2.2. Biological material e cyanobacterial culture and mussels
The exposure experiment was carried out with CYN-producing
cells of C. raciborskii (LEGE 97047). Cyanobacteria were cultured
to the death phase in Z8 medium (Kotai, 1972) (6 L flask) under
fluorescent light (light/dark cycle of 14/10 h) and a temperature of
25 ± 1 C. Live mussels, Mytilus galloprovincialis (Lamark, 1819),
with a mean shell length of 6.50 ± 0.56 cm (n ¼ 90) were purchased
in a Portuguese local market as for human consumption. The
mussels were acclimated for two weeks prior to the experiment in
20 L aquaria with seawater. During this period, the mussels were
fed twice a week with Chlorella vulgaris (105 cells/mL). The water
was renewed weekly.
2.3. Exposure and experiment
2.3.1. Mussels intoxication
The presence of CYN as well as the occurrence and growth of
C. raciborskii in marine ecosystems have not yet been documented.
However, recently, Vareli et al. (2012) found microcystins (freshwater
cyanotoxins) in concentrations ranging from 45 ± 2 to
141.5 ± 13.5 ng/g in specimens of M. galloprovincialis collected from
Mediterranean Sea, NW Greece. This raised attention for the potential
negative impacts of freshwater cyanotoxins in marine
aquaculture in Mediterranean estuaries. Therefore, in this study it
was attempted to simulate a contamination of estuarine or seashore
ecosystem with senescent toxic cyanobacterial blooms. Mussels
were intoxicated with cyanobacterial crude extract (cells and culture
medium) for four days in static conditions (z10e15 mg/L of
CYN per day). During the exposure experiment, the physical and
chemical conditions of the seawater were as follows: temperature
17.5 ± 1.3 C, salinity 32.84 ± 0.58‰ and pH 8.25 ± 0.23.