=Paper=
{{Paper
|id=Vol-1498/HAICTA_2015_paper92
|storemode=property
|title=Isolation of Toxic Marine Cyanobacteria and Detection of Microcystins in Thermaikos Gulf in Central Macedonia in Greece
|pdfUrl=https://ceur-ws.org/Vol-1498/HAICTA_2015_paper92.pdf
|volume=Vol-1498
|dblpUrl=https://dblp.org/rec/conf/haicta/KalaitzidouFPET15
}}
==Isolation of Toxic Marine Cyanobacteria and Detection of Microcystins in Thermaikos Gulf in Central Macedonia in Greece==
https://ceur-ws.org/Vol-1498/HAICTA_2015_paper92.pdf
Isolation of Toxic Marine Cyanobacteria and Detection
of Microcystins in Thermaikos Gulf in Central
Macedonia in Greece
Maria Kalaitzidou1, George Filliousis2, Evanthia Petridou3, Vangelis Economou4,
Alexandros Theodoridis5, Panagiotis Angelidis6
1
Region of Central Macedonia Greece, General Directorate of Rural Economy and Veterinary,
Directorate of Rural Economy and Veterinary of Regional Unit of Thessaloniki, Veterinary
Department, Greece, e-mail: M.Kalaitzidou@pkm.gov.gr
2
Laboratory of Microbiology and Infectious Diseases, School of Veterinary Medicine, Faculty
of Health Sciences, Aristotle University of Thessaloniki, Greece,
e-mail: georgefilious@vet.auth.gr
3
Laboratory of Microbiology and Infectious Diseases, School of Veterinary Medicine, Faculty
of Health Sciences, Aristotle University of Thessaloniki, Greece, e-mail: epetri@vet.auth.gr
4
Laboratory of Hygiene of Food of Animal Origin, School of Veterinary Medicine, Faculty of
Health Sciences, Aristotle University of Thessaloniki, Greece, e-mail: boikonom@vet.auth.gr
5
Laboratory of Animal Production Economics, School of Veterinary Medicine, Faculty of
Health Sciences, Aristotle University of Thessaloniki, Greece, e-mail: alextheod@vet.auth.gr
6
Laboratory of Ichthyology, School of Veterinary Medicine, Faculty of Health Sciences
Aristotle University of Thessaloniki, Greece, e-mail: panangel@vet.auth.gr
Abstract. The presence of toxic marine cyanobacteria and secondary
metabolites, microcystins, were studied in Thermaikos Gulf in Central
Macedonia in Greece, during the period from March 2013 to March 2014.
Toxic marine cyanobacteria were isolated in marine agar and identified with
PCR using primers based on 16S rDNA. The presence of microcystins in water
extraction was detected by immunoassay (competitive ELISA). The
concentration was ranged from 0.15 to 5ppm. It was observed that populations
of toxic marine cyanobacteria were increasing during spring and early winter
and there was a correlation to the physical and chemical parameters of the
water. The percentage of microcystins was 20.8 % and there were significant
differences (p<0.05) between the areas and the seasons.
Keywords: cyanobacteria, microcystins, water bloom, Thermaikos Gulf.
1 Introduction
Cyanobacteria (blue-green algae) occur in freshwater, brackish and marine
environments (Lawton and Cood, 1991). Toxic species can be potentially hazardous
for animal and public health, since the toxins known as cyanotoxins are produced
during eutrophication periods (Hitzfeld et al.; 2000). Poisoning by cyanotoxins has
been described in humans and animals (Carmichael, 1997; Chorus et al., 2010).
Cyanotoxins act as inhibitors of protein phosphatases 1and 2A (PP1 and PP2A),
832
inducing apoptosis and necrosis of the hepatocytes (Dawson, 1998). Moreover they
cause diarrhea and skin irritations (Chernoff et al., 2002; Martins et al., 2005).
Microcystins, which are cyclic heptapeptides, are cyanotoxins that exhibit toxic
activity due to a unique amino acid, Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-
phenyl-4,6-dienoic acid) (Codd et al., 1999; Hitzfeld et al., 2000). More than 70
analogues of microcystins have been described (MacElhiney & Lawton, 2005).
