I. Teneva, D. Belkinova, I. Dimitrova-Dyulgerova, M. Vlaknova, R. Mladenov
Plovdiv University “Paisii Hilendarski”, Faculty of Biology, Department of Botany, Plovdiv, Bulgaria
Correspondence to: Rumen Mladenov
E-mail: rummlad@uni-plovdiv.bg
ABSTRACT
Some species of Cyanoprokaryota produce toxins that affect animals and humans. Most of the freshwater basins in Bulgaria, including dams, are relatively well studied in terms of the phytoplankton composition, but the data for presence of cyanotoxins are limited. The aim of our study was to evaluate the diversity, distribution and quantitative development of the phytoplankton as well as the presence of cyanotoxins in the public reservoir Vacha. We have collected water and phytoplankton samples from Vacha reservoir at different time points. All water samples were analyzed for presence of cyanotoxins by ELISA, and tested for cytotoxicity on cell cultures in vitro. Physicochemical parameters, including water temperature, pH, total nitrogen and total phosphorus were measured. Algae, belonging to seven divisions (Cyanoprokaryota, Chlorophyta, Xantophyta, Dinophyta, Euglenophyta, Bacillariophyta and Criptophyta) were identified. A potentially toxic cyanoprokaryote Aphanizomenon flosaquae was detected in blooming concentrations in July and August 2008 as well as in July 2009 together with Microcystis aeruginosa. The water sample collected in August 2008 contained 0.25 ppb microcystins/nodularins. The total microcystins/nodularins concentration in the water samples collected in September 2009 was 0.5 ppb. The viability of HeLa cells was affected mainly after 48 h of exposure to the collected water samples.
Keywords: Cyanoprokaryota, cyanotoxins, water quality, monitoring, phytoplankton structure, Vacha Dam
Introduction
Cyanoprokaryota or blue-green algae occur worldwide and grow in eutrophic (nutrient-rich) freshwaters. Some species of Cyanoprokaryota produce toxins that affect animals and humans. Cyanoprokaryotic toxins in lakes, ponds, and dugouts in various parts of the world have long been known to cause poisoning in animals and humans. One of the earliest reports of their toxic effects was in China 1000 years ago (3). Most spread cyanotoxins are hepatotoxins and neurotoxins. Hepatotoxins (which affect the liver) are produced by some strains of the genera Microcystis, Anabaena, Oscillatoria, Nodularia, Nostoc, Cylindrospermopsis and Umezakia. Neurotoxins (which affect the nervous system) are produced by some strains of Aphanizomenon and Oscilatoria. Strains from the species Cylindrospermopsis raciborskii may also produce toxic alkaloids, causing gastrointestinal symptoms or kidney disease in humans. The human health risks associated with these cyanotoxins are an increasing concern to water managers worldwide. Many of the water basins in Bulgaria are used as a source of drinking water, irrigation water and fishing. Taking in account the aforementioned risks of contamination, there is an urgent need for continuous monitoring of water quality in terms of pollution with cyanotoxins. Most of the freshwater basins in Bulgaria, including dams, are relatively well studied in terms of the phytoplankton composition, but the data for presence of cyanotoxins are limited. There is one report about presence of microcystins in water samples from 15 Bulgarian reservoirs and lakes (5). Data showed that the concentration of total microcystins (MC-LR, MC-RR and MC-YR) in the biomasses ranged from 8 to 1070 µg.g-1 (d.w.). Our previous studies on phytoplankton communities and monitoring of cyanotoxins in Trakiets reservoir showed presence of saxitoxins (0.01 ng/mL) as well as microcystins (0.18 µg.L-1 ) in collected water samples (8). Linking the phytoplankton assemblages of water basins with the presence of cyanotoxins and water quality will give an opportunity to establish a good system for monitoring of the phytoplankton communities and their toxic potential. The National Action Program on the Environment and Health 2008-2013 of the Council of Ministers pointed out that in terms of bathing water and standing surface waters, which is used as a source of drinking water, there is not any information about the risk of cyanoprokaryotic blooms and their potential to produce cyanotoxins. In the coming years, as EU member, Bulgaria have to fulfill the obligations for monitoring and implementation of effective programs aiming to restore safe water quality, based on the approach for health risk assessment. One of the priorities will be to ensure the safety of drinking and recreational waters in terms of contamination with microparasites and formation of cyanoprokaryotic toxic blooms. Taking in account the main priorities and the lack of investigations in this direction in Bulgaria, the aim of our study was to evaluate the diversity, distribution and quantitative development of the phytoplankton as well as the presence of cyanotoxins in the public reservoir Vacha.
