Microcystins cause acute hepatotoxicity and chronic liver tumor promotion. This study presents the results of HPLC DAD analyses and their LC-MS confirmation of samples from five Bulgarian water bodies (reservoirs Stoudena, Pchelina, Bistritsa and lakes Dourankoulak, Vaya). The total concentration of microcystins in water samples ranged from 0.1 to 26.5 μg/l. The amount of microcystins in the biomasses ranged from 11.4 to 49.6 μg/g (d.w.). The high percent of positive samples in which the most toxic microcystin-LR is recorded, can serve as a strong alarm for the necessity of a serious study and relevant discussion of the problem with responsible authorities at national level.
Cyanoprokaryotic/cyanobacterial blooms in surface waters resulting generally from anthropogenic pollution with nitrogen and phosphorous are a well-known worldwide problem. Adverse health effects for humans and animals are associated with cyanobacterial toxins such as microcystins and nodularin [
In the summer months of 2011 sampling was carried out in five Bulgarianwater bodies of health and conservational importance (
Three liters of water from surface zone were collected in plastic bottles and stored frozen prior to sample preparation. In parallel, biomass samples were collected from water bodies and scum was taken in the littoral shallows with plankton net (20 µm). The biomasses were stored frozen and portions of them were defrosted and dried at room temperature prior to the analysis of toxins.
Water samples for biological analyses were collected in 1 liter glass bottles at the same points as the samples for toxins and immediately fixed with 2% - 4% formaldehyde. In the laboratory samples were concentrated by sedimentation.
The qualitative analysis was based on taxa determination according to the current taxonomic literature [
Stored water samples were frozen and defrosted three times to provide cell lysis. After that samples were filtered through nylon membrane filter 0.45 µm (Alltech). Extraction of microcystins and nodularin from water samples (1.5 to 2.l) 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 and HPLC-MS systems.
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.
The HPLC-MS system including Agilent 1200 Series coupled with HCT ultra ion trap detector (Bruker) was used for confirmation of DAD results. Toxins were analyzed on a Supelco Analytical Ascentis C18 column (50 mm × 3 mm, 3 µm, Supelco). The binary gradient of mobile phase consisted of 99% Chromasolve water, 1% acetonitrile + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B) (linear increase from 25% B at 0 min to 70% B at 5 min, 70% B to 6 min, 25% B at 6.1 min and stop time 10 min), the flow rate was 0.5 ml/min, the temperature 40˚C. Toxins were identified by Auto MS/MS mode.
Purified microcysin −LR, −RR, −YR, −LA and nodularin (stock concentration of 10 µg/ml, Abraxis) were used as external standards.
During the study, presence of microcystins was proved for three of the five investigated water bodies. Their concentrations are represented in
The concentration of intra + extracellular microcystins in water samples ranged from 0.1 to 26.5 µg/l. The amount of total microcystins in the biomasses ranged from 11.4 to 49.6 µg/g (d.w.). Three of the studied microcystins (−RR, −YR, −LR) were found in Lake Dоurankоulak in the reservoir Pchelina only microcystin-RR was detected and in the reservoir Stоudena only microcystin-LR was identified. The results obtained from HPLC DAD did not indicate presence of microcystin-LA and nodularin.
