Brahim Oudra

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.

Physiological changes in Triticum durum, Zea mays, Pisum sativum and Lens esculenta cultivars, caused by irrigation with water contaminated with microcystins: a laboratory experimental approach.

The aim of the present study was to investigate the effect of exposure to a microcystin (MC)-containing extract from a cyanobacteria bloom on growth, development, mineral nutrient accumulation, and photosynthetic activity of Triticum durum, Zea mays, Pisum sativum and Lens esculenta cultivars. The MCs in the extract, identified by HPLC and/or mass spectrometry (MS) were: MC-RR, -LR, -YR, -(H4)YR, -WR, and -FR. Plant growth and development was tested along 30 exposure days. After this period, MC-extract caused a clear reduction in plant growth and productivity, as well as deleterious effects on development and Photosystem II activity, measured by Fv/Fm fluorescence. However, the chlorophyll (a + b) content hardly varied, and the accumulation of Na+, K+, Ca2+, P and N was enhanced. All the effects observed were plant species, MC concentration, and exposure-time dependent. Relative accumulation of each MC variant greatly varied among plant species and plant organ. The data obtained supports the idea that the use of surface water containing MCs for crop irrigation can affect both plant yield and quality, and secondly, that MC accumulation in edible plants might pose a potential risk for human and animal health, if the MC intake exceeded the recommended tolerable limits.

Effects of cyanobacteria producing microcystins on seed germination and seedling growth of several agricultural plants

The effects of cyanobacteria aqueous extracts containing Microcystin-LR (MC-LR) on the seed germination and growth of Pisum sativum, Lens esculenta, Zea mays and Triticum durum were investigated. Experiments were carried out on a range of doses of the extract (equivalent to 0, 1.6, 2.9, 5.8, 8.7 and 11.6 μ g MC-LR/mL). The results confirm that these plants were sensitive to cell-free extracts of a toxic Microcystis and that germination inhibition was dose dependent. One-way analysis of variance (ANOVA) showed that P. sativum is the most sensitive tested species with a 97% germination rate reduction and L. esculenta was the most resistant. At the 8th day, the exposure to the microcystins (MC) resulted in a significant decrease of plant epicotyls length, roots length and a net inhibition of lateral root formation. It is concluded that MC could affect also terrestrial plants seedling germination and growth. Therefore, the use of water for irrigation contaminated by MC could exert negative biochemical effects on seed and plant metabolism which might influence the agricultural crops.

Allelopatic effects of cyanobacteria extracts containing microcystins on Medicago sativa–Rhizobia symbiosis

The eutrophication of water leads to massive blooms of cyanobacteria potentially producers of highly toxic substances: cyanotoxins, especially microcystins (MC). The contamination of water used for irrigation by these toxins, can cause several adverse effects on plants and microorganisms. In this work, we report the phytotoxic effects of microcystins on the development of symbiosis between the leguminous plant Medicago sativa (Alfalfa) and rhizobia strains. The exposure of rhizobial strains to three different concentrations 0.01, 0.05 and 0.1 μg MC ml(-1) led to decrease on the bacteria growth. The strains of rhizobia Rh L1, Rh L2, Rh L3 and Rh L4 reduced their growth to, respectively, 20.85%, 20.80%, 33.19% and 25.65%. The chronic exposure of alfalfa seeds and seedlings to different MC concentrations affects the whole stages of plant development. The germination process has also been disrupted with an inhibition, which reaches 68.34% for a 22.24 μg MC ml(-1). Further, seedlings growth and photosynthetic process were also disrupted. The toxins reduced significantly the roots length and nodule formation and leads to an oxidative stress. Thus, the MCs contained in lake water and used for irrigation affect the development of symbiosis between M. sativa and Rhizobia.

