Lake Burgas or Lake Vaya is the largest natural lake in Bulgaria (27 - 28 km²) which, together with Atanasovsko Lake and Mandrensko Lake, is part of the Burgas Lake Complex. It is situated in the immediate vicinity of Burgas - the second largest city on the Bulgarian Black Sea coast, which is an important industrial and transport centre (Fig. 1).

Figure 1.  

Location of the Lake Vaya (Bulgaria) and sampling stations. The red dot represents the position of the lake sampling stations : 1 – Vaya-East , 2 – Vaya-West.

Lake Vaya is very shallow (with an average dept of 1-1.5 m), separated from the sea by a strip of sand (Kumluka) and connected to it through a narrow channel. At its southern edge, the Lake forms a shallow bay which, in the past, had a connection with Mandrensko Lake. The rivers Aytoska, Sandardere and Chukarska flow into its western part. The water in the Lake is slightly salty with significant seasonal and annual fluctuations. It is surrounded by a strip of reeds which, in the western part, forms a large and dense massif.

The physicochemical parameters (temperature, dissolved oxygen, oxygen saturation, pH, conductivity, salinity) were measured in situ by using field devices of the brand WTW (Xylem Analytics Germany Sales GmbH & Co. KG, WTW, Weilheim, Germany) (Table 1).

Phytoplankton samples were collected five times during the growing season (April-September) of 2018 from two points of Lake Vaya: East (42°29'00.6''N, 27°26'16.7''E) and West (42°30'38.6''N; 27°22'03.6''E). Station Vaya-East is located in the area of the open water surface in the eastern part of the Lake. Station Vaya-West is located in the western part of the Lake, near the reed strip and is characterized by intense growth of submerged macrophytes. Due to the polymictic nature of the Lake, the sampling was done from the subsurface layer in accordance with sampling, preservation and sample preparation standards (ISO 5667-1,3: 2012). Phytoplankton samples were preserved with formalin (4% final concentration). Along with the phytoplankton samples, additional samples were taken for measurement of chlorophyll-a. Its concentration was measured spectrophotometrically, following the international standard ISO 10260:1992. During the sampling in September, from the same points, we also collected water samples without fixation to analyse for presence of cyanotoxins.

The taxonomic composition of the phytoplankton (in live and preserved samples) was determined by using a light microscope Amplival (magnification up to 1000x). For identification of the taxa, well-established cyanobacteria identifier books and floras were used. The phytoplankton count was performed with an inverted microscope Inverso (Medline Scientific, Chalgrove, Oxon, UK), equipped with a high definition digital camera using the Utermöhl method (Utermöhl 1958). For numerous species, at least 100 specimens were counted (Lund et al. 1958). The algal biovolume was calculated on the basis of formulas for geometric shapes (Hillebrand et al. 2002). The translation between biovolume and fresh biomass was performed according to Wetzel and Likens (2000). The full list for taxonomic composition and abundance of the phytoplankton species by stations is presented in Suppl. material 1.

The ecological status (ES) was determined by the HLPI method (Borics et al. 2018), intercalibrated for common intercalibration type L–EC1 (Lowland very shallow hard water) with the following characteristics: altitude < 200 m; depth < 6 m; conductivity 300-1000 μS cm^–1; alkalinity 1-4 meq l^–1 HCO₃.

As a metric unit for taxonomic composition, the HLPI index contains the Q index (Padisák et al. 2006), based on the functional groups (FGs) of the phytoplankton. The FGs of the phytoplankton species were determined following Reynolds et al. (2002), Padisák et al. (2009) and Borics et al. (2015). Only those FGs, which have a relative biovolume > 5%, were identified.

Collected non-fixed water samples in September were analysed for the presence of the cyanotoxins microcystins/nodularins, cylindrospermopsin and saxitoxins by using the Microcystins/Nodularins (ADDA), Cylindrospermopsin and Saxitoxin (PSP) ELISA Kits (Abraxis LLC, Warminster, PA, USA). The microcystins/nodularins test is an indirect competitive ELISA for the congener-independent detection of microcystins and nodularins, based on the recognition of microcystins, nodularins and their congeners by specific antibodies. The cylindrospermopsin and saxitoxin tests are direct competitive ELISA’s for detection of cylindrospermopsin and saxitoxins. They are also based on the recognition of cylindrospermopsin or saxitoxins by specific antibodies.