Although microcystins have been widely studied in fresh and brackish waters, the
bibliography is limited concerning microcystins in marine waters (Lawton and Cood,
1991). The presence of microcystins in the marine environment has been reported in
seawater from the Atlantic Ocean, the Caribbean, the Pacific, the Indian Ocean, the
Arabian Sea the Marmara Sea, and the Mediterranean Sea (Martins et. al., 2005; Taş
et al., 2006). In Greece there is only a report from Amvrakikos Gulf, which
confirmed the presence of microcystins in marine waters at levels ranging from 0.003
to 19.8 ng 1-1 (Vareli et al., 2012).
Although intense human activity such as fishing, shellfish farming and tourism
take place in the Thermaikos Gulf, there are no data concerning toxic marine
cyanobacteria. Thermaikos is a semi-closed gulf located in the northwest of Aegean
Sea, with a surface of 5.100 km2. It is one of the major productive areas of Central
Macedonia and generally Greece. The north limit is the bay of Thessaloniki with
maximum depths up to 27m (Nikolaidis et al., 2006), the west is the region of Pieria
and the east is the peninsula of Kassandra. In the south limit the gulf opens to
Aegean Sea with a maximum depth up to 90m. Thermaikos Gulf is enriched by three
big rivers, Axios, Loudias, and Aliakmonas and a small one, Gallikos. The annual
runoff mainly from November to May amounts to 150 m3s-1 (Ganoulis, 1988). The
sediment fluxes from the surrounding areas are 500 tn km-2 per year (Poulos et al.,
2000). These include nitrate and phosphate salts due to fertilizer application in the
adjacent crops.
The possible impact of cyanotoxins on people practicing recreational or
occupational activities in Thermaikos gulf is of concern. Many cases of skin and eyes
irritations, diarrhea, asthma and allergic reactions have been reported after swimming
in marine waters, where cyanobacteria bloom had occurred (Chorus et al., 2000;
Stewart et al., 2009). In addition toxic marine cyanobacteria constitute an
occupational hazard for fishermen, water sports teachers, cleaners and maintainers of
coasts, fish and shellfish farmers and divers (Stewart et al., 2009). Also it is reported
that toxic marine cyanobacteria inhibit grazing of zooplankton (Figueiredo et al.,
2004) and may be toxic to it and to crustaceans (Carmichael, 1992). Studies in
mussel’s embryos report that cyanobacterial extracts from Synechocystis spp. and
Synechococcus spp. caused total inhibition of embryogenesis (Martin et al., 2007).
This report is of importance for shellfish production of Thermaikos gulf since it
provides 90% of mussel production annually of Greece.
The morphological characteristics of Thermaikos Gulf, the enrichments and the
climate conditions of North Greece, especially of Thessaloniki can induce
eutrophication problems mostly near the shallow coastal zones (Koukaras, 2004;
Nikolaidis et al., 2006). The scope of this research was to identify the toxic marine
cyanobacteria and to detect the presence of microcystins in Thermaikos Gulf.
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2 Materials and methods
2.1 Field Sampling and Handling
During 2013, 120 water samples were collected from Thermaikos gulf every three
months, with the exception of the winter months. The sampling points were selected
according to their ecological and economic importance, with selected points having
intense fishing, shellfish farming and recreational activities. The sites were: 1.
Chalastra area in Thessaloniki region (40o 32΄ 20.12΄΄N and 22o 44΄56.63΄΄E) 2.
Aggelochori area in Thessaloniki region (40o 29΄30.05΄΄N and 22o 49΄11.79΄΄E) 3.
Makrigialos area in Pieria region (40o24΄57.98΄΄ N and 22o37΄14.93΄΄E) 4. Klidi area
in Imathia region (40o28΄37.03΄΄N and 22ο39΄58.94΄΄E). Sampling was performed by
a portable hose sampler, which was submerged at a depth of 2 m. After receiving the
water column, 500ml of marine water were transferred into a sterile flask. Salinity,
oxygen saturation, pH and temperature of the water were measured in the field with
an YSI 556 handheld multiparameter instrument (YSI Incorporated, Ohio, USA).
