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Administrative data

activated sludge respiration inhibition testing
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted 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:
trans-dimethyl 1 ,4-cyclohexanedicarboxylate
trans-dimethyl 1 ,4-cyclohexanedicarboxylate
Constituent 2
Chemical structure
Reference substance name:
1,4-dimethyl cyclohexane-1,4-dicarboxylate (trans)
Cas Number:
Molecular formula:
1,4-dimethyl cyclohexane-1,4-dicarboxylate (trans)
Details on test material:
- Name of test material (as cited in study report): DMCD
- Analytical purity: 99.9%
- Isomers composition: trans isomer
- Lot/batch No.: 910521

Test solutions

Details on test solutions:
No stock solutions could be prepared at the required concentrations in distilled water owing to the sparing so lubility of the test chemical. Instead, at the start of the study 500, 250, 50, 25, and 12.5 mg of test chemical were placed in the appropriate test beakers to provide the theoretical final test
concen tra tions required in the medium.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge microorganisms were obtained from the Van Lare Treatment Plant, Rochester, New York, a domestic wastewater treatment plant. Viability of the microorganisms was confirmed on receipt in the laboratory and activity checked by means of a reference (positive control) substance. On return to the laboratory, a small amount of the fresh sludge was weighed, oven-dried, and reweighed. A calculation was made from these results to determine the amount of wet sludge that must be suspended in laboratory dilution water (LDW) in order to obtain an activated sludge with a mixed liquor suspended solids (MLSS) level of approximately 4 g/L. Next, the sludge was washed twice with LDW using the following method: after centrifuging at approximately 7000 x g for 15 minutes, the supernatant was decanted and the remaining sludge solids resuspended in LDW and mixed well. This procedure was repeated twice. The final sludge suspension was prepared in LDW at an MLSS of approximately 4 g/L and then 25 mL of each of the two synthetic feed stock solutions were added per liter of sludge suspension. The pH of the suspension was checked and, if necessary, buffered to pH 6.0 - 8 .0 using a
5% sodium bicarbonate solution. The sludge suspension was aerated overnight at 20 ± 2 C.

Study design

Total exposure duration:
3 h

Test conditions

Test temperature:
20 ± 2°C
Nominal and measured concentrations:
25, 50, 100, 500, and 1000 mg/L nominal
Details on test conditions:
The final sludge suspension was prepared in LDW at an MLSS of approximately 4 g/L. This level gives a final sludge concentration of 1.6 g/L in the test medium. The test vessels used were 1-liter beakers. At time "0", 8 mL of each synthetic feed stock solution were placed in a 200 mL volumetric flask and brought to volume with distilled water. This mixture was placed in the Negative Control beaker. Next, 100 mL of distilled water were added to the beaker, and then, 200 mL of sludge inoculum were added to give a final volume of 500 mL. Aeration at 1 L/min was started using a pipet as aeration device. At time "12 min" (12 minutes is an arbitrary, but convenient, interval) the above was repeated for the first test chemical beaker. The process was repeated at 12-minute intervals to give a series of theorectical concentrations of the test substance in the test vessels. The reference substance was tested on each batch of microbial inoculum in the same way, except that 100 mL of the reference substance stock solution were placed in the fifth beaker instead of 100 mL of water. The process was repeated with different volumes of the reference substance stock solution (diluted with water to 100 mL) to provide a total of 3 reference substance concentrations. Finally, a second Negative Control was prepared. Oxygen consumption was measured and recorded after an aeration time of 3 hours at 20 ± 2°C. A standard BOD measuring bottle was filled from the contents of the first beaker and the respiration rate was measured over a period of up to 10 minutes using a dissolved oxygen meter hooked up to a recording chart. This determination was repeated on the contents of each vessel at 12-minute intervals so that the exposure time in each beaker was three hours.
Reference substance (positive control):

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
The top dose tested, 1000 mg/L, resulted in 4.1% inhibition which is not considered significant due to the experimental error of the test.
Results with reference substance (positive control):
The two negative control respiration rates were within 15% of each other and the 3-hour EC50 of 3,5-dichlorophenol was in the range 5-30 mg/L. These results meet the criteria for a valid test.

Applicant's summary and conclusion

Validity criteria fulfilled:
Executive summary:

A 3-hour activated sludge respiration inhibition test (OECD 209) was performed using activated sludge from a domestic wastewater treatment plant. The sludge microorganisms were exposed to five concentrations of trans-DMCD. The respiration rate, expressed as oxygen consumption by the microbes in mg O2 per liter per hour, was measured under defined conditions following the 3-hour exposure period. Inhibition values were calculated by comparing test respiration rates to negative control rates. A graph of percent inhibition versus concentration was plotted. An NOEC value of 1000 mg/L, the highest dose tested, was obtained for trans-DMCD .