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Microcystis Aeruginosa Bloom and the Occurrence of Microcystins (heptapeptides Hepatotoxins) from an Aquaculture Pond in Gazipur, Bangladesh

M. S. Ahmed
University of Dhaka, Department of Zoology, Laboratory of Aquatic Resource Management, Dhaka 1000, Bangladesh.

S. Hiller
University of Jena, Institute of Nutrition, Dornburger Street 25, 07743 Jena, Germany. * Corresponding Author: Tel.: +81.99 2556721; Fax: +81.99 2864133; E-mail: ms2ahmed@yahoo.com

B. Luckas
University of Jena, Institute of Nutrition, Dornburger Street 25, 07743 Jena, Germany

Abstract/ Executive Summary:

Bangladesh is a tropical country of large alluvial plain with 1.3 million freshwater ponds and lakes and has a proper environment for luxuriant growth of cyanobacteria. Algal bloom of Microcystis aeruginosa occurred in an aquaculture pond in Gazipur, Dhaka. Bloom sample was collected and filtered through a glass fiber filter. Methanol-water extract of filtered cells were analyzed by high-performance liquid chromatography (HPLC) with UV, MS and MS-MS detection, detected three types of microcystins viz., Microcystin-RR, Microcystin-YR and Microcystin-LR and those were confirmed by HPLC-MS. The amount of MC-LR was the highest (33.2 μg L^-1) followed by MC-RR (9.03 μg L^-1) and MC-YR (5.23 μg L^-1). The concentration of microcystins was well above the WHO provisional guideline value of 1 μg L^-1 MC-LR. Further, investigations need to characterize other types of microcystins from bloom-forming cyanobacteria and their effect on human health and cultured fish in Bangladesh.

Keywords: Microcystis aeruginosa, Microcystin, HPLC, Algal bloom, Bangladesh.

Location: Gazipur district, Bangladesh

Start Date:

End Date:

Program Area: Risk Management in Agriculture

Commodity: Fish farming, Pond, Health hazard

Objectives:

To determine the isolation and characterization of microcystins from a natural bloom of M. aeruginosa occurring in an aquaculture pond in Gazipur, Dhaka.

Methodology:

The study pond is located in Gazipur district (90°21′ E longitude 24°00′ N latitude) 20 km north from Dhaka city. The pond is 0.1 ha in size and stocking with catfish, Pangasius pangasius. Algal bloom (M. aeruginosa) was initiated in the fast week of March, 2005 and the highest cell density (95% Microcystis) was recorded on 10 March 2005. The bloom sample was collected with plankton net of 20 μm mesh size. A portion of (5 ml) of the concentrated samples were filtered through an 0.45 μm glass fiber filter (Whatman GF/C, 47 mm diameter) and dried in an oven at 60-80°C. Dried filters covered with algae cells were transported to the Alfred Wegner Institute, Sylt, Germany for analysis. Extraction: GF/C filters and 1.0 ml of a mixture of wate and methanol (50:50; v:v) was sonicated for 20 minutes and centrifuged (3,000 g). The supernatant was filtered through a nylon filter with 0.45 μm pore size. Chemical Analysis: The HPLC/UV determination of microcystins was carried out following the methods of Lawton et al. (1994) with some modifications (Hummert et al., 2001a; C18 column: Phenomenex prodigy, ODS (3), 250 x 4.6 mm, 5 μm, mobil phases: acetonitrile /water/0.05% TFA). Detection of microcystins was done by the use of an UV detector (Shimadzu SPD10AV; λ=238 nm). HPLC/MS and HPLC/MS-MS analysis were applied to ensure the identity of the toxin peaks in the chromatograms. The HPLC was coupled by means of an electrospray interface to a single quadrupol mass spectrometer (API 150, PE Sciex Instruments, Canada) and additionally to a triple quadrupol mass spectrometer (API 365, PE Sciex Instruments, Canada). The detection was carried out in selected ion monitoring (SIM) mode using LC/MS and multiple reactions monitoring mode (MRM) using LC/MS-MS (Hummert et al., 2001b). Microcystins and Nodularin Standards: Standards of Microcystin-RR, Microcystin-LR, Microcystin-YR, Microcystin-LA and Nodularin were purchased from Calbiochem/Novabiochem (La Jolla, CA, USA). Chemicals: HPLC grade acetonitrile and HPLC grade methanol were purchased from Baker (Deventer, Netherlands). Water was purified to HPLC grade quality with a Millipore-Q RG Ultra Pure Water System (Millipore, Milford, USA).

Source of Information: Turkish Journal of Fisheries and Aquatic Sciences 8: 37-41 (2008)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In the original bloom sample, the cell density of M. aeruginosa was 6.22 x 108 cells L^-1. During the bloom, dissolved oxygen, free carbon dioxide and nitrite nitrogen of pond water was recorded as 4.5, 16.0 and 0.68 mg L^-1. The pH was 8.5 and the water temperature was between 20-24ºC. HPLC analysis of M. aeruginosa extract showed three peaks, the retention time of which agreed well with standard MC-RR, MC-YR and MC-LR. The results of HPLC-MS revealed the identification of three variants of microcystins, according to their corresponding molecular weight: MC-LR (at m/z 995.0 [M+H]+ ), MC-RR (at m/z 519.5 [M+2H]2+) and MC-YR (at m/z 1045.0 [M+H]+ ). In M. aeruginosa sample, the amount of MC-LR was the highest (33.2 μg L^-1) followed by MC-RR (9.03 μg L^-1) and MCYR (5.23 μg L^-1). A small amount of MC-LA was also detected. Welker et al. (2004) in a study at three different regions in Bangladesh detected microcystins in 39 ponds, mostly together with varying abundance of potentially microcystin-producing genera such as Microcystis, Planktothrix and Anabaena. Total microcystin concentrations in their study ranged between <0.1 and up to >1000 μg L^-1, and more than half of the positive samples contained high concentrations of more than 10 μg L^-1. Our results clearly showed that the concentration of microcystins well above the WHO provisional guideline value of 1 μg L^-1 MC-LR. In Australia, a safety factor for tumor promotion is 1.0 μg microcystins or nodularins L^-1 (Falconer et al., 1994). In Canadian drinking water maximum accepted concentration for MC-LR is 0.5 mg L^-1 and for other microcystins, 1 μg L^-1 of total microcystins (Carmichael, 1995). The concentration of microcystins was well above the WHO provisional guideline value of 1 μg L^-1 MC-LR. Further, investigations need to characterize other types of microcystins from bloom forming cyanobacteria and their effect on human health and cultured fish in Bangladesh.

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