Characterization of cyanobacteria microcystins (cyanotoxins) blooming in the Dams of Northern Morocco

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


INTRODUCTION
The problem of water quality degradation in dams and reservoirs is due essentially to different sources of pollution that cause nutritive elements (nitrogen and phosphorus) enrichment causing the anarchic development of algae, which indicates an advanced state of water quality degradation. Furthermore, soil erosion brings additional elements that may accelerate the alteration of water quality (Issaka and Ashraf, 2017;Rose et al., 2010). The ecosystem imbalance caused by such phenomenon promotes the development of algae, in particular blue algae (cyanobacteria) which are responsible for the organoleptic and esthetic alteration of water, as well as the production of cyanotoxins within these waters (self-purification phenomenon). Previous works on lakes and reservoirs located in warm climate zones, such as dams in the Mediterranean, show that the latter are distinguished by particular hydrological, physico-chemical and biological characteristics (Loudiki, 1990;Loudiki et al., 1994;Cherifi and Loudiki, 2002). Among the determining factors, the unpredictability of the climate (flash floods, droughts and very variable low water levels) and the irregularity of rainfall and erosion materials play a predominant role.
Morocco is a Mediterranean country characterized by a semi-arid climate (Perrin et al., 2014, Ouhamdouch et al. 2019, with clear spatiotemporal disparities in rainfall towards the southern region of the country. Moreover, the country is likely to experience 20% on average net reduction in rainfall by the end of this century (IPCC, 2007); this will boost cyanobacteria in inland water bodies such as dams (Gophen, 2021).
In Morocco, the supply of drinking water is mainly ensured by rainfall collected in water reservoirs or dams. This strategic approach was adopted since the 1940s, in order to mobilize water resources through the construction of several large dams to provide drinking water and other services (El Ghachtoul et al., 2005). Nevertheless, these dams recognize in the summer period phenomena of eutrophication due to nutrient enrichment mainly nitrogen and phosphorus (El Ghachtoul et al., 2005).
In fact, recent climate change and anthropogenic impact on water environment by either intense withdrawal and diversion or chemical pollution and nutrient enrichment promoted a worldwide proliferation of cyanobacteria blooms often harmful to ecological and human health (Paerl, 2016). Certainly, the massive proliferation of cyanobacteria in dam waters is increasingly frequent phenomenon worldwide (Huisman et al., 2018), accompanied by the release of toxic substances in the form of secondary metabolites (cyanotoxins). These cyanotoxins cause harmful ecological, health and socio-economic effects leading to a degradation of water quality and a reduction in the productivity of the aquatic environment (Wiegand and Pflugmacher, 2005;Jacoby and Kann, 2007;Plaas and Paerl, 2016).
Most cyanotoxins are called Microcystins. According to some studies the first Microcystin was isolated from Microcystis aeruginosa (Carmichael, 1992a;Namikoshi et al., 1992Namikoshi et al., , 1995. Dittmann and Börner (2005) and Hotto et al. (2007) indicated that Microcystins are toxins produced by the most common cyanobacteria and are present in most of the world's water reservoirs. These cyanotoxins are structurally cyclic heptapeptide amino acids (Van Apeldoorn et al., 2007;Shimizu, 2014). According to the structure of Microcystins established by Chorus et al. (1999) the toxicity role of the acid group (3-amino-9methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) recognized under the name Adda is primordial. In addition, several studies reveal the carcinogenic potential of Microcystins under long-term exposure (Yu, 1995;Codd, 2000;Carmichael et al., 2001;ADH, 2006). Different Microcystins have been isolated from several species of cyanobacteria and several of them can be produced by a single bloom (Zastepa et al., 2017). Studies to date have focused solely on assessing the level of cyanotoxins in blooms rather than characterizing the water-soluble fractions of the toxins (Ai et al., 2020). In this context, this study is a qualitative approach to determine the degree of toxicity and the toxicological aspect of cyanotoxins in the studied reservoirs in order to setup a monitoring program for cyanobacteria blooms and the management of cyanotoxins produced in water bodies in Northern Morocco. The specific objectives are to extract and characterize the intracellular Microcystins using gas chromatography coupled with mass spectrometry analysis.

Sampling method
Sampling was performed in the morning (around 10:00 am) on a monthly basis, from June to December 2017. Three sampling points were selected separately following the long transect of the reservoir lake. We collected approximately 50 liters of surface water from each water reservoirs. The water samples were carried in sterilized special plastic laboratory bottles of 5 L. To allow a good conservation, these bottles were transported in a cooler box of 4°C. These raw water samples were used for isolation and purification of cyanobacteria, which requires a preliminary culture in synthetic media (Saoudi, 2008).

