Vol. 15(3), pp. 124-141, March 2021 DOI: 10.5897/AJEST2020.2967 Article Number: 19C08BB66258 ISSN: 1996-0786 Copyright ©2021 African Journal of Environmental Science and Author(s) retain the copyright of this article http://www.academicjournals.org/AJEST Technology

Full Length Research Paper

Characterization of () blooming in the Dams of Northern Morocco

Mustapha Ouhsassi1*, El Ouardy Khay1, Anass El Laghdach2, Farid Ben Abdelouahab3, Abdeltif El Ouahrani1, Mohamed Idaomar1 and Jamal Abrini1

1Laboratory of Biology and Health, Biotechnology and Applied Microbiology, Faculty of Sciences, Abdelmalek Essaadi University, Tétouan, Morocco. 2Laboratory of Water, Environmental Studies and Analysis, Department of Chemistry, Faculty of Sciences, Abdelmalek Essaâdi University, Tétouan, Morocco. 3Laboratory Materials and Radiation, Department of Physique, Faculty of Science, Tétouan, Faculty of Science, Abdelmalek Essaâdi University, BP 2121, Tétouan 93002, Morocco.

Received 10 December, 2020; Accepted 17 February, 2021

Cyanobacteria thrive in eutrophic freshwaters and impose a serious problem for the management of water bodies. Some Cyanobacteria species impose even a risk for public health due to the production of intracellular toxins. This study is a qualitative approach to determine the degree of toxicity and the toxicological aspect of cyanotoxins in order to setup a monitoring program for cyanobacteria blooms and the management of cyanotoxins thriving in three water bodies in Northern Morocco. Water samples were collected from three major water reservoirs/dams near the city of Tétouan (SMIR, BELMEHDI and NAKHLA). These water samples were screened for possible Cyanobacteria using specific culture media (BG13 & Z8). Three cyanobacteria species ( aeruginosa, Pseudanabaena galeata and Oscillatoria tenuis) were isolated, purified and lyophilized. Using gas chromatography coupled with mass spectrometry, nine types of microcystins were characterized namely: (MC-LR); (MC-YR); (MC-LA); (MC-FR); (MC-RF), [Mser7]MC-LR; [Dha7]MC-LR; MC-YAba; and [Mser7]MC-YR. Our results strongly recommend and urge different stakeholders to consider the various health risks potentially generated by these toxins during water use and management. In addition, this study is a contribution to raise awareness of the toxicological aspect of the cyanobacteria inhabiting the water bodies of Northern Morocco.

Key words: Blue algae, gas chromatography coupled to mass spectrum, bio-toxins, water dam.

INTRODUCTION

The problem of water quality degradation in dams and pollution that cause nutritive elements (nitrogen and reservoirs is due essentially to different sources of phosphorus) enrichment causing the anarchic

*Corresponding author. E-mail: [email protected].

