Identification and quantification of cyanobacterial toxins (microcystins) in two Moroccan drinking-water reservoirs (Mansour Eddahbi, Almassira).

Mansour Eddahbi (MED) (30 degrees 55'N, 6 degrees 53'W) and Almassira (ALM) (31 degrees 95'N, 6 degrees 72'W) are two Moroccan lake reservoirs located at an arid and semi-arid hydrographic basin, respectively. Both are used for irrigation, recreational activities and drinking-water production. This paper deals with the characterization and quantification of microcystins (MC) from two Microcystis aeruginosa blooms occurring in those reservoirs. The toxicity of the blooms was confirmed and evaluated by both mouse and Artemia bioassays. The calculated LD50 values revealed that the MED bloom had a medium toxicity (LD50=358 mg kg(-1) body weight), whereas the ALM bloom had low toxicity (LD50=829 mg kg(-1) body weight). The 24-h LC50 values were 1.88 and 4.15 mg ml(-1) for the MED and ALM blooms, respectively, using Artemia assay. The identification and quantification of MC variants were carried out by high performance liquid chromatography (HPLC) equipped with a photodiode array detector, and HPLC coupled to mass spectrometry. The MC content, as Microcystin-LR (MC-LR) equivalents, was higher in the MED bloom (64.4 microg g(-1) dry weight) than in the ALM bloom (9.9 microg g(-1) dry weight). Five MC variants were identified in the MED cyanobacteria bloom (MC-RR, MC-YR, MC-LR, MC-FR, and MC-WR) and only one (MC-LR) in the ALM bloom. The results show that the occurrence of toxic cyanobacteria blooms in the studied reservoirs may be regarded as a health hazard; therefore, cyanotoxin monitoring in them is highly recommended.

Heterotrophic and mixotrophic growth of Micractinium pusillum Fresenius in the presence of acetate and glucose: effect of light and acetate gradient concentration.

The main objective of this study was to determine the importance of secondary mechanism of organic carbon utilization (mixotrophic and heterotrophic modes) in addition to CO2 fixation (photoautotrophic mode) in the green alga, Micractinium pusillum Fresenius (chlorophyta), isolated from a waste stabilization pond. The growth was studied in the presence of acetate and glucose. The incorporation rate of 14C- acetate was measured in the light and in the dark at different concentrations. Finally, in order to underline the role of photosynthesis and respiration processes in the acetate assimilation, the effect of two specific metabolic inhibitors, a specific inhibitor of photosystem II (DCMU) and an uncoupler respiratory (DNP), has been studied. The obtained results showed that M. pusillum grows in the presence of organic substrates, i.e., glucose and acetate, in the light (mixotrophic growth) as well as in the dark (Heterotrophic growth). The growth was much more important in the light than in the dark and more in the presence of glucose than of acetate. In the light, the presence of acetate led to a variation of growth parameters mumax, iotaopt, and beta. The effect of acetate gradient on the growth of the microalga was severe as soon as its concentration in the medium was higher. The acetate uptake followed a Michaelis-Menten kinetic in the light as well as in the dark. The capacity of assimilation was slightly higher in the dark. The utilization of DNP and DCMU indicates that acetate incorporation is an active process depending on both anabolic (photosynthesis) and catabolic (respiration) metabolisms, corroborating the model of the Michaelis-Menten kinetic.

Detection and quantification of microcystins from cyanobacteria strains isolated from reservoirs and ponds in Morocco.

In Morocco, the occurrence of toxic cyanobacteria blooms is confirmed in some water bodies used for recreational and/or as drinking water reservoirs. According to WHO recommendations, the establishment of a monitoring program for microcystins is a necessity. This paper presents toxicological studies of 19 toxic cyanobacteria strains of Microcystis, Synechocystis, Pseudanabaena, and Oscillatoria. These strains were isolated from various water bodies including natural lakes, reservoirs, and ponds located in central regions of Morocco. The isolation, culture, and biomass production of these strains was made on Z8 or BG13 media under laboratory controlled conditions. The hepatotoxicity of cyanobacterial lyophilized material was confirmed by mouse bioassays. The amount of microcystins produced by each strain was determined by the enzyme-linked immunosorbent assay (ELISA). The detection and identification of microcystin variants was performed by high performance liquid chromatography (HPLC) with photodiode array detection. Almost all strains showed medium to high toxicity, the estimated LD50 i.p. mice bioassay ranged between 28 to 350 mg/kg body weight. The concentrations of microcystins varied between 2.16 to 944 micrograms/g and 26.8 to 1884 micrograms/g dry weight determined by ELISA and HPLC, respectively. The screening of bloom-forming and microcystin producer cyanobacteria strains in these fresh water bodies leads us to propose the need for the establishment of a survey of cyanobacteria and a cyanotoxin-monitoring program.

Bioactive compounds from Pseudanabaena species (Cyanobacteria).

Bioactive products of the multicellular filamentous benthic cyanobacterium, Pseudanabaena species, isolated from wastewater stabilization ponds at Marrakech, were tested against some pathogenic micro-organisms. Extracellular and intracellular products released by this blue-green alga in the stationary, growth phase, reduced the survival of Escherichia coli, Salmonella sp., Staphylococcus aureus and Candida albicans, and stimulated the growth of non-O1 V. cholerae, whereas no significant effect was noted on the growth of Candida tropicalis. Intracellular substances (methanolic extract) had a stronger inhibitory effect on these micro-organisms than that produced by extracellular substances. These antibacterial and antifungal substances may have a pharmacological value. They may have an important ecological effect on the composition of the bacterial community in Marrakech stabilization ponds when blooms of Pseudanabaena species occurred in this sewage treatment ecosystem during hot periods.

Isolation, characterization and quantification of microcystins (heptapeptides hepatotoxins) in Microcystis aeruginosa dominated bloom of Lalla Takerkoust lake-reservoir (Morocco).

This paper presents the first data on the identification, characterization and quantification of microcystins isolated from both an extract of a cyanobacteria natural bloom, collected from a eutrophic Moroccan reservoir (Lalla Takerkoust, Marrakesh) and an isolated strain cultivated under laboratory conditions. The isolation and purification of toxins was performed by reverse phase HPLC and then characterized by amino acid analysis and fast atom bombardment mass spectrometry (FAB-MS). Chemical characterization of the toxins from the bloom revealed variants of microcystins such as Mcyst-LR, Mcyst-RR, Mcyst-YR and [D-Asp3]Mcyst-LR. However, the Microcystis aeruginosa strain produced only Mcyst-RR. Using an ELISA assay the total microcystin contents of eight bloom samples collected from 1994 to 1997 ranged from 0.7 to 8.8 microg/mg of lyophilized material. The two isolated Microcystis strains contained higher amounts of microcystins (0.65 microg/ mg of dry weight) than the Pseudanabaena strains (0.021 microg/mg of dry weight). Our results show that the presence of cyanobacteria toxins in water used for drinking in a North African country may be regarded as an health hazard. These results are a contribution to the knowledge of the biogeography of toxic cyanobacteria and their toxins, namely in north African countries.