implications for the quantitative determination of these toxins in food webs. Furthermore, the detection methods for microcystins, involved determination of the amount of toxins in methanol extracts of homogenized tissue via either a direct protein phosphatase (PPase) inhibition assay or an antibody assay, can underestimate the concentration of microcystins in the tissue sample. Nevertheless, a new analytical approach capable of detecting both free and covalently bound microcystin fractions has been described (Williams et al., 1997a, b; Kaya and Sano, 1999). This analytical methodology is based upon the chemically unique nature of the Adda [(2S, 3S, 8S, 9S)-3-amino-9methoxy-2,6,8-trimethyl-10-phenyl-4,6-dienoic acid] residue of the microcystins. It has been showed that Lemieux oxidation of pure microcystin-LR (MCYST-LR), or MCYST-LR present in methanol extracts of cyanobacteria blooms, gives 2-methyl-3-methoxy-4-phenylbutanoic acid in high yield (Sano et al., 1992). This last butanoic acid represents a unique marker for the presence of microcystins and, therefore, it can be readily isolated from Lumieux oxidation reactions and detected at low concentrations by GC/MS (Williams et al., 1997a, b; Kaya and Sano, 1999). In October 2005, 12 freshwater turtles were found dead in a small area (0.5 ha) of Lake Oubeira, Algeria. The present study is the first attempt to evaluate the contribution of cyanobacterial poisoning to the turtle deaths. The quantification of microcystins in animal samples was achieved using both the protein phosphatase assay and the Lemieux oxidation GC/MS method. The identification of the different microcystin variants in the cyanobacterial bloom sample was achieved by the LC/MS technique. 2. Materials and methods 2.1. Study site and characterisation of the natural diet of terrapin species Lake Oubeira (361530 N and 0081230 E, Fig. 1) located in north-eastern Algeria is the home to El Kala National Park (PNEK) and has an average elevation of 25 m above sea level. It is the first largest freshwater lake in Algeria, and has an estimated surface area of 2200 ha and a maximum depth of about 4 m. In 1984, the lake was included as a wetland under the Ramsar Convention (Ramsar Convention Official Website, 2007, www.ramsar.org). Lake Oubeira is an important natural reserve for migratory birds and wildfowl species. Two species of terrapin are also present in this lake, E. orbicularis (Linnaeus, 1758) and M. leprosa (Schweigger, 1812). The E. orbicularis is carnivorous and carrion feeder. Its diet consists of molluscs, watery insects, fish corpses even of frogs, tritons, or tadpoles (Lebboroni and Chelazzi, 1991; Kotenko, 2000). However, M. leprosa is an omnivorous species, with prevalence carnivore. Its natural diet is highly biased towards filamentous algae, insect larvae,
earthworms, molluscs, small amphibians and tadpoles, and various aquatic plants. It is also known to be particularly partial to carrion.
2.2. Sample collection In October 2005, cyanobacterial samples were hand collected on one site (Fig. 1) from off the northern shore of Lake Oubeira were scum formation was visible. The bloom samples were fixed in formalin (5% final volume) for cyanobacteria determination under light microscopy, or dried and stored at 20 1C for later cyanobacterial toxin analysis. Dominant species of cyanobacteria were determined using Koma´rek and Anagnostidis (1999) based on cell structure and dimension, and colony morphology, and mucilage characteristics and photographically documented using an Eclipse TE200 light microscope (Nikon, Paris, France). The cell diameter was determined for 50 cells (10 cells from five different colonies of each morphospecies of Microcystis). Because of avian influenza and banning access by the health authorities to the PNEK, and in particular Lake Oubeira, only 2 (out of the observed 12) turtles of each species were allowed to be used for the toxin analysis. The two terrapin species were identified according to Fritz (1992, 1996); Duguy and Baron (1998) and Kotenko (2000). The two fresh carcasses of each terrapin species E. orbicularis and M. leprosa were necropsied and the livers were weighed and examined externally for abnormalities. The terrapin tissues (viscera, liver, and meet) were immediately frozen at 20 1C and then freeze-dried for later microcystins analysis.