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Ouhsassi et al. 125

development of algae, which indicates an advanced state (2007) indicated that Microcystins are toxins produced by of water quality degradation. Furthermore, soil erosion the most common cyanobacteria and are present in most brings additional elements that may accelerate the of the world's water reservoirs. These cyanotoxins are alteration of water quality (Issaka and Ashraf, 2017; Rose structurally cyclic heptapeptide amino acids (Van et al., 2010). The ecosystem imbalance caused by such Apeldoorn et al., 2007; Shimizu, 2014). According to the phenomenon promotes the development of algae, in structure of Microcystins established by Chorus et al. particular blue algae (cyanobacteria) which are (1999) the toxicity role of the acid group (3-amino-9- responsible for the organoleptic and esthetic alteration of methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) water, as well as the production of cyanotoxins within recognized under the name Adda is primordial. In these waters (self-purification phenomenon). addition, several studies reveal the carcinogenic potential Previous works on lakes and reservoirs located in of Microcystins under long-term exposure (Yu, 1995; warm climate zones, such as dams in the Mediterranean, Codd, 2000; Carmichael et al., 2001; ADH, 2006). show that the latter are distinguished by particular Different Microcystins have been isolated from several hydrological, physico-chemical and biological species of cyanobacteria and several of them can be characteristics (Loudiki, 1990; Loudiki et al., 1994; Cherifi produced by a single bloom (Zastepa et al., 2017). and Loudiki, 2002). Among the determining factors, the Studies to date have focused solely on assessing the unpredictability of the climate (flash floods, droughts and level of cyanotoxins in blooms rather than characterizing very variable low water levels) and the irregularity of the water-soluble fractions of the toxins (Ai et al., 2020). rainfall and erosion materials play a predominant role. In this context, this study is a qualitative approach to Morocco is a Mediterranean country characterized by a determine the degree of toxicity and the toxicological semi-arid climate (Perrin et al., 2014, Ouhamdouch et al. aspect of cyanotoxins in the studied reservoirs in order to 2019), with clear spatiotemporal disparities in rainfall setup a monitoring program for cyanobacteria blooms towards the southern region of the country. Moreover, the and the management of cyanotoxins produced in water country is likely to experience 20% on average net bodies in Northern Morocco. The specific objectives are reduction in rainfall by the end of this century (IPCC, to extract and characterize the intracellular Microcystins 2007); this will boost cyanobacteria in inland water bodies using gas chromatography coupled with mass such as dams (Gophen, 2021). spectrometry analysis. In Morocco, the supply of drinking water is mainly ensured by rainfall collected in water reservoirs or dams. MATERIALS AND METHODS This strategic approach was adopted since the 1940s, in order to mobilize water resources through the Study site construction of several large dams to provide drinking water and other services (El Ghachtoul et al., 2005). Water samples were collected from three major water bodies Nevertheless, these dams recognize in the summer supplying drinking water to the city of Tetouan (northern Morocco). period phenomena of due to nutrient The three dams are Smir (35°40'46.9 "N 5°23'33.8 "W) (Figure 1a), Belmehdi (35°42'33.2 "N 5°30'28.0 "W) (Figure 1b) and Nakhla enrichment mainly nitrogen and phosphorus (El Ghachtoul (35°42'01.9 "N 5°30'11.7 "W) (Figure 1c). Their characteristics are et al., 2005). noted in Table 1. These reservoirs are experiencing the In fact, recent climate change and anthropogenic phenomenon of eutrophication due to the accumulation of two impact on water environment by either intense withdrawal highest nutrients (nitrogen and phosphorus), which lead to the and diversion or chemical pollution and nutrient proliferation of cyanobacteria blooms, a sign of water quality degradation. enrichment promoted a worldwide proliferation of cyanobacteria blooms often harmful to ecological and human health (Paerl, 2016). Certainly, the massive Sampling method proliferation of cyanobacteria in dam waters is Sampling was performed in the morning (around 10:00 am) on a increasingly frequent phenomenon worldwide (Huisman monthly basis, from June to December 2017. Three sampling points et al., 2018), accompanied by the release of toxic were selected separately following the long transect of the reservoir substances in the form of secondary metabolites lake. We collected approximately 50 liters of surface water from (cyanotoxins). These cyanotoxins cause harmful each water reservoirs. The water samples were carried in sterilized ecological, health and socio-economic effects leading to special plastic laboratory bottles of 5 L. To allow a good a degradation of water quality and a reduction in the conservation, these bottles were transported in a cooler box of 4°C. These raw water samples were used for isolation and purification of productivity of the aquatic environment (Wiegand and cyanobacteria, which requires a preliminary culture in synthetic Pflugmacher, 2005; Jacoby and Kann, 2007; Plaas and media (Saoudi, 2008). Paerl, 2016). Most cyanotoxins are called Microcystins. According to Culture media and Isolation conditions some studies the first was isolated from Microcystis aeruginosa (Carmichael, 1992a; Namikoshi et Two culture media BG13 (Ferris and Hirsch, 1991) and Z8 (Kotai, al., 1992, 1995). Dittmann and Börner (2005) and Hotto et al. 1972; Rippka et al., 1979) were prepared in liquid and solid forms, 126 Afr. J. Environ. Sci. Technol.

Figure 1. Map showing geographic location of the three dams SMIR, BELMEHDI et NAKHLA in city Tetouan Northen morocco. Geographical Distribution and Location of Water Bodies and Sample Site (a) SMIR (b) Belmehdi (c ) Nakhla sampling locations.

Table 1. Morphometric and hydrological characteristics of the study reservoir lake.

Water reservoirs Characteristics Nakhla Smir Belmehdi Filling reservoir with water 1961 1991 2005 Reservoir Headwater Level - 43,5 49 capacity (mm3) 4,3 43 28,8 Annual flow of water (Mm3) 10 31 16 Area (Km²) 110 4 4,8 Maximum depth of reservoir (m) 15 30 50 Function Drinking water Drinking water Drinking water Stream of water Nakhla Smir Raouz City Tétouan Tétouan Tétouan

autoclaved for 20 min at 120°C and a pressure of 1.1 kg/cm². growing strain was transferred into new solid media, and this step is Afterwards, we added 100 mg of cycloheximide per litter to both repeated until purified strains were observed. These purified strains liquid and solid culture media using a sterile syringe with a 0.2 µm were then transferred to sterile bottles containing 100 ml of liquid porosity acrodisc, to remove most eukaryotes and obtain a culture media (BG13 and Z8). These bottles were sealed with a monoalgal culture. stopper of 0.2 µm porosity filter to allow airflow. By repeating these Water samples were filtered through Millipore membranes with a manipulations 3 to 4 times, we obtained an axenic strain after 12 porosity of 45 µm by means of a pressurized vacuum pump (Figure weeks. The purified strains would then be subject to a preliminary 2). These membranes were transferred into two liquid media BG13 morphological identification. Once verified, we transferred the (Ferris and Hirsch 1991) and Z8 (Kotai, 1972; Rippka et al., 1979), axenic strains to a vial containing 2L of sterile culture medium with pH adjusted to 8, and incubated for three weeks under ambient (BG13 and Z8) under illumination and continuous aeration to obtain temperature (20 - 25°C), fluorescent lamps of 2000 lumens intensity a large mass of each strain (massification phase). Two weeks later, and a photoperiod of 12 h. Afterwards, these membranes were the biomass of each cyanobacterial strains collected during the transferred into Petri dishes containing solid media (BG13 and Z8) massification phase was centrifuged (4000 g, 20 min), then for purification phase. After three weeks, a tiny fragment of the lyophilized and stored at -20°C f or cyanotoxins extraction. Ouhsassi et al. 127

Figure 2. Culture and isolation method of cyanobacteria sampled from the studied raw water reservoirs.