The LC-MS method shows m/z 995.7 at the retention time of microcystin-LR and m/z 1045.7 at the retention time of microcystin-YR. According to Poon et al. [
№ | Water source/sample type | Sampling date | Taxa of Cyanoprokaryota found | Biomass (mg/l) | Microcystins |
---|---|---|---|---|---|
1 | Stoudena | Drinking water reservoir for the town Pernik | |||
water | 03/08/2011 | Unidentified picoplankton | 0.01 | n.d. | |
water | 28/09/2011 | Raphidiopsismediterranea | <0.001 | −LR 0.1 (µg/l) | |
2 | Dourankoulak | Lake for sport, fishing and recreation | |||
water | 29/07/2011 | Leptolyngbya foveolarum Merismopedia hyalina Merismopedia tenuissima Microcystis wesenbergii Pseudanabaena mucicola Chroococcus sp. Unidentified colonial coccal cells | 0.011 0.008 0.002 6.873 0.118 <0.001 0.001 | −RR 12.7µg/l −YR 5.5µg/l −LR 8.3µg/l (RR + YR + LR)/LR = 26.5/8.3 | |
biomass | 29/07/2011 | −RR 22.1 µg/gd.w. −YR-trace −LR 27.5 µg/gd.w. (RR + LR)/LR = 49.6/27.5. | |||
3 | Pchelina | Reservoir for sport, fishing and recreation | |||
water | 03/08/2011 | Aphanizomenonsp. juv. Unidentified non-heterocystous filaments | 0.097 0.007 | −RR 0.5 µg/l | |
biomass | 03/08/2011 | −RR 11.4 µg/gd.w. | |||
water | 28/09/2011 | Aphanizomenonsp. ster. Planktolyngbyalimnetica | 0.772 <0.001 | n.d. | |
biomass | 28/09/2011 | n.d. | |||
4 | Vaya | Lake for sport, fishing and recreation | |||
water | 03/08/2011 | Anabaenopsiselenkinii Aphanizomenongracile Cylindrospermopsisraciborskii Oscillatoriacf. simplicissima Oscillatoriasp. Unidentifiednon- heterocystousfilaments | 2.919 8.041 9.294 <0.001 53.274 0.318 | n.d. | |
biomass | 03/08/2011 | n.d. | |||
5 | Bistritsa | Reservoir for sport, fishing and recreation | |||
water | 03/08/2011 | Microcystiswesenbergii | <0.001 | n.d. | |
water | 28/09/2011 | Anabaena spp. Aphanothece spp. | 0.1 <0.001 | ||
biomass | 28/09/2011 | n.d. |
of the CID in-source area fragmentation of microcystin −RR molecular ion at m/z 1038.6 in capillary exit area. The DAD results are qualitatively confirmed as follows: presence of microcystin −RR in water and biomass samples of Lake Dоurankоulak and reservoir Pchelina, presence of microcystin −LR in water and biomass samples in Lake Dоurankоulak and in a water sample in the drinking water reservoir Stoudena, and presence of microcystin −YR in water and biomass samples of Lake Dоurankоulak.
Results for the mountain drinking water reservoir Stoudena showed lack of toxins at first sampling but at the second date, 0.1 mg/l microcystin-LR was found (
The concentration of the toxin is 10 times lower than WHO limit of 1 µg/l. However, the fact that one of the most toxic cyanotoxins has been detected in a drinking water reservoir, can serve as “alarm” and clearly shows that this water body has to be studied and monitored in the future. In 2004 and 2005 microcystins were not found there [
During this study the highest microcystin concentration was found in Dourankoulak Lake, where strong algal bloom was observed and proved by classical light microscopic counts (
Positive results were obtained also in the reservoir Pchelina where only microcystin –RR in water and biomass from first sampling was found (
In Lake Vaya microcystins were not detected in 2011. The same was the situation in 2005, but in 2004 microcystins −RR, −YR and −LR were detected there with (RR + YR + LR)/LR ratio in biomass 4/1 [
Microcystins were not detected in water of the reservoir Bistritsa nor in 2011, neither in 2005. However, the identification of extracellular microcystins in water sampled in 2004 ((RR + YR + LR)/LR = 2/1) also suggests monitoring of the necessarily this reservoir for cyanotoxins [
The detected concentrations in Bulgarian water bodies are close to the quantities reported from Turkey [
Nevertheless of the fact that the recorded MC concentrations were lower in comparison with some other European countries, the high percent of positive samples in which the most toxic microcystin-LR is recorded, can serve as a strong alarm for the necessity of a serious study and relevant discussion of the problem with responsible authorities at national level. Observations of water bodies at risk and monitoring actions for limitation and control of cyanobacterial toxic blooms are urgently needed, in combination with increased attention to the effects of cyanobacterial toxins on human health and health of aquatic ecosystems in Bulgaria.
Authors are thankful for the financial support of MESY scientific projects DDVU-02/77 and DFNI-T01/5 NANOSORBLAB. 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. The data cited in Stoyneva et al. 2013 [