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activated sludge respiration inhibition testing
Type of information:
other: experimental result from similar substance
Adequacy of study:
key study
Study period:
1993-10-11 to 1993-10-18
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Sufficiently well documented study according to OECD 209 and under GLP.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:

Test material

Constituent 1
Reference substance name:
BY29 - similar substance 01
BY29 - similar substance 01

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
Concentration: 10, 32, 100, 320, 1000 mg/L

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Microbial source: activated sludge from a water treatment works treating mainly domestic sewage (date of sludge batch: 11.10.1993, date of test: 13.10.1993)- Temperature: 20.1 °C- Water: dechlorinated mains water- Preparation of inoculum for exposure: The sludge is allowed to settle and the supernatant liquor decanted. When the sludge is not used immediately, and for a period up to a maximum of four days, it is maintained aerated at a temperature of 20°C using an incubator. Synthetic sewage feed solution is added at the end of each working day. - Pretreatment: Before the start of the test the solids content of the sludge is determined and a suspension prepared containig 4 g solid per litre. The test beakers are prepared containing synthetic sludge feed. The appropriate volume of stock solution, and dilution water to a final volume of 300 mL. A volume of 200 mL diluted sludge is then added, the beaker placed in the water bath and aerated continuously at a constant rate for the duration of the test. Aeration of the test beakers is started at suitable time intervals, to allow for the oxygen measurement.- Initial biomass concentration: not reported, normalized to 4 g d.w./L

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
30 min
Post exposure observation period:
10 min, for measurement of dissolved oxygen concentration

Test conditions

Test temperature:
20.1 °C
Dissolved oxygen:
4.65 mg/L to 8.40 mg/L (initial)
Nominal and measured concentrations:
Concentration (nominal): 10, 32, 100, 320, 1000 mg/L
Details on test conditions:
TEST SYSTEM- Test vessel: 1-L beakers/BOD bottles- Type: open - Material: glass, 500 mL- Aeration: yes- No. of vessels per control (replicates): 2- Biomass loading rate: not reported, normalized to 4 g d.w./LTEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: dechlorinated mains water- Total organic carbon:- Particulate matter: 4 g d.w./L
Reference substance (positive control):
3,5 dichlorophenol

Results and discussion

Effect concentrations
30 min
Dose descriptor:
Effect conc.:
59 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
The results of this study show that the test item caused a significant inhibitory effect on the respiration rate of activated sludge after a contact period of 30 min. The estimated EC50 value was 59 mg/L.
Results with reference substance (positive control):
EC50 (3 h) 21 mg/L

Any other information on results incl. tables

Table 1: Respiration rates of activated sludge exposed to the test item. Contact time 30 min

Concentration of sample (mg/L)  Respiration rate (mg O2/L h)  Inhibition (%)
 0  mean 31.4  -
 10  24.4  22.1
 32  19.4  38.3
 100  12.0  61.7
 320  7.9  74.9
 1000  3.7  88.3

Applicant's summary and conclusion

Validity criteria fulfilled:
The 30 min EC50 value for the test item is determined to be 59 mg/L.
Executive summary:

To investigate potential harmful effects of the test item on environmentally important microorganisms, a study according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test) was performed under GLP. When exposed to the test substance at concentrations of 10, 32, 100, 320, 1000 mg/L, the activated sludge exhibited significantly lower respiration rates. The EC50 after 30 min was determined to be 59 mg/L.