2.3. LC/MS analysis of the cyanobacterial bloom extract The LC/MS technique was carried out on an Ion Trap LC/MS system (Bruker, France) and a Hewlett Packard (France) HP-1100 Series system equipped with a binary solvent pump coupled to an analytical work station. The bloom material was dried (50 1C, 1 h) and then extracted three times (overnight at 4 1C) with 100 mL/g of 75% (v/v) aqueous methanol. Extracts were centrifuged (2000g, 10 min.) and supernatants pooled and diluted to a 20% methanol final concentration. The total microcystin content per phytoplankton biomass was then evaluated by the PP2A inhibition assay as described below. The 20% (v/v) aqueous methanol extract was then preconcentrated over a Bakerbond SPE cartridge (Baker, The Netherlands) according to the method described by Maatouk et al. (2002). The toxin fraction after solid phase extraction was then evaporated to dryness under nitrogen and dissolved in 1 mL of 50% (v/v) aqueous methanol. This last fraction was then chromatographed on an Inertsil ODS (150 mm 2 mm i.d., 5 mm) stainless steel column with a guard column LiChrosorb RP-18 (35 mm 4.6 mm, 5 mm) both from Chrompack (Les Ulis, France) using acetonitrile (solvent A) and water with 0.5% formic acid (solvent B) as mobile phase. The gradient program selected for LC/MS was set: 0 min 20% A, 10 min 40% A, 15 min 60% A, 20 min 80% A, and 25 min 100% A, flow-rate 0.3 mL/min. The ES/MS interface in positive mode operated at 350 1C gas temperature, 13 L/min drying gas flow, 40,000 Pa nebulizer gas pressure and 4000 V capillary voltage. Fullscan LC/MS chromatograms were obtained by scanning from m/z 300–1100 with a scan time of 0.75 s.
2.4. Methanol extraction of terrapin tissues and protein phosphatase analysis for free microcystins One portion of the frozen dried tissues of each terrapin was extracted at room temperature for 12 h in methanol: 5, 10, and 20 mL/g for liver, viscera, and muscle, respectively. The different extracts were filtered (GF/C, Millipore, France) and the residues were again re-extracted two times with fresh methanol in the same conditions stated above. The combined methanol extracts were evaporated to dryness in vacuo on a rotary evaporator. The resultant brown oils were dissolved in methanol and kept at 20 1C until analysis. The different tissue extracts of each terrapin were then assayed for the ability to inhibit the catalytic subunit of
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Fig. 1. Map showing the location of the study area. The star indicates the sampling site of cyanobacterial bloom samples and the carcasses of the two terrapin species Emys orbicularis and Mauremys leprosa.
PP2A phosphatase, and the equivalent unit quantity of MCYST-LR was determined according to Bouaı¨ cha et al. (2002).
2.5. Lemieux oxidation of terrapin tissues A quantity of 100 mg of each dried terrapin tissues (viscera, liver, and muscle) was subjected to exhaustive Lemieux oxidation (Williams et al., 1997a, b; Ott and Carmichael, 2006). After 3 h of oxidation reaction (Ott and Carmichael, 2006), the erythro-2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) was converted to its methyl ester using 12% trifluoroborate in methanol according to Kaya and Sano (1999).
