Principle
The respiration inhibition test for activated sludge is investigating potential effects of test substances or waste water on mixed bacterial populations, in particular in aerobic wastewater treatment plants.

Abstract
Activated sludge is exposed to a serial dilution of the test substance or waste water for 30 min and/or 3 hours. A control without test substance is studied in parallel. The inhibitory effect of the test substance on the respiration rate in the test vessels is expressed as percentage of the mean respiration rate of the controls. As test result, the 50% effect concentration related to the oxygen consumption (EC₅₀) is determined. A parallel test with N-allylthiourea can be performed to determine the inhibition of heterotrophic respiration and nitrification.

Endpoints
Total respiration, heterotrophic respiration, nitrification

*) DIN standards have been withdrawn.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 (LID_L-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.

Principle
The nitrification inhibition test with activated sludge determines the potential impact of test substances or wastewater samples on nitrifying bacteria, which oxidise ammonium to nitrate. Due to their long generation time, these bacteria are particularly sensitive, a fact that can lead to disturbances in the nitrification process of wastewater treatment plants.

Abstract
A serial dilution of the test substance is prepared within the relevant concentration range. In parallel, a negative control without the test substance and a reference substance (nitrification inhibitor N-allylthiourea) are tested. To each test vessel, a defined amount of activated sludge, the nutrient medium and the test item are given. After an incubation time of 4 hours, the concentrations of ammonium, nitrite and nitrate are determined. By comparing the oxidised N-compounds in the test flasks with those in the blank sample it is possible to calculate the percentage inhibition for each test concentration.

Endpoint
Concentration of oxidised N-compounds (nitrate and nitrite)

Principle
The test assesses effects of water soluble and chemical bonded pollutants of natural sample material such as soil, sludge and waste on the dehydrogenase activity of Arthrobacter globiformis.

Abstract
The toxicity is determined by measuring the dehydrogenase activity of Arthrobacter globiformis using the redox dye resazurine. Dehydrogenases, as respiratory chain enzymes of microorganisms, are essential components for biological transformations. Because the dehydrogenase activity correlates with the metabolic activity of the cells, the quantity of Resorufin is a measure for the toxicity of the test substance. The increase of Resorufin is determined by measuring the fluorescence every 15min for a period of one hour. To determine the inhibition of the dehydrogenase activity the rate of increase of resorufin in the sample is related to the rate of increase of resorufin in a control.

Principle
Assessing the potential toxicity of a test material by determining the production of biogas from the anaerobic digestion of sewage sludge.

Abstract
Aliquots of a mixture of anaerobically digesting sludge and a degradable substrate solution are incubated alone and simultaneously with a range of concentrations of the test material in sealed vessels for up to 3 days. The amount of gas (methane plus carbon dioxide) produced is measured by the increase in pressure in the bottles. The percentage inhibition of gas production is calculated from the amounts produced in the respective test and control bottles. The EC₅₀ and other effective concentrations are calculated from plots of percentage inhibition against the concentration of the test chemicals or its logarithm.

Test substance properties
The test is applicable to substances which are soluble or insoluble in water, including volatile substances, as well as wastewaters, sludges or other environmental samples.