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

to new culture medium (20 ml) once every 30 days. The strains were
added to the algal culture collection of at Genetics Laboratory,
School of Veterinary Medicine, Complutense University of Madrid.

The damage caused by crude oil on these Arroyo Minero strains
was measured using a toxicity test. We also used the Scenedesmus
intermedius SiD1 wild-type strain (from algal collection of Genetics
Laboratory, School of Veterinary Medicine, Complutense University
of Madrid) as a control. SiD1 strain was isolated from a pristine lake
in Doñana National Park (Huelva, Spain), which never has been
exposed neither to crude oil nor petroleum derivates. Crude oil
collected in Arroyo Minero as well as a standard of petroleum (Fluka
Analytical Petroleum special standard. Sigma-Aldrich Chemie,
GmbH, Ch-9471, Buchs, Germany) were used at concentration of
12% v/v diluted in BG-11 medium. The mixing of petroleum and
culture medium, was obtained by sonication with Vibra Cell (Sonics
& Materials Inc Danbury CT, USA), maintaining the tubes within a
bucket of crushed ice during the process. The pattern for sonication
was of 4 pulses of 10 seconds each, with a power of 40 watts and a
frequency of 16 KHz. Measuring crude oil effect on photosynthetic
performance we performed the toxicity test. The change in the
effective quantum yield of photosystem II (FPSII) was measured on
three replicates of both exposed and control cultures using a ToxY-
PAM fluorimeter (Walz, Effeltrich, Germany). The measures were
performed after 48 hours of petroleum exposure. Effective quantum
yield was calculated as follows:

FPSII = (F’m - Ft) / F’m

where F’m and Ft are the maximum and the steady-state fluorescence
of light-adapted cells, respectively (29).

3. RESULTS

Apparently, the natural spill of crude oil of Arroyo Minero is a
complex and variable mix of crude oil, gas and water (Figure 1a, 1b).
Total hydrocarbons concentration in the spill area was 11,7% v/v.
Water in the spill area was at 19 ºC, pH 8.1 and 694 µS conductivity.
There is a strong evaporation of the more volatile parts of the crude
oil with an intense odour to hydrocarbons in all the area.

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