Acyanobacterial harmful algal bloom (CyanoHAB) has occurred in May 2015 in the Delta of Axios River, in Thessaloniki, in the region of Central Macedonia, in Northern Greece. The bloom expanded to the coastline of Thermaikos Gulf near the areas of Chalastra and Kimina. Toxic Anabaena sp. was isolated from the water samples. Microcystin-RR(50ppb) was confirmed with immunoassay method for microcystins (Adda specific ELISA) and Liquid Chromatography- Mass Spectrometry (LC-MS).In this area monitoring of microcystins and its algal blooms are only experimentally applied by a satellite system. This research investigated the toxin and microcystin cells in the local coastal water and emphasize to the satellite remote sensing, as a precaution, to reduce and avoid impacts on animals' health, Public Health and ecosystems, after harmful algal blooms.
Cyanobacteria are ancient gram negative, prokaryotic, photosynthetic
microorganisms, which are distributed globally in fresh, brackish and marine waters and
terrestrial environments Toxic species can be potentially hazardous for animals’ and
public health, especially during the “water bloom phenomenon”, since they produce
secondary metabolites, the cyanotoxins
In Greece CyanoHABs have been reported mostly in lakes
In late May 2015 after expansion of a cyanobacterial blooming to the coastline of Thermaikos Gulf, surface water samples were collected from three sampling places, one near the Delta of Axios, one near Chalastra and one near Kimina (40o 32΄ 39.22΄΄Βand 22o 45΄ 16.74΄΄Ε, 40ο30΄ 12.60΄΄Βand 22ο42΄ 19.40΄΄Ε, 40ο31΄ 13.94΄΄Βand 22ο41΄ 43.29΄΄Ε) (Figure 1). During each sampling temperature, salinity, dissolved oxygen and ph of the surface water were recorded by anYSI 556 handheld multiparameter instrument (YSI Incorporated, Ohio, USA).To investigate the presence of cyanobacteria500ml of surface water were aseptically sampled, hold in a sterile 500ml flask and transferred in the laboratory in insulated cold boxes. One hundred and fifty milliliters (150ml) were filtered through filters with 0.45µm pore diameter (PALL CORPORATION, Michigan) and 150ml were filtered through Whatman GF/C filters (Sigma–Aldrich, Germany). The second filter was kept frozen at -80oC, until toxin detection.
Filtered water samples through filters with 0.45µm pore diameter, were incubated on Marine Agar and cyanobacteria growth was obtained according to Kalaitzidou et al., 2015.Cyanobacteria from colonies showing typical morphology were observed in an optical microscope (Olympus CH30), after Gram staining. The taxonomy system according to Anagnostidis and Komárek (1985) was used to classify cells showing typical morphology. Immobilecells that were long, filamentous with heterocysts were characterized as presumptive Anabaena sp. Bacterial cultures were further purified on Marine agar. DNA from bacteria of a single colony per plate was extracted and further analyzed with polymerase chain reaction, using specific primers for the amplification of the cyanobacterial 16S rRNA fragments (Forward primer CYA359F,
Kimina
Chalastra Delta of Axios
On examination day the sampled water that was filtered through Whatman GF/C filters and then stocked at -80oC, was defrost at 2oC, dissected and extracted with methanol 75% (Merck, Germany). The extraction method was previously described by Kalaitzidou et al., 2015. The extracts were first examined for microcystins with a commercial immunoassay method for microcystins (Adda specific) ELISA kit (Enzo Life Sciences Inc, USA). The detection limit was 0.10ppb microcystin-LR analogues. Samples with concentration over than 1ppb were considered positive and were further examined by Liquid Chromatography- Mass Spectrometry (LC-MS) (ACQUITY UPLC I-Class System, WATERS, USA).
The results of our research confirmed the presence of the freshwater toxic strain of Anabaena sp. only in one out of the three sampling places(Delta of Axios River). (figure 2)In this sampling place the LC-MS was positive for microcystin-RR (figure 3). The toxin concentration in water samples was 50ppb. Fig. 3. LC/MS settings for microcystin-RR. The retention time was 6.08, precursor ion was 519.8, quantifier ion was 135.1 and the UV absorption spectrum was 239nm.
It is suggested that the harmful algal bloom existing this period in this place was
favored by the climate conditions and the physicochemical parameters. Water
temperature was 16oC, salinity was 15psu and ph was 8.38. Moreover some fish and
crustaceans mortality was observed in the area. The detection of Anabaena sp. in
algal blooms have also been reported in the Baltic Sea, demonstrating that higher
flow rates within estuaries of rivers and coastlines may cause expansion of the
blooms to the marine environments (Engströ-Öst, and Mikkonen, 2011). Discharges
of freshwater into coastal marine environments inducing toxic cyanobacteria bloom
have been described also in Adriatic Sea in Lake Occhito
The impact of harmful cyanobacterial blooms is of great concern, since toxic
species can kill fish, seabirds and marine mammals and enter the human food chain.
Moreover nontoxic species cause economic losses due to the damage of the
ecosystem, the reduction of the fisheries and water resources and the recreational
activities
In Greece satellite monitoring systems are still experimental. In Thermaikos gulf chlorophyll-a (Chl-a), total suspended matter and sea surface temperature, were monitored by Medium Resolution Imaging Spectrometer (MERIS) and Moderate Resolution Imaging Spectroradiometer (MODIS) satellite systems. The results provided spatial and temporal patterns for the water quality parameters, showing that the increased Chl-a concentration, especially during late spring and summer, may induce high algal concentration (Monachouet al., 2014). In Ionian Sea an experimental program is implemented in cooperation between Greek and Albanian governments, with the code name “SAIMON”. The aim of the program is to monitor by “satellite real time monitoring” the eutrophication risk for the marine waters over the Greek-Albanian cross border area (http://saimon.getopendata.gr).
In conclusion typical monitoring strategies can detect but not predict cyanobacterial blooms. Satellite remote sensing is considered as a promising technique for tracking and detecting cyanobacterial harmful algal blooms. It can be implemented with traditional monitoring microbiological and chemical methods to reduce costs and working hours in the laboratories. It provides advantages, such as satisfactory spatial and temporal patterns, frequency and duration of exceeding nutrient criteria parameters, area mapping and collecting of useful data to control blooming and implement mitigation strategies.