All manipulations (transplantation, purification, and massification) et al., 2017). The composition of fatty acid is used as a were carried out under a laminar flow hood at 25°C. The materials phylogenetic marker for cyanobacteria (Kenyon and Stanier, 1970; necessary for these manipulations (Pasteur pipettes, platinum wire, Murata et al., 1992; Sarsekeyeva et al., 2014; Los and Mironov, and others) were sterilized. The species were observed, measured 2015). and morphologically identified using a light microscope according to criteria-based using several specialized cyanobacterial florae and a multitude of works dealing specifically with these Extraction and pre-purification of Microcystins organisms (Bourrelly, 1985; Lund and Lund, 1995, Komárek and Anagnostidis, 2005; Komárek, 2016). This identification focused on the definition of many morphological criteria according to universally To extract and purify Microcystins, we followed the method accepted identification keys: described by Lawton et al. (1994). For each study site, the lyophilizate (250 mg) was recovered in the stationary phase from (i) The color and structure of cyanobacteria (unicellular or colonial), each pure culture. For each lyophilizate, the extraction is done (ii) The shape of the colony or trichome, three times with 70% methanol (Figure 3). After each extraction, the (iii) The size of the cells suspensions were centrifuged (4000 × g, 10 min, +4°C). The total (iv) The presence or absence of gelatinous sheath (color, extract was diluted with ultrapure water (milli-Q, Millipore) to obtain appearance and size), akinetes, heterocysts and gas vacuoles an extract with 20% methanol. For pre-purification of microcystins, (pseudovacuoles). the final extract was passed through an ODS silica gel column (Sep-Pak Vac C18, Waters Corporation, Milford, MA, USA). The Furthermore, the morphological identification was confirmed by last recovered fraction containing the microcystins is completely analyzing the conservative fatty acid composition profile according evaporated at 40°C, dissolved in 1 mL methanol /ultrapure water to previous studies (Passaquet et al., 1989; Hayakawa et al., 2002; (50:50, v/v), and filtered through a 0.2 µm filter (Acrodisc, Nylon, Thajuddin and Subramanian, 2005; Sharathchandra and Gelman Sciences Inc.) before being analyzed by gas Rajashekhar, 2011; Mahapatra and Ramachandra, 2013; Ouhsassi chromatography coupled with a mass spectrometer (GC-MS). 1 Gelman Sciences Inc.)128 before Afr. being J. Environ. analyzed Sci. byTechnol. gas chromatography coupled with a mass

2 spectrometer (GC-MS).

conservative fatty acid method, allows identifying three 250 mg of lyophilized major cyanobacteria species with the same cyanobacterial strain characteristics at the level of the studied water bodies (SMIR, BELMEHDI and NAKHLA):

Microcystis aeruginosa, belonging to , isolated from Smir (1R), Belmehdi (4R) and Nakhla (7R), is a unicellular cyanobacterium with spherical colonies; it is grouped as an envelope and floated using gaseous

vacuoles (Figure 4a).

Pseudanabaena galeata, belonging to

mortar mortar Pseudanabaenaceae, isolated from SMIR (2R), grinding BELMEHDI (5R) and NAKHLA (8R), is a filamentous cyanobacterium with solitary, mobile and without sheathing trichomes. The cells are distant from each other and joined by a gelatinous bridge. There are no kinetes or heterocysts (Figure 4b). 3-fold extraction by 70% methanol Oscillatoria tenuis, belonging to Oscillatoriaceae, isolated from SMIR (3R), BELMEHDI (6R) and NAKHLA (9R), is a filamentous cyanobacteria with a free, solitary and sheathless trichome (Figure 4c). The movement and helical displacement of the apex are characteristic of this genus. The analysis of GC-MS chromatograms revealed the presence of a wide variety of Microcystins in all Centrifugation studied cyanobacteria strains. The data (Tables 2 to 4) (4000xg,10 min, 4°C) regroup Microcystins elaborated by M. aeruginosa, P. galeata and O. tenuis isolated from the three studied dams: SMIR, BELMEHDI and NAKHLA. The chromatograms show the retention times recorded by different microcystins elaborated by M. aeruginosa Dilution of the extract to (Figure 5), and they are:

20% methanol (i) MC-LR and a demethylated form MC-YAab in the case of SMIR reservoir (Figures 6a and 6b); (ii) MC-RF and two demethylated forms MC-YAab and [Dha7]MC-LR in the case of BELMEHDI reservoir (Figures 9b to d); Solution of the fraction in a (iii) MC-LA and a demethylated form MC-YAab in the water-methanol volume case of NAKHLA reservoir (Figure 12b and c). (50:50, v:v) In the case of P. galeata, the retention times are shown respectively for the three studied dams (Figure 5). The elaborated Microcystins by this species are:

(i) MC-YR and a demethylated form [Mser7]MC-YR Analysis by gas chromatography (Figure 7a and b) in the case of SMIR reservoir; coupled to a mass spectrometer (ii) MC-YR and a demethylated form MC-YAab (Figure 3 10a) in the case of BELMEHDI dam;

4 Figure 3. ProtocolFigure 3. ofProtocol the cyanobacterial of the cyanobacterial microcystin microcystin extraction(iii) MC-LA and a demethylated form MC-YAba (Figure 5 extraction. 13a and 13b) in the case of NAKHLA reservoir.