2.6. Preparation of the methyl ester of MMPB The methyl ester of the MMPB used as a standard was prepared as follows: cyanobacterial cells from a bloom of Microcystis spp. were collected as a thick scum from the water surface in the lake Oubeira (Algeria). The bloom material was dried (50 1C, 1 h) and then extracted three times (overnight at 4 1C) with 100 mL/g of 75% (v/v) aqueous
methanol. Extracts were centrifuged (2000g, 10 min) and supernatants pooled and diluted to a 20% methanol final concentration. The 20% (v/v) aqueous methanol extract was then preconcentrated over a Bakerbond SPE cartridge (Baker, The Netherlands) according to the method described by Maatouk et al. (2002). The toxin fraction after solid phase extraction was then evaporated to dryness under nitrogen and dissolved in 0.4 mL of 1 M K2CO3, and was reacted with 10 mg of sodium metaperiodate and 0.4 mL of 0.024 M potassium permanganate as described in Kaya and Sano (1999). After 3 h of oxidation reaction (Ott and Carmichael, 2006), the MMPB was extracted with 10 ml of n-hexane and then converted to its methyl ester using 14% trifluoroborate in methanol as described in Kaya et al. (1985). The methyl ester of MMPB was then purified using high performance liquid chromatography on a HP1090 LC system (Agilent Technologies, France) coupled to a diode array detector, and a Partisil silica (150 4.6 mm i.d., 5 mm) column using n-hexane with 20% ethyl acetate as mobile phase, flow-rate 1 mL/min and detection absorbance at 254 nm. The purified methyl ester of MMPB was then analyzed by capillary GC/MS as described below, and its mass spectrum was compared to that reported by Kaya and Sano (1999).
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2.7. GC/MS analysis and quantification of the methyl ester of MMPB from oxidized terrapin tissues
triplicate using the m/z 190.10, 131.00, and 75.05 calibration ions at retention time of 14.73 min.
Capillary GC/MS analysis was carried out on a HP-5890 (HewlettPackard) gas chromatograph linked to HP-5973 Mass Selective Detector. A HP-1 capillary column (0.32 mm 30 m) was used with helium as carrier gas (flow-rate: 1.2 mL/min; splitless). The program rate for the analysis was 80 1C (1 min) to 250 1C at 6 1C/min. The other conditions were as follows: source 190 1C, injector temperature 250 1C, detector temperature 250 1C, and interface temperature 280 1C. Calibration curve was established for the standard, methyl ester of the MMPB, with a retention time of 14.73 min. Curve was developed in triplicate on a SIM (Singal Ion Monitoring) mode for the three strong fragment ions with m/z 190.10, 131.00, and 75.05 of the methyl ester of the MMPB standard. The concentration of the standard injected ranged from 0.1 to 50 ng in 1.0 mL of n-hexane. Oxidized tissue extracts of terrapins were then analyzed in
3. Results 3.1. Characterisation of dominant cyanobacteria and the dead terrapin species Based on the microscopic analyses, the cyanobacteria communities were dominated by the genus Microcystis. Nine morphotypes of Microcystis (Fig. 2) showing diverse variations in individual cell sizes, colony morphology and size, and mucilage characteristics were observed in the cyanobacterial bloom sample collected in October 2005 in
Fig. 2. Different morphotypes of the genus Microcystis observed in the cyanobacterial bloom sample collected in Lake Oubeira in October 2005.
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Table 1 Morphological characteristics of Microcystis colonies (morphospecies) observed in Lake Oubeira (October 2005) Morphospecies
Diameter of cells (mm)
Shape of colonies
Cell density
Mucilaginous margin
A
5.1 (4–6)
Densely
Distinct, not very wide margin around cells
B C
4.83 (4–6) 5.7 (4.5–6)
More or less spherical, with one distinct hole Spherical Shaped arc
Distinct, not very wide margin around cells Distinct, not very wide margin around cells
D E F
5 (4.5–6) 3.26 (2.5–3.5) 4 (3–4.5)
Shaped stick Irregular, later becoming rounded or elongated Two subcolonies with distinct holes
G
4.5 (4–5)
H
5.4 (5–6)
I
4.5 (4–5)
Elongated and irregular in outline, spheroidal lobate Irregular in outline and composed of small subcolonies Elongate colonies
Densely Very densely and irregular agglomerated Densely Densely aggregated in all the mucilage surface Very densely and irregular agglomerated cells Densely and irregular agglomerated cells Densely packed cells Densely and irregular agglomerated cells
Distinct, with delimited smooth margin Indistinct, diffuse, slightly and irregularly enveloping cells there is no wide mucilaginous envelope Distinct, not very wide margin around cells
Distinct, not very wide margin around cells Colourless, very thick and distinctly exceeding the outline of cell cluster by more than 20 mm Distinct, not very wide margin around cells
Diameter of cells: median (typical range).
peripheral fringes of the lake or it covered the entire lake, and in this case the mortality of the terrapin could probably reach several tens of individuals. Necropsy revealed liver crumbling in M. leprosa, but not E. orbicularis. The fresh weights of recuperated livers are 2.1070.07 and 16.0570.35 g, respectively. 3.2. Microcystins quantification and variants identification in the cyanobacterial bloom extract
Fig. 3. Surface bloom of Microcystis spp. in Lake Oubeira (Algeria) associated with freshwater turtle deaths.