Materials and Methods
Site description and physicochemical water quality analysis Vacha Dam (geographic coordinates 41°93’25’’N and 24°43’75’’E) (Fig. 1) is one of the biggest dams in Bulgaria and largest on the river Vacha – the second longest river in Southern Bulgaria, after river Arda. The dam is over 30 km long and is one of the deepest. Vacha Wall Dam is the highest in Bulgaria – 144.5 meters. Except for energy production, fishing and irrigation, it is used as a drinking
water source for part of the Plovdiv region. Physicochemical parameters, including water temperature, pH, total nitrogen and total phosphorus were measured on field by a portable photometer pHotoFlex® (WTW GmbH, Weilheim, Germany) or in the laboratory. Sample collection and phytoplankton analysis For qualitative analyses of the phytoplankton, samples were collected three times per year (July, August and September) in 2008 and 2009 from the surface layer (0.5 m) with a 20 µm mesh size net at a fixed collection station and preserved in 4% formaldehyde. For phytoplankton counting, samples were collected with Meyer bottles of 1L and preserved with Lugol solution. Phytoplankton analyses were performed from both, fresh and conserved (in 4% formaldehyde) samples, with an inverted microscope (PZO Poland) according to Lund et al. (4), using sedimentation chambers for phytoplankton identification and cell density estimation. Water samples for chemical and toxicological analysis were collected at the same time as the phytoplankton samples from the same sampling points.
In vitro cytotoxicity tests
HeLa cells (human cervical epithelial adenocarcinoma, ATCC CCL-2) were used for the in vitro tests. Cells were
cultured in 75 cm2 flasks in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, UK), supplemented with 10% (v/v) heat inactivated fetal calf serum (FCS, PAA Laboratories, Austria), 100 U/ml penicillin and 100 g/ml streptomycin (Sigma, Steinheim, Germany), at 37C with 5% CO2 in air and high humidity. Cell viability was measured with the trypan blue exclusion test prior to seeding. Cells were plated in 96-well tissue culture plates at a density of 1.5×104 per 200 L DMEM with 10% FCS. After 24 h (to allow the attachment of the adherent cells) the cultures were exposed to 10% of water samples. Control wells were prepared by adding equal amounts of Millipore water to the culture medium. The cells were exposed for 24 h or 48 h prior to analysis. After the desired time of exposure with water samples, 20 l of MTT solution (5 mg/ml in PBS) were added directly to each well and incubated at 37°C for 3 h. Thereafter, the supernatant was discarded and 0.1 ml of dimethylsulfoxide (DMSO) was added to each well in order to facilitate solubilization of the formazan product. After 15 min at room temperature the plates were shaken, and absorbance was read at 570 nm in a SPECTRAmax PLUS microplate spectrophotometer.
Analysis of cyanotoxins by ELISA
Saxitoxins
The water samples were analyzed by the Ridascreen™ saxitoxin ELISA kit (R-Biopharm, Darmstadt, Germany). This is a competitive ELISA for the quantitative analysis of saxitoxin and related toxins based on the competition between the free toxins from samples or standards and an enzyme-conjugated saxitoxin for the same antibody. The mean lower detection limit of the Ridascreen™ saxitoxin assay is about 0.010 ppb (µg.L-1 ).
Microcystins and nodularins
The analysis of the water samples for presence of microcystins and nodularins was performed using the Microcystins ELISA (Abraxis LLC, Warminster, PA). As for the saxitoxin ELISA, this is a quantitative, competitive immunosorbent assay that allows the congener-independent detection of microcystins and nodularins in water samples. The limit of detection of the Microcystins ELISA is 0.10 ppb (µg.L-1 ).