In the case of O. tenuis, the retention times recorded by different Microcystins are shown in Figure 5. The RESULTS elaborated Microcystins are:

7 The morphological identification, confirmed by (i) MC-LA and a demethylated form [Mser ]MC-LR 1 trichome (Figure 4c). The movement and helical displacement of the apex are Ouhsassi et al. 129

2 characteristic of this genus.

3 4 a b c

5 FigureFigure 4. 4Microscopic. Microscopic plates of cyanobacteriaplates of cyanobacteria species: (a) M. aeroginosa species: (b) P.galeata (a) M. ( caeroginosa) O. tenuis isolated (b from) P.galeata SMIR, BELMEHDI (c) O. and NAKHLA dams.

6 tenuis isolated from SMIR, BELMEHDI and NAKHLA dams.

Table 2. Variants of Microcystins elaborated by Microsystis aeruginosa on different water bodies.

Body of water Analytical reference strain Microcystin identified m/z[M+H]+ calculated m/z[M+H]+ measured MC-LR 995.5 995.95 SMIR 1R MC-YAba 974.5 974.36

MC-FR et MC-RF 1029.5 1029.64 BEMEHDI 4R [Dha7]MC-LR 981.5 981.90

MC-YAba 974.5 974.36 NAKHLA 7R MC-LA 910.5 910.64 MC-YAba 974.5 974.25

Table 3. Variants of Microcystins elaborated by Pseudanabaena galeata on different water bodies.

Body of Analytical Microcystin m/z [M+H]+ m/z[M+H]+ water reference strain identified calculated measured MC-YR 1045.5 1045.06 SMIR 2R [Mser7]MC-YR 1063.84 1063.5 BELMEHDI 5R MC-YAba 974.5 974.11 MC-LA 910.5 910.58 NAKHLA 8R MC-YAba 974.5 974.34

Table 4. Variants of Microcystins elaborated by Oscillatoria tenis on different water bodies.

Body of water Analytical reference strain Microcystin identified m/z [M+H]+ calculated m/z [M+H]+ measured [Mser7]MC-LR 1013.5 1013.23 SMIR 3R MC-LA 910.5 910.58 BELMEHDI 6R MC-YAba 974.5 974.52 [Mser7]MC-LR 1013.5 1013.15 NAKHLA 9R MC-YAba 974.5 974.38 130 Afr. J. Environ. Sci. Technol.

Figure 5. Chromatogram of retention time.

(Figure 8a and 8b) case of SMIR dam; molecular ion, usually called a molecular ion peak. The (ii) a single demethylated form MC-YAba (Figure 11a) accompanying signals represent ion fragments. For case of BELMEHDI dam; example, dissociation of MC-LR exhibits a molecular ion (iii) two demethylated forms MC-YAba and ([Mser7]MC- peak break ([M.H]+, 995.5546), dissociation of MC-RR LR (Figure 14a and 14b) case of Nakhla dam. exhibits a molecular ion peak break ([M.H]+, 1038.5), and dissociation of MC-YR exhibits a molecular ion peak break ([M.H]+, 1045.5358). DISCUSSION Some authors have shown that cyanobacteria blooms in Morocco appear in summer and reach their maximum Characterization of cyanotoxins has been performed proliferation in October with a significant annual variability (Poon et al., 1993; Diehnelt et al., 2005, 2006; Miles et in biomass. Sbiyyaa (1997) and Oudra et al. (1998) al., 2013) using GC-MS or LC-MS or other mass indicated that the main species responsible for blooms in spectrometry methods, which use precise mass Moroccan dams is attributed to three main species: M. measurements showing the connectivity of amino acids in aeruginosa, M. aeruginosa flos-aquae, and P. muscicola. different Microcystins. By standardizing the base peak (Malki, 1994) are reported from the Al Massira reservoir. intensity to 100%, the appearance of a mass spectrum M. aeruginosa proliferates regularly and dominates the becomes independent of the absolute amount of sample. phytoplankton each year between November and Thus, mass spectra can be compared even when they December with a toxic bloom of cyanobacteria. Oudra et have been generated from different sample quantities al. (2002a) reported that several species of and/or different instruments. A list of m/z values (m = Microcystaceae often bloom in numerous dams, and are mass; z = atomic number) and intensity is useful for a therefore the most studied and geographically the most more detailed analysis of a spectrum. The signal resulting distributed. from molecular dissociation has an ion mass (m/z) and a The toxicity of a single bloom is a function of time and spectrum that normally reflects the corresponding space. In this study, the monitoring of cyanobacterial Ouhsassi et al. 131

1

185LC01R #82 RT: 3.50 AV: 1 SB: 32 5.08-6.72 , 3.11-4.22 NL: 2.85E5 F: - c ESI Full ms [ 50.00-2000.00] 366.99 280000