Lake Oubeira (Table 1). According to Koma´rek and Anagnostidis (1999), morphospecies A, F, G, and I can be assigned as Microcystis aeruginosa, morphospecies E as M. panniformis, and morphospecies I as M. botrys. However, morphospecies B, C, and D are likely young stages of known species and, therefore, could not be unequivocally determined to species level. Twelve dead terrapins (eight individuals of the species M. leprosa and four individuals of the species E. orbicularis) were observed in a small area (0.5 ha) where a scum of Microcystis spp. bloom was observed on only one accessible shore of Lake Oubeira (Fig. 3). Due to the large surface area of the lake (2200 ha) and the difficulty of access to all the places, sampling did not covered large parts of this lake. Therefore, we cannot confirm if the bloom is limited to the
The total microcystin content per phytoplankton biomass was estimated to 1.12 mg MCYST-LR equivalents/g dried bloom material. The identification of microcystin variants in this extract was carried out by LC/MS analysis. Positive-ion mass spectra are shown in Fig. 4. In the mass range of m/z 300–1100 Da numerous peaks were detected, of which m/z 995.7, 1038.5, and 1045.5 [M+H]+ ions assigned these as MCYST-LR, microcystin-RR (MCYSTRR), and microcystin-YR (MCYST-YR), respectively. 3.3. Microcystin concentrations in terrapin tissues Table 2 summarizes the total levels of MCYST-LR equivalents in terrapin liver, viscera and muscle tissues as determined using the GC/MS after Lemieux oxidation as well as the levels of methanol extractable MCYST-LR equivalents as determined using the Protein phosphatase inhibition (PP2A) assay. The PP2A inhibition assay analysis of methanol extracts of the fresh carcasses of the two terrapin species tissues showed very low levels of microcystins. In contrast, the Lemieux oxidation method showed a high concentration of microcystins. Of the total amount of MCYST-LR equivalents calculated in the two terrapin tissues, up to 99% was detected by the Lemieux
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Fig. 4. LC/MS spectra of the crude extract of Microcystis spp. bloom sample (October 2005) exhibit parent ions [M+H]+ of MCYST-LR (m/z: 995.7), MCYST-YR (m/z: 1045.5), and MCYST-RR (m/z: 1038.5).
Table 2 Comparison of the Lemieux oxidation-GC/MS method and the protein phosphatase (PP2A) inhibition assay for the analysis of microcystins in viscera, liver, and muscle tissues of fresh carcasses of two terrapin species Emys orbicularis and Mauremys leprosa collected in Lake Oubeira (Algeria) in October 2005 Terrapin tissues
Viscera Liver Muscle Total
PP2A assays (mg MCYST-LR equivalent/g dw)
Lemieux oxidation-GC/MS (mg MCYST-LR equivalent equivalent/g dw)
Emys orbicularis
Mauremys leprosa
Emys orbicularis
Mauremys leprosa
0.006 (1.95%) 0.300 (97.7%) 0.001 (0.35%) 0.307
0.02 (6.77%) 0.27 (87%) 0.02 (6.23%) 0.31
37.19 (52.8%) 23.84 (33.8%) 9.40 (13.4%) 70.43
90.25 (6.98%) 1192.80 (92.2%) 10.13 (0.82%) 1293.18
Each value represents an average amount of microcystins in the organs of two individuals. The value in the brackets represents the percentage of microcystins in each terrapin tissue compared to the total level of microcystins.
