A mixture of: 7,9,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecane-1,16-diylprop-2-enoate; 7,7,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecan-1,16-diylprop-2-enoate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 27, 2008

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Sincethe test substance was hardly soluble in water test concentrations were prepared separately in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).
Two 1 litre test bottles were filled with 250 ml of test substance mixtures with initial loading rates of 200 mg/l. These mixtures were stirred in closed bottles for 26 hours. Subsequently, 16 ml synthetic sewage feed, 200 ml sludge and Milli-RO water up to 500 ml were added resulting in a loading rate of 100 mg/l. This loading rate was tested in duplicate. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (4.3 g/l of sludge, as used for the test). The pH was 6.2 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

After the exposure period, oxygen consumption was measured and recorded for approximately 10 minutes.
Measurement of pH.

Hardness:

not applicable

Test temperature:

18.2 - 19.5°C

pH:

7.2 - 7.5

Dissolved oxygen:

7.3 - 9.0 mg/l

Salinity:

not applicable

Nominal and measured concentrations:

a loading rate of 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: glass, approximately 300 ml oxygen bottles (measuring) and 1 litre test bottles (exposure).
- Aeration: continuously during exposure
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: 200 ml activated sludge


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: none

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The respiration rate from each vessel, in mg O2/l/hr, was calculated from the linear part of the respiration curve, which was generally between 2.5 and 6.5 mg O2/l.
If applicable the EC50 value can be determined.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Slight inhibition of respiration rate of the sludge was recorded at 100 mg test substance per litre. The duplicate measurement confirmed the result of the first measurement (values of 12 and 18% inhibition only were recorded). Therefore, no further testing was needed.
Hence, the EC50 of the test substance exceeded a loading rate of 100 mg/l (based on nominal concentrations).

Results with reference substance (positive control):

Three concentrations were tested: 5.0, 12 and 30 mg/l.
Results:
Conc. 5.0 mg/l - respir. inhibition 40 %
Conc. 12.0 mg/l - respir. inhibition 77 %
Conc. 30.0 mg/l - respir. inhibition 91 %

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the EC50 of the test substance exceeded a loading rate of 100 mg/l (based on nominal concentrations). Slight inhibition of respiration rate of the sludge was recorded at 100 mg test substance per litre. The duplicate measurement confirmed the result of the first measurement (values of 12 and 18% inhibition only were recorded).

Executive summary:

A study was conducted to determine the toxicity of the test substance on activated sludge microorganisms according to OECD Guideline 209, EU Method C.11 and ISO 8192, in compliance with GLP. Two 1 L test bottles were filled with 250 mL of test substance mixtures with initial loading rates of 200 mg/L. These mixtures were stirred in closed bottles for 26 h. Subsequently, 16 mL synthetic sewage feed, 200 mL sludge from a predominantly domestic sewage source and Milli-RO water up to 500 mL were added resulting in a final loading rate of 100 mg/L. This loading rate was tested in duplicate. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-h exposure period. Thereafter, oxygen consumption was measured and recorded for approximately 10 min. Slight inhibition of respiration rate of the sludge microorganisms was observed. The duplicate measurement confirmed the result of the first measurement (12 and 18% inhibition respectively). Therefore, no further testing was needed. Since all criteria for acceptability of the test were met, this study was considered to be valid. Under the study conditions, the EC50 of the test substance was determined to be greater than 100 mg/L (based on nominal concentrations) (Desmares-Koopmans, 2008).

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance on activated sludge microorganisms according to OECD Guideline 209, EU Method C.11 and ISO 8192, in compliance with GLP. Two 1 L test bottles were filled with 250 mL of test substance mixtures with initial loading rates of 200 mg/L. These mixtures were stirred in closed bottles for 26 h. Subsequently, 16 mL synthetic sewage feed, 200 mL sludge from a predominantly domestic sewage source and Milli-RO water up to 500 mL were added resulting in a final loading rate of 100 mg/L. This loading rate was tested in duplicate. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-h exposure period. Thereafter, oxygen consumption was measured and recorded for approximately 10 min. Slight inhibition of respiration rate of the sludge microorganisms was observed. The duplicate measurement confirmed the result of the first measurement (12 and 18% inhibition respectively). Therefore, no further testing was needed. Since all criteria for acceptability of the test were met, this study was considered to be valid. Under the study conditions, the EC50 of the test substance was determined to be greater than 100 mg/L (based on nominal concentrations) (Desmares-Koopmans, 2008).