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Stability of toxigenic Microcystis blooms
Harmful Algae 8 (2009) 377–384
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Stability of toxigenic Microcystis blooms Satya Prakash a, Linda A. Lawton b, Christine Edwards b,* a b
Department of Botany, Banaras Hindu University, Varanasi 221005, India School of Pharmacy and Life Sciences, The Robert Gordon University, St. Andrew Street, Aberdeen AB25 1HG, UK
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 April 2008 Received in revised form 25 August 2008 Accepted 25 August 2008
This is the first detailed study on the occurrence of cyanobacterial toxins in India, where we studied five eutrophic, temple ponds in the vicinity of Varanasi city, Uttar Pradesh, which continuously supported blooms of Microcystis sp. for several years. Bloom material from all five ponds was sampled bi-monthly from September 2003 to August 2004. Analysis of extracts by high-performance liquid chromatography (HPLC) indicated that microcystin-RR (MC-RR) was present all year round at high concentrations (311– 1540 mg/g, dry weight), posing a significant health hazard especially since all five ponds are widely used for bathing, washing, cattle drinking supply, irrigation and recreation. In addition, there was unusually low temporal variation in concentration of MC-RR in each pond, <20% variation in four out of five ponds throughout the year. Characterization of microcystin composition of several bloom samples from this study by HPLC–PDA/ MS confirmed that additional microcystins were present in many of the samples. The rarely reported, MCAR was frequently detected in bloom samples from three of the ponds (Adityanagar, Durgakund and Sankuldhara), where it typically represented 20% of the microcystin pool. MC-WR was frequently found in samples from Adityanagar and Sankuldhara, representing 5–10% of the microcystin pool. MC-LR, along with the previously unreported MC-AHar, each represented approximately 5% of the microcystin pool when present. Bloom samples from each pond had a characteristic microcystin profile, when sampled from 2003 to 2006, suggesting persistent species/strain domination. The perennial and consistent nature of the toxic Microcystis blooms in these ponds is highly unusual, in contrast to the commonly encountered temporal and spatial variation of toxigenic and non-toxigenic species. Laboratory isolates from several ponds were shown to produce microcystins, showing similar microcystin composition to the original bloom material. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Cyanobacteria Microcystins Perennial blooms
1. Introduction Cyanobacteria frequently form nuisance blooms (benthic and planktonic) in freshwater bodies in all continents across the globe. Many of these organisms produce toxins, presenting a hazard to animal and human health. Microcystins, a large group of cyclic peptides (>85 variants), are the most commonly occurring toxins, produced by a growing number of cyanobacterial genera including; Microcystis, Anabaena, Nostoc, Plantothrix, Oscillatoria (Sivonen and Jones, 1999). Whilst microcystins have been associated with a number of acute toxicoses (Zurawell et al., 2005; Van Apeldoom et al., 2007), there is also increasing evidence from epidemiological studies, supporting the long-term chronic exposure resulting in primary liver cancer (Ueno et al., 1996; Zhou et al., 2002). This potential chronic toxicity led the WHO to establish a guideline of
* Corresponding author. Tel.: +44 1224 262839; fax: +44 1224 262828. E-mail address: [email protected] (C. Edwards). 1568-9883/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2008.08.014
1 mg/L as a maximum concentration of MC-LR in drinking water (WHO, 1998). More recently, a review of all data on toxicity of MC-LR by the International Agency for Research on Cancer (IARC) resulted in this compound being classified as a potential carcinogen (Group 2B) in June 2006 (Grosse et al., 2006). Insufficient data were available to classify other microcystins or nodularin but it would be pertinent to assume they are potential carcinogens. Whilst there have been lengthy investigations into the occurrence of toxic cyanobacteria in many countries, there are only a few brief reports on their occurrence in India. However, several studies have indicated that many ponds in Varanasi, Uttar Pradesh, are eutrophic or hypertrophic and frequently support cyanobacterial blooms, mostly Microcystis sp. (Parker et al., 1997; Singh et al., 2001). A recent study, based on mouse bioassay, did reveal that indeed hepatotoxic Microcystis blooms occurred in several eutrophic ponds in Varanasi, however, the extent of a potential hazard was not investigated in detail (Tyagi et al., 2006). Many of these freshwater ponds in the Varanasi area are essential for bathing, clothes washing, recreation, cattle drinking
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and irrigation. The presence of toxic cyanobacterial blooms is a health hazard with potential exposure via multiple routes. Direct exposure may result from oral ingestion of microcystins via contaminated water or inhalation during recreation/other direct water use. Indirect exposure via food is also a potential route for chronic exposure and there are numerous reports of accumulation of microcystins in fish and shellfish as well as detection in crops irrigated with contaminated water (Van Apeldoom et al., 2007). Five eutrophic ponds, known to support Microcystis blooms were selected for a detailed study monitoring cyanobacterial species, microcystin occurrence, environmental and meteorological parameters. This manuscript provides a detailed study on the occurrence of microcystins in five of these ponds.
UV detector (2487), a Rheodyne 7725i manual injector. Data acquisition and processing were achieved using Millenium-32 (Waters, Milford, USA). Samples (20 mL) were separated on Novapak C18 silica column (150 mm 4.6 mm i.d., 5 mm particle size; Waters, Milford, USA). The mobile phase used was methanol/ 0.05 M potassium phosphate buffer adjusted to pH 3 (57:43), at a flow rate 1 mL/min for 24 min (Tsuji et al., 1997). Microcystin variants were identified by characteristic retention time and UV absorbance at 238 nm. Microcystins were quantified as microcystin equivalents based on MC-LR (Alexis Corporation, Lausen, Switzerland).
2. Materials and methods
Selected freeze-dried samples from all five ponds in the study were extracted in 70% methanol (50 mg dry weight per mL solvent). Microcystins in these extracts were initially identified by HPLC with photodiode array (PDA) detection. A Waters Alliance 2695 solvent delivery system equipped with a 2996 PDA (Waters, Elstree, UK). Separation was effected on a Sunfire C18 column (2.1 mm i.d. 150 mm long; 3.5 mm particle size) which was maintained at 40 8C. Mobile phase was Milli-Q water (A) and acetonitrile (B) both containing 0.05% TFA. Test samples (20 mL) were separated using a gradient increasing from 15% to 60% B over 25 min at a flow rate of 0.3 mL/min followed by ramp up to 100% B and re-equilibration over the next 10 min. Eluent was monitored from 200 to 400 nm with a resolution 1.2 nm. Instrument control, data acquisition and processing were achieved using Empower v2.0. Microcystins were identified based on retention time and comparison of UV spectra to a range of standards, and by characteristic spectra where standards were not available. Known and unknown microcystins were quantified using MC-LR as an external standard at 238 nm (Alexis Corporation, Lausen, Switzerland). Further characterization of microcystins was achieved using an identical system equipped with ZQ 2000 MS detector in series. MS analyses were all performed in positive ion electrospray mode, scanning from m/z 100 to 1600 with a scan time of 2 s and interscan delay of 0.1 s. Ion source parameters; sprayer voltage, 3.07 kV; cone voltage, 100 V; desolvation temperature, 300 8C; and source temperature, 100 8C. Instrument control, data acquisition and processing were achieved using Masslynx v4.1. Cyanotox software v1.5.3 (Hyphen MassSpec Consultancy, Leiden, Netherlands) was used to support tentative identification of microcystins where no standards were available. This program allows prediction of fragment ions for microcystins based on the fragmentation of the singly charged molecular ion [M+H]+, using electrospray tandem mass spectrometry.
