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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Aug 2016
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:
GLP compliance:
yes (incl. QA statement)
Staatliches Gewerbeaufsichtsamt, Hildesheim, Germany (01.02.2016)
Details on test solutions:
- Method: 1000 mg/L were pipetted directly into Erlenmeyer flasks
- Controls: test medium without test or reference item
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: municipal sewage treatment plant, Hildesheim, Germany
- Pretreatment and preparation of inoculum for exposure: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10%. The sludge was used within 24 h after sampling.
- Dry solids concentration of sludge: 2.71 g/L
- Dry solids concentration in test solution: 1.36 g/L
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
19.1 °C
activated sludge: 7.4
synthetic waste water: 7.27
Nominal and measured concentrations:
nominal: control, 1000 mg/L
Details on test conditions:
- Test vessel: Erlenmeyer flasks, DIN 12380
- Type: closed with an active carbon trap that allows air exchange
- Size, headspace, fill volume: Test vessel volume: 1000 mL; Test volume: 500 mL; Headspace: 500 mL
- Aeration: shaking aeration to keep the dissolved oxygen concentration above 60 - 70% saturation and to maintain the sludge flakes in suspension. The flasks were closed with an active
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 1.36 g/L dry solids concentration
- Nutrients provided for bacteria: synthetic waste water according to OECD guideline 209
- Nitrification inhibitor used: N-allylthiourea (non-GLP preliminary range finding test)

- Source/preparation of dilution water: demineralised water
- pH: measured prior to test start in activated sludge as well as in the synthetic waste water
- temperature: Room temperature was recorded continuously. Temperature in the measuring cell was determined once during the measurement.

- Adjustment of pH: no
- Details on termination of incubation: After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 5 min on a flat bed recorder.

Oxygen concentration: at the end of 3 h exposure, during at least 5 min

- Spacing factor for test concentrations: limit test at limit concentration of 1000 mg/L (nominal)
- Range-finding study: yes
- Test concentrations range-finding study: 10, 100, and 1000 mg/L, 2 replicates each and addtionally 2 replicates with 1000 mg/L to study the inhibition of the nitrification using Allylthiourea ATU
- Results used to determine the conditions for the definitive study: The heterotrophic respiration as well as the nitrification were not inhibited. Therefore, the definitive study was carried out with the limit concentration of 1000 mg/L.
Reference substance (positive control):
Copper (II) sulphate pentahydrate p.a.
3 h
Dose descriptor:
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: yes, test substance is not miscible in water
- Blank controls oxygen uptake rate: 17.2 mg O2/g*h
- Coefficient of variation of oxygen uptake rate in control replicates: 3.5%
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h) = 102 mg/L, 95% Confidence Interval = 98.4 - 106 mgL
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculation. When running a One Way Analysis of Variance a Normality test and an Equal Variance test werd done first. The p-value for both tests was 0.05 and the α-value 0.05.
The EC-value of the reference item was calculated by linear regression using software GraphPad Prism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.


The validity criteria for the present study are summarized in Table 1. The mean respiration rate of the blank controls was slightly below the validity criterion set out by the guideline but was not considered to be of concern for the quality and validity of the present study. Variations in oxygen uptake by activated sludge are common and influenced by seasonal fluctuations, sewage flow etc. Moreover, the EC50 value of the reference item was in the required range. Therefore, the outcome of this study was not expected to be affected by this minor deviation (Table 1).

Table 1: Validity criteria for OECD 209.

Criterion from the guideline


Validity criterion fulfilled

The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

The mean specific oxygen uptake rate of the control replicates was 17.2 mg O2/g*h

The slightly lower oxygen uptake rate was considered to reflect common variation in uptake rates and was not expected to impact the quality and integrity of the study. Moreover, the EC50 value of the reference item was in the required range.

The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.

The coefficient of variation of the oxygen uptake rates in the control replicates was 3.5%.




In the preliminary test, no nitrification inhibition was observed at 1000 mg/L. Therefore, nitrification inhibition was not evaluated in the definitive test.

In the definitive test, the test substance did not show any inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L.

Validity criteria fulfilled:
The mean respiration rate of the blank controls was slightly below the validity criterion. For further details please refer to "Any other information on results incl. tables".
The test resulted in a NOEC (3 h) of ≥ 1000 mg/L (nominal, OECD 209, activated sludge), indicating that the substance does not inhibit the respiration of activated sludge of municipal sewage treatment plants up to the saturation level.

Description of key information

NOEC (3 h) ≥ 1000 mg/L (nominal, OECD 209, municipal sewage treatment plant)

Key value for chemical safety assessment

Additional information

One study is available, in which the toxicity of test item to activated sludge microorganisms was assessed according to OECD 209 and GLP.

In the static respiration inhibition test, a limit test item concentration of 1000 mg/L was inoculated with 3.0 g/L ± 10% mg/L activated sludge of a municipal treatment plant for 3 h. Control and reference replicates were run in parallel. The limit concentration was previously established in a preliminary range-finding study, which also indicated no inhibition of nitrification by the test substance. After 3 h contact time the respiration rates of the control, reference and test item replicates were measured during at least 5 min and statistically compared.

The mean inhibition of respiration by the test item was 4% after 3 h of exposure, indicating no inhibitory effects of the test item on activated sludge respiration at the limit concentration. The reference item confirmed the suitability of the inoculum. The NOEC (3 h) was ≥ 1000 mg/L (nominal).