Effect of different microcystin profiles on toxin bioaccumulation in common carp (Cyprinus carpio) larvae via Artemia nauplii

In this study, a 12-day growth trial was conducted to compare the effect of the variation in microcystin (MC) composition in two Microcystis aeruginosa bloom samples on the growth performance and MC accumulation/transfer in the common carp (Cyprinus carpio L.) larvae. Fish were fed Artemia salina nauplii that had been preexposed to extracts from two M. aeruginosa natural blooms with different microcystins (MCs) profiles. Bloom A had MC-LR as major toxin (74.05%) while bloom B had a diversity of MC (MC-RR; MC-(H4)YR; MC-YR; MC-LR; MC-FR; MC-WR) with no dominance of MC-LR. Newly-hatched Artemia nauplii were exposed separately to the two M. aeruginosa extracts A and B (100 microg L(-1)EqMC-LR) for 2h. The MC concentration in the nauplii was 73.60+/-7.88ngEqMC-LRg(-1)FW (n=4, mean+/-SE) for bloom A and 87.04+/-10.31ngEqMC-LRg(-1)FW for bloom B. These contaminated nauplii were given at the same ration to different groups (A and B) of fish larvae. Larval weight and length from day 9 were significantly different between groups A and B, and in both cases lower than that of a control group fed non-exposed nauplii. MCs accumulation by larvae, inversely correlated with the growth performance, was also significantly different between groups A and B (37.43+/-2.61 and 54.55+/-3.01ngEqMC-LRg(-1) FW, respectively, at the end of the experimental period). These results indicate that MC profile of a bloom may have differential effects on toxin accumulation/transfer and toxicity.

CyanoHAB Occurrence and Water Irrigation Cyanotoxin Contamination: Ecological Impacts and Potential Health Risks

The world-wide occurrence of harmful cyanobacteria blooms “CyanoHAB” in fresh and brackish waters creates problems for all life forms. During CyanoHAB events, toxic cyanobacteria produce cyanotoxins at high levels that can cause chronic and sub-chronic toxicities to animals, plants and humans. Cyanotoxicity in eukaryotes has been mainly focused on animals, but during these last years, data, related to cyanotoxin (mainly microcystins, MCs) impact on both aquatic and terrestrials crop plants irrigated by water containing these toxins, have become more and more available. This last cited fact is gaining importance since plants could in a direct or indirect manner contribute to cyanotoxin transfer through the food chain, and thus constitute a potent health risk source. The use of this contaminated irrigation water can also have an economical impact which appears by a reduction of the germination rate of seeds, and alteration of the quality and the productivity of crop plants. The main objective of this work was to discuss the eventual phytotoxicity of cyanotoxins (microcystins) as the major agricultural impacts induced by the use of contaminated water for plant irrigation. These investigations confirm the harmful effects (ecological, eco-physiological, socio-economical and sanitary risk) of dissolved MCs on agricultural plants. Thus, cyanotoxin phytotoxicity strongly suggests a need for the surveillance of CyanoHAB and the monitoring of water irrigation quality as well as for drinking water.

Compensatory Growth Induced in Zebrafish Larvae after Pre-Exposure to a Microcystis aeruginosa Natural Bloom Extract Containing Microcystins

Early life stage tests with zebrafish (Danio rerio) were used to detect toxic effects of compounds from a Microcystis aeruginosa natural bloom extract on their embryolarval development. We carried out the exposure of developing stages of fish to complex cyanobacterial blooms containing hepatotoxic molecules - microcystins. Fish embryo tests performed with the bloom extract containing 3 mg·L−1 Eq microcystin-LR showed that after 24 h of exposure all fish embryos died. The same tests performed with other diluted extracts (containing 0.3, 0.1 and 0.03 mg·L−1 Eq microcystin-LR) were shown to have an influence on zebrafish development and a large number of embryos showed malformation signs (edema, bent and curving tail). After hatching the larvae were transferred to a medium without toxins to follow the larval development under the new conditions. The specific growth of the pre-exposed larvae was significantly more important than that of the control larvae. This may represent a compensatory growth used to reduce the difference in size with the control fish noted after hatching.