2.1. Sampling and sample sites Five eutrophic ponds; Lakshmikund, Karmadeshwar, Adityanagar, Durgakund and Sankuldhara in Varanasi, Uttar Pradesh, India (258200 N, 838010 E) were selected for investigation as they frequently supported cyanobacterial blooms. Surface bloom samples were collected twice monthly from September 2003 to August 2004. Other samples were collected at intermittent intervals from 2004 to 2006. The bloom samples were harvested from the shoreline using a nylon net (200 mm mesh size), lyophilized and stored at 20 8C. 2.2. Laboratory isolation and culture of Microcystis from bloom samples Microcystis species in blooms samples were identified according to the systematic keys provided by Desikachary (1959). Adhering mud and other contaminants were removed carefully and the specimens were washed with sterile water. In order to reduce contaminants, two-step centrifugation of inocula (150 g for 30 min, at room temperature, followed by higher-speed centrifugation; 1000–4000 g for 5 min, at room temperature) and repetitive sub-culturing of single cyanobacterial cell, in solid and liquid culture media alternately, were made as suggested by Shirai et al. (1989, 1991). Microcystis colonies were disaggregated by vortex mixing in de-ionized water, as described by Parker (1982). After the two-step centrifugation of inocula, the surface layer was withdrawn with a micropipette and plated on modified Jagar medium (Parker, 1982). After incubation for 10–12 days in light (22 mE/(m2 s)), a contaminant-free individual colony was selected with the help of fine-bore glass capillary, under binocular microscope (magnification 10–25) and transferred to culture tubes containing 10 mL modified J-medium. Cultures were incubated at 25 1 8C and illuminated with full spectrum fluorescent lights where the light intensity at the surface of the flasks was 22 mE/(m2 s). The cultures were shaken manually 3–4 times daily. 2.3. Extraction and analysis of microcystins Lyophilized cyanobacterial samples (20 mg) were extracted in 10 mL of 70% methanol, with ultrasonication for 15 min followed by intermittent shaking for 1 h. Debris was removed by centrifugation for 5 min at 8830 g. The pellet was re-extracted and the combined supernatants were evaporated to dryness at 30 8C using a rotary evaporator (Perfit, India). The residue was resuspended in 1.0 mL 80% methanol and filtered (0.22 mm cellulose–acetate; Axiva, India) prior to analysis by high-performance liquid chromatography (HPLC). Equipment included a Waters 600 E quaternary pump, coupled with variable wavelength
2.4. Characterisation of microcystins
3. Results 3.1. Detection of microcystin-RR Analysis of extracts from surface scum samples by HPLC revealed that MC-RR was the predominant microcystin in all five ponds and most notably, was present in all five ponds at every sampling throughout the year long study (Fig. 1). The highest concentration of MC-RR was detected in Lakshmikund (1540 mg/g dry weight) in November and lowest concentration was detected in June (880 mg/g dry weight; Fig. 1A). Throughout the year, the mean concentration of MC-RR was 1131 216 mg/g dry weight representing less than 20% variation. Similar patterns showing only slight fluctuation in MC-RR concentrations were detected in samples from the other four ponds in the study, albeit with lower maximum and minimum values. In Karmdeshwar, Adityanagar, and Durgakund
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Fig. 1. Detection of MC-RR in cyanobacterial biomass from five freshwater ponds: Lakshmikund (A), Karmdeshwar (B), Adityanagar (C), Durgakund (D) and Sankuldara (E) from September 2003 to August 2004. Data plotted are the mean of three replicates S.D.
ponds (Fig. 1B, C and D, respectively), maximum concentrations of MC-RR ranged from 1083 to 528 mg/g dry weight in November/ December whereas highest concentrations in samples from Sankuldhara (792 mg/g dry weight) occurred in April. As in Lakashmikund, there was less than 20% variation in MC-RR concentration throughout the year in Karmdeshwar, Adityanagar and Sankuldara ponds compared to 32% in Durgakund pond.
3.2. Identification and characterisation of microcystins in bloom extracts Characterisation of the microcystin composition of a selection of the bloom extracts by HPLC–PDA and HPLC–PDA/MS detection confirmed that the predominant microcystin was always MC-RR (2) with m/z of 1038 (Table 1). UV and MS spectral data revealed
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Table 1 Structural assignments of microcystins frequently identified in bloom extracts from five ponds Ion structure +
[M+H] [M+H CO]+ [M+H H2O]+ [M+2H 135]+ [MeAsp-Arg-Adda-Glu+H]+ [Glu-Mdha-Ala-Leu-MeAsp-Arg+H]+ [Glu-Mdha-Ala-Ala-MeAsp-Arg+H]+ [MeAsp-Arg-Adda+H]+ [Mdha-Ala-Leu-MeAsp-Arg+H]+ [Mdha-Ala-Ala-MeAsp-Arg+H]+ [Mdha-Ala-Trp-MeAsp-Arg+H]+ [Glu-Mdha-Ala-Ala-MeAsp+H]+ [Glu-Mdha-Ala-Ala-MeAsp+H COOH]+ [Arg-MeAsp-Ala+H]+ [C11H15O-Glu-Mdha+H]+ [Glu-Mdha+H]+ [PhCH2CHOMe]+
[Dha7]MC-RR (1)
MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-WR (5)
MC-AHar (6)
1024 – 1006 –
1038 – 1020 – – – – 599 –
995 987 – 861 728 682 – 599 553
953 925 – 819 728 – 640
1068 1040 – 934 – – – 599 –
967 940
– – – –
– 511
833 – 654
525 626
–
–
–
– 199 135
375 213 135
375 213 135
484 440 357 375 213 135
–
484 440
375 213 135
375 213 135
Fragmentation data based on cone voltage 100 eV.
that several other microcystins were present in some of the extracts. Collision-induced dissociation (CID) spectra obtained at a cone voltage of 100 V, provided increased fragmentation enabling structural assignment as summarised in Table 1. The most polar microcystin [Dha7]MC-RR (1) with m/z of 1024 was confirmed by retention time and spectral data compared to that of a previously characterised standard. MC-LR (3) was detected in some samples although concentrations were always low. One of the most commonly occurring microcystins was tentatively identified as MC-AR (4) in the absence of a reference compound. CID spectra gave a m/z of 953 and included fragment ions at m/z 640 and 511, indicative of [Glu-Mdha-Ala-Ala-MeAsp-Arg+H]+ and [Mdha-AlaAla-MeAsp-Arg+H]+, respectively. Identification as MC-AR was further supported by the use of Cyanotox, which enabled prediction of potential fragment ions for MC-AR, by replacing leucine with alanine in the MC-LR amino acid sequence. Another microcystin commonly occurring in these samples was MC-WR (5), where retention time, UV and CID fragmentation correlated with authentic material. A sixth microcystin occasionally detected was tentatively identified as MC-AHar (Har = homoarginine) (6) based CID spectra where most of the fragments corresponded to those obtained for MC-AR apart from those containing arginine where m/ z of 654 and 525 were present compared to m/z 640 and 511 in MCAR. As with MC-AR, this tentative identification was supported by use of Cyanotox, where fragment ions were predicted when arginine was replaced by homoarginine in the MC-AR amino acid sequence. Whilst several microcystin variants were detected in all the extracts from the five ponds, microcystin profiles were different for each pond. All the bloom extracts from Lakshmikund pond contained predominantly MC-RR with low concentrations of MC-LR and AR (Table 2). Bloom extracts from Durgakund pond, typically contained MC-RR, MC-LR and MC-AR, where MC-RR was
again the predominant microcystin, representing 71.5% of the microcystin pool. However, MC-AR, was the second major microcystin in these extracts representing around 20% of the microcystin pool whereas MC-LR represented 3% (Table 2). Bloom extracts from Adityanagar and Sankuldara, had very similar profiles, once again MC-RR was the predominant toxin (approx. 70%), however, MC-WR and MC-AR were always present in significant proportions (10% and 20%, respectively; Table 2). In contrast, only MC-RR was detected in extracts from Karmdeshwar Pond, and at much lower concentrations as illustrated by the example in Table 2. 3.3. Persistence of microcystins in extracts from Microcystis blooms HPLC–PDA/MS analysis of extracts from bloom samples from Durgakund and Sankuldara ponds from October to December in 2005 revealed that microcystins were present on all occasions and once again, MC-RR was the predominant toxin (Tables 3 and 4). The toxic profiles observed for Microcystis blooms from these two ponds in 2003 (Table 2) were consistent with those observed in 2005. In the samples from Durgakund pond MC-RR represented 67–73% of the toxin pool, followed by MC-AR (16–30%) with MC-LR and MC-AHar representing minor components (Table 3). Whilst the major microcystin in extracts from Sankuldara pond was MCRR, representing 57–62% of the toxin pool, MC-AR and MC-WR contributed 20% and 15%, respectively to the total microcystin concentration, and MC-AHar was consistently present as a minor component (Table 4). It is interesting to note that there was little variation in the concentration of microcystins at each sampling apart from that collected from Sankuldara in December 2005, where there was a fivefold decrease in microcystin concentration although the proportions of those present were consistent with those in previous samplings.
