Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP test, according to OECD guideline with acceptable modifications of the test system
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
: closed system used to avoid loss by volatilisation; more diluted cell and substrate concentration; use of parallel test and control reactors; concentration of K2HPO4 was increased to increase the buffering capacity
GLP compliance:
Analytical monitoring:
Test organisms (species):
activated sludge
Details on inoculum:
laboratory culture simulating domestic sewage - Two microorganisms sources were used.
OECD substrate stock solution:
peptone: 16g
beef extract: 11 g
urea: 3g
NaCl: 0.7g
CaCl2.2H2O: 0.4g
MgSO4.7H2O: 0.2g
K2HPO4: 28g
the concentration of K2HPO4 was increased 10-fold from the value recommended by OECD to increase the buffering capacity of the medium during the test.
Test type:
Water media type:
Limit test:
Total exposure duration:
30 min
Test temperature:
24 to 26°C
6.7 to 7.3
Dissolved oxygen:
The dissolved oxygen (DO) concentration was monitored throughout the entire test and rearetion was performed only when the DO concentration dropped below 40% of saturation.
Nominal and measured concentrations:
Not given
First test was performed at water solubility (490 mg/L).
2nd test: no details on on variety of concentration tested
Details on test conditions:
Appropriate quantities of cell suspension and OECD substance solution were added to two 150-mL beakers to give cell and substrate SCOD concentrations of approximately 75 mg/l and 375 mg/L, respectively, after addition of the test compound.
These concentrations were chosen to ensure a linear oxygen uptake rate throughout the test and minimize the need of reaeration. Beakers were stirred with magnetic stirrers and aerated using Pasteur pipets for approximately 7 minutes to ensure oxygen saturation and a stable exogenous oxygen consumption rate. Stirring and aeration were then stopped, the pipets were removed, and the test compound (inhibitor) was added to the test reactor , while an equal quantity of water was added to the control reactor. The polytetrafluoroethylene plugs, containing DO probes, were then inserted so that headspace was eliminated. Stirring was resumed and strip chart recorders were started to record oxygen concentrations. Measurement was continued for 5 minutes and the average utilization rate in each vessel was determined from the strip chart plots.
The test vessel rate was then expressed as a percent of the rate in the control and labeled the immediate response to the test compound. Stirring was continued for 21 additional minutes while DO concentration monitoring continued. If the DO concentration dropped below 40% of saturation it was returned to saturation by inserting the pipet in the reactor (adjusting the plug as needed) and very gently aerating. The pipet was again removed and the plug was readjusted to eliminate any head space. Typically, only one reaeretion was required.
The rate in the test vessel was expressed as percent of the rate in the control and labeled the 30-minute response to test comppound.
Reference substance (positive control):
Key result
30 min
Dose descriptor:
Effect conc.:
140 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Results with reference substance (positive control):
Both sources of biomass gave similar responses with EC50 values ranging from 4 to 10 mg/L

The EC 50 (30 min) of 3,5-dichlorophenol is in the accepted range.

Validity criteria fulfilled:
not specified
validity criteria can not be confirmed as data of oxygen uptake are not available
The EC50 obtained was 140 mg/L, nominal.
Executive summary:

Volskay and Grady (1988).

In a respiration inhibition test according to the OECD test guideline 209, modified in order to avoid the volatilisation of the test compound, activated sludge was exposed to o-chlorobenzene in a closed system during 30 minutes. The highest concentration tested was that of the water solubility. The EC50 obtained was 140 mg/L, nominal.

Description of key information

30min-EC50 (activated sludge) = 140 mg/L (highest tested concentration); According to OECD TG 209; Volskay and Grady (1988)

Key value for chemical safety assessment

EC50 for microorganisms:
140 mg/L

Additional information

Several studies on the toxicity of chlorobenzene to aquatic bacteria were summarised in the assessment of the BUA report 54 (1990). The available tests are mainly studies with one species and not a mixture as stipulated in the OECD test guideline i.e. with activated sludge, furthermore tests were performed following different methods: Microtox tests with fluorescent bacteria, growth inhibition tests with Pseudomonas putida or protozoa according to a German standard method (8 days test period), determination of MIC, nitrification inhibiton tests, inhibition of the dehydrogenase activity test, and finally an anaerobic digestion test.

A comparison between the literature data, obtained with different methods, show differences up to two orders of magnitude. This is due to the different sensitivity of the test organisms and the different methodological approach.

The only test with activated sludge (Volskay and Grady, 1988) was taken as the key study, since it is of good quality and it is the most representative test for this trophic level, furthermore it is the type of test recommended by the OECD. Lower EC50 results were obtained in the microtox studies, however, they must be considered as other informations (see separate entries).