Culture media and Isolation conditions
Two culture media BG13 (Ferris and Hirsch, 1991) and Z8 (Kotai, 1972;Rippka et al., 1979) were prepared in liquid and solid forms,  Tétouan Tétouan Tétouan autoclaved for 20 min at 120°C and a pressure of 1.1 kg/cm². Afterwards, we added 100 mg of cycloheximide per litter to both liquid and solid culture media using a sterile syringe with a 0.2 µm porosity acrodisc, to remove most eukaryotes and obtain a monoalgal culture. Water samples were filtered through Millipore membranes with a porosity of 45 µm by means of a pressurized vacuum pump ( Figure  2). These membranes were transferred into two liquid media BG13 (Ferris and Hirsch 1991) and Z8 (Kotai, 1972;Rippka et al., 1979), with pH adjusted to 8, and incubated for three weeks under ambient temperature (20 -25°C), fluorescent lamps of 2000 lumens intensity and a photoperiod of 12 h. Afterwards, these membranes were transferred into Petri dishes containing solid media (BG13 and Z8) for purification phase. After three weeks, a tiny fragment of the growing strain was transferred into new solid media, and this step is repeated until purified strains were observed. These purified strains were then transferred to sterile bottles containing 100 ml of liquid culture media (BG13 and Z8). These bottles were sealed with a stopper of 0.2 µm porosity filter to allow airflow. By repeating these manipulations 3 to 4 times, we obtained an axenic strain after 12 weeks. The purified strains would then be subject to a preliminary morphological identification. Once verified, we transferred the axenic strains to a vial containing 2L of sterile culture medium (BG13 and Z8) under illumination and continuous aeration to obtain a large mass of each strain (massification phase). Two weeks later, the biomass of each cyanobacterial strains collected during the massification phase was centrifuged (4000 g, 20 min), then lyophilized and stored at -20°C f or cyanotoxins extraction. All manipulations (transplantation, purification, and massification) were carried out under a laminar flow hood at 25°C. The materials necessary for these manipulations (Pasteur pipettes, platinum wire, and others) were sterilized. The species were observed, measured and morphologically identified using a light microscope according to criteria-based taxonomy using several specialized cyanobacterial florae and a multitude of works dealing specifically with these organisms (Bourrelly, 1985;Lund andLund, 1995, Komárek andAnagnostidis, 2005;Komárek, 2016). This identification focused on the definition of many morphological criteria according to universally accepted identification keys: (i) The color and structure of cyanobacteria (unicellular or colonial), (ii) The shape of the colony or trichome, (iii) The size of the cells (iv) The presence or absence of gelatinous sheath (color, appearance and size), akinetes, heterocysts and gas vacuoles (pseudovacuoles).

Extraction and pre-purification of Microcystins
To extract and purify Microcystins, we followed the method described by Lawton et al. (1994). For each study site, the lyophilizate (250 mg) was recovered in the stationary phase from each pure culture. For each lyophilizate, the extraction is done three times with 70% methanol (Figure 3). After each extraction, the suspensions were centrifuged (4000 × g, 10 min, +4°C). The total extract was diluted with ultrapure water (milli-Q, Millipore) to obtain an extract with 20% methanol. For pre-purification of microcystins, the final extract was passed through an ODS silica gel column (Sep-Pak Vac C18, Waters Corporation, Milford, MA, USA). The last recovered fraction containing the microcystins is completely evaporated at 40°C, dissolved in 1 mL methanol /ultrapure water (50:50, v/v), and filtered through a 0.2 µm filter (Acrodisc, Nylon, Gelman Sciences Inc.) before being analyzed by gas chromatography coupled with a mass spectrometer (GC-MS).

RESULTS
The morphological identification, confirmed by conservative fatty acid method, allows identifying three major cyanobacteria species with the same characteristics at the level of the studied water bodies (SMIR, BELMEHDI and NAKHLA): Microcystis aeruginosa, belonging to Microcystaceae, 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 Pseudanabaenaceae, isolated from SMIR (2R), 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).