Table 2 Microcystin concentrations (percentage composition in parentheses) in crude extracts of bloom material from five ponds sampled in November 2003 Concentration of microcystin (mg g dry weight)
Lakshmikund Karmdeshwar Adityanagar Durgakund Sankuldhara nd, not detected.
[Dha7]MC-RR (1)
MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-WR (5)
MC-AHar (6)
nd nd nd nd nd
1519 126 760 972 958
42 (2.7) nd nd 46 (3.4) nd
2 (0.1) nd 217 (19.9) 304 (22.4) 276 (20.1)
13 (0.8) nd 63 (5.8) nd 140 (10.2)
nd nd 49 (4.5) 37 (2.7) nd
(96.4) (100) (69.8) (71.5) (69.7)
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Table 3 Microcystin concentrations (percentage composition) in extracts of bloom material from Durgakund Pond in 2005 Concentration of microcystin (mg g dry weight)
September October November December
[Dha7]MC-RR (1)
MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-WR (5)
MC-AHar (6)
nd nd nd nd
810 460 990 991
62 15 124 65
181 222 368 372
nd nd nd nd
52 29 60 61
(73.3) (63.3) (64.2) (66.6)
(5.6) (2.1) (8.0) (4.4)
(16.4) (30.6) (23.9) (25.0)
(4.7) (4.0) (3.9) (4.0)
nd, not detected.
Table 4 Microcystin concentrations (percentage composition) in extracts of bloom material from in Sankuldara in 2005 Concentration of microcystin (mg g dry weight)
September October November December
[Dha7]MC-RR (1)
MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-WR (5)
MC-AHar (6)
nd nd nd nd
1140 1650 1350 190
nd nd nd nd
401 589 472 63
310 364 308 36
150 145 59 18
(57.0) (60.0) (61.7) (61.9)
(20.0) (21.4) (21.6) (20.5)
(15.5) (13.2) (14.1) (11.7)
(7.5) (5.3) (2.7) (5.9)
nd, not detected.
Perennial consistency of toxin occurrence and profile was clearly observed in bloom samples from Durgakund pond in September in 2004 and 2006, once again revealing the presence of microcystins (Fig. 2). Interestingly in the extracts from 2004 and 2006, qualitative and quantitative composition of microcystins
was very similar to that detected in 2003 and 2005. Once again a characteristic toxin profile was observed, where MC-RR was the major component representing 65–75% total microcystin, whilst MC-AR composed 16–22% with minor components MC-LR and MCAHar representing 5–8% and 4–5%, respectively (Table 5).
Fig. 2. Chromatograms (238 nm) of crude extracts of cyanobacterial biomass from Durgakund Pond in September 2004 (A), September 2005 (B) and September 2006 (C). ([Dha7]MC-RR (1), MC-RR (2), MC-LR (3), MC-AR (4), MC-WR (5) and MC-AHar (6).
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Table 5 Microcystin concentrations (percentage composition) in cyanobacterial biomass collected from Durgakund pond Year
Concentration of microcystin (mg g dry weight) MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-AHar (6)
September 2004 September 2005 September 2006
689 (68.5) 810 (73.3) 528 (70.4)
65 (6.5) 62 (5.6) 59 (7.9)
206 (20.5) 181 (16.4) 163 (21.7)
46 (4.5) 52 (4.7) nd
nd, not detected.
Fig. 3. Chromatograms (238 nm) of crude extracts from laboratory cultured Microcystis from Lakshmikund (A), Karmdeshwar (B) and Adityanagar (C). ([Dha7]MC-RR (1), MCRR (2), MC-LR (3), MC-AR (4), MC-WR (5) and MC-AHar (6).
The isolate from Lakshmikund pond produced [Dha7]MC-RR and MC-RR, where MC-RR was produced at a high concentration, 1.6 mg/ g dry weight (Fig. 3). It is interesting that this isolate had a toxin profile consistent with bloom extracts studied from this pond, suggesting species dominance. In contrast, there were no microcystins detected in the isolate from Karmdeshwar Pond suggesting
3.4. Laboratory isolation of species from bloom material Successful isolation of laboratory cultures from bloom material was achieved for three out of the five ponds. Single isolates from Lakshmikund, Karmdeshwar and Adityanagar in 2004 were identified as Microcystis sp., but exhibited a variety of toxin profiles.
Table 6 Microcystin concentrations (percentage composition) in extracts from laboratory isolates Concentration of microcystin (mg g dry weight)
Lakshmikund Karmdeshwar Adityanagar Durgakund Sankuldhara
[Dha7]MC-RR (1)
MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-WR (5)
MC-AHar (6)
45 (2.7) nd 54 (3.5) n/a n/a
1623 (97.3) nd 1290 (82.8) n/a n/a
nd nd nd n/a n/a
nd nd 110 (7.1) n/a n/a
nd nd 90 (5.8) n/a n/a
nd nd 14 (0.9) n/a n/a
nd, not detected; n/a, laboratory isolation of cultures from bloom material was unsuccessful.