The samples were transferred in the laboratory in insulated cold boxes. 150 ml were
filtered through filters with 0.45μm pore diameter (PALL CORPORATION, 600
South Wagner Road Michigan). One filter was used for culture and one was stored in
freezer at -80oC until microcystin detection.
Fig. 1. Sampling areas in Thermaikos Gulf
2.2. Cyanobacteria isolation and identification
After filtration the filters were placed on Marine Agar growth medium (CONDA
S.A. Torrejon de Ardoz, Madrid Spain) supplemented with imipenem (50mg l-1,
BIO-RAD, München, Germany) (Ferris and Hirsch, 1991) and kanamycin (50mg l-1,
BIO-RAD, München, Germany). The petri dishes were incubated up to 21 days at
25oC under cool white fluorescent light (700LUX), with a 14:10 light:dark cycle and
monitored for growth at the seventh, tenth, fourteenth and twenty first day of
incubation. Colonies showing typical morphology were observed in an optical
microscope (Olympus CH30) after Gram staining. The morphology characteristics
were used to characterize cells as to Synechocystis spp. according to Anagnostidis
834
and Komárek (1985). In brief cells that were coccoid, nanoplaktonic morphology
with a diameter of 1 to 4 μm, organized as single cells, doublets of dividing cells, and
cloverleaf-type cell aggregates were characterized as presumptive.
Fig. 2. Marine cyanobacteria cells after Gram staining.
Colonies showing typical morphology were harvested and passaged in Marine
Agar. The cultures were considered axenic after two passages, and further identified
with PCR, using specific primers for the amplification of the cyanobacterial 16S
rDNA fragments (Forward primer 27f 5’-AGA GTT TGA TCM TGG CTC AG,
reverse primer 1525r 5’-AAG GAG GTG WTC CAR CC) (Svenning et al., 2005).
2.3 Sample Analysis for Microcystins
For microcystins detection the frozen filters were dissected and extracted with
methanol (Merck, Germany). The extraction was performed with a 75% aqueous
solution of methanol, since it is reported to be the most suitable for microcystins
extraction (Rodriguez et al., 2005). The filters were cut in squares pieces of 2-3 mm
acne and put in 10 ml methanol overnight. The supernatant was transferred into 50
ml tubes, 10 ml of methanol 75% were added in the pellets. Then they were heated at
55oC for 15 min, centrifuged at 3000rpm for 20min at 4oC and the supernatant was
collected and transferred into the 50ml tube. This procedure was repeated once more,
the extracts were pooled and concentrated using rotary evaporation until dry,
dissolved in 1ml methanol 75% and filtered through syringe filters.
The extracts were examined for microcystins with the commercial immunoassay
method Microcystins (Adda specific) ELISA kit according to `manufacturer’s
instructions (Enzo Life Sciences Inc, USA). The detection limit was 0.10ppb
microcystin-LR analogues. The absorbance was read at 450nm using a microplate
ELISA photometer (DAS model A3, Italy). Calculation of microcystin concentration
was performed according to the standard curve plotted against standard
concentrations of 0.15, 0.40, 1.00, 2.00, 5.00 ppb of microcystin-LR analogues.
835
2.4 Statistical Analysis
The presence of cyanotoxins was measured as the percentage of microcystins at
concentrations higher than 1 ppb, within the selected samples. The relation of the
toxins frequencies to region and season was evaluated using the chi-square test of
independence (χ2-test). The chi-square test of independence was applied to determine
whether there is a significant association between two variables or not. Analysis of
Variance (ANOVA) was performed to evaluate possible effects of season and region,
as well as their interaction, on toxins concentration, temperature, ph and salinity.
Differences between mean values of specific factors were evaluated using the
Duncan’s new multiple range test. The statistical analysis was conducted using the
SPSS software program and significance was declared at p ≤0.05, unless otherwise
noted.
Fig. 3. PCR detection of 16S rDNA of cyanobacteria.
