*) DIN standards have been withdrawn. The Wastewater Ordinance however refers to them and therefore they are still valid.
Principle
The luminescent bacteria test is a static short-term test for the investigation of wastewater or aqueous solutions of test substances. Marine bacteria of the species Vibrio fischeri (= Photobacterium phosphoreum) emit a natural light ("bioluminescence") during normally functioning metabolism, which can be measured by a luminometer. A disturbance of the metabolism (e. g. caused by the action of chemicals) reduces the bacterial light intensity. The reduction of the light intensity after an exposure time of 30 minutes and with regard to the control tests is taken as test criterion.
A) Single substance testing
A dilution series of a test substance is prepared with NaCl and 106 bacteria per ml are added. At 0 and 30 minutes the intensity of luminescence is measured and the percentage of inhibition compared to the control is calculated. The concentration-effect relationship can be calculated from the test data by means of a regression analysis and by log-probit-transformation of the obtained parameter pairs. From this, the 50% effect concentration (EC50) related to light emission can be determined.
B) Waste water testing
The bioluminescence test is mainly used for wastewater assessment. NaCl and 106 luminescent bacteria are added to a dilution series taken from the wastewater sample. At 0 and 30 minutes the intensity of luminescence is measured and the percentage of inhibition compared to the control is calculated. The Lowest Ineffective Dilution, at which the inhibitory effect on the light intensity is below 20 %, is given as the test result (LIDL-value). Hereby, LID corresponds to the dilution factor or the reciprocal value of the respective volume proportion of the wastewater (No. 404 Wastewater Ordinance and DIN 38412-L34).
C) Chronic toxicity
For testing of chronic toxicity, Vibrio fischeri is exposed to a serial dilution of a test substance or waste water for 7 h and the inhibition of the cell growth of the bacteria is determined by turbidity measurements at 436 nm (DIN 38412-L37). Test design corresponds to the Pseudomonas growth inhibitory test.