oxidation method. The total amount of MCYST-LR equivalents detected by the PP2A assay in the viscera, liver and muscle tissues of the species E. orbicularis (0.307 mg/g dw) was similar than that detected in the species M. leprosa (0.31 mg/g dw). Up to 97.7% and 87% of these total amounts were accumulated in the liver tissues of E. orbicularis and M. leprosa, respectively. However, the total amount of microcystins detected by Lemieux oxidation was higher in the species M. leprosa (1293.2 mg/g dw)
than that detected in the species E. orbicularis (70.4 mg/g dw), with up to 92.2% of this total amount. In contrast, the total amount of microcystins E. orbicularis was distributed in the viscera (52.8%), then the liver (33.8%), and muscle (13.4%) tissues. In the viscera tissue of the fresh carcass of the species E. orbicularis, the concentration of MCYST-LR equivalents detected using the GC/MS after Lemieux oxidation was 6200 times than that detected using the PP2A assay, and representing 52.8% and 1.95% of total
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microcystin levels, respectively. However, in the viscera tissue of the fresh carcass of the species M. leprosa, although the concentration of MCYST-LR equivalents detected using the GC/MS after Lemieux oxidation was approximately 4500 times than that detected using the PP2A assay, these concentrations represent similar percentage of total microcystin levels (Table 2) when calculated using the two analytical methods. 4. Discussion Animal mortalities due to cyanobacterial toxins can occur by two routes: through consumption of cyanobacterial cells from the water, or indirectly through consumption of other animals that have themselves fed on cyanobacteria and accumulated cyanotoxins. Consequently, there is considerable potential for toxic effects to be magnified in aquatic food chains. The massive development of cyanobacteria is common in warm countries and in eutrophic reservoirs. Within the North-African basin, in Egypt (Abdel-Rahman et al., 1993; Brittain et al., 2000; Mohamed et al., 2003) and in Morocco (Oudra et al., 2001, 2002; Sabour et al., 2002) microcystins have been detected in reservoirs during the warmest months particularly in summer and in autumn. In Algeria, the presence of toxic cyanobacterial blooms dominated by the genus Microcystis was first established in Lake Oubeira in 2000–2001 (Nasri et al., 2004). The results of 2001 study showed that microcystin concentrations in the lake water ranged between 3 and 29,163 mg MCYST-LR equivalents per liter with a peak in August 2001. In October 2005, the death of two species of freshwater turtle was observed in this lake, during a Microcystis spp. bloom (Fig. 3). As shown in Fig. 2 and in Table 1, morphospecies A, F, H, and I, morphospecies E, and morphospecies H can assign as M. aeruginosa, M. panniformis, and M. botrys, respectively. However, the three other morphospecies B, C, and D observed in the bloom samples could not be unequivocally determined to species level and thus were grouped as Microcystis spp. In previous studies, Nasri et al. (2004) reported that eight morphospecies were observed in a bloom of Microcystis in Lake Oubeira in which only four morphospecies: M. aeruginosa, M. ichthyoblabe, M. wesenbergii, and M. parasitica have been clearly distinguished. It is inferred that terrapins died of cyanobacterial hepatotoxicoses due to the ingestion of microcystins. Indeed, during this period microcystins were detected in cyanobacterial sample from Lake Oubeira and in the liver, viscera, and muscle tissues of dead terrapins. The total microcystin content per phytoplankton biomass evaluated with the methanol extraction-PPase methodology was 1.12 mg MCYST-LR equivalents/g dried bloom material. The analysis of this bloom extract by the LC/MS technique demonstrated the presence of three microcystin variants (Fig. 4): MCYST-LR, MCYST-YR, and MCYST-RR. However, it is possible that the turtles have already been weakened by other anthropogenic
