The hemolytic activity of C. marina and C. ovata was quantified using rabbit blood erythrocytes following Eschbach et al. and Ling and Trick [61,107]. Erythrocytes were directly obtained from the rabbit’s ear (New Zealand White rabbit), washed twice with phosphate- buffered saline (PBS) and stored at 4 °C for up to 7 days. For hemolysis analysis, the erythrocytes were washed again and diluted to a final concentration of 5% (v/v) in PBS. The previously prepared C. marina or C. ovata suspension of prepared erythrocytes (150 µL) was mixed into a 1 mL centrifuge tube, and set as test samples (Ae). The same amount of algal suspension, incubated in PBS, served as control (Aa) to account for algal background absorbance. The complete lysis of erythrocytes (exposed to 2% digitonin) served as positive control (Ap) and the prepared erythrocytes were the negative control (An). All samples were incubated for 5 h at 25 °C under a light intensity of 100 µmol m−2 s−1. Then, the samples were centrifuged at 3000 rpm, 25 °C for 10 min. A volume of 200 µL of the supernatant from each tube was transferred to a 96-well microplate (Corning, Glendale, AZ, USA) and the released hemoglobin absorbance was measured at 414 nm in a Microplate Reader (Biotek Synergy HT, Winooski, VT, USA).

Hemolytic activity was expressed as a percentage (%) according to Ling and Trick [61]:

where Ae, Aa, An and Ap are the absorption at 414 nm of the sample incubated with algae + erythrocytes (test samples), algae only (background), healthy erythrocytes (negative control), and lysed erythrocytes (positive control), respectively.

The hemolytic 50% effective concentration of C. marina and C. ovata, EC50, was first established by dose-effect simulation. Concentrations of 3 × 103, 7.5 × 103, 1.5 × 104, 3 × 104, 6 × 104, 1 × 105, and 2 × 105 cells mL−1 were used. A final EC50 value of 5 × 104 cells mL−1 for C. marina or C. ovata was obtained. Therefore, all toxin samples were prepared to yield a final test concentration of 5 × 104 cell mL−1. Thus, ~10 to 20 mL of C. marina or C. ovata from each treatment were centrifuged at 3000 rpm at 4 °C for 10 min. Pellets were resuspended in assay buffer [107] to yield 5 × 104 cells mL−1, and the suspension was ultrasonicated (Sonifier 540, Branson, Brookfield, CT, USA) on ice at 10% cycle (650 W) for 50 s (2 s pulse on, 1 s pulse off), to be ready for the hemolysis assay. Toxin production rate was calculated over the entire growth cycle of C. marina or C. ovata, by dividing the percent difference by the number samplings days, expressed in units of% hemolytic activity of 5 × 104 cells mL−1 per day.

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