This study presented the results of HPLC-DAD microcystin and nodularin analyses from five Bulgarian water bodies (reservoirs Stoudena, Pchelina and Bistritsa, and lakes Dourankoulak and Vaya) carried out in 2012-2014, as a continuation of our work from 2004 to 2005 and in 2011, and first data on microcystins in the lake Momin Brod were detected in the period 2012-2014. The total concentration of microcystins in water samples ranged from 0.1 to 1.8 μg/l and their amount in the concentrated biomasses from net samples ranged from 1.35 to 296 μg/g (d.w.). The presence of the most toxic microcystin-LR was recorded in all studied lakes and reservoirs, where also microcystins RR and YR were detected, but no microcystin-LA and nodularin were found.
Cyanoprokaryotes (=cyanobacteria = blue-green algae) are photosynthetic prokaryotic organisms which have inhabited the Earth for approximately 3.5 billion years [
Sampling was carried out in six Bulgarian water bodies of health and conservational importance, five of which are mostly used for sports, fishing and recreation and one is a drinking water reservoir (
№ | Water source/ sample type/data | Taxa of Cyanoprokaryota found | Biomass [mg/l] | Microcystins [µg/l for WSs; µg/g d.w. for “biomasses” from NSs] | N [mg/l] | P [mg/l] | N/P |
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1 | Lake Momin Brod | Site of recreational and conservational importance | |||||
WS 19/08/2012 | Microcystis aeruginosa M. wesenbergii [+Aphanocapsa cf. delicatissima; Chroococcus cf. aphanocapsoides; Merismopedia glauca; M. tenuissima; cf. Planktolyngbya sp.] | 0.2 0.6 | LR - 1 YR - 0.3 LR + YR/LR = 1.3/1 = 1.3 | n.a. | n.a. | n.a. | |
NS 19/08/2012 | n.a. | n.a. | LR - 23 YR - 4 LR + YR/LR = 27/23 = 1.17 | n.a. | n.a. | n.a. | |
WS 21/10/2013 | Microcystis aeruginosa M. wesenbergii Merismopedia tenuissima cf. Planktolyngbya sp. | 3.5 0.2 0.001 0.01 | RR- 0.4 YR- 0.4 LR- 1 RR + YR + LR/LR = 1.8/1 = 1.8 | 5.32 | 0.07 | 76 | |
2 | Reservoir Stoudena | Drinking water reservoir for the town Pernik | |||||
WS 31/07/2012 | [unidentified picoplankton] | <0.001 | n.d. | 2.31 | 0.07 | 33 | |
WS18/09/2012 | Dolichospermum viquerii [+unidentified picoplankton] | 8.8 | n.d. | 2.57 | 0.05 | 51 | |
WS 12/08/2013 | 0 | 0 | n.d. | 4.67 | 0.04 | 117 | |
WS14/10/2013 | Microcystis sp. [unidentified filamentous species (<1 μm] | LR -0.1 | 6.35 | 0.05 | 127 | ||
NS14/10/2013 | n.a. | n.a. | RR - 2 YR- 0.8 LR- 8.1 l RR + YR + LR/LR = 10.9/8.1 = 1.35 | - | - | - | |
WS02/10/2014 | [Microcystis sp.; unidentified filamentous species (<1 μm] | n.d. | 6.53 | 0.03 | 218 | ||
3 | Reservoir Pchelina | Reservoir for sport, fishing and recreation | |||||
WS31/07/2012 | Microcystis sp. cf. Pseudanabaena catenata akinetes [Woronichinia fusca] | 0.1 0.1 0.1 | n.d. | 3.45 | 0.07 | 49 | |
NS31/07/2012 | n.a. | n.a. | trace | - | - | - | |
WS18/09/2012 | cf. Anabaena sp. st. [Planktothrix sp.; unidentified picoplankton] | 10.4 | n.d. | 4.21 | 0.07 | 60 | |
NS18/09/2012 | n.a. | n.a. | trace | ||||
WS12/08/2013 | 0 | 0 | n.d. | 4.20 | 0.09 | 47 | |
NS12/08/2013 | n.a. | n.a. | n.d. | - | - | - |
WS14/10/2013 | Microcystis sp. [unidentified filamentous species (<1 μm; unidentified picoplankton] | 0.01 | n.d. | 5.60 | 0.10 | 56 | |
---|---|---|---|---|---|---|---|
NS 14/10/2013 | n.a. | n.a. | n.d. | - | - | - | |
water 02/10/2014 | akinetes [Spirulina major] | 6.2 | RR- 0.07 YR- 0.13 LR - 0.17 RR + YR + LR/LR = 0.37/0.17 = 2.18 | 7.47 | 0.15 | 50 | |
NS02/10/2014 | n.a. | n.a. | RR- 91.5 YR- 71.8 LR- 132.8 RR + YR + LR/LR = 296.1/132.8 = 2.23 | - | - | - | |
4 | Reservoir Bistritsa | Reservoir for sport, fishing and recreation | |||||
