Within the buy Navitoclax reclaimed land, irrigation water is pumped from a waterworks located near station R1, however irrigation of the vegetable field in the newly reclaimed land is limited as the annual precipitation of Isahaya district is 10–20% higher than the rest of Japan. On the other hand, wetland rice cultivation carried out on previously reclaimed land is heavily reliant on irrigation. The water for rice cultivation originates from small irrigation ponds, from which it is pumped up to each rice field. Beginning in September 2009, we expanded our environmental monitoring of MC content to include the irrigation

ponds, as well as the water pumped out of these ponds for irrigation. An enzyme-linked immunosorbent assay (ELISA) was used to measure total MC levels

(dissolved plus cell-bound) in surface water and pore water of the sediment, as described previously (Umehara et al., 2012). Several grams of target organ or whole specimen were frozen at −30 °C and freeze dried at below −40 °C. Lyophilized samples were homogenized and extracted three times with 10–20 mL BuOH:MeOH:H2O (1:4:15) solution for ∼24 h at 4 °C with stirring. Extracts were centrifuged for 20 min at 1600g, filtrates were collected, and 5 mL filtrate was centrifuged again for 1 h at 72,000g at −4 °C. Then the supernatant was filtered with a 0.45 μm mesh nitrocellulose filter. Then 3 mL supernatant was concentrated on octadecil-silane cartridges (C18), washed with 20 mL distilled water followed click here by 20 mL 20%

methanol, and eluted with 20 mL 100% methanol. This final fraction was again concentrated on a silica cartridge, washed with 20 mL 100% methanol, and eluted with 20 mL H2O:TFA:methanol (10:0.1:89.9 v/v) solution ( Harada et al., 1988 and Tsuji et al., 1994). Next, the methanol in the elute was vaporized using a rotary evaporator, and the remaining solution was lyophilized and dissolved by distillation of 1 mL. Finally, the MC content of the solution was measured by ELISA. Total MC levels were determined using a commercial Microcystin ELISA Kit (WAKO Pure Chemicals Industry, Ltd., Japan). After Glycogen branching enzyme 2011, these analyses were supplemented with new analyses using a different but similar ELISA kit (Microcystin Plate Kit, Beacon Analytical Systems Inc., USA). The monoclonal antibody used in the ELISA recognizes the Adda residue of MCs, which may be related to toxicity. The analytical curves of both kits are drawn based on the standard of MC-LR, but the susceptibilities to different MC homologs varies. Therefore, we measured each sample using both ELISA kits to determine the correlation between kits (W = 1.03B, r2 = 0.914, n = 22; W: value by Wako’s kit, B: value by Beacon’s kit). This difference was considered to be within the margin of error of detection for the scanner used. Blooms of M.