Contributed Article Dynamics and toxicity of Anabaena aphanizomenoides (Cyanobacteria) waterblooms in the shallow brackish Oued Mellah lake (Morocco)

Abstract The structure and abundance of phytoplankton communities were investigated during 1997 to 1999 in Oued Mellah, a shallow brackish and hypertrophic lake, with particular regard to Anabaena aphanizomenoides dynamics. Important events of algal blooms were observed mostly by the cyanobacteria Microcystis ichthyoblabe, Anabaena aphanizomenoides and Oscillatoria chlorina and by the ichthyotoxic haptophyceae Prymnesium parvum. Anabaena aphanizomenoides proliferated during late summer after the Microcystis ichthyoblabe blooms. The percentage of Anabaena aphanizomenoides of the phytoplankton biomass varied from 88 to 94 percent during bloom periods. Maximum biomass was 146 and 120 mg fresh weight l−1 during the 1997 and 1999 summer periods, respectively. The main environmental factors leading to the ecological success of A. aphanizomenoides were high temperature (25–28ˆC), high incident light intensities (1488–1912 μ E m−2 s−1, high nutrient deficiency (0 μ g P-PO−4 l−1; 0–0.18 mg N-NO3 l−1) and decrease of alkalinity (329–494 mg HCO3 − l−1). The toxicity of the Anabaena aphanizomenoides bloom was evaluated by bioassays and analyses. The lethal dose50 of the bloom sample tested in mice was 254 mg DW kg−1 body weight while toxicity (24 h LC−50) in the brine shrimp Artemia salina was 3.68 mg DW ml−1. The low microcystin content (3.28 μ g g DW −1) determined by ELISA was not consistent with the tested bioassays and is suggestive of the presence of other toxic compounds in the bloom extracts. Four toxic fractions were separated by HPLC-PDA and identified as microcystins according to their UV spectra. The production of microcystins by Anabaena aphanizomenoides bloom was confirmed by the analysis of the isolated strain which, in Z8 medium under controlled laboratory conditions, produced three variants of microcystins, two of them being similar to those produced by the natural bloom.

Effect of light and temperature on the population dynamics of two toxic bloom forming Cyanobacteria – Microcystis ichthyoblabe and Anabaena aphanizomenoides

The effect of light and temperature on the growth of Microcystis ichthyoblabe and Anabaena aphanizomenoides, isolated from the subtropical Oued Mellah lake, Morocco (33°30′N–07°20′W), were investigated in batch culture. Growth rates at 66 light–temperature combinations were determined and fitted with different mathematical models. The results show that the two Cyanobacteria grow at all light intensities and temperatures, except at 10 °C for A. aphanizomenoides, where the growth was strongly limited. The μmax of M. ichthyoblabe increased with temperature from 0.56 d−1 at 10 °C to 1.32 d−1 at 35 °C. At all tested temperatures, a relative photoinhibition within the studied range of irradiance was observed and the photosensitivity was thermodependent. For Anabaena, the obtained μmax ranged between 0.07 d−1 at 10 °C and 1.46 d−1 at 35 °C, and a weak photoinhibition was observed at 15 °C. The positive correlation between μmax and Iopt (r2≥0.93) indicates a close interaction between light and temperature on the cyanobacteria growth. The results obtained in this work suggest that the growth of these two species is possible under low light and low temperature.

Effects of the microcystin profile of a cyanobacterial bloom on growth and toxin accumulation in common carp Cyprinus carpio larvae

A 12 day growth trial was conducted to compare the effect of the variation in microcystins (MC) composition of two bloom samples of Microcystis aeruginosa on the growth performance and microcystin accumulation in common carp Cyprinus carpio larvae. Two M. aeruginosa natural bloom samples with different MC profiles were collected and larvae were exposed to cyanobacterial cells through their diet. Three diets, a basal control diet and two diets prepared from the basal diet plus the same toxins content (60 ng MC g(-1) diet) of each cyanobacterial bloom, were given at the same ration level to three groups of larvae during the experimental period. Larval mass and standard length from day 9 were significantly different between cyanobacterial treatments and in both cases lower than that of the control. The MC accumulation by larvae, inversely correlated with the growth performance, was also significantly different between cyanobacterial treatments (26.96 v. 17.32 ng g(-1) at the end of the experimental period). These results indicate that MC variants profile may have effects on the toxin uptake and toxicity. To date, this is the first laboratory study to show that fish accumulate MC depending on the toxin profile of the cyanobacterial bloom.