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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-05-6 to 2010-07-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure:
Fresh samples of activated sludge are withdrawn on June 22th, 2010 from the sewage treatment plant Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany, which is mainly fed with municipal wastewater. On return to the laboratory the sludge was washed three times with isotonic solution which is in accordance with the guideline.
- Pretreatment: Subsequently, mixed liquor suspended solids level of the sludge was
adjusted on about 4.0 g/L. The solution was kept aerated at 20 ± 2 °C until test start.
- Initial biomass concentration: The concentration used in the test was 1.61 g dry mass/litre.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
18-22 °C
pH:
7.2
Nominal and measured concentrations:
Nominal concentrations of test substance: 95.3, 171.5, 308.6, 555.6, and 1,000 mg/l.
Nominal concentrations of the reference substance: 4, 12, and 36 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flask
- Material, size, headspace, fill volume: 1 liter volume glass flasks. Fill volume of 500 ml
- Aeration: Contents were aerated during incubation (0.75 l/min)
- No. of vessels per concentration (replicates):1
- No. of vessels per control (replicates):2 and 1 control withouth inoculum

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized water free from inhibitory conentrations of toxic substances was used.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration rates were measured and recorded over a period of 10 minutes.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 95.3, 171.5, 308.6, 555.6, and 1,000 mg/l.
- Results used to determine the conditions for the definitive study: Results from range finding study indicated that the EC50 is somewhere in the range of 500-600 mg/l.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
564 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL = 364-887 mg/l
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
308.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
555.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The EC50 of 3,5-dichlorophenol for respiration inhibition was found to be within the range of 5.0 and 30.0 mg/l.
Reported statistics and error estimates:
Numerical values in this report are frequently rounded to a smaller degree of precision (number of digits) than were used in the actual calculation. Minor differences in results obtained from calculations with such rounded values in comparison to those obtained with higher precision values are possible. They are, however, well within the limits of the experimental accuracy and thus of no practical concern.
- Calculation of the respiration rates and the percent inhibition of these rates was performed according to the guideline and listed in a table.
- The test results were statistically analyzed using the Probit-analysis of the computer program (ToxRat) assuming log-normal distribution of the values.

Table 1: Inhibition (%) of the respiration rate by the test item

Test Concentration

95.3 mg/l

171.5 mg/l

308.6 mg/l

555.6 mg/l

1000 mg/l

4.4%

12.6%

8.1%

47.2%

94.2%

Validity criteria fulfilled:
yes
Remarks:
The validity crieria of the test were fulfilled. The pH of the test medium remained in the range of 6.0 -8.0, the incubation temperature remained in the range of 18 -22 °C, the EC50 of the reference substance (3,5 -dichlorphenol) was in the the accepted r
Conclusions:
In a test according to EU Method C.11, the 3h EC50 of the test substance is 564 mg/l with a 95% confidence limit of 364 -887 mg/l. The NOEC was found to be 308.6 mg/l and the LOEC 555.6 mg/l.
Executive summary:

In a test according to EU Method C.11, the 3h EC50 of the test substance is 564 mg/l with a 95% confidence limit of 364 -887 mg/l. The NOEC was found to be 308.6 mg/l and the LOEC 555.6 mg/l.

Description of key information

The 3h EC50 of the test substance is 564 mg/l. The NOEC was found to be 308.6 mg/l and the LOEC 555.6 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:
564 mg/L

Additional information

In a test according to EU Method C.11, the 3h EC50 of the test substance is 564 mg/l with a 95% confidence limit of 364 -887 mg/l. The NOEC was found to be 308.6 mg/l and the LOEC 555.6 mg/l.