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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19th May 1993
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
Analytical monitoring:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A single stock solution was prepared by dissolving 25 mg in 1000 mL of acetone. The final concentration of the stock solution was 25.0 mg/L. To the test flasks, 2.5, 7.5, 22.5, 67.5 and 202.5 mL of the stock solution was added for the test to achieve test concentrations of 125, 375, 1125, 3375 and 10125 mg/L (nominal), respectively. The abiotic control recieved 202.5 mL of the 24 mg/mL stock in acetone. The vehicle blank was prepared with 202.5 mL of acetone. All flasks were placed in a warm water bath uder a hood, and a stream of nitrogen was fed to each flask with a distributing apparatus made of rummer tubing, T-connectors, and Pasteur pipets. The flasks were removed for use when no more acetone was visible. Each flask was checked for the odour of residual acetone. No residual acetone was detected in any of the test flasks.
Test organisms (species):
activated sludge
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge was obtained from the Columbia Wastewater Treatment Plant in Columbia, Missouri on the 17th May 1993. The plant receives predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sludge was washed three times by centrifuging and resuspending in well water. The mixed liquor solids (MLSS) level was determined by filtering three well mixed 2 mL aliquots of the sludge through glass-fibre filters, followed by drying in a drying oven overnight. The increased weight of the filters was used to determine the suspended solids level.
The pH of the culture was measured using a pH pen. The pH was 8.5, outside of the acceptable range of the protocol. Phosphate salts were added to the water to adjust the MLSS and the pH. 14.9 g KH2PO4 and 17.1 g K2HPO4. These weights result in a final concentration of 3.39 g/L KH2PO4 and 3.89 g/L K2HPO4 in the final volume of sludge culture which was 4.4 L. The pH of the sludge after addition of the phosphate buffer was 7.1.
The MLSS was adjusted to approximately 4 g/L by the addition of phosphate buffered well water. The sludge was maintained in aerated vessels and synthetic sewage feed (50 mL/L/day) was added to the sludge until use. Synthetic sewage feedstock was produced using the following:
16.0 g Peptone
11.0 g Beef extract
3.0 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g KH2PO4
Added to 1 L of well water
- Initial biomass concentration: 4 g/L
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
10 minutes
Test temperature:
19-21 °C
Initial 7.6-7.9
Final: 8.0-8.4
Nominal and measured concentrations:
125, 375, 1125, 3375 and 10125 mg/L (nominal)
Details on test conditions:
- Test vessel: 1 L Erlenmeyer flasks
- Material, size, headspace, fill volume: 500 mL
- Aeration: The sludge was aerated prior to use.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 1
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 4 g/L MLSS
- Nutrients provided for bacteria: Synthetic feed
- Source/preparation of dilution water: Well water
16 mL of synthetic feed was diluted to 300 mL with well water in an Erlenmeyer flask. 200 ml of the activated sludge were added. This was the 1st control. Contact flask preparation was performed at 15 minute intervals to allow 10 minutes for respiration at the end of the 3 hour contact period. The five test flasks were prepared with the test material and treated in the same manner as the control flasks. Three flasks were dosed with the reference compound stock solution (0.5037 mg/L) to achieve 10, 20 and 40 mg/L concentrations. An addition flask was prepared at a concentration of 10125 mg/L of test material with no active sludge. This served as an abiotic control. A vehicle control flask was prepared using the same method.
- Adjustment of pH: the pH was adjusted as using a phosphate buffer
- Details on termination of incubation: After a 3 hour contact period, the contents of the flask from control 1 were poured into a BOD bottle and the respiration rate was measured for 10 minutes. The respiration rate determination was conducted for each flask after a contact time of 3 hours.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The temperature was measured through using a circular chart recorded which is part of the environmental control chamber. The temperature remained between 19-21 °C during the study.
The respiration rate was plotted by the recorder trace as mg O2/l/minute over a 10 minute interval. These values were multiplied by 60 to obtain mg O2/L/hr. To calculate the percent inhibition at a particular concentration, the respiration rate was expressed as a percentage of the average control rates calculated using the following:
% inhibition = (1 – ((2Rs)/(Rc1 + Rc2))) x 100
Rs = Respiration rate at given concentration
Rc1 = Respiration rate of control 1
Rc2 = Respiration rate of control 2
A median effective concentration (EC50) calculation and statistical analysis for the reference compound were performed by employing a computerised EC50 program. This program calculates the EC50 and it’s 95 % confidence limit using binomial, moving average, and probit tests.
A graph of percent inhibition versus reference compound concentration was plotted. An EC50 value was determined for the reference compound by regression analysis of the data points.
Reference substance (positive control):
A stock was prepared by dissolving 0.5193 mg in 10 mL 1 N NaOH solution. This was diluted to 30 mL using reagent water and 1 NH2SO4 to incipient precipitation. This was then diluted with 1000 mL of reagent water to give 0.5037 mg/mL.
Key result
3 h
Dose descriptor:
Effect conc.:
> 10 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: estimated
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: A resdiue of the test material was found at the bottom of the flasks of all test concentrations except 125 mg/L. In addition, a heavy foaming was observed throughout the 3 hour contact period in all of the flasks containing the test material (this was visibly less in the 125 mg/L flask). The foam in all of the flasks containing the test substance except the 125 mg/L flasks was such that it was forced out of the test system via the exhaust tube of the flask
- Effect concentrations exceeding solubility of substance in test medium: The test substance was not soluble in water. This may have limited the concentrations of test substance in the test solutions.
- Blank controls oxygen uptake rate: No significant oxygen was observed in the abiotic control.
Results with reference substance (positive control):
- Results with reference substance valid?
- Relevant effect levels: EC50 12 mg/L (8-15 mg/L 95 % CI)
The EC50 value for the reference material was within 5-30 mg/L range.
Reported statistics and error estimates:
Analysis of the results showed that the individual respiration rates of the two control flasks were within 15 % of the average respiration rate of the control flasks.
An EC50 value for the test material could not be estimated over the concentrations tested because no trend toward increasing inhibition was observed. The results did indicate that the test substance does inhibit the respiration of the sludge to a variable extent at nominal concentrations up to 10125 mg/L. It was not possible to estimate a no observed effect concentration (< 10 % inhibition) from the data because no trend was observed over the full range of concentrations tested. An EC50 for the test material was estimated to be greater than 10000 mg/L.

Table 1: Results


Respiration rate (mg O2/L/hr)

% Inhibition

Control 1


Control 2




Average ± 15 %

(33.1, 44.7)

Vehicle blank



Test Material

125 mg/L



375 mg/L



1125 mg/L



3375 mg/L



10125 mg/L



Abiotic control 10125 mg/L



Reference substance 3,5-dichlorophenol

10 mg/L



20 mg/L



40 mg/L




Validity criteria fulfilled:
Due to foaming and the insolubility of the test material, it was not possible to determine a no observed effect concentration due to a lack of trend over all the concentrations tested, however it was observed that some inhibition occurred during the study. An EC50 of 10000 mg/L was estimated.
Executive summary:

The toxicity of the test material to microorganisms of the test material was determined in an activated sludge respiration inhibition assay conducted in accordance with OECD 209.

Due to foaming and the insolubility of the test material, it was not possible to determine a no observed effect concentration due to a lack of trend over all the concentrations tested, however it was observed that some inhibition occurred during the study. An EC50 of 10000 mg/L was estimated.

Description of key information

The estimated EC50 of the test material was determined to be >10000 mg/L in the key study conducted in accordance with OECD 209.

Key value for chemical safety assessment

EC50 for microorganisms:
10 000 mg/L

Additional information