=Paper=
{{Paper
|id=Vol-2030/HAICTA_2017_paper4
|storemode=property
|title=Cyanobacterial Harmful Algal Bloom in the Delta of Axios River in the Northern Greece: Impacts and Monitoring
|pdfUrl=https://ceur-ws.org/Vol-2030/HAICTA_2017_paper4.pdf
|volume=Vol-2030
|authors=Maria Kalaitzidou,George Filiousis,Evanthia Petridou,Vangelis Economou,Alexandros Theodoridis,Panagiotis Angelidis
|dblpUrl=https://dblp.org/rec/conf/haicta/KalaitzidouFPET17
}}
==Cyanobacterial Harmful Algal Bloom in the Delta of Axios River in the Northern Greece: Impacts and Monitoring==
https://ceur-ws.org/Vol-2030/HAICTA_2017_paper4.pdf
Cyanobacterial harmful algal bloom in the Delta of Axios
River in the Northern Greece: Impacts and monitoring
Maria Kalaitzidou1, George Filliousis2, Evanthia Petridou2,Vangelis Economou2,
Alexandros Theodoridis2, Panagiotis Aggelidis2
1
Veterinary Directorate of Central Macedonia, Department of Public Health
email: M.Kalaitzidou@pkm.gov.gr
2
Department of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of
Thessaloniki, Greece,email: panangel@vet.auth.gr
Abstract. 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.
Keywords: cyanobacteria, microcystins, algalbloom, Thermaikos Gulf,
satellite remote sensing.
1 Introduction
Cyanobacteria are ancient gram negative, prokaryotic, photosynthetic micro-
organisms, 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 (Whitton and Potts, 2002). The problem of
cyanobacterial harmful algal blooms (CyanoHABs) has risen the last decades
(O’Neil et al., 2012). Climate changes and human activities, such as the intensive use
of fertilizers with nitrates and phosphates salts and the farm animals’, industrial and
urban wastes running off into the coastal waters, have significant role in the
proliferation of harmful cyanobacterial blooms (Paerl and Huisman, 2009).
In Greece CyanoHABs have been reported mostly in lakes (Gelis et al., 2006), but
there in not enough data in marine environments. In late May 2015 during research
sampling for toxic cyanobacteria in Axios River, it was found that there was a bloom
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near the Delta of the river. It was decided to take water samples from the Delta and
the surrounding area to search for the potential presence of cyanobacteria toxic
species. Axios is the largest river in the region of Central Macedonia. Its annual
runoff into Thermaikos Gulf comes up to 279m3s-1, enriching the shallow coastal
zones (Nikolaidis et al., 2006). The Delta of the river and its surrounding land are
important wetland, protected by the Ramsar Convention (Jones et al.,
2012).Thermaikos is a semi-closed gulf located in the northwest of Aegean Sea, with
intensive fishing, mussel farming and recreational activities (Koukaras, 2004). The
expansion of toxic cyanobacterial blooms into the shallow coastal waters in
Thermaikos Gulf, could affect the ecosystem, the fishing and farming activities and
the public health. The scope of this research was to detect the presence of toxic
cyanobacteria after blooming and to propose its monitoring systems, beyond the
typical sampling and microbiological and chemical technics.
2 Materials and Methods
2.1 Field Sampling
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.
2.2 Isolation and Identification of Cyanobacteria
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,
29
5΄-GGG GAA TYT TCC GCA ATG GG-3΄ and reverse primer CYA781R, 5΄-GAC
TAC TGG GGT ATC TAA TCC CAT T-3΄ (Nübel et al., 1997)
Chalastra
Kimina
Delta of Axios
Fig. 1. Sampling places in the Delta of Axios River
2.3 Sample Analysis for Microcystins
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).
3 Results and Discussion
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. 2. Anabaena sp. cells and PCR detection of 16S rRNA of cyanobacteria.
30
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 (De Pace et al., 2014). In
the case of Adriatic Sea the expansion of the blooming affected the farming of the
Mediterranean mussel Mytilus galloprovincialis, as microcystins were detected in
these mussels in high concentration.
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(Anderson, 2009). These impacts led the United States Environmental
Protection Agency (US EPA) to issue guideline for monitoring criteria for inland and
marine waters using satellite remote sensing (Gibsonet al., 2000). Typical monitoring
systems based on sampling water cost (Corrales and Maclean, 1995). Monitoring by
satellite remote sensing can be the solution to transmit on time data and permit
efficient alerts (Kutser et al., 2006).
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
31
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.
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