260000

240000 (a) 220000

200000

180000

160000

140000 Intensity

120000

100000 1378.51 80000 1913.52

1579.83 60000 406.18 1041.02 1140.57 270.92 MC-LR 1462.57 40000 172.76 742.51 1783.67 525.88 995.93 760.00 20000

0 200 400 600 800 1000 1200 1400 1600 1800 2000 2 m/z 185LC01R #89-102 RT: 3.80-4.31 AV: 7 SB: 15 4.35-4.91 , 3.07-3.76 NL: 1.01E6 F: + c ESI Full ms [ 50.00-2000.00] 702.47 1000000 974.36 950000 414.63 900000

850000 430.65 MC-YAba 800000 838.48 1110.27 (b) 750000

700000 686.41 650000

600000

550000

500000 Intensity 450000 1246.18 400000 240.42 350000 470.97 1382.13 300000 550.41 199.53 250000

200000 398.64 1518.12 257.93 1653.98 150000 183.41 100000 1789.91 1925.72 50000

0 200 400 600 800 1000 1200 1400 1600 1800 2000 3 m/z

Figure 6. Analysis performed on extracts of cyanobacterial bloom Microcystis aeruginosa (1R) isolated 4 fromFigure the waters 6. Analysis of SMIR Dam. performed (a, b ) Mass onspectrum extracts m/z showing of cyanobacterial the peak of the Microcystin bloom fraction:Microcystis aeruginosa MC-LR (995.93): [M+H]+ and MC-YAba (974.36) : [M+H]+. 5 (1R) isolated from the waters of SMIR Dam. (a, b ) Mass spectrum m/z showing the peak of

6 the Microcystin fraction: MC-LR (995.93) : [M+H]+ and MC-YAba (974.36) : [M+H]+. strains toxicity revealed that the studied species generate Microcystis were demonstrated for the first time in the a wide variety of cyanotoxins. Toxic blooms of the genus Oued el Mellah dam in 1997 (Loudiki et al., 2002). As 132 Afr. J. Environ. Sci. Technol.

185LC02R #109 RT: 4.66 AV: 1 SB: 29 4.74-5.78 , 2.80-4.18 NL: 4.10E5 F: + c ESI Full ms [ 50.00-2000.00] 275.49 400000 (a) 380000 360000 340000 252.16 320000 300000

280000 332.26 260000 240000 220000 236.71

200000 Intensity 180000 160000 140000 452.59 513.65 120000 178.99 350.63 100000 MC-YR 429.33 541.03 718.30 80000 605.76 147.37 765.12 60000 632.10 785.80 965.95 1045.06 40000 796.31 902.27 1069.76 139.56 838.37 20000

0 100 200 300 400 500 600 700 800 900 1000 1100 m/z

185LC02R #112 RT: 4.79 AV: 1 SB: 37 4.74-6.34 , 2.80-4.31 NL: 3.55E5 F: - c ESI Full ms [ 50.00-2000.00] 378.83 340000 (b) 320000

300000

280000

260000

240000

220000

200000

180000

Intensity 160000

140000 460.62

120000 324.61 [Mser7]MC-YR 100000

80000 476.45 1160.71 60000 646.39 1063.84 613.54 836.47 1696.70 748.55 998.24 1229.39 1390.63 1630.05 40000 1481.72 308.78 20000

0 200 400 600 800 1000 1200 1400 1600 m/z

Figure 7. Analysis performed on extracts of cyanobacterial blooms Pseudanabaena galeata (2R) isolated from the waters of Smir Dam. (a,b) corresponding Mass spectrum m/z showing the peak of the Microcystin fraction: MC-YR (1045.06): [M+H]+. [Mser7]MC-YR (1063.84): [M+H]+.

well as in other Moroccan aquatic systems, particularly in cyanobacterial blooms in stagnant waters (Douma et al., dams intended to provide drinking water to urban 2010). In fact, in the studied water bodies, Microcystis populations. Species of this genus are isolated from other spp produces more cyanotoxins compared to other Moroccan water reservoirs, including Imfout, Takerkoust, species. Thus, Microsystis aeruginsa produces MC-LR and Almassira (Oudra et al., 2001a, 2001b; Loudiki et al., and MC-YAba in SMIR dam; MC-FR, MC-RF, and 2002; Sabour, 2002). This genus is known to form [Dha7], MC-LR in BELMEHDI dam; and MC-YAba, MC- Ouhsassi et al. 133

185LC03R #72-83 RT: 3.11-3.54 AV: 6 SB: 32 3.67-5.08 , 1.73-2.98 NL: 2.01E5 F: + c ESI Full ms [ 50.00-2000.00] 309.69 200000 190000 (a) 851.15 180000

170000

160000

150000

140000 689.19 130000

120000

110000

100000 Intensity 90000

80000 364.96 70000

60000 527.16 50000 1013.23 40000 1567.76 30000 439.16 1165.07 1280.77 1395.53 1473.48 1620.22 20000 549.95 884.54 1130.92 814.59 10000

0 400 600 800 1000 1200 1400 1600 m/z

185LC03R #108 RT: 4.61 AV: 1 SB: 43 4.52-6.46 , 1.99-3.71 NL: 3.48E6 F: - c ESI Full ms [ 50.00-2000.00] 644.81 3400000 (b) 3200000