DISCUSSION
Characterization of cyanotoxins has been performed (Poon et al., 1993;Diehnelt et al., 2005Diehnelt et al., , 2006Miles et al., 2013) using GC-MS or LC-MS or other mass spectrometry methods, which use precise mass measurements showing the connectivity of amino acids in different Microcystins. By standardizing the base peak intensity to 100%, the appearance of a mass spectrum becomes independent of the absolute amount of sample. Thus, mass spectra can be compared even when they have been generated from different sample quantities and/or different instruments. A list of m/z values (m = mass; z = atomic number) and intensity is useful for a more detailed analysis of a spectrum. The signal resulting from molecular dissociation has an ion mass (m/z) and a spectrum that normally reflects the corresponding molecular ion, usually called a molecular ion peak. The accompanying signals represent ion fragments. Some authors have shown that cyanobacteria blooms in Morocco appear in summer and reach their maximum proliferation in October with a significant annual variability in biomass. Sbiyyaa (1997) and Oudra et al. (1998) indicated that the main species responsible for blooms in Moroccan dams is attributed to three main species: M. aeruginosa, M. aeruginosa flos-aquae, and P. muscicola. (Malki, 1994) are reported from the Al Massira reservoir. M. aeruginosa proliferates regularly and dominates the phytoplankton each year between November and December with a toxic bloom of cyanobacteria. Oudra et al. (2002a) reported that several species of Microcystaceae often bloom in numerous dams, and are therefore the most studied and geographically the most distributed.
The toxicity of a single bloom is a function of time and space. In this study, the monitoring of cyanobacterial   well as in other Moroccan aquatic systems, particularly in dams intended to provide drinking water to urban populations. Species of this genus are isolated from other Moroccan water reservoirs, including Imfout, Takerkoust, and Almassira (Oudra et al., 2001a(Oudra et al., , 2001bLoudiki et al., 2002;Sabour, 2002). This genus is known to form cyanobacterial blooms in stagnant waters (Douma et al., 2010). In fact, in the studied water bodies, Microcystis spp produces more cyanotoxins compared to other species.  LA, and MC-YAba in NAKHLA dam. These varieties of cyanotoxins are recognized by their acute toxicity. Studies have revealed that the toxicity of Microcystis is due to the disposition of the gene coding for microcystin (Carmichael, 1995;Rouhiainen et al., 1995;Dittmann et al., 1997).
P. galaeta filamentous species is also incriminated in dam waters and produces cyanotoxins also recognized            bodies and when the winter is not very cold (Sas, 1989;Oudra et al., 2002a;Loudiki et al., 2002). O. tenuis is also inventoried in Moroccan water bodies (Oudra et al., 2002a). This species develops variant cyanotoxins also recognized by its acute toxicity. In our study, it is found in SMIR ([Mser7]MC-LR, MC-LA), in BELMEHDI (MC-YAba), and in NAKHLA ([Mser7]MC-LR and MC-YAba). This species is less frequent than the other species but it also has the capacity to generate large varieties of cyanotoxins.
Microcystis, Pseudanabaena and Oscillatoria species are the most common and potentially toxic cyanobacteria  found in Moroccan freshwater and are the main producers of cyanotoxins. This is also evident in Euro-Mediterranean countries (Filatova et al., 2020), such as in France (Lac de Grand-Lieu), Portugal (Vasconcelos et al., 1996;Moreira et al., 2020), Spain (Quesada et al., 2004), Greece (Christophoridis et al., 2018), and Italy (Bruno et al., 1992), African countries such as Algeria (Saoudi et al., 2017), Ethiopia (Major et al., 2018), and Nigeria (Kadiri et al., 2020). The presence of microcystins in freshwater species of the genus Oscillatoria is not only an ecological problem, but also presents a health risk when water is used for  drinking purposes (Lindholm et al., 1989). On the other hand, some strains, known for their toxicological potential, are considered to be of biotechnological interest, such as Anabaena variabilis. This species is used for its hydrogen production (Yoon et al., 2006) and can remove phenols and its derivatives (Hirooka et al.,  2003) as well as heavy metals (Nagase et al., 2005) from the environment and industrial wastewater (Yoon et al., 2006).

Conclusion
This study contributes to the knowledge of the systematics and biogeography of toxic cyanobacteria and Their toxins quality in the water bodies of Northern Morocco. It is a qualitative analysis of cyanotoxins produced by cyanobacteria species thriving in three water reservoirs near the City of Tetouan, namely SMIR, BELMEHDI and NAKHLA. The results show that the water bodies of Northern Morocco are exposed to cyanobacterial proliferation exposing these water bodies to numerous variants of Microcystins (MC-YR,