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VICTORIA LÓPEZ-RODAS Y COLS. AN. R. ACAD. NAC. FARM.

after further incubation for 60 days, some cultures became increased
in density again, due to the growth of cells that were resistant to the
toxic effect of TBT.

The approach for understanding adaptation of microalgae to TBT
is to analyze the rare variants that proliferate after the massive
destruction of the sensitive cells by this selective agent. Fluctuation
analysis is the appropriate procedure to discriminate between TBT-
resistant cells arising by rare spontaneous mutations occurring
randomly during replication of organisms prior to exposure to TBT
and TBT-resistant cells arising through specifically acquired
adaptation induced by TBT (reviewed in 31).

Genetic adaptation by rare spontaneous mutation is the mecha-
nism allowing adaptation of microalgae to TBT contamination. The
large fluctuation in number of resistant cells detected in the set 1
experiment in contrast to the scarce fluctuation in set 2 controls,
unequivocally demonstrates that these resistant cells arose by rare
spontaneous single mutations (which mainly occur prior to TBT
exposure) and not through direct and specific adaptation in response
to TBT. Results of fluctuation suggest that only one gene is implica-
ted in the TBT-resistance process. If several genes are involved in
TBT resistance then we should be unable to detect TBT-resistant
cells growing in flaks of set 1 experiment because the scarce proba-
bility of occurrence for several mutations at the same time. Conse-
quently, microalgae can adapt to antifouling paints much more ra-
pidly by single mutations that if the ability to survive require multiple
mutations. Several evidences suggest that single spontaneous muta-
tion at one locus can achieve adaptation of mesophile microalgae to
other severe anthropogenic contaminants including antibiotics (32,
33), herbicides (34-36) other potent biocides (37), and crude oil (38,
39). Even, microalgae are able to rapid adaptation to heavy metals
(which are the toxic compounds of antifouling paints) by mean of
single pre-selective mutations (40-43). Resistant mutants of microa-
lgae are usually used to manufacture specific biosensors (44, 45).
This capability is perhaps a consequence of microalgae ability for
adaptation to hostile natural environments by mechanisms based on
single mutations (46-50).

The DcM1 strain was widely used to measure mutation rates
from sensitivity to resistance to several anthropogenic and natural

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