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VOL. 76 (2), 189-208, 2010 THE IGNORED STOWAWAYS: WORLDWIDE...

2.2. Experimental organism

Laboratory experiments were performed with the unicellular
Chlorophyta (strain Dc1M), from the Algal Culture Collection of the
Universidad Complutense (Madrid). The strain was grown axenically
in cell culture flasks (Greiner, Bio-One Inc., Longwood, NJ, USA)
with 20 ml of Sigma algal culture medium (Sigma, Aldrich Chemie,
Taufkirchen, Germany), at 20º C under continuous light of 60 mol
photom m–2 s–1 over the waveband 400-700 nm. It was maintained in
mid-long exponential growth by serials transfers of a cell inoculum
to fresh medium (once every month). Prior to the experiments, the
cultures were re-cloned (by isolating a single cell) to avoid including
any previous spontaneous mutants accumulated in the cultures.
Cultures were maintained as axenic as possible and only cultures
without detectable bacteria were used in the experiments.

2.3. Toxicity test: effect of TBT on growth rate
2.3. and photosynthesis performance

A stock solution of TBT (Sigma) was prepared in BG-11 medium
to obtain serial dilutions of 0, 1, 3, 10, and 30 ppm. Each
experimental culture was inoculated with 6 × 106 cells from mid-log
exponentially growing cultures. Two replicates of each concentration
of TBT, as well as two unexposed controls, were prepared.

The toxic effect of the TBT was estimated by calculating
acclimated maximal growth rate (m) in mid-log exponentially
growing cells, derived from the equation:

Nt = N0 e mt (21)

where t = 7 days, the time that cultures were exposed to different
dose of TBT, and Nt and N0 are the cell numbers at the end and at the
start of the experiment, respectively. Experiments and controls were
counted blind (i.e., the person counting the test did not know the
identity of the tested sample), using a Uriglass settling chamber (Bio-

siga, Cona, Italy) and an inverted microscope (Axiovert 35, Zeiss).

The effective quantum yield (F PSII) was also measured in
triplicates of experiments and controls of both species using a ToxY-

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