All ELISA tests were performed in accordance with manufacturers' instructions. The intensity of the blue colour is inversely proportional to the concentration of microcystins, cylindrospermopsin or saxitoxins present in the sample. The coloured reactions were recorded by measuring the absorbance at 450 nm on a microplate reader ELx800™ (BioTek Instruments Inc., Winooski, VT, USA). The concentrations of the samples are determined by interpolation using the standard curve constructed with each run. The detection limit of the Microcystins/Nodularins ELISA kit is 0.15 ppb (μg.l^–1). The mean lower detection limit of the cylindrospermopsin assay is about 0.05 ppb (μg.l^–1). The detection limit of the Saxitoxin ELISA kit is 0.02 ppb (μg.l^–1).

Lake Vaya is a shallow, polymictic lake whose salinity varies slightly from 0.4 to 0.5‰ (Table 1). It is characterized by low transparency, which decreases from 0.5 m during the spring to 0.1 m at the end of the summer due to the growth intensity of the phytoplankton community. The proximity of the rivers that flow into the Lake determine the lower transparency of the Vaya-West station. The waters of the Lake are alkaline and, in July, the pH reaches 10.8. Over the entire study period, we found oxygen supersaturation of the surface layer up to > 200% during the summer months.

Analyzing the phytoplankton composition of Lake Vaya from April to September 2018, we identified 100 taxa belonging to eight phytoplankton groups (Cyanobacteria, Chlorophyta, Charophyta, Ochrophyta, Bacillariophyta, Euglenophyta, Dinophyta and Cryptophyta), amongst which Cyanobacteria were dominant during July and September (Fig. 2, Table 2).

Figure 2.  

Seasonal succession in Lake Vaya according to the biovolume: (A) –Vaya-East, (B) – Vaya-West.

In both parts of the Lake, the phytoplankton numbers/total biovolume increased steadily from spring (April, May) to autumn (September) (Fig. 3). From July to September, phytoplankton abundance tripled in the eastern part (up to 1400x10⁶ cells l^–1) and doubled in the western part of the Lake (up to 744x10⁶ cells l^–1) (Fig. 3A). A similar situation was observed for the overall biovolume (Fig. 3B). In both parts of the Lake, the July and September values were significantly higher than those from April to June (Fig. 3B). From April to September, the biovolume increased from 1 mm³ l^–1 to 57 mm³ l^–1 at the Vaya-East station and from 3 mm³ l^–1 to 69 mm³ l^–1 at the Vaya-West station.

Figure 3.  

Total phytoplankton abundance (A) and total biovolume (B) during the study period.

During the study period, the two parts of the Lake (East and West) showed different qualitative and quantitative composition of the phytoplankton. In the eastern part, a higher phytoplankton density was observed. Ochrophytes, which dominated there in April, together with the cyanobacteria and chlorophytes, were not represented in the western part of the lake. The eastern part was richer in cyanobacterial species with a relative biovolume greater than 5% and there was a bloom of Planktothrix agardhii during the summer and autumn. The other dominant cyanobacterial species were representatives of the orders Nostocales (Anabaenopsis elenkinii, Aphanizomenon klebanii and Dolichospermum flos-aquae) and Synechococcales (Snowella litoralis), while dominant in the western part were representatives of the Synechococcales (Limnothrix redekei, Phormidesmis molle) and Oscillatoriales (Planktothrix agardhii, Planktothrix isothrix). The greater taxonomic diversity amongst the dominant diatoms (five species) and euglenophytes (eight species) in the western part is noticeable compared to the eastern part, where the two divisions were represented by two species each. Interestingly, the diatom species dominating in the eastern part in May and June (Aulacoseira granulata, Cyclotella meneghiniana) were much better represented in the western part in July and September, while their biovolume decreased in the eastern part. Chlorophytes (Coelastrum microporum, Oocystis marssonii, Pandorina morum) dominated in both parts of the Lake mainly in spring and cyanobacteria (Planktothrix agardhii) during summer and autumn (Table 2).

In June, euglenophytes (dominated by Euglenaformis proxima), diatoms (Aulacoseira granulata and Cyclotella meneghiniana) and chlorophytes (Pseudopediastrum boryanum) were most abundant in the eastern part of the Lake, while only euglenophytes and diatoms were present in the western part (Table 2, Fig. 4 and Fig. 5). In July, the dominance of euglenophytes and diatoms was replaced by that of cyanobacteria: Planktothrix agardhii in blooming concentration, Anabaenopsis elenkinii, Aphanizomenon klebahnii and Dolichospermum flos-aquae in Vaya-East; Planktothrix agardhii, P. isothrix and Phormidesmis molle in Vaya-West. This trend continued in September and was more pronounced in the eastern part of the Lake.