3000000 378.86 2800000

2600000

2400000 910.58 2200000 MC-LA

2000000

1800000

Intensity 1600000

1400000

1200000 1176.63 628.69 1000000 894.58

800000 550.76 780.59 1046.49 600000 476.63

400000 1698.16 362.78 1311.95 1442.54 1820.30 200000 1630.39 268.96 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

Figure 8. Analysis performed on extracts of cyanobacterial bloom Oscillatoria tenuis (3R) isolated from the waters of Smir Dam: (a, b) corresponding Mass spectrum showing the peak of the Microcystin + + fraction:Figure 8.Analysis[Mser7]MC- LRperformed m/z 1013.23 on :extracts [M+H] (c) of andcyanobacterial MC-LA m/z 910.58 bloomOscillatoria : [M+H] . tenuis(3R) isolated from the

waters of Smir Dam:(a, b)corresponding Mass spectrum showing the peak of theMicrocystin fraction: [Mser7]MC-LR m/z 1013.23 : [M+H]+(c)and MC-LA m/z 910.58 : [M+H]+. LA, and MC-YAba in NAKHLA dam. These varieties of by their acute toxicity either in SMIR (MC-YR, cyanotoxins are recognized by their acute toxicity. [Mser7]MC-YR), BELMEHDI (MC-YAba) and NAKHLA Studies have revealed that the toxicity of Microcystis is (MC-LA and MC-YAba). Studies in this context affirm that due to the disposition of the gene coding for microcystin several species of Pseudanabaenaceae have been (Carmichael, 1995; Rouhiainen et al., 1995; Dittmann et inventoried in Moroccan water bodies, including al., 1997). Pseudanabaena mucicola. This family is dominant in P. galaeta filamentous species is also incriminated in lakes and shallow ponds, and this dominance can dam waters and produces cyanotoxins also recognized sometimes persist throughout the year in turbid water 134 Afr. J. Environ. Sci. Technol.

(a) MC-YAba

(b)

MC-RF

185LC04R #106 RT: 4.51 AV: 1 SB: 28 4.56-5.51 , 2.29-3.67 NL: 5.59E5 F: - c ESI Full ms [ 50.00-2000.00] 378.85 550000

500000 (c)

450000

400000

350000

300000 Intensity 250000

200000 362.80 [Dha7]MC-LR

1206.33 150000

514.77 716.23 1171.50 100000 900.29 836.30 981.90 1294.12 476.51 612.44 678.16 1014.30 50000 210.81 284.88 1369.84

0 200 400 600 800 1000 1200 m/z

Figure 9. chromatograms of the analysis performed on extracts of cyanobacterial efflorescence Microcystis aeruginosa (4R) isolated from the waters of Belmehdi Dam (a, b and c) corresponding Mass spectrum showing the peak of the Microcystin fraction: MC-YAba m/z (974.36): [M+H]+, MC-RF m/z 1029.64:[M+H]+ and [Dha7]MC-LR m/z 981.90:[M+H]+. Ouhsassi et al. 135

185LC05R #97 RT: 4.14 AV: 1 SB: 26 4.45-5.57 , 2.59-3.63 NL: 1.76E5 F: + c ESI Full ms [ 50.00-2000.00] 414.60

170000

160000 958.10 430.63 821.88 150000

140000 240.38 130000 670.37 120000

110000 974.11

100000 702.39 398.67 1213.68 90000

Intensity 80000

70000 360.59

60000 153.56 1365.81 50000 1467.90 550.72 1652.64 1869.98 40000 1771.87 30000 1991.53 20000

10000

0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

FigureFigure 10: Analysis 10. Analysis performed performedon extracts of oncyanobacterialefflorescencePseudanabaena extracts of cyanobacterial efflorescence galeata(5R) isolatedPseudanabaena from the waters galeata of Belmehdi (5R) Dam:corresponding isolated from Mass the spectrum waters showing of Belmehdi the peak of Dam:the Microcystincorresponding fraction: Mass MC -spectrumYAba m/z (974.11) showing : [M+H]+. the peak of the Microcystin fraction: MC-YAba m/z (974.11): [M+H]+.

185LC06R #89-115 RT: 3.80-4.92 AV: 14 SB: 40 5.00-6.51 , 1.90-3.71 NL: 3.84E5 F: + c ESI Full ms [ 50.00-2000.00] 275.42 380000

360000

340000

320000

300000 702.45

280000

260000 414.66 974.42 MC-YAba 240000

220000 686.37 822.29

200000

Intensity 180000 252.36 1110.23 160000

140000 199.51 120000 550.50 100000 1246.23 1382.21 80000 183.45 60000 1518.08

40000 1653.96 20000 1789.98 1926.51 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

FigureFigure 11 :11. Analysis Analysis performed performed on extracts of on cyanobacterialbloomOscillatoria extracts of cyanobacterial tenuis(6R) bloom isolated Oscillatoria from the tenuiswaters of (6R) Belmehdi isolated Dam:(a)corresponding from the waters Mass spectrum of Belmehdi showing the Dam: peak of(a) the corresponding Microcystin fraction: Mass MC-YAba m/z (974.42):[M+H]+. spectrum showing the peak of the Microcystin fraction: MC-YAba m/z (974.42):[M+H]+. 136 Afr. J. Environ. Sci. Technol.