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that the Microcystis population was dominated by non-toxic strains. The isolate from Adityanagar produced a microcystin profile consistent with that observed in bloom material (Fig. 3c), although MC-RR represented 83% and MC-AR and MC-WR each represented 7% and 6%, respectively with MC-AHar and [Dha7]MC-RR representing only minor components (Table 6). This variation in proportions is most likely the result of different conditions within the laboratory (e.g. media composition, light and temperature) and that they are from a single isolate not a naturally mixed population as in the ponds. 4. Discussion This is the first detailed study on the occurrence of microcystins in India and it has demonstrated that MC-RR was present in cyanobacterial extracts from September 2003 to August 2004 in all five ponds investigated. In addition, there was little variation in MC-RR concentration throughout the year in contrast to published studies. Very few investigations have reported perennial cyanobacterial blooms. In one of the early and most detailed pieces of work, Wicks and Thiel (1990) studied the bloom dynamics in Hartbeesport Dam, South Africa from January 1985 to May 1987. This lake supported persistent blooms of Microcystis but not persistent toxicity which was considered most likely to be due to seasonal variation between toxic and non-toxic species. This was supported by a 4-year study in Lake Suwa, Japan, which clearly indicated that temporal variation of microcystins was closely related to the composition of Microcystis species (Park et al., 1998). In most of these and other studies the occurrence of toxic blooms was seasonal, however, in the investigation presented here, toxic Microcystis species were present throughout the year, and at all samplings in subsequent years. The presence of extra-cellular microcystins in the water column all year round was recently reported in Lake Taihu, China where toxic Microcystis was present from April to January (Song et al., 2007). Analysis of microcystin composition in cells collected at four sampling sites, showed qualitative and quantitative differences between the sites, most likely reflecting species/strain variation. Several detailed studies in Europe have shown that Planktothrix sp. and microcystins have been present all year round, indicating that the perceived seasonality of toxic blooms in temperate climates is no longer clear-cut (Briand et al., 2002; Messineo et al., 2006). However, all these studies reports seasonal variation in species and microcystin concentrations as in earlier studies. It is most likely that the observed year round presence of toxic Microcystis is common in tropical and sub-tropical climates and lack of information reflects lack of investigation in these countries. Spatial and temporal variation of toxic species and the toxins they produce is considered a normal scenario, however, in the present study, the converse seems to be occurring in four out of the five ponds almost representing a large, continuous cultures. This was reflected by the similarity of the isolated cultures compared to the bloom material from which they were isolated. In many studies in temperate climates, MC-LR has been reported to be the predominant microcystin, compared to warmer climates, where MC-RR often predominates. This has been supported by laboratory experiments, where strains producing both MC-LR and MC-RR, produced higher concentrations of MC-LR at lower temperatures and higher concentrations of MC-RR at higher temperatures (Rapala et al., 1997). However, it is unusual, for MC-LR to be a relatively minor component of the toxin pool. The detection of MC-WR and MC-AR as a significant proportion of the microcystin pool is also unusual, and most likely reflects a unique
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strain. It is interesting that MC-WR was the predominant microcystin, along with MC-RR and MC-YR in a strain of Microcystis aeruginosa, isolated from Lake Thanh Cong in Vietnam (Hummert et al., 2001). No MC-LR was detected on this occasion. Clearly the Microcystis strains occurring in the Varanasi ponds broaden the spectrum of known geographical diversity and warrant further investigation. 5. Conclusions This data indicates that there is a significant health hazard in the five ponds studied and it is most likely that this scenario is widespread in tropical and semi-tropical countries. Although there are no reports of toxic events associated with the ponds in Varanasi, this is most likely due to lack of awareness of the risks and symptoms of microcystin intoxication. Similarly, long-term chronic effects may not be traced back to exposure again due to lack of awareness of the potential hazards. It is clear from the study that these essential water resources require rapid remedial action, however, this is just one of many challenges in a developing country. Along with the apparent, perennial species domination within each pond, the cyanobacteria themselves clearly warrant further investigation from a genetic and biochemical perspective, in an effort to aid our understanding of strain-to-strain microcystin production. Acknowledgements The authors are thankful to the Council of Scientific and Industrial Research (CSIR, New Delhi, India), for financial assistance to first author (Satya Prakash) in the form of Junior as well as Senior Research Fellowships and to the Department of Botany, BHU, Varanasi for necessary facilities during the study. We would also like to thank Dr Jens Dahlmann, Applied Biosystems, Germany for providing a copy of the Cyantox software.[SS] References Briand, J.F., Robillot, C., Quiblier-Llobe´ras, C., Bernard, C., 2002. A perennial bloom of Planktothrix agardhii (cyanobacteria) in a shallow eutrophic French lake; limnological and microcystin production studies. Arch. Hydrobiol. 153, 605–622. Desikachary, T.V., 1959. Cyanophyta, I.C.A.R. Monographs on Algae, New Delhi. p. 686. Grosse, Y., Baan, R., Straif, K., Secretan, B., Ghissassi, F.El., Cogliano, V., 2006. Carcinogenicity of nitrate, nitrite, and cyanobacterial peptide toxins. Lancet Oncol. 7, 628–629. Hummert, C., Dahlmann, J., Reinhardt, K., Dang, H.Ph.H., Luckas, B., 2001. Liquid chromatography–mass spectromemtry identification of microcystins in Microcystis aeruginosa strain from Lake Thanh Cong, Hanoi, Vietnam. Chromatographia 54, 569–575. Messineo, V., Mattei, D., Melchiorre, S., Salvatore, G., Bogialli, S., Salzano, R., Mazza, R., Capelli, G., Bruno, M., 2006. Microcystin diversity in a Plantothrix rubescens population from Lake Albano (Central Italy). Toxicon 48, 160–174. Park, H.-D., Iwami, C., Watanabe, M.F., Harada, K.-I., Okino, T., Hayashi, H., 1998. Temporal variabilities of the concentrations of intra- and extracellular microcystin and toxic Microcystis species in a hypertrophic lake, Lake Suwa, Japan (1991–1994). Environ. Toxicol. Water Qual. 13, 61–72. Parker, D.L., 1982. Improved procedures for the cloning and purification of Microcystis cultures (Cyanophyta). J. Phycol. 18, 471–477. Parker, D.L., Kumar, H.D., Rai, L.C., Singh, J.B., 1997. Potassium salts inhibit growth of the cyanobacterium Microcystis spp. in pond water and defined media: implications for control of microcystin-producing aquatic blooms. Appl. Environ. Microbiol. 63, 2324–2329. Rapala, J., Sivonen, K., Lyra, C., Niemela¨, S.I., 1997. Variation of microcystins, cyanobacterial hepatotoxins, in Anabaena spp. as a function of growth stimuli. Appl. Environ. Microbiol. 63, 2206–2212. Shirai, M., Matsumaru, K., Ohotake, A., Takamura, Y., Aida, T., Nakano, M., 1989. Development of a solid medium for growth and isolation of axenic Microcystis strains (Cyanobacteria). Appl. Environ. Microbiol. 55, 2569–2571. Shirai, M., Ohotake, A., Sano, T., Matsumoto, S., Sakamoto, T., Sato, A., Aida, T., Harada, K.I., Shimada, T., Suzuki, M., Nakano, M., 1991. Toxicity and toxins of
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natural blooms and isolated strains of Microcystis spp.(Cyanobacteria) and improved procedure for purification of cultures. Appl. Environ. Microbiol. 57, 1241–1245. Singh, D.P., Tyagi, M.B., Kumar, A., Thakur, J.K., Kumar, A., 2001. Antialgal activity of a hepatotoxic-producing cyanobacterium, Microcystis aeruginosa. World J. Microb. Biot. 17, 15–22. Sivonen, K., Jones, G., 1999. Cyanobacterial toxins. In: Chorus, I., Bartram, J. (Eds.), Toxic Cyanoabacteria in Water. E and FN Spon, London, pp. 41–111. Song, L., Chen, W., Peng, L., Wan, N., Gan, N., Zhang, X., 2007. Distribution and bioaccumulation of microcystins in water columns: a systematic investigation into the environmental fate and the risks associated with microcystins in Meiliang Bay, Lake Taihu. Water Res. 41, 2853–2864. Tsuji, K., Watanuki, T., Kondo, F., Watanabe, M.F., Nakazawa, H., Sujuki, M., Uchida, H., Harada, K.I., 1997. Stability of microcystins from cyanobacteria-IV. Effect of chlorination on decomposition. Toxicon 35, 1033–1041. Tyagi, M.B., Singh, D.P., Kumar, A., Jha, P.N., Sinha, R.P., Kumar, A., 2006. Hepatoxicity of Microcystis aeruginosa strains growing as blooms in certain eutrophic ponds. EXCLI J. 5, 66–78.
