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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-01-22 to 2018-02-01
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
according to guideline
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Source and lot/batch No.of test material: 20141015
- Expiration date of the lot/batch: 2019-01-31
- Purity test date: 2017-01-31
- Purity: 99.35%

- Storage condition of test material: Room temperature. Tightly closed container in a dry and well-ventilated place
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data
Analytical monitoring:
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and is reflected in the GLP compliance statement.
Details on sampling:
- Sampling method: For measurement of the respiration rate a well-mixed sample of each incubation mixture was poured into a 250 mL glass BOD bottle after three hours incubation time, and was not further aerated. Subsequently, the dissolved oxygen (DO) concentration was measured with a self-stirring DO sensor attached to an oxygen meter and was continuously recorded for a period of up to 10 minutes or until the oxygen concentration fell below 2 mg/L.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the results of the range-finding test, no appropriate stock solution (e.g. of 5 g/L) could be prepared due to the low water solubility of the test item. Therefore, the following test item amounts were weighed by means of an analytical balance and transferred to the designated test vessels with 234 mL deionized water:

Nominal test item concentration Amount of test item [mg per vessel]
[mg/L] Replicate 1 Replicate 2 Replicate 3
10 5.04 5.01 5.05
32 16.04 16.03 16.13
100 50.06 50.04 50.11
320 160.1 160.2 160.1
1000 500.2 500.5 500.3

The test item was mixed into the deionized water by intense stirring for approximately 5 minutes at room temperature to finely disperse the test item. No emulsifiers or solvents were used. After stirring, the appearance of the test media was evaluated and the pH measured. The pH of the test dispersions was adjusted with a 0.1M NaOH solution from the pH range 6.8-7.0 to the range 7.0-7.2. Then, 16 mL of synthetic sewage feed and 250 mL of the activated sludge inoculum were added.
- Controls: yes, the control group was maintained under identical conditions but not exposed to the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All loading rates were clearly above the water solubility limit of JEFFSOL® AG-1700 under the test conditions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: aerobic activated sewage sludge from the aeration stage of the local wastewater treatment plant, ARA Birs (Birsfelden / Switzerland), which treats predominantly domestic sewage.
- Pretreatment: The aerobic activated sewage sludge was washed three times by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in chlorine free tap water. Aliquots of the homogenized final sludge suspension were weighed, thereafter dried and the dry weight of the suspended solids was determined. During the holding period of one day prior to use, the sludge was fed once with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use. Before use, on the next day, the pH of the activated sludge inoculum was measured to be 7.3.
- Initial biomass concentration: The suspended solids concentration was equal to 3.0 g/L prior to use.

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
After 3 h of exposure, oxygen consumption was recorded for apprimately 10 minutes.
Not reported
Test temperature:
* The pH of the complete incubation mixture was measured in each vessel at the start and end of the incubation period. At start the pH was in the range of 7.2-7.6 and at the end in the range of 7.7-8.0.
* For the test item incubation mixtures the pH was measured also before the addition of synthetic sewage feed and activated sludge. The pH was adjusted at this step from 6.8-7.0 to 7.0-7.2 with a diluted NaOH solution.
Dissolved oxygen:
Just before measurement of the respiration rates, at the end of the incubation period, the dissolved oxygen concentrations were at least 6.1 mg/L
Not reported
Not reported
Nominal and measured concentrations:
Range-Finding Test: 10, 100 and 1000 mg/L
Final test: 10, 32, 100, 320, 1000 mg/L
Details on test conditions:
- Test vessel: 2 L beakers to contain 500 mL of incubation mixture were used.
- Aeration: Magnetic stirrers and followers, covered with inert material, were used to assure the aeration and mixing during incubation.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): The suspended solids concentration was equal to 3.0 g/L prior to use.

- Source/preparation of dilution water: Deionized water, containing less than 1 mg/L DOC (dissolved organic carbon) was used except where chlorine free tap water is specified.

- Adjustment of pH: The pH of the test dispersions was adjusted with a 0.1M NaOH solution from the pH range 6.8-7.0 to the range 7.0-7.2.
- Photoperiod and Light intensity: The test was conducted under normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen uptake rate of each sample was determined from the linear part of the respiration curve normally in the range of 2-7 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes.

- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
Key result
3 h
Dose descriptor:
Effect conc.:
358 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% C.L. 73-not determined due to mathematical reasons mg/L
Key result
3 h
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data
- Blank controls oxygen uptake rate: The specific respiration rates for the two blank control replicates prepared at the beginning of the exposure period were 39 and 35 mg O2 per dry weight of activated sludge per hour, for the two blank controls prepared after the reference item group were 42 and 45 mg O2/gh* and for the two replicates prepared at the end of the test were 43 and 51 mg O2/gh. All six values are higher than the 20 mg O2/gh stipulated in the guidelines.
- Coefficient of variation of oxygen uptake rate in control replicates: The resulting coefficient of variation of oxygen uptake rate in the control replicates has a value of 13 % and is therefore, as stipulated in the guidelines, not more than 30 %.

* The specific respiration rate, Rs, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour [mg/gh]

- EC10 (95 % confidence limits): 96 (n.d. – 213)
- EC20 (95 % confidence limits): 151 (n.d. – 295)
- EC80 (95 % confidence limits): 852 (432 – n.d.)
Results with reference substance (positive control):
The 3 hour EC50 of the reference item 3,5 dichlorophenol (positive control) was calculated to be 5.9 mg/L with 95 % confidence limits from 1.4 to 11 mg/L. The 3 hour EC50 is within the guidelines-recommended range of 2–25 mg/L, confirming the suitability of the activated sludge used.
Reported statistics and error estimates:
The percentage inhibition of the respiration was plotted against logarithm of the test substance concentration using suitable software (ToxRat® Professional).
The NOEC was derived by per-vessel statistics using the Williams t-test (one sided smaller, α = 0.05).
The 3-hour EC10, EC20, EC50 and EC80 values and their 95 % confidence limits were, as far as possible, derived by Probit Analysis.
Validity criteria fulfilled:
An activated sludge respiration inhibition test (according to OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h EC50 = 358 mg/L. In conclusion, the test substance was not toxic to activated sludge at or below a loading rate of 100 mg/L. The results of the study can be considered reliable without restriction.
Executive summary:

The inhibition of the respiration rates for all tested concentrations was in the range of -4.9 to 76 % compared to the mean control. For three lower tested concentration of 10, 32 and 100 mg/L the respiration rate of activated sludge after the incubation period of 3 hours was in the range of the control (i.e. mean values of 0.2, -2.3 and -1.4 %, respectively compared to the control). Therefore, up to and including the concentration of 100 mg/L the test item had no statistically significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours (result of a Williams t-test,one-sided smaller,a = 0.05). At the higher test item concentrations of 320 and 1000 mg/L the test item had an increasing statistically significant inhibitory effect, with mean values of 56 and 75 % inhibition, respectively. A clear concentration-effect relationship on the inhibition of the respiration rate was observed.

Description of key information

The study of Eisner (2018), investigating the toxicity of butyl benzoate to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage. The 3-h EC50 was = 358 mg/L. The 3-h NOEC was 100 mg/L. Up to and including the concentration of 100 mg/L the test item had no statistically significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours.

Key value for chemical safety assessment

EC50 for microorganisms:
358 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information