WS31/07/2012 | 0 | 0 | n.d. | 2.75 | 0.03 | 92 | |
WS18/09/2012 | Microcystis sp. [cf. Dolichospermum macrosporum; unidentified filamentous species (<1 μm] | 0.2 | n.d. | 2.95 | 0.03 | 98 | |
WS12/08/2013 | 0 | 0 | n.d. | 3.73 | 0.09 | 41 | |
WS(*from rest water after draining of the reservoir) 14/10/2013 | Microcystis sp. | n.a. | LR- 0.6 | 3.55 | 0.06 | 59 | |
WS 02/10/2014 | 0 | 0 | n.d. | 4.67 | 0.04 | 117 | |
5 | Lake Dourankoulak | Lake for sport, fishing and recreation; conservational importance | |||||
WS12/07/2012 | Microcystis wesenbergii [Pseudanabena mucicola; Woronichinia fusca; unidentified picoplankton] | 4.2 | n.d. | 4.20 | 0.32 | 13 | |
WS8/08/2012 | Microcystis wesenbergii [Pseudanabena mucicola; Woronichinia compacta; W. fusca; unidentified picoplankton] | 6.2 | n.d. | 4.75 | 0.30 | 16 | |
WS18/08/2013 | Microcystis botrys M. cf. flos-aquae M. wesenbergii | 0.6 7.2 2.7 | RR- 0.1 YR- 0.9 LR- 0.2 RR + YR + LR/LR = 1.2/0.2 = 6 | 4.20 | 0.16 | 26 | |
NS18/08/2013 | n.a. | n.a. | RR- 103.2 YR- 47.6 LR- 63.5 RR + YR + LR/LR = 214.3/63.5 = 3.37 | - | - | - |
6 | Vaya | Lake for sport, fishing and recreation | |||||
---|---|---|---|---|---|---|---|
WS 02/08/2012 | Anabaena knipowitschii Anabaena sp. Aphanizomenon cf. yezoense Dolichospermum compactum Planktothrix agardhii [cf. Aphanocapsa sp.; Anabaenopsis elenkinii; Aphanizomenon klebahnii] | 15.1 7.7 3.5 2.1 2.5 | n.d. | 12.12 | 0.97 | 13 | |
WS16/08/2012 | Aphanizomenon cf. yezoense Aphanizomenon sp. st Planktothrix agardhii unidentified filamentous species [cf. Planktothrix isothrix; Planktolyngbya limnetica] | 21.2 11.1 73.2 1.5 | n.d. | 17.73 | 1.02 | 17 | |
WS21/08/2013 | Anabaena sp. Anabaenopsis elenkinii Cylindrospermopsis raciborskii Planktothrix agardhii | 3.7 5.9 30.3 164.7 | n.d. | 18.25 | 1.75 | 10 | |
NS21/08/2013 | n.a. | n.a. | n.d. | - | - | - |
died water bodies are included with the following identification numbers: Momin Brod―IBW8307, Stoudena― IBW1060, Pchelina―IBW 1039, Bistritsa―IBW 1067, Dourankoulak―IBW8825, and Vaya―IBW0191, in the Bulgarian Wetlands Inventory [
Samples have been taken in summer and late summer-early autumn periods (July-October) of 2012-2014 (
Water samples for microscopical analyses of the phytoplankton were collected in 1 liter glass bottles at the same points as the samples for toxins and immediately fixed with 10 ml formaldehyde (35% - 38%). In the laboratory samples were concentrated to 30 ml by standard sedimentation procedure (>48 hours).
For microscopic analyses only quantitative water samples of 1.5 l (sedimented to 30 ml) were processed. The qualitative analysis was based on taxa determination according to the current taxonomic literature [
Determination of these nutrients started in 2013-2014. The total nitrogen (N) was performed using Kjeldahl method by “Kjeltec 2100”, Foss Tecator technique. Analysis of total phosphorous (P) was done by spectrophoto-
metric method at wavelength 700 nm. The role of N and P biogenes as growth limiting factors was evaluated according to the total nitrogen/total phosphorus ratio (N/P) scale proposed by Forsberg et al. [
Stored water sample were frozen and defrosted three times to provide cell lysis. Afterwards samples were filtered through nylon membrane filter 0.45 µm (Alltech). Extraction of MC and nodularin from water samples was performed by solid-phase extraction with Empore Extraction Disks C-18 (Varian). Toxins were eluted with methanol. Eluates were dried by gentle stream of nitrogen, re-dissolved in 500 µl of 50% methanol (v/v), filtered through 0.22 µm PTFE syringe filters (ALBET LabScience) and analyzed by HPLC.