Ueno, Y., Nagata, S., Tsutsumi, T., Hasegawa, A., Watanabe, M.F., Park, H.D., Chen, G.C., Chen, G., Yu, S.Z., 1996. Detection of microcystins, a blue-green algal hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary liver cancer in China, by highly sensitive immunoassay. Carcinogenesis 17, 1317–1321. Van Apeldoom, M.E., Van Egmond, H.P., Speijers, G.J.A., Bakker, G.J.I., 2007. Toxins of cyanobacteria. Mol. Nutr. Food Res. 51, 7–60. World Health Organization, 1998. Guidelines for drinking water quality, 2nd ed. Addendum to vol. 2. Health criteria and other supporting information. World Health Organization, Geneva, 95–110. Wicks, R.J., Thiel, P.G., 1990. Environmental factors affecting the production of peptide toxins in floating scums of the cyanobacterium Microcystis aeruginosa in a hypertrophic African reservoir. Environ. Sci. Technol. 24, 1413–1418. Zhou, L., Yu, H., Chen, K., 2002. Relationship between microcystin in drinking water and colorectal cancer. Biomed. Environ. Sci. 15, 166–171. Zurawell, R.W., Chen, H., Burke, J.M., Prepas, E.E., 2005. Hepatotoxic cyanobacteria: a review of the biological importance of microcystins in freshwater environments. J. Toxicol. Environ. Health 8 (1), 1–37.
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Harmful Algae journal homepage: www.elsevier.com/locate/hal
Stability of toxigenic Microcystis blooms Satya Prakash a, Linda A. Lawton b, Christine Edwards b,* a b
Department of Botany, Banaras Hindu University, Varanasi 221005, India School of Pharmacy and Life Sciences, The Robert Gordon University, St. Andrew Street, Aberdeen AB25 1HG, UK
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 April 2008 Received in revised form 25 August 2008 Accepted 25 August 2008
This is the first detailed study on the occurrence of cyanobacterial toxins in India, where we studied five eutrophic, temple ponds in the vicinity of Varanasi city, Uttar Pradesh, which continuously supported blooms of Microcystis sp. for several years. Bloom material from all five ponds was sampled bi-monthly from September 2003 to August 2004. Analysis of extracts by high-performance liquid chromatography (HPLC) indicated that microcystin-RR (MC-RR) was present all year round at high concentrations (311– 1540 mg/g, dry weight), posing a significant health hazard especially since all five ponds are widely used for bathing, washing, cattle drinking supply, irrigation and recreation. In addition, there was unusually low temporal variation in concentration of MC-RR in each pond, <20% variation in four out of five ponds throughout the year. Characterization of microcystin composition of several bloom samples from this study by HPLC–PDA/ MS confirmed that additional microcystins were present in many of the samples. The rarely reported, MCAR was frequently detected in bloom samples from three of the ponds (Adityanagar, Durgakund and Sankuldhara), where it typically represented 20% of the microcystin pool. MC-WR was frequently found in samples from Adityanagar and Sankuldhara, representing 5–10% of the microcystin pool. MC-LR, along with the previously unreported MC-AHar, each represented approximately 5% of the microcystin pool when present. Bloom samples from each pond had a characteristic microcystin profile, when sampled from 2003 to 2006, suggesting persistent species/strain domination. The perennial and consistent nature of the toxic Microcystis blooms in these ponds is highly unusual, in contrast to the commonly encountered temporal and spatial variation of toxigenic and non-toxigenic species. Laboratory isolates from several ponds were shown to produce microcystins, showing similar microcystin composition to the original bloom material. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Cyanobacteria Microcystins Perennial blooms
1. Introduction Cyanobacteria frequently form nuisance blooms (benthic and planktonic) in freshwater bodies in all continents across the globe. Many of these organisms produce toxins, presenting a hazard to animal and human health. Microcystins, a large group of cyclic peptides (>85 variants), are the most commonly occurring toxins, produced by a growing number of cyanobacterial genera including; Microcystis, Anabaena, Nostoc, Plantothrix, Oscillatoria (Sivonen and Jones, 1999). Whilst microcystins have been associated with a number of acute toxicoses (Zurawell et al., 2005; Van Apeldoom et al., 2007), there is also increasing evidence from epidemiological studies, supporting the long-term chronic exposure resulting in primary liver cancer (Ueno et al., 1996; Zhou et al., 2002). This potential chronic toxicity led the WHO to establish a guideline of
* Corresponding author. Tel.: +44 1224 262839; fax: +44 1224 262828. E-mail address: [email protected] (C. Edwards). 1568-9883/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2008.08.014
1 mg/L as a maximum concentration of MC-LR in drinking water (WHO, 1998). More recently, a review of all data on toxicity of MC-LR by the International Agency for Research on Cancer (IARC) resulted in this compound being classified as a potential carcinogen (Group 2B) in June 2006 (Grosse et al., 2006). Insufficient data were available to classify other microcystins or nodularin but it would be pertinent to assume they are potential carcinogens. Whilst there have been lengthy investigations into the occurrence of toxic cyanobacteria in many countries, there are only a few brief reports on their occurrence in India. However, several studies have indicated that many ponds in Varanasi, Uttar Pradesh, are eutrophic or hypertrophic and frequently support cyanobacterial blooms, mostly Microcystis sp. (Parker et al., 1997; Singh et al., 2001). A recent study, based on mouse bioassay, did reveal that indeed hepatotoxic Microcystis blooms occurred in several eutrophic ponds in Varanasi, however, the extent of a potential hazard was not investigated in detail (Tyagi et al., 2006). Many of these freshwater ponds in the Varanasi area are essential for bathing, clothes washing, recreation, cattle drinking
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and irrigation. The presence of toxic cyanobacterial blooms is a health hazard with potential exposure via multiple routes. Direct exposure may result from oral ingestion of microcystins via contaminated water or inhalation during recreation/other direct water use. Indirect exposure via food is also a potential route for chronic exposure and there are numerous reports of accumulation of microcystins in fish and shellfish as well as detection in crops irrigated with contaminated water (Van Apeldoom et al., 2007). Five eutrophic ponds, known to support Microcystis blooms were selected for a detailed study monitoring cyanobacterial species, microcystin occurrence, environmental and meteorological parameters. This manuscript provides a detailed study on the occurrence of microcystins in five of these ponds.
UV detector (2487), a Rheodyne 7725i manual injector. Data acquisition and processing were achieved using Millenium-32 (Waters, Milford, USA). Samples (20 mL) were separated on Novapak C18 silica column (150 mm 4.6 mm i.d., 5 mm particle size; Waters, Milford, USA). The mobile phase used was methanol/ 0.05 M potassium phosphate buffer adjusted to pH 3 (57:43), at a flow rate 1 mL/min for 24 min (Tsuji et al., 1997). Microcystin variants were identified by characteristic retention time and UV absorbance at 238 nm. Microcystins were quantified as microcystin equivalents based on MC-LR (Alexis Corporation, Lausen, Switzerland).
