Evaluation of the Impact of Natural Microcystin-LR Extract on Brine Shrimp as an Invertebrate Bioassay Model

Abstract

Frequent occurrences of toxic cyanobacteria dominantly Microcystis aeruginosa bloom arebecoming a nuisance problem in Sri Lankan reservoirs in conformity with its globalenvironmental and health issues. Most of cyanobacteria produce heat stable cyanotoxinswhich have potent health impacts. Many researchers have focused on pure cyanobacterialtoxins and little was known about the toxic effect of natural cyanobacteria. The present studywas carried out to investigate the effect of natural cyanobacterial toxins on Artemia salina(Brine shrimp) as a bioassay. The toxin, Microcystin LR was extracted from bloom samplescollected from Beira and Borelesgamuwa Lakes. Both water bodies were dominated bycyanobacterium M. aeruginosa, which may contain toxic and non toxic strains. Density of M.aeruginosa at the sampling occasion in Beira Lake was 9.64x105cells/l and inBoralesgamuwa Lake was 5.31x105 cells/l respectively. One liter of lake water was filteredthrough GF/C glass microfiber filters (47 mm) and the filter disks with entrappedcyanobacterial cells were ultrasonicated with 20 ml of 100% methanol for 5 minutes and thenthe methanolic extract was filtered and HPLC-DAD analysis was done to quantify theMicrocystin (MC)-LR. MC-LR in Beira Lake was 17.13±0.02 μg/ml. In BorelesgamuwaLake MC-LR was not detected at the sampling occasion. Methanol in the extract wasevaporated and subsequently a known weight of residue was re-suspended in 2 ml of filteredsea water to make stock solutions of crude extracts to use for the bioassay. Artemia eggs werepurchased and allowed to hatch with continuous aeration in filtered sea water undercontinuous white fluorescent light for 18 h. After incubation newly hatched naupli of Artemiawere collected. For the bioassay about 30 Artemia naupli were inoculated into each well in96-well microplate. To confirm the initial number of naupli in each well, counting wasperformed under the microscope. MC-LR standard and extracts from Beira andBoralesgamuwa Lakes were used to prepare toxin (MC-LR) at final concentration series of0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0 and 50.0 μg/ml. Number of dead individuals were counted ineach well after 6, 12, 18, 24 and 48 hours incubation. EC50 (50% effective concentration)values were assessed using non-linear regression by Graph Pad Prism software. EC50 value ofMC-LR standard was 4.78 μg/ml after 6 h incubation. However, EC50 values ofcyanobacterial crude extracts from Beira and Boralesgamuwa lakes at the same incubationperiod was 4.98 and 9.83 μg dry weight/ml respectively. The results showed that the standardMC-LR and cyanobacterial crude extracts from Beira Lake had almost similar EC50 valuewhile Boralesgamuwa Lakes was significantly deviated and having higher (p<0.05) EC50value. The higher toxicity of Beira Lake crude extract was due to presence of MC-LR in theextract. As confirmed by the HPLC, MC-LR was not detected in the extract prepared fromBoralesgamuwa Lake. Though the extracts showed toxicity effect on Artemia reveal that thetoxicity may due to other cyanotoxin variants present in the extracts. Further, it was detectedthat decreasing tendency of EC50 values along with the increasing incubation periods for allextracts and standard.