185LC07R #110 RT: 4.70 AV: 1 SB: 31 2.55-3.75 , 4.53-5.82 NL: 8.45E5 F: - c ESI Full ms [ 50.00-2000.00] 378.86 (a) 800000

750000

700000

650000

600000

550000

500000

450000

Intensity 400000

350000

300000

250000 362.86 644.78 200000 284.87 894.55 150000 514.55 MC-YAba 476.56 628.70 1030.61 100000 596.60 878.34 910.64 998.16 444.78 732.53 852.81 1058.81 1152.24 50000 248.86 154.94 764.71 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z 185LC07R #95 RT: 4.05 AV: 1 SB: 35 4.53-5.69 , 1.94-3.71 NL: 5.45E5 F: + c ESI Full ms [ 50.00-2000.00] 702.51 822.17 838.43 500000 414.64 974.25 450000 199.38 (b)

400000 240.25 MC-LA 430.56

350000 470.81

300000 1093.88 1246.10

Intensity 250000

534.29 200000

150000 1518.12 183.55 398.68 1365.70 1796.16 100000 297.35 1620.83

50000 1931.74

0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

FigureFigure 12: 12. Analysis Analysis performed performed on on extracts extracts ofcyanobacterial of cyanobacterial efflorescence efflorescence Microcystis Microcystis aeruginosa(7R) aeruginosaisolated from (7R) the isolated waters of from Nakhla the Dam: waters (a, b)corresponding of Nakhla Dam: Mass (a, bspectrum) corresponding showing Massthe peak of the spectrumMicrocystins showing fractions: the peakMC-YAba of the m/z Microcystins (974.25):[M+H fractions:] and MC MC-LA-YAba m/z (910.64): m/z (974.25 [M+H]+.): [M+H and MC-LA m/z (910.64): [M+H]+.

bodies and when the winter is not very cold (Sas, 1989; BELMEHDI (MC-YAba), and in NAKHLA ([Mser7]MC-LR Oudra et al., 2002a; Loudiki et al., 2002). and MC-YAba). This species is less frequent than the O. tenuis is also inventoried in Moroccan water bodies other species but it also has the capacity to generate (Oudra et al., 2002a). This species develops variant large varieties of cyanotoxins. cyanotoxins also recognized by its acute toxicity. In our Microcystis, Pseudanabaena and Oscillatoria species study, it is found in SMIR ([Mser7]MC-LR, MC-LA), in are the most common and potentially toxic cyanobacteria Ouhsassi et al. 137

185LC08R #106 RT: 4.52 AV: 1 SB: 34 4.56-5.69 , 1.90-3.66 NL: 1.07E6 F: - c ESI Full ms [ 50.00-2000.00] 378.77 1652.40 (a) 1050000 628.79 1000000 950000 644.71 900000 850000 800000 612.88 750000 700000 650000 600000 550000 910.58 MC-YAba

Intensity 500000 894.56 450000 362.83 400000 350000 878.44 300000 460.82 1034.71 1611.49 250000 1522.01 1864.80 764.58 1388.41 284.91 200000 1144.76 1264.09 150000 100000 50000 268.93 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z 185LC08R #95 RT: 4.05 AV: 1 SB: 36 4.91-6.16 , 2.46-4.22 NL: 1.14E6 F: + c ESI Full ms [ 50.00-2000.00] 702.45

1100000 974.34 MC-LA (b) 1050000

1000000 430.68 950000 838.34 900000 1110.34 850000 800000 686.24 750000 414.66 700000 650000 600000

550000 Intensity 500000 1518.15 450000 239.94 1246.20 400000 350000 300000 550.50 250000 1382.05 1914.86 200000 398.64 150000 183.45 1653.90 100000 258.12 1779.47 50000 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

FigureFigure 13: 13. AnalysisAnalysis performed performed on on extracts extracts of cyanobacterial of cyanobacterialefflorescence efflorescence Pseudanabaena Pseudanabaena galeata(8R) galeataisolated (8R) from isolated the waters from of theNakhla waters Dam: of (a, Nakhla b)corresponding Dam: (a, bMass) corresponding spectrum showing Mass the spectrum peak of the + showingMicrocystin the peakfraction: of the MC Microcystin-YAba m/z (974.34): fraction: [M+H]+MC-YAba and m/z MC -(974.34):LA m/z (910.58): [M+H] [M+H]+.and MC -LA m/z (910.58): [M+H]+.

found in Moroccan freshwater and are the main et al., 1992), African countries such as Algeria (Saoudi et producers of cyanotoxins. This is also evident in Euro- al., 2017), Ethiopia (Major et al., 2018), and Nigeria Mediterranean countries (Filatova et al., 2020), such as in (Kadiri et al., 2020). France (Lac de Grand-Lieu), Portugal (Vasconcelos et The presence of microcystins in freshwater species of al., 1996; Moreira et al., 2020), Spain (Quesada et al., the genus Oscillatoria is not only an ecological problem,

2004), Greece (Christophoridis et al., 2018), and Italy (Bruno but also presents a health risk when water is used for 138 Afr. J. Environ. Sci. Technol.