2. Materials and methods
Selected freeze-dried samples from all five ponds in the study were extracted in 70% methanol (50 mg dry weight per mL solvent). Microcystins in these extracts were initially identified by HPLC with photodiode array (PDA) detection. A Waters Alliance 2695 solvent delivery system equipped with a 2996 PDA (Waters, Elstree, UK). Separation was effected on a Sunfire C18 column (2.1 mm i.d. 150 mm long; 3.5 mm particle size) which was maintained at 40 8C. Mobile phase was Milli-Q water (A) and acetonitrile (B) both containing 0.05% TFA. Test samples (20 mL) were separated using a gradient increasing from 15% to 60% B over 25 min at a flow rate of 0.3 mL/min followed by ramp up to 100% B and re-equilibration over the next 10 min. Eluent was monitored from 200 to 400 nm with a resolution 1.2 nm. Instrument control, data acquisition and processing were achieved using Empower v2.0. Microcystins were identified based on retention time and comparison of UV spectra to a range of standards, and by characteristic spectra where standards were not available. Known and unknown microcystins were quantified using MC-LR as an external standard at 238 nm (Alexis Corporation, Lausen, Switzerland). Further characterization of microcystins was achieved using an identical system equipped with ZQ 2000 MS detector in series. MS analyses were all performed in positive ion electrospray mode, scanning from m/z 100 to 1600 with a scan time of 2 s and interscan delay of 0.1 s. Ion source parameters; sprayer voltage, 3.07 kV; cone voltage, 100 V; desolvation temperature, 300 8C; and source temperature, 100 8C. Instrument control, data acquisition and processing were achieved using Masslynx v4.1. Cyanotox software v1.5.3 (Hyphen MassSpec Consultancy, Leiden, Netherlands) was used to support tentative identification of microcystins where no standards were available. This program allows prediction of fragment ions for microcystins based on the fragmentation of the singly charged molecular ion [M+H]+, using electrospray tandem mass spectrometry.
2.1. Sampling and sample sites Five eutrophic ponds; Lakshmikund, Karmadeshwar, Adityanagar, Durgakund and Sankuldhara in Varanasi, Uttar Pradesh, India (258200 N, 838010 E) were selected for investigation as they frequently supported cyanobacterial blooms. Surface bloom samples were collected twice monthly from September 2003 to August 2004. Other samples were collected at intermittent intervals from 2004 to 2006. The bloom samples were harvested from the shoreline using a nylon net (200 mm mesh size), lyophilized and stored at 20 8C. 2.2. Laboratory isolation and culture of Microcystis from bloom samples Microcystis species in blooms samples were identified according to the systematic keys provided by Desikachary (1959). Adhering mud and other contaminants were removed carefully and the specimens were washed with sterile water. In order to reduce contaminants, two-step centrifugation of inocula (150 g for 30 min, at room temperature, followed by higher-speed centrifugation; 1000–4000 g for 5 min, at room temperature) and repetitive sub-culturing of single cyanobacterial cell, in solid and liquid culture media alternately, were made as suggested by Shirai et al. (1989, 1991). Microcystis colonies were disaggregated by vortex mixing in de-ionized water, as described by Parker (1982). After the two-step centrifugation of inocula, the surface layer was withdrawn with a micropipette and plated on modified Jagar medium (Parker, 1982). After incubation for 10–12 days in light (22 mE/(m2 s)), a contaminant-free individual colony was selected with the help of fine-bore glass capillary, under binocular microscope (magnification 10–25) and transferred to culture tubes containing 10 mL modified J-medium. Cultures were incubated at 25 1 8C and illuminated with full spectrum fluorescent lights where the light intensity at the surface of the flasks was 22 mE/(m2 s). The cultures were shaken manually 3–4 times daily. 2.3. Extraction and analysis of microcystins Lyophilized cyanobacterial samples (20 mg) were extracted in 10 mL of 70% methanol, with ultrasonication for 15 min followed by intermittent shaking for 1 h. Debris was removed by centrifugation for 5 min at 8830 g. The pellet was re-extracted and the combined supernatants were evaporated to dryness at 30 8C using a rotary evaporator (Perfit, India). The residue was resuspended in 1.0 mL 80% methanol and filtered (0.22 mm cellulose–acetate; Axiva, India) prior to analysis by high-performance liquid chromatography (HPLC). Equipment included a Waters 600 E quaternary pump, coupled with variable wavelength
2.4. Characterisation of microcystins
3. Results 3.1. Detection of microcystin-RR Analysis of extracts from surface scum samples by HPLC revealed that MC-RR was the predominant microcystin in all five ponds and most notably, was present in all five ponds at every sampling throughout the year long study (Fig. 1). The highest concentration of MC-RR was detected in Lakshmikund (1540 mg/g dry weight) in November and lowest concentration was detected in June (880 mg/g dry weight; Fig. 1A). Throughout the year, the mean concentration of MC-RR was 1131 216 mg/g dry weight representing less than 20% variation. Similar patterns showing only slight fluctuation in MC-RR concentrations were detected in samples from the other four ponds in the study, albeit with lower maximum and minimum values. In Karmdeshwar, Adityanagar, and Durgakund
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Fig. 1. Detection of MC-RR in cyanobacterial biomass from five freshwater ponds: Lakshmikund (A), Karmdeshwar (B), Adityanagar (C), Durgakund (D) and Sankuldara (E) from September 2003 to August 2004. Data plotted are the mean of three replicates S.D.
ponds (Fig. 1B, C and D, respectively), maximum concentrations of MC-RR ranged from 1083 to 528 mg/g dry weight in November/ December whereas highest concentrations in samples from Sankuldhara (792 mg/g dry weight) occurred in April. As in Lakashmikund, there was less than 20% variation in MC-RR concentration throughout the year in Karmdeshwar, Adityanagar and Sankuldara ponds compared to 32% in Durgakund pond.
3.2. Identification and characterisation of microcystins in bloom extracts Characterisation of the microcystin composition of a selection of the bloom extracts by HPLC–PDA and HPLC–PDA/MS detection confirmed that the predominant microcystin was always MC-RR (2) with m/z of 1038 (Table 1). UV and MS spectral data revealed
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Table 1 Structural assignments of microcystins frequently identified in bloom extracts from five ponds Ion structure +
[M+H] [M+H CO]+ [M+H H2O]+ [M+2H 135]+ [MeAsp-Arg-Adda-Glu+H]+ [Glu-Mdha-Ala-Leu-MeAsp-Arg+H]+ [Glu-Mdha-Ala-Ala-MeAsp-Arg+H]+ [MeAsp-Arg-Adda+H]+ [Mdha-Ala-Leu-MeAsp-Arg+H]+ [Mdha-Ala-Ala-MeAsp-Arg+H]+ [Mdha-Ala-Trp-MeAsp-Arg+H]+ [Glu-Mdha-Ala-Ala-MeAsp+H]+ [Glu-Mdha-Ala-Ala-MeAsp+H COOH]+ [Arg-MeAsp-Ala+H]+ [C11H15O-Glu-Mdha+H]+ [Glu-Mdha+H]+ [PhCH2CHOMe]+
[Dha7]MC-RR (1)
MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-WR (5)
MC-AHar (6)
1024 – 1006 –
1038 – 1020 – – – – 599 –
995 987 – 861 728 682 – 599 553
953 925 – 819 728 – 640
1068 1040 – 934 – – – 599 –
967 940
– – – –
– 511
833 – 654
525 626
–
–
–
– 199 135
375 213 135
375 213 135
484 440 357 375 213 135
–
484 440
375 213 135
375 213 135
Fragmentation data based on cone voltage 100 eV.