Figure 4.  

Phytoplankton abundance of the taxonomic groups (A) and biovolume of the taxonomic groups (B) in Lake Vaya during September.

Figure 5.  

Seasonal succession of the relative biovolume of the main FGs (%) in Vaya-East (A) and Vaya-West (B).

The cyanobacterial bloom and the detected microcystins and cylindrospermopsin in the water samples from September encouraged us to perform a detailed study of the phytoplankton during this month.

In September, cyanobacteria showed the highest numbers (1365.7x10⁶ cells l^–1) in the eastern part of the Lake, followed by chlorophytes (24.7x10⁶ cells l^–1), cryptophytes (4.41x10⁶ cells l^–1) and diatoms (2.65x10⁶ cells l^–1). Euglenophytes (0.69x10⁶ cells l^–1) and ochrophytes (0.10x10⁶ cells ^–1) were observed in relatively low numbers (Fig. 4A; Suppl. material 1). The total phytoplankton biovolume in this part of Lake Vaya was 57.5 mm³ l^–1. Values of the biovolume followed the same trend as the numbers being the highest for cyanobacteria (42.7 mm³ l^–1), followed by chlorophytes (5.59 mm³ l^–1) and cryptophytes (3.76 mm³ l^–1) (Fig. 4B; Suppl. material 1).

In Vaya-West, during September, cyanobacteria showed again the greatest numbers (674.6x10⁶ cells l^–1). Unlike the eastern part, however, the second place was occupied by diatoms (35.79x10⁶ cells l^–1), which displaced chlorophytes (24.26x10⁶ cells l^–1) to third place. The following taxonomic groups were euglenophytes (5.7x10⁶ cells l^–1), ochrophytes (0.76x10⁶ cells l^–1), dinophytes (0.32x10⁶ cells l^–1) and cryptophytes (0.26x10⁶ cells l^–1) (Fig. 4B; Suppl. material 1). The total phytoplankton biovolume in the western part of Vaya-Lake in September was 68.6 mm³ l^–1 dominated again by cyanobacteria (21.19 mm³ l^–1), followed by diatoms (19.18 mm³ l^–1) and euglenophytes (18.30 mm³ l^–1).

The application of the HPLI method showed that both stations of the Lake (Vaya-East and Vaya-West) are eutrophied (Table 3). The ecological status of Vaya-East is moderate, while the Vaya-West ecological status is of lower quality class – poor. The bad ecological status of Vaya-West is due to the significantly higher mean value of chlorophyll-a, as the values of the taxonomic index (Q index or normalized Ecological Quality Ratio /EQR/ of the Q index) at both stations do not differ significantly.

Effects of the eutrophication, which immediately affect the primary producers, were present at both (Vaya-East and Vaya-West) points – intensive, prolonged blooming of eutrophic cyanobacteria (Fig. 4). There is evidence that the dominant cyanobacterial species in blooming concentrations produce cyanotoxins. The physicochemical parameters confirmed the bad quality of the water – alkaline pH, low transparency and oxygen supersaturation of the surface layers during the day (Table 1).

During the study period, 14 main FGs were identified in Vaya-East and 13 FGs in Vaya-West (Fig. 5). Eight of them were common to both stations: C, F, J, P, S1, W1, X2 and Y. Except for functional group F, the others are typical inhabitants of nutrient-rich conditions in turbid eutrophic lakes (Padisák et al. 2009).

Although located in the same Lake, the two stations had different seasonal succession of the main FGs, which reflects the specifics of the two habitats. At Vaya-West station, there is an increased growth of macrophytes (Potamogeton pectinatus, Lemna minor) and the seasonal succession takes place in the following sequence: W2→W1,G→W1→S1,W1→W1,S1 (Fig. 5). Dominants or subdomains throughout the growing season were Euglenophyta from FGs W1 and W2, with their strongest development in June (70% relative biovolume). Other flagellated species from FGs G (green algae) and Y (cryptophytes) were co-dominants in May. After the peak of Euglenophyta in June, the presence of FGs tolerant to reduced light conditions increased in the middle and late summer. This is FG S1 (Planktothrix spp.) that is attached to persistently mixed layers, in which light is increasingly the limiting constraint (Padisák et al. 2009). The presence of Tc (Phormidesmis) and diatoms from MP (Navicula recens) is explained by the abundance of emergent macropytes (Table 2). The lack of stratification and continuous mixing are favourable for the growth of heavy diatoms from C (Cyclotella meneghiniana) and P (Aulacoseira granulata).