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toxicants and this increases their susceptibility to cyanotoxin poisoning. Since microcystins covalently bind to the protein phosphatases (PP1 and PP2A), conventional methods such as organic extraction only extract the unbounded portions of the toxin, which will lead to a false determination of total toxin concentration. Thus, in this study, a comparison of microcystin concentrations as determined by the Lemieux oxidation-GC/MS methodology and the methanol extraction-PPase methodology has been used to look for evidence of covalently bound forms of microcystins in the viscera, liver, and muscle tissues of two died species of freshwater turtles, M. leprosa and E. orbicularis. Free microcystins was detected in the liver, viscera and in the muscle tissues of the fresh carcasses of the two terrapin species, and the highest concentrations were located in the liver tissues (Table 2). Free microcystins in the visceral tissues of E. orbicularis was present in concentration 11 times the concentration in the muscle tissue (Table 2). However, free microcystins in the visceral tissues of M. leprosa was present in concentration approximately similar to that in muscle tissue (Table 2). In contrast, when we determine both free and bound microcystin concentrations, the results obtained for these two species (Table 2) show that greater than 99% of the total level of microcystins was not detected by the methanol extraction-PPase methodology. In fact, microcystins are known to interact with PP1 and PP2A by a two-step mechanism involving initial rapid reversible binding and inactivation of PPase activity followed by slower formation of an irreversible covalent linkage between toxin and enzyme (Mackintosh et al., 1995; Runnegar et al., 1995). Williams et al. (1997b) demonstrated with Atlantic salmon, which had received an i.p. injection of MCYST-LR, that the PPase inhibition assay is compromised as more than 60% of the total microcystin burden is bound covalently and irreversibly in the liver tissue. In the same work, authors showed that the Lemieux oxidation-GC/MS method detected 10,000-fold greater microcystin concentrations in Cypress Island Dungeness crab larvae than did the methanol extraction-PPase method. In the present study, the total level of microcystins detected by the Lemieux oxidation technique in the tissue samples of the terrapin species M. leprosa was approximately 4000-fold greater than that detected by the PP2A assay. However, in the species E. orbicularis was approximately 230-fold greater. The difference in the total level of microcystins observed between these two species can be explained by their natural diet. The species E. orbicularis is carnivorous and carrion feeder. Its diet consists of molluscs, watery insects, fish corpses even of frogs, tritons, or tadpoles. Thus, this terrapin species can accumulate microcystins particularly by feeding on aquatic organisms that contained bound covalently microcystins. In fact, as shown in Table 2, the highest concentration of toxin detected by the Lemieux oxidation-GC/MS method, indicating of free and covalently bound microcystins, in the terrapin species
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E. orbicularis was observed in the viscera, with 52.8% of the total toxin levels, and was approximately 6200 times the concentration detected by the methanol extraction-PPase methodology (1.95% of the total toxin levels). However, M. leprosa is an omnivorous species, with prevalence carnivore. Its natural diet is highly biased towards filamentous algae, insect larvae, earthworms, molluscs, small amphibians and tadpoles, and various aquatic plants. Thus, this terrapin species can accumulate microcystins particularly by direct uptake of free cyanobacterial cell toxins from the water. Although the toxin concentration detected in the viscera of the species M. leprosa by the Lemieux oxidation-GC/MS method (90.25 mg MCYST-LR equivalents/g dw) was higher than that detected by the methanol extraction-PPase methodology (0.02 mg MCYSTLR equivalents/g dw), these concentrations represent similar percentage of the total toxin levels with the two analytical methods (6.77% and 6.98%, respectively). As shown in Table 2, up to 92.2% of the total microcystin levels were accumulated in the liver tissue of the species M. leprosa. The indication of hepatotoxicoses, consistent with microcystins poisoning, was provided both by the accumulation of microcystins in the liver tissues and by the crumbling of the liver observed after the necropsy examination of the fresh carcass of this species. In consequence, the liver weight of the species M. leprosa (2.1070.07 g) was 8 times lower when compared to that of E. orbicularis (16.0570.35 g). In fact, toxicity of microcystins is mediated through the active