Results and Discussion
Physicochemical parameters
The environmental variables of the investigated reservoir are given in Table 1. In 2008 the water temperature at the surface ranged from 21.5°C in September to 25°C in August. pH values were between 7.1 and 7.7. Total nitrogen (TN) concentrations varied from 0 mg.L-1 in July and August to 2.5 mg.L-1 in September. Total phosphorus (TP) concentrations were from 0 to 0.07 mg.L-1 in August. TN/TP ratio in September was 125, which indicates phosphorus limitation on a community level. In 2009 the water temperature at the surface ranged from
21°C in July to 24°C in August. pH values were between 6.5 and 7.5. Total nitrogen (TN) concentrations in dam wall varied from 0 mg.L-1 in July and August to 0.2 mg.L-1 in September. At the same time the total nitrogen concentration in the dam tail varied from 17.8 mg.L-1 in July to 0 mg.L-1 in August. Total phosphorus (TP) concentrations were from 0.40 mg.L-1 in July (dam tail) to 0.01 mg.L-1 in August (dam wall). TN/TP ratio in September was 1.67 (dam wall) and 40 (dam tail), which indicates again phosphorus limitation on a community level in the dam tail and nitrogen limitation at the dam wall.
Physicochemical variables of the surface water in Vacha Dam (2008 and 2009)
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Taxonomic composition and structure of the phytoplankton community
In this study, algae belonging to seven divisions (Cyanoprokaryota, Chlorophyta, Xantophyta, Dinophyta, Euglenophyta, Bacillariophyta and Criptophyta) were identified. During the first study period (July, August and September, 2008), Chlorophyta represented 53% in July, 56% in August and 48% in September from the total phytoplankton, followed by Bacillariophyta (16% in July, 18% in August and 26% in September), Cyanoprokaryota (9% in July, 13% in August and 12% in September), Euglenophyta (8% in July, 8% in August and 5% in September), Dinophyta (10% in July, 5% in August and 5% in September) and Xantophyta (4% in July, lack in August and 4% in September) (Fig. 2A). During the second study period (July, August and September, 2009), Chlorophyta represented 56% in July, 42% in August and 48% in September from the total phytoplankton, followed by Bacillariophyta (7% in July, 29% in August and 26% in September), Cyanoprokaryota (19% in July, 17% in August and 15% in September), Criptophyta (6% in July, 8% in August and 7% in September), Dinophyta (6% in July, 4% in August and 4% in September) and Euglenophyta (6% in July) (Fig. 2B). In 2009, species from the division Xantophyta were not detected. Instead, the division Criptophyta has been presented with a relative high number. Our data shown that Vacha reservoir has relatively low phytoplankton diversity and stable taxonomic composition. The green algae have a dominant role in the summer phytoplankton. Similar data have been shown for other Bulgarian lakes and reservoirs as well (1, 2, 6, 7). The Cyanoprokaryota group was relative pure represented (8%- 13% in 2008 and 15%-19% in 2009) (Fig. 2). In 2008, total density of Cyanoprokaryota varied within a range from 2.25×106 cells/L in July to 1.61×106 cells/L in September, and the biomass from 0.17 mg/L in July to 0.07 mg/L in September. In 2009, the values for total density varied from 964×106 cells/L in July to 9.37×106 cells/L in September and the biomass from 77.142 mg/L in July to 0.062 mg/L in September (Table 2). The quantitative development of Cyanoprokatyota also showed substantial differences during the three investigated months of 2009. The potentially toxic cyanoprokaryote Aphanizomenon flos-aquae was detected in the formed algal bloom in July and August 2008 (Table 2). Microcystis aeruginosa was presented with relative high density during the whole investigated period in 2008 together with Phormidium sp. in July and Oscillatoria tenuis in September. These three species are also good candidates for toxin production. More details related to species composition and quantitative characteristics of Cyanoprokaryota are given in Table 2.
Species composition and quantitative characteristics of Cyanoprokaryota in Vacha Dam (2008).
| Months | Species composition (2008)
|
Density (106 cells/L) | Biomass (mg/L) | Total density (106 cells/L) | Total biomass (mg/L) |
| July | Aphanizomenon flos-aque (L.) Ralf (bloom)
Microcystis aeruginosa (Kütz.) Kütz. Phormidium sp. |
1 480 000 650 000
120 500 |
0.12
0.05 – |
2 250 500 |
0.17 |
| August | Aphanizomenon flos-aque (L.) Ralfs (bloom) Gomphosphaeria lacustris Chod.