3 Results and Discussion
Our research confirmed the presence of toxic marine cyanobacteria in Thermaikos
gulf by culturing of cyanobacteria in a novel medium, marine agar supplemented
with imipenem and kanamycin. We found that the combination of imipenem and
kanamycin at the final concentrations of 50mg l-1 helped to reduce saprophytic
microbial flora and greatly enhanced the easiness and possibility of obtaining axenic
cultures. ELISA showed that concentrations of microcystins varied from 0.15 ppb in
late summer and late autumn to 5.00 ppb in spring and early autumn. April, May and
June were the months with higher toxin concentrations. It was found that 20.8% of
the samples had concentrations higher than 1 ppb (Table 1). The percentage of
positive samples were higher in Aggelochori and Makrigialos areas (36.7% and
26.7% respectively), while Chalastra and Imathia had lower ones (13.3% and 6.7%
respectively).
The number of positive samples varied had shown a seasonal variation (Table 2).
In Chalastra and Aggelochori the number of positive samples was higher during
836
spring (3 and 6 positive samples respectively). On the other hand in Makrigialos the
number of positive samples was higher during autumn (5 samples). This can be
explained by the position of the sampling points in regard of the discharges of Axios
River. The outfall of Axios is near the Chalastra and Aggelochori sampling points
which are situated in the greater vicinity of it. Whereas the Imathia (approximately 6
km) and especially Makrigialoas (approximately 14 km) . The sampling points in
these areas are located near to Delta of Axios, which is the second most polluted
river in Greece (Skoulidis, 1993). The discharges of Axios are higher during spring
reaching the 279 m3 sec-1 (Poulos et al., 2000; Koukaras, 2004) since they are
connected to the snowmelt from mountains in FYROM (Poulos et al., 2000). In
addition the gulf streams which are south-south east during spring and summer
(Koukaras, 2004), transfer big amounts of water masses from Chalastra to
Aggelochori.
Table 1. Presence of toxins in marine waters for each region
Regions Presence of toxins N (%)
Chalastra 4/30 (13.3%)
Makrygialos 8/30 (26.7%)
Imathia 2/30 (6.7%)
Aggelochori 11/30 (36.7%)
Total 25/120 (20.8%)
Table 2. Presence of toxins in marine waters in each region according to seasons
Seasons
Regions Spring Summer Autumn Total
Chalastra 3/10 1/10 0/10 4/30
Makrigialos 2/10 1/10 5/10 8/30
Imathia 0/10 1/10 1/10 2/30
Aggelochori 6/10 3/10 2/10 11/30
Total 11/40 6/40 8/40 25/120
The test of independence show that there is a significant association between the
concentration of the toxins and the region (p-value= 0.02). Significant differences in
microcystins concentrations were observed within areas in spring and autumn as it is
shown in Table 3.
Larger microcystin concentrations were observed in samples from Aggelochori
sampling point. This can be explained by the proximity of the sampling point to
urban waste treatment facility, where all the sewage of Thessaloniki, the second
bigger city in Greece, is processed.
Regarding the physical and chemical characteristics of the water, temperature
ranged from 13oC to 23oC depending on the season, ph from 7.80 to 8.79 and salinity
from 34.8 to 38‰. Oxygen saturation was 75-80%. Tables 4 and 5 show the mean
temperature values between areas in different seasons and significant differences of
ph and salinity according to seasons respectively. Statistical analysis has revealed
837
significant effects of the physicochemical characteristics to the growth of toxic
cyanobacteria, especially during spring and autumn.
Table 3. Microcystin concentration differences within areas according to the season (ppb)
Regions
Seasons Chalastra Makrygialos Imathia Aggelochori
Mean±SD Mean±SD Mean±SD Mean±SD
Spring 1.55±2.05ac 1.06±1.42ab 0.35±0.22b 2.25±1.53c
Summer 0.58±0.71a 0.75±0.21a 0.41±0.29a 1.15±0.88a
Autumn 0.60±0.29a 1.75±1.72b 0.67±0.39a 0.97±0.38a
ab a b
Total 0.91±1.31 1.18±1.31 0.47±0.33 1.45±1.16a
* Different uppercase letters indicate statistically significant differences.
