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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-11-24 - 2008-12-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministry for environment, agriculture and consumerism of North Rhine-Westphalia
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analogue material was used.
Analytical monitoring:
no
Details on sampling:
No details available.
Vehicle:
no
Details on test solutions:
No details are available.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Type: mixed population of aquatic microorganisms (activated sludge)
- Origin: aeration tank of a domestic sewage treatment plant (Municipal STP Cologne - Stammheim)
- Date of collection: 2008-11-24
- Preparation: Sludge was settled and the supernatant was decanted. Centrifugation was performed for 15 min at 4500 rpm and 20 °C. The supernatant was decanted. Approx. 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was first dissolved in synthetic medium and than filled up to a defined end volume with deionised water. Before start of the test 10 mL of the sludge suspension were sucked of to obtain a concentration of activated sludge of 2 g/L (dry weight) suspended solids.
- Storage of sludge: aeration of the activated sludge at 20 °C +/- 2 °C, daily fed with synthetic medium
- pH of the suspension before application: 7.7
- Synthetic sewage feed: A synthetic waste water feed was made by dissolving the following amounts of substance in 1 L of water: 16.0 g peptone, 11.0 g meat extract, 3.0 g urea, 0.7 g NaCl, 0.4 g CaCl2 x 2H2O, 0.2 g MgSO4 x 7H2o, 2.8 g K2HPO4
- pH of the synthetic sewage feed: 7.5 +/- 0.5
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
No post exposure observation period.
Hardness:
No details are available.
Test temperature:
Test item: 18.5 °C (1000 mg/L)
18.7 °C (100 mg/L)
18.8 °C (10 mg/L)
Reference: 18.6 °C (10 and 20 mg/L)
18.7 °C (5 mg/L)
Controls: 18.8°C and 18.9 °C
Physico-chemical oxygen consumption control: 18.8 °C
pH:
Test item: 8.0 (10 and 1000 mg/L)
8.1 (1000 mg/L)
Reference: 8.0 (5 mg/L)
8.1 (10 and 20 mg/L)
Controls: 8.1 (both)
Physico-chemical oxygen consumption control: 7.5
Dissolved oxygen:
Test item: 10 mg/L: start: 6.1 mg O2/L; end: 3.1 mg O2/L
100 mg/L: start: 6.2 mg O2/L; end: 3.0 mg O2/L
1000 mg/L: start: 6.5 mg O2/L; end: 3.0 mg O2/L
Reference: 5 mg/L: start: 6.4 mg O2/L; end: 4.6 mg O2/L
10 mg/L: start: 7.6 mg O2/L; end: 6.3 mg O2/L
20 mg/L: start: 7.6 mg O2/L; end: 7.2 mg O2/L
Control 1: start: 6.5 mg O2/L; end: 3.3 mg O2/L
Control 2: start: 6.4 mg O2/L, end: 3.6 mg O2/L
Physico-chemical oxygen consumption control: start and at the end: 7.8 mg O2/L
Salinity:
No details are available.
Nominal and measured concentrations:
Test item concentrations: 10, 100 and 1000 mg/L
Test item concentration in physico-chemical oxygen consumption control: 1000 mg/L
Concentration of the reference compound: 5, 10 and 20 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type: closed
- Material, size, headspace, fill volume: 300 mL
- Aeration: performed through a glass tube at 50 - 100 L/h with clean oil-free air
- Controls: 2
- Physico-chemical oxygen consumption control: 1

Before use the pH of the activated sludge was measured and adjusted to pH 6 - 8. 8 mL of the synthetic medium and 100 mL of activated sludge were added to the dissolved test item. The mixture was filled up with deionised water to 250 mL and aerated at 20 °C +/- 2°C.

The exposure medium with the reference substance was prepared by adding 8 mL of the synthetic medium, 100 mL of activated sludge and a defined amount of the stock solution to achieve the test concentrations and was filled up to 250 mL with deionised water.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The test item showed 0.0 % respiration inhibition of activated sludge at the highest test item concentration of 1000 mg/L.
Results with reference substance (positive control):
EC50: 9.2 mg/L
(Valid result since the EC50 of the reference substance have to be in the range of 5 - 30 mg/L)
Reported statistics and error estimates:
No statistics and error estimates are reported.
Validity criteria fulfilled:
yes
Remarks:
All validity criteria of the test method were met.
Conclusions:
The study is regarded as a valid guideline study with certificated GLP compliance. No toxic effects against bacteria were observed since no respiratory inhibition was observed.
Executive summary:

The acute toxicity of bis-(2,6 -diisopropylphenyl)carbodiimide to microorganisms was tested according to OECD guideline 209 and EU Method C.11 in a static freshwater test (Neuhahn, 2008). The method assessed the effect of the test item by measuring the respiration rate under defined conditions in the presence of different test concentrations. The study was conducted under certificated GLP compliance. A mixed population of aquatic microorganisms (activated sludge), taken from the aeration tank of a domestic sewage treatment plant (Municipal STP Cologne - Stammheim) was used as inoculum. An incubation period of 3 hours was chosen with aeration through a glass tube at 50 - 100 L/h using clean oil-free air. Two controls without test item were included in the test design, one at the start and the other at the end of the test series. Each batch of activated sludge was checked using 3.5 -Dichlorophenol as reference compound. The physico-chemical oxygen consumption control was carried out, since some substances may consume oxygen by chemical reactivity. Direct weighings were prepared to give the different test item concentrations, which were 10, 100 and 1000 mg/L. 3.5 -Dichlorophenol was used in the concentrations of 5, 10 and 20 mg/L. 1000 mg/L test item was included in the physico-chemical oxygen consumption control. No analytical methods were applied. Analysis of the respiration rates at the end of the test gave the following result: EC50> 1000 mg/L. No respiration inhibition occured at the highest test item concentration. The EC50value of the reference compound was measured to be 9.2 mg/L, thus being in the validity range of 5 - 30 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1988-03-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, with documentation deficiencies
Qualifier:
according to guideline
Guideline:
other: ISO 8192-1986
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analogue material used.
Analytical monitoring:
not specified
Details on sampling:
No details on sampling available.
Vehicle:
no
Details on test solutions:
- Method: direct weigh of the substance
Test organisms (species):
activated sludge
Details on inoculum:
- Activated sludge: 6.0 g TS/L
- Culture medium: according to ISO 8192-1986; Chapter 4.2
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
No post exposure observation period reported.
Hardness:
No details available.
Test temperature:
No details available.
pH:
No details available.
Dissolved oxygen:
No details available.
Salinity:
No details available.
Nominal and measured concentrations:
100, 1000 and 10000 mg/L
Details on test conditions:
- Test vessel: 3 L laboratory vessel (OECD)
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorphenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
>= 10 000 mg/L
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: No further details available.
Details on results:
No further details available.
Results with reference substance (positive control):
74 % inhibition after 3 h.
Reported statistics and error estimates:
No details available.
Validity criteria fulfilled:
yes
Remarks:
Validity criteria of test guideline fulfilled, however, documentation sparsely.
Conclusions:
The study is regarded as a valid guideline study with certificated GLP compliance. No toxic effects against bacteria were observed since almost no respiratory inhibition was observed.
Executive summary:

The acute toxicity of bis-(2,6 -diisopropylphenyl)carbodiimide to microorganisms was tested according to OECD Guideline 209 in a static freshwater test (Kanne, 1988). Activated sludge (activation not further specified) was used as inoculum and the incubation period of 3 hours was chosen with substance concentrations of 100, 1000 and 10000 mg/L. 3,5 -Dichlorophenol was used as reference compound and an inhibition of 74 % was reported. The EC50 value is stated to be greater than or equal to 10000 mg/L, with the remark that the substance was water insoluble.

Description of key information

1) Toxicity to microorganisms: EC50 (3h) > 1000 mg/L for activated sludge based on respiration rate (OECD 209, GLP)

2) Toxicity to microorganisms: EC50 (3h) > 10000 mg/L for activated sludge based on respiration rate (OECD 209, GLP)

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The acute toxicity of bis(2,6-diisopropylphenyl)carbodiimide to microorganisms was tested according to OECD Guideline 209 and EU Method C.11 in a static freshwater test (Neuhahn, 2008). The method assessed the effect of the test item by measuring the respiration rate under defined conditions in the presence of different test concentrations. The study was conducted under certificated GLP compliance. A mixed population of aquatic microorganisms (activated sludge), taken from the aeration tank of a domestic sewage treatment plant (Municipal STP Cologne - Stammheim) was used as inoculum. An incubation period of 3 hours was chosen with aeration through a glass tube at 50 - 100 L/h using clean oil-free air. Two controls without test item were included in the test design, one at the start and the other at the end of the test series. Each batch of activated sludge was checked using 3,5-Dichlorophenol as reference compound. The physico-chemical oxygen consumption control was carried out, since some substances may consume oxygen by chemical reactivity. Direct weighing was prepared to give the different test item concentrations, which were 10, 100 and 1000 mg/L. 3,5-Dichlorophenol was used in the concentrations of 5, 10 and 20 mg/L. 1000 mg/L test item was included in the physico-chemical oxygen consumption control. No analytical methods were applied. Analysis of the respiration rates at the end of the test gave the following result: EC50 > 1000 mg/L. No respiration inhibition occurred at the highest test item concentration. The EC50 value of the reference compound was measured to be 9.2 mg/L, thus being in the validity range of 5 - 30 mg/L.

Supporting data are given by a further acute toxicity test according to OECD Guideline 209 (Kanne, 1988). The EC50 value is stated to be greater than or equal to 10,000 mg/L after 3 h incubation time, with the remark that the substance was water insoluble.