Microcystis aeruginosa (Kütz.) Kütz. Synechococcus elongatus (Näg.) Näg. |
750 800
94 700 138 000 1 350 000 |
0.06
– 0.01 0.04 |
2 333 500 |
0.11 |
| September | Gomphosphaeria lacustris Chod.
Microcystis aeruginosa (Kütz.) Kütz. Oscillatoria tenuis Ag. Synechococcus elongatus (Näg.) Näg. |
230 400
380 000 145 400 860 000 |
–
0.03 0.02 0.02 |
1 615 800 |
0.07 |
Species composition and quantitative characteristics of Cyanoprokaryota in Vacha Dam (2009).
| Months | Species composition (2009)
|
Density (106 cells/L) | Biomass (mg/L) | Total density (106 cells/L) | Total biomass (mg/L) |
| July | Aphanizomenon flos-aque (L.) Ralf (bloom) Aphanothece clathrata W. et G.S. West Chroococcus limneticus Lemm. | 964 275 000 single cells single cells | 77.142
– – |
964 275 000 |
77.142 |
| August | Aphanizomenon flos-aque (L.) Ralfs
Aphanocapsa planctonica (G.M. Smith) Kom. et Anagn. Aphanothece clathrata W. et G.S. West Microcystis aeruginosa (Kütz.) Kütz. |
single cells
1 354 500
1 257 750 single cells |
–
0.010
0.005 – |
2 612 250 |
0.015 |
| September | Aphanizomenon flos–aquae (L.) Ralfs
Aphanocapsa planctonica (G.M. Smith) Kom. et Anagn. Aphanothece clathrata W. et G.S. West Microcystis aeruginosa (Kütz.) Kütz. |
single cells
6 192 000
3 182 000 single cells |
–
0.050
0.012 – |
9 374 000 |
0.062 |
Aphanizomenon flos-aquae was presented in the phytoplankton during the whole investigated period in 2009 (July, August and September), but only in July it was in blooming concentrations (Table 3). In August and September 2009, Microcystis aeruginosa was a part of the species composition. Other represented species with a relative high number of density were Aphanocapsa planctonica and Aphanothece clathrata Assessment of the toxic potential of Cyanoprokaryota In vitro cytotoxicity of water samples Water samples collected in July, August and September (2008 and 2009) were tested for cytotoxicity. HeLa cells were used as a test system. The duration of exposure was 24 and 48 hours. After 24h of exposure, all water samples, collected in 2008 did not show any cytotoxic effect (July, August) and even a weak stimulatory effect (September) was detected (Fig. 3). After 24h treatment of the cells with water samples collected in 2009, weak cytotoxic effects (10-12% toxicity) were measured (Fig.4). In contrast, after 48 hours of exposure, all samples (2008 and 2009) showed quite significant cytotoxic effects ranged between 20% (July and August, 2008) and 30% toxicity (July, 2009) (Figs. 3, 4). These data correlated with the presence of a potentially toxic cyanoprokaryote Aphanizomenon flos-aquae detected in bloom concentrations in July and August 2008 as well as in July 2009.
ELISA tests
Performed ELISA test for microcystins/nodularins showed presence of these toxins in the sample collected in August 2008 (0.25 ppb) (Fig. 5) as well as in the samples collected in September 2009 from the wall and the tail of Vacha Dam, 0.4 ppb and 0.5 ppb respectively (Fig 6). No saxitoxins were detected in the water samples collected in 2008 and 2009 from the Vacha reservoir.
Conclusions
Our data showed that the species composition and quantitative characteristics of Cyanoprokaryota in Vacha reservoir correlated with the obtained in vitro cytotoxic effects and presence of cyanotoxins detected by the microcystins/nodularins ELISA test. The study underlines that a toxicity assesment is necessary for risk assesment of the water for contamination with cyanotoxins. Although cyanotoxins were detected in concentrations lower than the recommended levels for drinking water, there is an urgent need from monitoring programs of Cyanoprokaryota and their toxins in the reservoirs used as sources of drinking water.
Acknowledgments
This work was financially supported by a Marie Curie European Reintegration Grant to Ivanka Teneva (Contract No. MERG-CT-2007-210514).
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