Registration Dossier

Administrative data

activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 1993-11-15 to 1994-07-08
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with agreed test protocols following OECD Method 209. Acceptable restriction was that analytical measurements on the test material were not conducted.
Reason / purpose:
reference to same study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Principles of method if other than guideline:
not applicable.
GLP compliance:

Test material

Details on test material:
- Substance type: brown viscous liquid
- Physical state: liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: 133386-002
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: at room temperature in the dark
- Other: none
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: 1, 10, 100, 1000, and 10000 ppm
- Sampling method: The sludge/microbial inoculum was exposed to the test material. To measure the rate of oxygen consumption for the activated sludge after exposure to the test materials a Model 51 Yellow Springs Instruments dissolved oxygen meter was used. The oxygen meter was fitted with a BOD bottle probe, with a standard sensitivity membrane and an external output to a Fisher Recordal strip chart recorder. To measure the consumption of oxygen, a sub-sample of the exposed sludge was placed in a clean BOD bottle, the change in oxygen concentration over time was measured with the YSI meter and probe, and the results were recorded on the strip chart recorder.
- Sample storage conditions before analysis: none

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: As the test material was considered insoluble, no stock solution was prepared and the material concentrations were 1, 10, 100, 1000, 10000. Thus for 1 mg/L, 0.0005 g was added to the test vessel; for 10 mg/L, 0.0050 g; for 100 mg/L 0.05 g; for 1000 mg/L 0.5 g; and for 10000 mg/L 5.0 g. Each vessel was set up at 15 min intervals.
- Eluate: The dilution water for this test was from the City of Franklin, Tennessee, water supply. The water was softened and dechlorinated prior to use.
- Differential loading: see above.
- Controls: After the last test material vessel was prepared, a vessel with no test material was prepared as a test control replicate.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: Activated sludge with associated aerobic organisms were obtained from the Cottonwood Subdivision Wastewater Treatment Plant located in Franklin, Tennessee.
- Method of cultivation: Immediately upon arrival, the sludge was aerated with low-pressure, oil-free air. The activated sludge organisms were fed a synthtic sewage feed at a rate of 50 mL per liter.
- Preparation of inoculum for exposure: Triplicate 4-mL samples of the mixed sludge were dried at 100°C in a Fisherbrand drying oven, until a constant weight was achieved. Based on these results the sludge was diluted to produce a dry weight per unit volume concentration of 4 g/L.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:

Test conditions

no data
Test temperature:
20 +/- 1°C
no data
Dissolved oxygen:
no data
not applicable
Nominal and measured concentrations:
nominal concentrations: 1, 10, 100, 1000, and 10000 mg/L
Details on test conditions:
- Test vessel: flint glass bottles
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 1 L
- Aeration: yes
- No. of organisms per vessel: no data
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: no data

- Adjustment of pH: no data
- Photoperiod: in the dark
- Light intensity: not applicable

- Spacing factor for test concentrations: 10
Reference substance (positive control):
3,5-dichlorophenol (97% pure)

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
> 10 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50(3h) = 16 mg/L, i.e. in the acceptable range of 5-30 mg/L.
- Other: none
Reported statistics and error estimates:

Any other information on results incl. tables

No other information.

Applicant's summary and conclusion

Validity criteria fulfilled:
The respiration rates of the sludge-associated microbes exposed to the 5 nominal concentrations of test item were 36.9, 31.7, 31.8, 29.5, and 20.2 mg O2/Lxhr respectively. The EC50 was calculated to be greater than 10000 mg/L.
Executive summary:

The potential impact of the substance on microbial metabolism, as represented by the consumption of oxygen, was investigated using the "Activated Sludge, Respiration Inhibition Test" as prescribed by OECD (1984) and detailed in WCC Protocol OECD 209 (Expanded Range Procedures). The test was performed on February 15, 1994. The test duration was a 3 -h exposure period to the test material followed by up to 10 minutes for the measurement of oxygen consumption. The study design was comprised of 5 nominal exposure concentrations: 1, 10, 100, 1000, and 10000 ppm; a duplicated control group; and an assessment of the sensitivity of the inoculum used in the test to a reference toxicant (3,5 -dichlorophenol).

The activated sludge respiration test with test item passed the quality control criteria for an acceptable test. The EC50 calculated for the reference toxicant was 16.2 mg/L, within the acceptable range of 5 to 30 mg/L. The 2 control replicates produced oxygen consumption rates within the required 15% of each other, 33.4 and 33.0 mg O2/L x hour.

The respiration rates of the sludge-associated microbes exposed to the 5 nominal concentrations of the substance were 36.9, 31.7, 31.8, 29.5, and 20.2 mg O2/Lxhr respectively. The EC50 was calculated to be greater than 10000 mg/L.