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Administrative data

activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between Sep 18th, 1992, and Dec. 14th, 1992.
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study; well-documented study report.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)

Test material

Details on test material:
- Physical state: liquid at room temperature
- Analytical purity: not provided.
- Lot/batch No.: 119860-006
- Stability under test conditions: stable
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Sampling and analysis

Analytical monitoring:
Details on sampling:
No data available.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Test Solutions:
As the test material was considered insoluble, no stock solution was prepared and the material was added directly to each test vessel by way of glass microscope cover slips. The study design was comprised of a single nominal exposure concentration (1000 ppm) test in duplicate, a duplicate control group, and an assessment of the sensitivity of the inoculum used in the test to a reference toxicant: 3,5-dichlorophenol.
Reference Solutions:
3,5-dichlorophenol (97% purity) stock solution was prepared as follows:
1.5g of 3,5-dichlorophenol was dissolved in 10 mL of a 1N NaOH solution which was subsequently diluted to 30 mL with WCC laboratory reagent grade water, placed on a magnetic stirrer and stirred with a teflon-coated stir bar, followed by the addition of 8 mL of 1N H2SO4 (ACS grade) . The solution was then brought to a total volume of 1 L with WCZ laboratory reagent grade water for a nominal concentration of 500 ppm.
In the test, the nominal reference toxicant concentrations were diluted to 10, 20, and 40 ppm.
- Eluate: not applicable.
- Differential loading: not applicable.
- Controls: The control group was maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle was used.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data available.

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: no.
- Method of cultivation: The activated sludge organisms were fed a synthetic sewage feed at a rate of 50 ml per liter.
- Preparation of inoculum for exposure: Activated sludge with and its associated aerobic organisms was obtained from the Cottonwood Subdivision Wastewater Treatment Plant located in Franklin, Tennessee. This wastewater treatment plant serves only the residential subdivision and its community pool.
One gallon of activated sludge was collected by the plant operator, picked up on the day of collection by WCC personnel and transported to the testing laboratory. Immediately upon arrival, the sludge was aerated with low-pressure, oil-free air. The activated sludge organisms were fed a synthetic sewage feed at a rate of 50 ml per liter.
Triplicate 4-ml samples of the mixed sludge were dried at 100 oC 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.
- Pretreatment: no.
- Initial biomass concentration: 4 g/L.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
Not applicable.

Test conditions

No data available.
Test temperature:
No data available.
Dissolved oxygen:
No dissolved oxygen data was available, but oxygen consumptions after various treatments were available and showed in the Result section.
Not applicable.
Nominal and measured concentrations:
Test material nominal concentration: 1000 ppm. Reference substance: 10, 20, and 40 ppm.
Details on test conditions:
-Test Vessels: the sludge/microbial inoculums were exposed to the test material in a 1-L glass serum bottles. Total volume within the test vessel for each test condition was 500 ml. Low pressure oil free air was delivered to each test vessels using a glass pipet. Air lines were connected to a pressurized manifold, where air pressure was controlled. All of the test vessels were held in a water-jacketed incubator in the dark during the 3 hour exposure period. The incubator was maintained at 20 oC.
-Number of Vessels and Concentrations (Range Finding Study): No data available.
-Number of Vessels and Concentrations (Definitive Study): duplicate.
Controls – duplicate
Reference – one each at 10, 20 and 40 ppm 3,5-dichlorophenol
Test Material –1000 ppm, nominal.

-Test Water: the dilution water for this test was from the City of Franklin, Tennessee, water supply. The water was softened and dechlorinated prior to use. Dechlorination was confirmed with the DPD colorimetric method.

- Adjustment of pH: No data available.
- Photoperiod: tested in the dark.
- Light intensity: not applicable.

During testing, each mixture was aerated with oil-free compressed air. After the 3 hour contact time, 300 mL of the sludge mixture was removed from the vessels and transferred into a BOD bottle for measurement of dissolved oxygen.

-Measuring Vessel/Oxygen Meter: BOD bottles contained a teflon- coated magnetic stir bar. The bottle was capped with the BOD bottle oxygen probe to eliminate air space and placed on a magnetic stirring plate. The sludge mixture was stirred at a constant rate and the consumption of oxygen over time was recorded on a strip chart recorder. The test material, control or reference toxicity concentration was written on the strip chart paper. The oxygen readings were recorded for ten minutes until a linear trace covering a sufficient range of oxygen concentrations was obtained.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Consumption of oxygen over time was recorded after 3 hours exposure.

- Range finding study: No data available.
- Results used to determine the conditions for the definitive study: No data available.
Reference substance (positive control):

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
ca. 2 433 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no observation.
- Effect concentrations exceeding solubility of substance in test medium: no data available.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- Relevant effect levels: 3-Hour EC50 value was determined to be 29 mg/L which was within the acceptable range of 5 to 30 mg/L.
- Other:
Reported statistics and error estimates:
Analysis of Consumption Data:
Only the linear section on the chart paper representing the oxygen consumption was used to evaluate the results. The chart readings were transformed into mg of O2 per liter consumed per hour (mg O2/L*hr). To calculate the two control respiration rates for each test material concentrations:
1 ─ 2 *100* RS/(Rc1+Rc2),
RS = oxygen consumption rate of the inoculum exposed to the test material;
Rc1 = oxygen consumption rate, control 1;
Rc2 = oxygen consumption rate, control 2.
In addition to the assessment of the test material, the reference toxicant results were similarly evaluated. The reference toxicant data were subject to probit analysis to calculate the EC50. To ensure quality control the two control consumption rates needed to be within 15% of each other and the 3 h EC 50 of the reference toxicant needed to be between 5 and 30 mg/L. Both of these conditions were met for the test reported herein.

Any other information on results incl. tables

Only one concentration of test substance was used. The correlation between the test substance concentration and the respiration rate was assumed to be linear and an extrapolated EC50 value was calculated.

The oxygen consumption rates of the 2 replicate controls were within 15% of each other and the EC50 value of the reference substance was between 5 – 30 mg/L meeting the guideline requirements for a valid test.

Table: Results:


Respiration Rate

% Inhibition

3-Hour EC50


98.4 (mean)



10 ppm



20 ppm



29 mg/L

40 ppm



Test Substance (1000 ppm)

77.9 (mean)


2,433 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
The test substance does not show any significant effect on the oxygen consumption of activated sludge. 3 h EC 50 is 2433 mg/L.
Executive summary:

The potential impact of test material 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 OECD209. The test duration was a three-hour exposure period to the test material followed by up to ten minutes for the measurement of oxygen consumption. The study design was comprised of a single nominal exposure concentrations1000 ppm, tested in duplicate; a duplicate 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 material passed the quality control criteria for an acceptable test. The EC50calculated for the reference toxicant was 29 rng/L, within the acceptable range of 5 to 30 mg/L. The two control replicates produced oxygen consumption rates within the required 15 % of each other, 90.0 and 106.7 mg O2/L*hr, which is also required for the validity of the test.

The oxygen consumption rates observed for the activated sludge microbe exposure to the test material was 57.6 and 98.2 mg O2/L*hr respectively. These values translate into inhibitions of 41.1% and 0% as compared to control. Statistical analysis of the results reveal that there does not exist a statistically significant difference between the oxygen consumption rates of the microbes exposed to the test material and the non-exposed control microbes. Given an average inhibition of 20.6% and assuming that the relationship between concentration and effect is linear, an extrapolated EC 50 value equals 2433 mg/L.