Extraction of toxins from dried biomass was performed by ultrasonification of 40 to 60 mg biomass in 1 ml of 50% methanol (v/v). After centrifugation the methanolic extracts were filtered through 0.22 µm PTFE syringe filters and analyzed by HPLC.
Analyses were performed by HPLC-DAD.
The HPLC-DAD system for quantitative and qualitative analyses includes Agilent 1200 Series coupled with Diode Array Detector (Agilent Technologies). Toxins were analyzed on a Supelcosil ABZ + Plus column (150 mm × 4.6 mm, 5 µm, Supelco). The binary gradient of mobile phase consisted of milli-Q water + 0.1% TFA (A) and acetonitrile + 0.1% TFA (B) (linear increase from 20% B at 0 min to 46% B at 25 min and stop time 30 min), the flow rate was 1 ml/min, the temperature 25˚C. Chromatograms at 238 nm were recorded and toxins were identified by the retention time and characteristic UV absorption spectra from 200 to 300 nm.
Purified MC-LR, -RR, -YR, and -LA (Abraxis) were used as external standards. The limit of determination for the quantitative water samples is 0.08 - 0.15 µg/l and for the net samples it is 5 - 10 µg/g.
During the study, MC-LA and nodularin were not found, but presence of other MCs (-LR, -YR, -RR) was proved for all investigated water bodies (except Lake Vaya) in the period 2013-2014, while in 2012 MCs were detected in measurable amounts only in the samples from the lake Momin Brod. The cyanoprokaryotic taxa found and their biomass, the MC concentrations together with the amounts of total nitrogen and phosphorous and the N/P ratio are shown in
The occurrence of MCs in the examined water bodies was irregular in terms of time (
In the samples from the lake Vaya, collected in 2012 and 2013, MCs were not detected. The same was the situation in 2011 and 2005 and only earlier, in 2004 -RR, -YR and -LR were found there [
During this study, MCs were not detected also in the reservoir Bistritsa, with one exception of the sample examined from water remains after the draining of the reservoir in 2013 (
Results from the sample analyses for the mountain drinking water reservoir Stoudena show general lack of MCs, with the exception of both quantitative water and net samples from October 2013 (
During this study MCs were found in Dourankoulak Lake only in the samples from 2013 (
Positive results were obtained from one sampling also in the reservoir Pchelina, where in October 2014 three MCs (-YR, -RR and -LR) in both quantitative water and net samples were found (
The results of N/P ratio analysis show its high value (ranging between 17 and 218―
The detected maximum concentrations of MCs from Bulgarian water bodies are lower in quantitative water samples, but higher in net samples, when a comparison with our previous study [
The results from this study, obtained during the period 2012-2014, show the presence of cyanotoxins MCs in five of the six investigated water bodies. When found, they ranged from 0.1 to 1.8 µg/l in quantitative water samples and from 1.35 to 296 µg/g d.w. in concentrated biomasses from net samples. Despite the fact that in 2012 MCs were not found in five of the all six studied water bodies and that the registered MCs were still lower in comparison with some other European countries, the presence of samples in which the most toxic MC-LR was recorded could serve as a strong alarm for the necessity of a serious study and relevant discussion of the problem with responsible authorities at national level with need of recognition of the problem as a new health risk factor. This statement conforms to our previous conclusions [
Authors are thankful for the financial support of MESY scientific projects DDVU-02/77 and DFNI-T01/5 NANOSORBLAB. One of the authors of this paper, B. Uzunov, participates in the Project № BG051PO001- 3.3.06-0045/18.07.2013 in Human Resources Development Operational Programme, to which thankfulness is due. The authors would like to acknowledge the European Cooperation in Science and Technology, COST Action ES 1105 “CYANOCOST―Cyanobacterial blooms and toxins in water resources: Occurrence, impacts and management” for adding value to this study through networking and knowledge sharing with European experts and researchers in the field.
VeraPavlova,MayaStoyneva-Gärtner,BlagoyUzunov,BlagoyUzunov,ZlatkaBratanova,AntoanetaLazarova,IrinaKaradjova, (2015) Microcystins-LR, -YR and -RR in Six Bulgarian Water Bodies of Health and Conservational Importance (2012-2014). Journal of Water Resource and Protection,07,1375-1386. doi: 10.4236/jwarp.2015.716111