that several other microcystins were present in some of the extracts. Collision-induced dissociation (CID) spectra obtained at a cone voltage of 100 V, provided increased fragmentation enabling structural assignment as summarised in Table 1. The most polar microcystin [Dha7]MC-RR (1) with m/z of 1024 was confirmed by retention time and spectral data compared to that of a previously characterised standard. MC-LR (3) was detected in some samples although concentrations were always low. One of the most commonly occurring microcystins was tentatively identified as MC-AR (4) in the absence of a reference compound. CID spectra gave a m/z of 953 and included fragment ions at m/z 640 and 511, indicative of [Glu-Mdha-Ala-Ala-MeAsp-Arg+H]+ and [Mdha-AlaAla-MeAsp-Arg+H]+, respectively. Identification as MC-AR was further supported by the use of Cyanotox, which enabled prediction of potential fragment ions for MC-AR, by replacing leucine with alanine in the MC-LR amino acid sequence. Another microcystin commonly occurring in these samples was MC-WR (5), where retention time, UV and CID fragmentation correlated with authentic material. A sixth microcystin occasionally detected was tentatively identified as MC-AHar (Har = homoarginine) (6) based CID spectra where most of the fragments corresponded to those obtained for MC-AR apart from those containing arginine where m/ z of 654 and 525 were present compared to m/z 640 and 511 in MCAR. As with MC-AR, this tentative identification was supported by use of Cyanotox, where fragment ions were predicted when arginine was replaced by homoarginine in the MC-AR amino acid sequence. Whilst several microcystin variants were detected in all the extracts from the five ponds, microcystin profiles were different for each pond. All the bloom extracts from Lakshmikund pond contained predominantly MC-RR with low concentrations of MC-LR and AR (Table 2). Bloom extracts from Durgakund pond, typically contained MC-RR, MC-LR and MC-AR, where MC-RR was
again the predominant microcystin, representing 71.5% of the microcystin pool. However, MC-AR, was the second major microcystin in these extracts representing around 20% of the microcystin pool whereas MC-LR represented 3% (Table 2). Bloom extracts from Adityanagar and Sankuldara, had very similar profiles, once again MC-RR was the predominant toxin (approx. 70%), however, MC-WR and MC-AR were always present in significant proportions (10% and 20%, respectively; Table 2). In contrast, only MC-RR was detected in extracts from Karmdeshwar Pond, and at much lower concentrations as illustrated by the example in Table 2. 3.3. Persistence of microcystins in extracts from Microcystis blooms HPLC–PDA/MS analysis of extracts from bloom samples from Durgakund and Sankuldara ponds from October to December in 2005 revealed that microcystins were present on all occasions and once again, MC-RR was the predominant toxin (Tables 3 and 4). The toxic profiles observed for Microcystis blooms from these two ponds in 2003 (Table 2) were consistent with those observed in 2005. In the samples from Durgakund pond MC-RR represented 67–73% of the toxin pool, followed by MC-AR (16–30%) with MC-LR and MC-AHar representing minor components (Table 3). Whilst the major microcystin in extracts from Sankuldara pond was MCRR, representing 57–62% of the toxin pool, MC-AR and MC-WR contributed 20% and 15%, respectively to the total microcystin concentration, and MC-AHar was consistently present as a minor component (Table 4). It is interesting to note that there was little variation in the concentration of microcystins at each sampling apart from that collected from Sankuldara in December 2005, where there was a fivefold decrease in microcystin concentration although the proportions of those present were consistent with those in previous samplings.
Table 2 Microcystin concentrations (percentage composition in parentheses) in crude extracts of bloom material from five ponds sampled in November 2003 Concentration of microcystin (mg g dry weight)
Lakshmikund Karmdeshwar Adityanagar Durgakund Sankuldhara nd, not detected.
[Dha7]MC-RR (1)
MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-WR (5)
MC-AHar (6)
nd nd nd nd nd
1519 126 760 972 958
42 (2.7) nd nd 46 (3.4) nd
2 (0.1) nd 217 (19.9) 304 (22.4) 276 (20.1)
13 (0.8) nd 63 (5.8) nd 140 (10.2)
nd nd 49 (4.5) 37 (2.7) nd
(96.4) (100) (69.8) (71.5) (69.7)
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Table 3 Microcystin concentrations (percentage composition) in extracts of bloom material from Durgakund Pond in 2005 Concentration of microcystin (mg g dry weight)
September October November December
[Dha7]MC-RR (1)
MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-WR (5)
MC-AHar (6)
nd nd nd nd
810 460 990 991
62 15 124 65
181 222 368 372
nd nd nd nd
52 29 60 61
(73.3) (63.3) (64.2) (66.6)
(5.6) (2.1) (8.0) (4.4)
(16.4) (30.6) (23.9) (25.0)
(4.7) (4.0) (3.9) (4.0)
nd, not detected.
Table 4 Microcystin concentrations (percentage composition) in extracts of bloom material from in Sankuldara in 2005 Concentration of microcystin (mg g dry weight)
September October November December
[Dha7]MC-RR (1)
MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-WR (5)
MC-AHar (6)
nd nd nd nd
1140 1650 1350 190
nd nd nd nd
401 589 472 63
310 364 308 36
150 145 59 18
(57.0) (60.0) (61.7) (61.9)
(20.0) (21.4) (21.6) (20.5)
(15.5) (13.2) (14.1) (11.7)
(7.5) (5.3) (2.7) (5.9)
nd, not detected.
Perennial consistency of toxin occurrence and profile was clearly observed in bloom samples from Durgakund pond in September in 2004 and 2006, once again revealing the presence of microcystins (Fig. 2). Interestingly in the extracts from 2004 and 2006, qualitative and quantitative composition of microcystins
was very similar to that detected in 2003 and 2005. Once again a characteristic toxin profile was observed, where MC-RR was the major component representing 65–75% total microcystin, whilst MC-AR composed 16–22% with minor components MC-LR and MCAHar representing 5–8% and 4–5%, respectively (Table 5).
Fig. 2. Chromatograms (238 nm) of crude extracts of cyanobacterial biomass from Durgakund Pond in September 2004 (A), September 2005 (B) and September 2006 (C). ([Dha7]MC-RR (1), MC-RR (2), MC-LR (3), MC-AR (4), MC-WR (5) and MC-AHar (6).
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Table 5 Microcystin concentrations (percentage composition) in cyanobacterial biomass collected from Durgakund pond Year
Concentration of microcystin (mg g dry weight) MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-AHar (6)
September 2004 September 2005 September 2006
689 (68.5) 810 (73.3) 528 (70.4)
65 (6.5) 62 (5.6) 59 (7.9)
206 (20.5) 181 (16.4) 163 (21.7)
46 (4.5) 52 (4.7) nd
nd, not detected.
Fig. 3. Chromatograms (238 nm) of crude extracts from laboratory cultured Microcystis from Lakshmikund (A), Karmdeshwar (B) and Adityanagar (C). ([Dha7]MC-RR (1), MCRR (2), MC-LR (3), MC-AR (4), MC-WR (5) and MC-AHar (6).
The isolate from Lakshmikund pond produced [Dha7]MC-RR and MC-RR, where MC-RR was produced at a high concentration, 1.6 mg/ g dry weight (Fig. 3). It is interesting that this isolate had a toxin profile consistent with bloom extracts studied from this pond, suggesting species dominance. In contrast, there were no microcystins detected in the isolate from Karmdeshwar Pond suggesting
3.4. Laboratory isolation of species from bloom material Successful isolation of laboratory cultures from bloom material was achieved for three out of the five ponds. Single isolates from Lakshmikund, Karmdeshwar and Adityanagar in 2004 were identified as Microcystis sp., but exhibited a variety of toxin profiles.
Table 6 Microcystin concentrations (percentage composition) in extracts from laboratory isolates Concentration of microcystin (mg g dry weight)
Lakshmikund Karmdeshwar Adityanagar Durgakund Sankuldhara
[Dha7]MC-RR (1)
MC-RR (2)
MC-LR (3)
MC-AR (4)
MC-WR (5)
MC-AHar (6)
45 (2.7) nd 54 (3.5) n/a n/a
1623 (97.3) nd 1290 (82.8) n/a n/a
nd nd nd n/a n/a
nd nd 110 (7.1) n/a n/a
nd nd 90 (5.8) n/a n/a
nd nd 14 (0.9) n/a n/a
nd, not detected; n/a, laboratory isolation of cultures from bloom material was unsuccessful.
