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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 Sep 1999 - 10 Sep 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Deviations:
yes
Remarks:
Less inoculum was used than as specified in OECD 209 (1984). However, as the validity criteria were met, this does not influence the results of the study.
Qualifier:
according to
Guideline:
other:
Version / remarks:
Procedure 'Amtsblatt der EG L 133 Teil C: Biologische Abbaubarkeit: Prüfung der Atmungshemmung' was used, which corresponds largely to OECD 209.
Deviations:
no
Principles of method if other than guideline:
Procedure 'Amtsblatt der EG L 133 Teil C: Biologische Abbaubarkeit: Prüfung der Atmungshemmung' was used, which corresponds largely to OECD 209.
GLP compliance:
yes
Specific details on test material used for the study:
Purity 98.1%
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct application of test substance to test vessel
- Differential loading: 560, 1000, 1800, 3200, 5600 mg/L
- Controls: Control without test item, abiotic control (no inoculum, 10 000 mg/L test item)
- The weighed test item was stirred overnight in ~130 ml deionized water.
- Composition of synthetic medium: not specified
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: activated sludge of a sewage treatment plant which is operated with municipal sewage (Wupperverband)
- Pretreatment: no pre-treatment
- Initial biomass concentration: 320 mg/L, which is lower than what OECD209 (1984) prescribes (i.e. 1.6 g/L). Due to the strong oxygen depletion of the used activated sludge, a lower biomass concentration was used.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
pH:
7.2-7.9
Dissolved oxygen:
Test solution start: 5.9-9.4 mg/L
Test solution end: 2.8-9.4 mg/L
Nominal and measured concentrations:
Nominal: 560, 1000, 1800, 3200, 5600 mg/L (abiotic control: 10 000 mg/L). Nominal concentrations are not analytically confirmed, thus effect concentrations are based on nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: not specified
- Aeration: constant aeration
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 320 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: not specified
- Light intensity: not specified
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorphenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
2 170 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
817 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Blank controls oxygen uptake rate: 26.6-29.1 mg/L/h, average 27.9 mg/L/h
- Coefficient of variation of oxygen uptake rate in control replicates: not specified in report, calculated based on individual respiration rates of controls: 6.3%. This meets the validity criterium, the respiration rates of the two controls are within 15% of each other.
Results with reference substance (positive control):
- Results with reference substance valid? Not specified. Inhibition lowest test concentration (2.5 mg/L): 0.0%, highest concentration (40 mg/L): 85.7%.
- Relevant effect levels: EC50: 13 mg/L, which is within the accepted range of 5-30 mg/L.
Reported statistics and error estimates:
Effect level determined using Probit analysis

Test item concentration (mg/L)

Respiration rate test item (mg/L/h)

Abiotic O2 depletion (mg/L/h)

Respiration rate abiotic O2 depletion (mg/L/h)

Inhibition (%)

560

26.6

0.0*

26.6

4.7

1000

24.0

0.0*

24.0

14.0

1800

16.8

0.0*

16.8

39.8

3200

8.3

0.0*

8.3

70.3

5600

3.0

0.0*

3.0

89.2

Control average

27.9

 

Control vessel 1

29.1

Control vessel 2

26.6

*: The abiotic O2 depletion was tested at test item concentration of 10000 mg/L. Because there was no abiotic O2 depletion found in this study, also lower concentrations of test item will not lead to O2 depletion (derived values).

Validity criteria fulfilled:
yes
Remarks:
Control respiration rates are within 15% of each other (i.e. 6.3%) and the EC50 of the reference substance is between 5-30 mg/L (i.e. 13 mg/L).
Conclusions:
Based on the measurements, an EC50 of 2170 mg/L was found for N,N-Bis-(hydroxyethyl)-m-toluidin in an activated sludge respiration inhibition test.
Executive summary:

N,N-Bis-(hydroxyethyl)-m-toluidin was tested in an activated sludge respiration inhibition test. The EC50 value for respiration inhibition was determined to be 2170 mg/L, the EC10 was 817 mg/L.

A method which corresponds closely to OECD 209 (1984) was used. A clear dose-response curve was found for the test item. The activated sludge showed good performance in the reference test and thus was acceptable for use. The test was performed under GLP and subject to a Quality Assurance check. Based on these reasons, the reliability of the study is evaluated as Klimisch Score 1, comparable to guideline study.

Description of key information

N,N-Bis-(hydroxyethyl)-m-toluidin was tested in an activated sludge respiration inhibition test. The EC50 value for respiration inhibition was determined to be 2170 mg/L, EC10 was 817 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
2 170 mg/L
EC10 or NOEC for microorganisms:
817 mg/L

Additional information

A method which corresponds closely to OECD 209 (1984) was used. A clear dose-response curve was found for the test item. The activated sludge showed good performance in the reference test and thus was acceptable for use. The test was performed under GLP and subject to a Quality Assurance check. Based on these reasons, the reliability of the study is evaluated as Klimisch Score 1, comparable to guideline study.