Biomarkers
Test on bacteria
PUFAs used in the test were commercial standards or natural purified
compounds extracted from cultures. The toxicity of PUFAs for bacteria was determined by the Microtox® test applied to the bioluminescent marine bacteria Photobacterium phosphoreum. Decrease in light emission in presence of several dilutions of the toxic substance was measured by a luminometer, and results expressed in term of Effective Concentration producing 50% light attenuation (EC50). The methodology
was adapted to organic solvent (methanol) according to Microbics
Corp (1992).
Test on algae
The effect of PUFAs on the primary production can be estimated by bioassay on a diatom growth. Chaetoceros gracile used as a model of forage
production and easy to grow in laboratory conditions, was cultivated in
f/2 medium, in 50 ml borosilicate glass tubes. Cultures were enriched
in different diluted PUFAs methanolic solutions added at 10 - 6 to the
sea water. Initial concentration was about 30 million cells per litre.
The diatom growth was followed every day by direct chlorophyllian
fluorescence measurement, untill the stationnary phase was reached.
Growth curves were compared to the control grown with pure methanol addition at 10 - 6 , considering the growth rate μ2 (Fukazawa et al., 1980).
Test on embryos and larvae
Mytilus sp. was conditionned according to Loosanoff & Davis (1963).
The gametes were directly introduced in the solutions to be tested
(Chapman & Long, 1983). The experiments were conduced in glass
vessel at 20°C, and embryo development was stopped after 48 h by
addition of formaldehyde solution. The result of embryo development
in sea water enriched with increasing quantities of PUFAs in methanol,
was compared to a control as above, for evaluation of normal shaped
shells.
Pecten maximus larvae (9 days old) were distributed in 2 1-glass vessels
containing ichthyotoxic contaminated sea water, sampled in Etang de
Diane in Autumn 1993 (Bodennec et al., 1994) when Gymnodinium
mikimotoi was quasi-monospecific (2.7 million.l - 1 , 90% of the algal
population). Initial concentration in larvae was 7 500 animals per litre.
The test was realised with total toxic sample (sea water + algal cells),
0.45 pm filtered sample, a Gymnodinium mikimotoi culture and a control
in (no-toxic) oceanic sea water. After three days, sea water was changed
every two days and larvae fed with forage algae: Pavlova lutherii, Isochrysis
galbana and Skeletonema costatum. Samples with initial toxic sea water
treatment received oceanic sea water. Shells were observed every two
days, during ten days.
Haemolytic test
The haemolytic test was applied to determine the cytolytic property
of the PUFAs, which can act by radical formation in presence of oxygen
55
Test on bacteria
PUFAs used in the test were commercial standards or natural purified
compounds extracted from cultures. The toxicity of PUFAs for bacteria was determined by the Microtox® test applied to the bioluminescent marine bacteria Photobacterium phosphoreum. Decrease in light emission in presence of several dilutions of the toxic substance was measured by a luminometer, and results expressed in term of Effective Concentration producing 50% light attenuation (EC50). The methodology
was adapted to organic solvent (methanol) according to Microbics
Corp (1992).
Test on algae
The effect of PUFAs on the primary production can be estimated by bioassay on a diatom growth. Chaetoceros gracile used as a model of forage
production and easy to grow in laboratory conditions, was cultivated in
f/2 medium, in 50 ml borosilicate glass tubes. Cultures were enriched
in different diluted PUFAs methanolic solutions added at 10 - 6 to the
sea water. Initial concentration was about 30 million cells per litre.
The diatom growth was followed every day by direct chlorophyllian
fluorescence measurement, untill the stationnary phase was reached.
Growth curves were compared to the control grown with pure methanol addition at 10 - 6 , considering the growth rate μ2 (Fukazawa et al., 1980).
Test on embryos and larvae
Mytilus sp. was conditionned according to Loosanoff & Davis (1963).
The gametes were directly introduced in the solutions to be tested
(Chapman & Long, 1983). The experiments were conduced in glass
vessel at 20°C, and embryo development was stopped after 48 h by
addition of formaldehyde solution. The result of embryo development
in sea water enriched with increasing quantities of PUFAs in methanol,
was compared to a control as above, for evaluation of normal shaped
shells.
Pecten maximus larvae (9 days old) were distributed in 2 1-glass vessels
containing ichthyotoxic contaminated sea water, sampled in Etang de
Diane in Autumn 1993 (Bodennec et al., 1994) when Gymnodinium
mikimotoi was quasi-monospecific (2.7 million.l - 1 , 90% of the algal
population). Initial concentration in larvae was 7 500 animals per litre.
The test was realised with total toxic sample (sea water + algal cells),
0.45 pm filtered sample, a Gymnodinium mikimotoi culture and a control
in (no-toxic) oceanic sea water. After three days, sea water was changed
every two days and larvae fed with forage algae: Pavlova lutherii, Isochrysis
galbana and Skeletonema costatum. Samples with initial toxic sea water
treatment received oceanic sea water. Shells were observed every two
days, during ten days.
Haemolytic test
The haemolytic test was applied to determine the cytolytic property
of the PUFAs, which can act by radical formation in presence of oxygen
55