The seasonal succession at Vaya-East, located in the open Lake, was dominated by other functional groups: J→F→W1→S1,H1 (Fig. 5). In April, the phytoplankton was dominated by FG J. According to Borics et al. (2012), the most characteristic elements of the phytoplankton in well-mixed open lakes are the chlorococcalean green algae (J). For a short time, FG F (Oocystis marssonii - 80.7%) was found as a monodominant in May, although, according to literature, FG F is typical for clear, deeply-mixed meso-eutrophic lakes (Reynolds et al. 2002). In June, the number of main FGs increases and the biovolume is distributed more evenly between diatoms (C, P) and flagellated species (X2, W1). From July to September, intensive blooms of eutrophic cyanobacteria S1 (Planktothrix agardhii) with subdomains H1 (Anabaenopsis elenkinii, Aphanizomenon klebahnii, Dolichospermum flos-aquae) and S_N (Raphidiopsis raciborskii) appeared and remained (Fig. 5, Suppl. material 1). A probable reason for the dominance of FGs S1, H1 and S_N is that they are strong light competitors (Reynolds 2006). The situation, in which, during the later succession state, the lakes are dominated by several species or a certain functional group of algae, is known as equilibrium states or steady-state assemblages (Naselli-Flores et al. 2003).

In the present study, 22 cyanobacterial species belonging to 18 genera have been identified. Many of the identified cyanobacteria were previously reported as producers of cyanotoxins – Dolichospermum flos-aquae, Microcystis aeruginosa, Microcystis wesenbergii, Planktothrix agardhii and Raphidiopsis raciborskii. Previous studies on the phytoplankton composition of Lake Vaya registered cyanobacterial blooms with dominance of Microcystis wesenbergii, Aphanizomenon gracile, Aphanizomenon flos-aquae and Dolichospermum spiroides (Stoyneva 2003).

During September 2018, a proven producer of cyanotoxins, Planktothrix agardhii, was found in blooming concentration in the eastern part of the Lake. Its biomass was 24.89 mg.l^–1, which exceeded twice the accepted WHO moderate risk threshold of 10 mg.l^–1. Other bloom-forming species, such as Anabaenopsis elenkinii (3.5 mg.l^–1), Aphanizomenon klebahnii (3.09 mg.l^–1), Dolichospermum flos-aquae (2.2 mg.l^–1) Microcystis aeruginosa (1.65 mg.l^–1), Raphidiopsis raciborskii (1.66 mg.l^–1) and Microcystis wesenbergii (1 mg.l^–1), were well-represented in a cyanobacterial bloom although their biomass was below the level of the bloom concentrations.

Planktothrix agardhii (9.3 mg.l^-1) was found also in the western part of the Lake at a concentration close to a moderate risk threshold of 10 mg.l^-1. Other cyanobacterial species represented in this part of the Lake were Anabaenopsis elenkinii (0.7 mg.l^-1), Aphanizomenon klebahnii (0.1 mg.l^-1), Dolichospermum flos-aquae (0.6 mg.l^-1) Microcystis aeruginosa (1.05 mg.l^-1), Phormidesmis molle (3.9 mg.l^-1) and Planktothrix isothrix (4.7 mg.l^-1). Microcystis wesenbergii was not detected in this part of the lake.

Our ELISA tests for cyanotoxins in September, showed 0.3 μg.l^–1 and 0.25 μg.l^–1 microcystins and 0.05 μg.l^–1 and 0.04 μg.l^–1 cylindrospermopsin in the eastern and western part, respectively. (Fig. 6). Saxitoxins were not detected. Previous analyzes for cyanotoxins from 2005 and 2011-2013 conducted by HPLC, did not detect the presence of cyanotoxins (Pavlova et al. 2014; Pavlova et al. 2015; Stoyneva-Gärtner et al. 2017).

Figure 6.  

Presence of microcystins (A) and cylindrospermopsin (B) in the water samples from September and tested by ELISA.

We have reported for the first time cylindrospermopsin from a Bulgarian water basin (Teneva et al. 2019).

The cyanotoxins detected in this study, albeit in quantities below the maximum levels, are a warning sign, even more so as Lake Vaya is located on the Via Pontica migratory road and is one of the most important stations for bird migration on the Bulgarian Black Sea coast.