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that the Microcystis population was dominated by non-toxic strains. The isolate from Adityanagar produced a microcystin profile consistent with that observed in bloom material (Fig. 3c), although MC-RR represented 83% and MC-AR and MC-WR each represented 7% and 6%, respectively with MC-AHar and [Dha7]MC-RR representing only minor components (Table 6). This variation in proportions is most likely the result of different conditions within the laboratory (e.g. media composition, light and temperature) and that they are from a single isolate not a naturally mixed population as in the ponds. 4. Discussion This is the first detailed study on the occurrence of microcystins in India and it has demonstrated that MC-RR was present in cyanobacterial extracts from September 2003 to August 2004 in all five ponds investigated. In addition, there was little variation in MC-RR concentration throughout the year in contrast to published studies. Very few investigations have reported perennial cyanobacterial blooms. In one of the early and most detailed pieces of work, Wicks and Thiel (1990) studied the bloom dynamics in Hartbeesport Dam, South Africa from January 1985 to May 1987. This lake supported persistent blooms of Microcystis but not persistent toxicity which was considered most likely to be due to seasonal variation between toxic and non-toxic species. This was supported by a 4-year study in Lake Suwa, Japan, which clearly indicated that temporal variation of microcystins was closely related to the composition of Microcystis species (Park et al., 1998). In most of these and other studies the occurrence of toxic blooms was seasonal, however, in the investigation presented here, toxic Microcystis species were present throughout the year, and at all samplings in subsequent years. The presence of extra-cellular microcystins in the water column all year round was recently reported in Lake Taihu, China where toxic Microcystis was present from April to January (Song et al., 2007). Analysis of microcystin composition in cells collected at four sampling sites, showed qualitative and quantitative differences between the sites, most likely reflecting species/strain variation. Several detailed studies in Europe have shown that Planktothrix sp. and microcystins have been present all year round, indicating that the perceived seasonality of toxic blooms in temperate climates is no longer clear-cut (Briand et al., 2002; Messineo et al., 2006). However, all these studies reports seasonal variation in species and microcystin concentrations as in earlier studies. It is most likely that the observed year round presence of toxic Microcystis is common in tropical and sub-tropical climates and lack of information reflects lack of investigation in these countries. Spatial and temporal variation of toxic species and the toxins they produce is considered a normal scenario, however, in the present study, the converse seems to be occurring in four out of the five ponds almost representing a large, continuous cultures. This was reflected by the similarity of the isolated cultures compared to the bloom material from which they were isolated. In many studies in temperate climates, MC-LR has been reported to be the predominant microcystin, compared to warmer climates, where MC-RR often predominates. This has been supported by laboratory experiments, where strains producing both MC-LR and MC-RR, produced higher concentrations of MC-LR at lower temperatures and higher concentrations of MC-RR at higher temperatures (Rapala et al., 1997). However, it is unusual, for MC-LR to be a relatively minor component of the toxin pool. The detection of MC-WR and MC-AR as a significant proportion of the microcystin pool is also unusual, and most likely reflects a unique
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strain. It is interesting that MC-WR was the predominant microcystin, along with MC-RR and MC-YR in a strain of Microcystis aeruginosa, isolated from Lake Thanh Cong in Vietnam (Hummert et al., 2001). No MC-LR was detected on this occasion. Clearly the Microcystis strains occurring in the Varanasi ponds broaden the spectrum of known geographical diversity and warrant further investigation. 5. Conclusions This data indicates that there is a significant health hazard in the five ponds studied and it is most likely that this scenario is widespread in tropical and semi-tropical countries. Although there are no reports of toxic events associated with the ponds in Varanasi, this is most likely due to lack of awareness of the risks and symptoms of microcystin intoxication. Similarly, long-term chronic effects may not be traced back to exposure again due to lack of awareness of the potential hazards. It is clear from the study that these essential water resources require rapid remedial action, however, this is just one of many challenges in a developing country. Along with the apparent, perennial species domination within each pond, the cyanobacteria themselves clearly warrant further investigation from a genetic and biochemical perspective, in an effort to aid our understanding of strain-to-strain microcystin production. Acknowledgements The authors are thankful to the Council of Scientific and Industrial Research (CSIR, New Delhi, India), for financial assistance to first author (Satya Prakash) in the form of Junior as well as Senior Research Fellowships and to the Department of Botany, BHU, Varanasi for necessary facilities during the study. We would also like to thank Dr Jens Dahlmann, Applied Biosystems, Germany for providing a copy of the Cyantox software.[SS] References Briand, J.F., Robillot, C., Quiblier-Llobe´ras, C., Bernard, C., 2002. A perennial bloom of Planktothrix agardhii (cyanobacteria) in a shallow eutrophic French lake; limnological and microcystin production studies. Arch. Hydrobiol. 153, 605–622. Desikachary, T.V., 1959. Cyanophyta, I.C.A.R. Monographs on Algae, New Delhi. p. 686. Grosse, Y., Baan, R., Straif, K., Secretan, B., Ghissassi, F.El., Cogliano, V., 2006. Carcinogenicity of nitrate, nitrite, and cyanobacterial peptide toxins. Lancet Oncol. 7, 628–629. Hummert, C., Dahlmann, J., Reinhardt, K., Dang, H.Ph.H., Luckas, B., 2001. Liquid chromatography–mass spectromemtry identification of microcystins in Microcystis aeruginosa strain from Lake Thanh Cong, Hanoi, Vietnam. Chromatographia 54, 569–575. Messineo, V., Mattei, D., Melchiorre, S., Salvatore, G., Bogialli, S., Salzano, R., Mazza, R., Capelli, G., Bruno, M., 2006. Microcystin diversity in a Plantothrix rubescens population from Lake Albano (Central Italy). Toxicon 48, 160–174. Park, H.-D., Iwami, C., Watanabe, M.F., Harada, K.-I., Okino, T., Hayashi, H., 1998. Temporal variabilities of the concentrations of intra- and extracellular microcystin and toxic Microcystis species in a hypertrophic lake, Lake Suwa, Japan (1991–1994). Environ. Toxicol. Water Qual. 13, 61–72. Parker, D.L., 1982. Improved procedures for the cloning and purification of Microcystis cultures (Cyanophyta). J. Phycol. 18, 471–477. Parker, D.L., Kumar, H.D., Rai, L.C., Singh, J.B., 1997. Potassium salts inhibit growth of the cyanobacterium Microcystis spp. in pond water and defined media: implications for control of microcystin-producing aquatic blooms. Appl. Environ. Microbiol. 63, 2324–2329. Rapala, J., Sivonen, K., Lyra, C., Niemela¨, S.I., 1997. Variation of microcystins, cyanobacterial hepatotoxins, in Anabaena spp. as a function of growth stimuli. Appl. Environ. Microbiol. 63, 2206–2212. Shirai, M., Matsumaru, K., Ohotake, A., Takamura, Y., Aida, T., Nakano, M., 1989. Development of a solid medium for growth and isolation of axenic Microcystis strains (Cyanobacteria). Appl. Environ. Microbiol. 55, 2569–2571. Shirai, M., Ohotake, A., Sano, T., Matsumoto, S., Sakamoto, T., Sato, A., Aida, T., Harada, K.I., Shimada, T., Suzuki, M., Nakano, M., 1991. Toxicity and toxins of
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