Toxicity measurements in concentrated water samples - Rivm

4.2.1 Bacteria
Bacteria are important for nutrient cycling in an ecosystem. Many studies have been performed with
the Microtox ® assay, which is among other microbiotests one of the most sensitive (Willemsen et al.,
1995). The bacteria used in this assay are sensitive to a wide range of substances, including metals,
pesticides, industrial chemicals and crude oils (Calow, 1993; Azur environmental www.azurenv.com ).
Microtox ® assay
The Microtox ® bioluminescence assay (Bulich, 1979) is a standardized test which was commercialized
in the early 1980s (Bulich and Isenberg; 1981; Microbics, 1992). The test organism is Vibrio fischeri
(synonym for the Photobacterium phosphoreum), a luminescent marine bacterium (Figure 4-1).
Luminescence in this species is a consequence of respiration. It diminishes as the bacterium is exposed
to a chemical which interferes with the cell metabolism.
The bacteria are stored in lyophilized form, and can be used after 5 minutes of rehydration. The
bacteria are incubated at 15 °C and exposed to a range of concentrations of the water extract in glass
cuvettes of 3 ml. The luminescence of the bacteria is measured with a Microtox toxicity meter (M 500)
after 5, 15 and 30 minutes of exposure.
The dose-response relation is estimated from the inhibition of luminescence of the bacteria by the
different dilutions, expressed as a percentage of the control. Toxicity values expressed as the
concentration at which 50 % of the organisms (EC50) are affected are calculated using the Azur
software package MicrotoxOmni .
Figure 4-1. Agar plates with luminescent Vibrio fischeri colonies.
4.2.2 Algae
Algae are primary producers in the aquatic ecosystem and are responsible for the uptake, storage and
deposition of nutrients. The growth inhibition test is sensitive to a large number of pollutants, including
metals, insecticides, industrial organic components and, especially, herbicides. Inhibition of
photosynthetic activity has been shown for surfactants, herbicides and oil products. In general,
photosynthetic activity has been a more sensitive indicator of effect than growth.
Algae PAM-test
The algae PAM test with the unicellular freshwater alga Pseudokirchneriella subcapitata (synonym for
Raphidocelis subcapitata and Selenastrum capricornutum; Figure 4-2) is an alternative to the
conventional algae growth test (ISO 8692, 2004). The endpoint of this test is the inhibition of
photosynthetic efficiency.
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