Our results show a seasonal growth of toxic marine cyanobacteria in Thermaikos
gulf during spring and early autumn. Seasonal distribution of cyanobacteria
especially in these seasons have been reported previously (Magalhães et al., 2003;
Taş et al., 2006; De Pace et al., 2014) that also report that cyanobacterial blooms in
fresh, brackish and marine waters are observed mainly during late spring and early
winter, especially in May or September.
Table 4. Temperature differences among seasons (oC) in each area
Regions
Seasons Chalastra Makrygialos Imathia Aggelochori
Mean±SD Mean±SD Mean±SD Mean±SD
Spring 13.30±0.67abc 12.70±0.48b 14.50±0.53c 14.00±1.33abc
Summer 19.70±2.58a 20.00±1.76ab 18.70±2.58a 21.50±1.78b
a c b
Autumn 16.00±3.16 19.50±1.58 20.60±2.76 18.40±0.52c
Total 16.33±3.53a 17.40±3.64b 17.93±3.35b 17.97±3.38b
* Different uppercase letters indicate statistically significant differences.
The high water temperatures generally reported in Greece can contribute in marine
cyanobacteria bloom, confirming that temperature is the most important physical
parameter which affects growth rates of bloom forming cyanobacteria (Davis et al.,
2009). Moreover the four rivers enrich the gulf through the runoff of large quantities
of nutrients, including nitrate and phosphate salts from adjacent farmlands,
increasing the risk. Discharges of freshwater (rivers or lakes) into coastal marine
environments inducing toxic cyanobacteria bloom, have been described in Marmara
Sea (Taş et al., 2006) and in Adriatic Sea from lake Occhito (De Pace et al., 2014).
Microcystin concentration in Thermaikos gulf is of great concern, since the upper
mean limit was 2.25±1.53 ppb in Aggelochori area during May. The larger
concentration observed were above the Tolerable Daily Intake (TDI) recommended
by the World Health Organization (0.04μg kg-1d-1). This concentration is higher than
the one in Amvrakikos Gulf (19.8 ppt), despite that Amvrakikos is a shallow gulf,
low salinity, with two major rivers (Louros and Arachthos) draining into this gulf
and considered the most polluted gulf in the Ionian Sea (Economou et al., 2007). In
Amvrakikos gulf high concentrations of microcystins were observed in mussels
838
Mytilus galloprovincialis, at levels ranging from 45±2 to 141.5±13.5 ppt w/w (Vareli
et al., 2012). Other studies from Mediterranean Sea confirmed the presence of
microcystins in marine environment. In Adriatic Sea the concentration of microcystin
in water was 0.61 ppb in May 2009, after a cyanobacterial bloom in Lake Occhito
(De Pace et al., 2014).There are several reports of microcystin accumulation in
seafood from the Baltic Sea (Luckas et al., 2005). In Sebetiba Bay in Brazil the
concentration of microcystins in marine waters due to Synechocystis spp. bloom in
June 1999 was 0.12 μg l-1.Microcystins were detected also in fisheries and
crustaceans with the largest concentration (0.198μg kg-1d-1) observed in a crab sample
in September and was 13 times above the upper limit posed by the World Health
Organization (Magalhães et al., 2003).
Table 5. ph and salinity (‰) differences according to seasons
Parameters
Seasons ph Salinity
Mean±SD Mean±SD
Spring 8.28±0.28a 35.10±0.76a
Summer 8.09±0.12b 34.50±2.08b
Autumn 8.00±0.25c 35.50±1.40a
Total 8.12±0.25 35.03±1.54b
* Different uppercase letters indicate statistically significant differences
In conclusion from our results it is found that marine cyanobacteria can be isolated
in marine agar medium with the addition of imipenem and kanamycin at a final
concentration of 50mg l-1. The presence of toxin producing marine cyanobacteria in
Thermaikos gulf is seasonable, especially during spring and winter. The physical and
chemical parameters of the water (temperature, ph and salinity) conducive to the
development of marine cyanobacteria. The high levels of microcystins especially
during late spring and early autumn are of awareness, since they are considered
hazardous for public health, aquatic animals and the marine ecosystem.
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