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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline, GLP study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
a stock solution (1000 mg of test substance a.i - per litre) was prepared 1 hour (approx.) before the start of the exposure, dissolving the test substance into dilution water ; pH of this stock solution was not adjusted.
Test organisms (species):
activated sludge
Details on inoculum:
• the sludge was used on the day of collection.
• the sludge was mixed during 1 minute in order to destroy the coarse particles.
• the concentration of suspended solids was evaluated from a small amount of sludge.
• the sludge was centrifuged and the suspended solids obtained were washed before to be resuspended for the preparation of the inoculum (centrifuged 3 times with demineralized water containing synthetic sewage for 5 minutes at 2000 r.p.m., with discarding of the supernatants)
• the suspended solids were then re-suspended into synthetic sewage so as to obtain a nominal concentration of suspended solids of approximately 4 grammes per litre in the inoculum
• this inoculum was kept aerated and at thermoregulated temperature (20°C ± 2°C) until its using for the preparation of the media.

Source: activated sludge from a sewage treatment plant treating predominantly domestic sewage (La Fouillouse (Loire) - France).
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
ranged from 20.0 °c to 24.1 °c
pH of the test media at the start of the study: ranged from 7.23 to 7.42
pH of the test media at the end of the study: ranged from 7.78 to 8.09
Dissolved oxygen:
Aeration: continuous aeration (air-flow : a rate of 10 to 20 bubbles per second approximately) and continuous magnetic stirring.
Nominal and measured concentrations:
12, 27, 61, 135, and 300 mg
Details on test conditions:
Sludge nominal concentration: 1,600 mg of suspended solids per litre of control, reference and test media.
Test volumes: 500 mL per flask
Containers: 1,000 mL beakers with covers

Measurements of the dissolved oxygen concentration over a period of 10 minutes (or until the concentration reaches 1 mg/l) after approximately 3 hours exposure, in 300 ml samples from each container.
These measurements were performed in appropriate BOD-flasks, with O2-electrode.
Reference substance (positive control):
3 h
Dose descriptor:
Effect conc.:
64 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: O2 consumption
Validity criteria fulfilled:
Taking into account the results obtained under the experimental conditions employed, these ones may be expressed as follows:
EC 50-3 hours: 64mg/L (a.i.)
Executive summary:

Compliance with OECD Guideline No. 209 (1984) and EEC Directive 88/302.L133-Part C (1988).

METHOD: Micro-organisms from activated sludge (1,600 mg of suspended solids per litre of medium) were exposed to a range of concentrations of test substance for 3 hours 12, 27, 61, 135, and 300 mg test substance (active matter) per litre of test medium,

- Two control media (without test substance) were tested, one at the beginning of the study, the other one at the end of the study.

- The dissolved oxygen concentration was measured in 300 mL samples from each medium after approximately 3 hours of exposure.

- A reference substance, 3,5 - dichiorophenol, was tested under the same conditions in order to control the sensitivity of the activated sludge (3 concentrations).

- The criteria of quality for the study, as notified in the OECD and EEC standards and relative to the level of oxygen consumption in the two control media and to the toxicity of 3,5 - dichlorophenol, were respected.

RESULTS: Taking into account the results obtained under the experimental conditions employed, these ones may be expressed as follows:

EC 50-3 hours: 64mg/L(active mater)

Description of key information

Taking into account the results obtained under the experimental conditions employed, these ones may be expressed as follows:
EC 50-3 hours: 64mg/L (a.i.)

Key value for chemical safety assessment

EC50 for microorganisms:
64 mg/L

Additional information