185LC09R #79 RT: 3.37 AV: 1 SB: 33 4.88-6.17 , 2.63-4.14 NL: 3.29E5 F: + c ESI Full ms [ 50.00-2000.00] 852.24 320000

300000 (a)

280000

260000

240000

220000 689.26 200000

180000 439.06 1013.15 [Mser7]MC-LR

160000 Intensity

140000 1782.04

120000 1883.61 787.85 100000 1975.61 1563.39 255.83 1176.40 80000 167.44 1505.28 400.04 60000 973.23 1254.46 1411.13 312.08 597.61 40000

20000

0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

185LC09R #95 RT: 4.06 AV: 1 SB: 33 4.88-6.17 , 2.63-4.14 NL: 8.68E5 F: + c ESI Full ms [ 50.00-2000.00] 414.68 850000 800000 (b) 750000

700000

650000

600000 430.68 550000

500000 199.32 702.49 450000 471.09 1110.27

Intensity 974.38 400000 MC-YAba 350000 822.20 686.31 300000 838.36 550.42 250000 240.36 183.49 200000 1246.14

150000 398.57 1486.79 100000 1365.63 1654.15 310.64 1915.26 50000 1757.50

0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

Figure 14. AnalysisFigure 14 performed. Analysis on performed extracts of on cyanobacterialbloomOscillatoria extracts of cyanobacterial bloom tenuis(9R)Oscillatoria isolated tenuis from the (9R) isolated from the waters of Nakhla Dam: (a, b) corresponding Mass spectrum waters of Nakhlashowing Dam:(a, the peak b)corresponding of the Microcystin Mass fractions: spectrum [Mser showing7]MC-LR the at peakm/z 1013.15:[M+H] of the Microcystin+ and : fractions: [Mser7]MCMC-LR -atYAba m/z1013.15:[M+H]+and à m/z 974.38:[M+H]+. : MC-YAba à m/z 974.38:[M+H]+.

drinking purposes (Lindholm et al., 1989). On the other interest, such as Anabaena variabilis. This species is hand, some strains, known for their toxicological used for its hydrogen production (Yoon et al., 2006) and potential, are considered to be of biotechnological can remove phenols and its derivatives (Hirooka et al., Ouhsassi et al. 139

2003) as well as heavy metals (Nagase et al., 2005) from Christophoridis C, Zervou SK, Manolidi K, Katsiapi M, Moustaka-Gouni the environment and industrial wastewater (Yoon et al., M, Kaloudis T, Hiskia A (2018). Occurrence and diversity of cyanotoxins in Greek lakes. Scientific Reports 8(1):1-22. 2006). Codd GA (2000). Cyanobacterial toxins, the perception of water quality, and the prioritisation of eutrophication control. Ecological Engineering 16(1):51-60. Diehnelt CW, Dugan NR, Peterman SM, Budde WL Conclusion (2006). Identification of Microcystin Toxins from a Strain of Microcystis aeruginosa by Liquid Chromatography Introduction into a This study contributes to the knowledge of the Hybrid Linear Ion Trap-Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. Analytical Chemistry 78(2): 501-512. systematics and biogeography of toxic cyanobacteria and Diehnelt CW, Peterman SM, Budde WL (2005). Liquid Their toxins quality in the water bodies of Northern chromatography–tandem mass spectrometry and accurate m/z Morocco. It is a qualitative analysis of cyanotoxins measurements of cyclic peptide cyanobacteria toxins. Trends in produced by cyanobacteria species thriving in three water Analytical Chemistry 24(7):622-634. Dittmann E, Börner T (2005). Genetic contributions to the risk reservoirs near the City of Tetouan, namely SMIR, assessment of microcystin in the environment. Toxicology and BELMEHDI and NAKHLA. The results show that the Applied Pharmacology 203(3):192-200. water bodies of Northern Morocco are exposed to Dittmann E, Neilan BA, Erhard M, Von Döhren H, Börner T (1997). cyanobacterial proliferation exposing these water bodies Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium to numerous variants of Microcystins (MC-YR, Microcystis aeruginosa PCC 7806. Molecular microbiology 26(4):779- [Mser7]MC-YR, MC-YAba, MC-LA, [Mser7]MC-LR, MC- 787. FR, MC-RF, [Dha7]MC-LR) produced by the three major Douma M, Ouahid Y, del Campo FF, Loudiki M, Mouhri Kh, Oudra B species (Microsystis aeruginosa, Pseudanabaena (2010). Identification and quantification of cyanobacterial toxins (microcystins) in two Moroccan drinking-water reservoirs (Mansour galeata, Oscillatoria tenis). These cyanotoxins are Eddahbi, Almassira). Environmental Monitoring and Assessment recognized by their acute toxicity and reflect a permanent 160(1-4):439-450. threat to the health of the consumer (drinking water) El Ghachtoul Y, Alaoui Mhamidi M, Gabi H (2005). Eutrophisation des mainly in late summer time. Thus, the need for action eaux des retenues des barrages Smir et Sehla (maroc): causes, conséquences et consignes de gestion. 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