transport of these toxins into hepatocytes by the bile acid organic anion transport system, followed by inhibition of eukaryotic serine/threonine protein phosphatases 1 and 2A (Eriksson et al., 1990). This inhibition leads to hyperphosphorylation of proteins associated with the cytoskeleton in hepatocytes (Toivola et al., 1997). Therefore, the rapid loss of the sinusoidal architecture and attachment to one another leads to the accumulation of blood in the liver, and death most often results from hemorrhagic shock. Indeed, acute poisoning, leading to death from massive hepatic hemorrhage, has been reported to occur in both animals and humans (Beasley et al., 1989; Jochimsen et al., 1998; Pouria et al., 1998). Even though the liver is the target organ for microcystins, these two species and particularly the species E. orbicularis were found to accumulate toxins in the viscera and muscle tissues also. Mohamed et al. (2003) reported that tilapia fish accumulated microcystins in the liver, kidney and muscle tissues. Furthermore, several studies have been reported that the liver is the major target organ for microcystins toxicities, it was shown to accumulate 20–70% of a radioactively labeled toxin dose (i.v.) in mice (Brooks and Codd, 1987; Falconer et al., 1986). 5. Conclusions A contribution of the cyanobacterial toxins to the deaths of the freshwater terrapin species E. orbicularis
and M. leprosa in Lake Oubeira (Algeria) during October 2005 is suggested by: (a) the presence of high concentrations of microcystins in the Microcystis spp. bloom sample occurred during the deaths of terrapins; (b) the presence of high concentrations of the cyanobacterial microcystins in terrapin viscera and liver contents; and (c) observations of the liver crumbling after the necropsy of the fresh carcass of the species M. leprosa. Intoxication with cyanobacterial microcystins could occur either by direct toxicoses from consuming contaminated water or indirectly by the food chain. Although the most usual route of human intoxication is drinking contaminated water, the accumulation of toxins in the food chain may increase the number of intoxications and heighten the long-term effects, including the risk of hepatocancer. Therefore, the presence of these hepatotoxins in Lake Oubeira led us to suggest that monitoring programs of cyanobacteria and their toxins should be implemented. Acknowledgments We acknowledge the critical comments of the referees and the help of Dr S. Ghouti (The language Department, University of Paris-Sud 11, UFR Pharmacy) for critically reading of the manuscript. We acknowledge the help of Dr. R. Zenki (Veterinary Doctor) for the necropsy of dead terrapins. This study was supported in France by grant from the Ministe`re de l’Enseignement Supe´rieur de la Recherche et de la Technologie (EA3542) and in Algeria by the National Agency for the Development of Research in Health (A.N.D.R.S. 03050004124); The National Observatory of Environment and Durable Development (O.N.E.D.D., Code 222), and a fellowship to Hichem Nasri from the Ministry for Higher Education and Scientific Research (M.E.R.S.), Algeria. We confirm that no studies involving humans or experimental animals were conducted in this work. References Abdel-Rahman, S., El-Ayouty, Y.M., Kamael, H.A., 1993. Characterization of heptapeptide toxins extracted from Microcystis aeruginosa (Egyptian isolate)—comparison with some synthesized analogs. Int. J. Pept. Protein Res. 41, 1–7. Beasley, V.R., Dahlem, A.M., Cook, W.O., Valentine, W.M., Lovell, R.A., Hooser, S.B., Harada, K.I., Suzuki, M., Carmichael, W.W., 1989. Diagnostic and clinically important aspects of cyanobacterial (blue-green algae) toxicoses. J. Vet. Diagn. Invest. 1, 359–365. Brittain, S., Mohamed, Z.A., Wang, J., Lehmann, V.K.B., Carmichael, W.W., Rinehart, K.L., El-Sharouny, H.M., 2000. Isolation and characterization of microcystins from a River Nile strain of Oscillatoria tenuis Agardh ex Gomont. Toxicon 38, 1759–1771. Brooks, W.P., Codd, G.A., 1987. Distribution of Microcystis aeruginosa peptide toxin and interactions with hepatic microsomes in mice. Pharm. Toxicol. 60, 187–191. Bouaı¨ cha, N., Maatouk, I., Vincent, G., Levi, Y., 2002. A colorimetric and fluorometric microplate assay for the detection of microcystin-LR in drinking water without preconcentration. Food Chem. Toxicol. 40, 1677–1683.
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