bis(2-ethylhexyl) (4-hydroxy-3,5-dimethoxybenzyl)malonate

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:

2007-05-10 to 2007-07-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

adopted 04 April 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

30 May 1988

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

N/A

Vehicle:

no

Details on test solutions:

The test item is not soluble in water. Therefore, the test item was directly dosed into each test flask and tap water was added. For complete emulsification of the test item, this mixture was stirred for about 24 hours at test temperature (18 - 22 °C) in the dark / diffuse light before adding the inoculum. The test flasks were tightly closed to avoid water loss by evaporation. After approximately 24 hours the test water containing the test item was slightly red coloured and the test item was not completely dissolved.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The activated sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. The latter procedure was repeated twice. The washed sludge was re-suspended in tap water and sewage feed (50 mL per L) was added. The sludge was kept at room temperature under continuous aeration until use for one day. Immediately before use, the dry weight of the activated sludge was determined, and diluted to 3.5 g/L with tap water. The pH of the activated sludge was 7.6 and therefore no adjustment was necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

17-20°C

pH:

7.6

Dissolved oxygen:

9.7 mg/L

Nominal and measured concentrations:

Nominal: 10, 32, 100, 320 and 1000 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 Liter
- Type (delete if not applicable): open
- Fill volume: 500 mL
- Aeration: yes, with compressed air (0.8 L per minute)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 3.5g/L
- Weight of dry solids per volume of reaction mixture per unit of time: 1.4 g/L
- Nutrients provided for bacteria: Synthetic sewage

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

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Details on termination of incubation:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
For the measurement of the respiration rate a well-mixed sample of each test medium was poured into a Karlsruher flask after exactly 3 hours incubation time, and was not further aerated. The oxygen concentration was measured with an oxygen electrode, and was recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption (in mg O2/L / minute) was determined from the most linear part of the respiration curve, and were determined in the range between approximately 9.7 - 3.3 mg O2/L.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125
- Test concentrations: 10, 32, 100, 320 and 1000

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

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

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

For details see executive summary

Results with reference substance (positive control):

For details see executive summary

For details see executive summary

Validity criteria fulfilled:

yes

Conclusions:

Up to the limit dose of 1000 mg/L, the test item did not inhibit the respiration rate of activated sludge. The 3-hour EC20and EC50 are clearly higher than 1000 mg/L under the present test conditions.

Executive summary:

Purpose

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions.

Study design

The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations (10, 32, 100, 320 and 1000 mg/L) of the test item after an incubation period of 3 hours. This study was conducted according to Good Laboratory Practice (GLP) and the method applied followed the OECD Guideline for Testing of Chemicals, Section 2, No.209: "Activated Sludge, Respiration Inhibition Test", adopted April 04, 1984.

3,5-Dichlorophenol was used as a positive control.

 

Results

The following nominal concentrations of the positive control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 32, 10 and 3.2 mg/L. In comparison to the controls the respiration rate of the activated sludge was moderately inhibited by 15.3 % at the lowest nominal concentration of 3.2 mg/L. At the nominal concentrations of 10 and 32 mg test item/L, the respiration rate was inhibited by 49.2% and 79.7 %, respectively.

All validity parameters were fulfilled in this study:

- Inoculum control: The respiration rates of the two controls did not differ by more than 15%.

- Reference item: The 3h EC50 of the refernce item 3,5-Dichlorophenol was determined to be 10.8 mg/L and is thus in the range of 5 to 30 mg/L for the used activated sludge batch.

- Dissolved oxygen: The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 7.3 mg O2/L and thus >6.5 mg/L

In comparison to the inoculum controls the respiration rate of the activated sludge was slightly enhanced by 5.1% and 1.7% at the two lowest nominal test concentrations of 10 and 32 mg/L, respectively. At the next higher concentrations of nominal 100 and 320 mg/L, the respiration rate was moderately enhanced by 22%: At the highest nominal test concentration of 1000 mg/L an activation of activated sludge respiration of 32% was measured. Concentrations exceeding 1000 mg/L nominal were not tested.

Based on measured inhibition rates, the 3-hour EC₂₀and ECs0 could not be quantified because up to the highest nominal test concentration of 1000 mg/L no inhibition was noted after three hours incubation. Nevertheless, the 3 -hour EC₂₀and EC₅₀are clearly higher than 1000 mg/L under the present test conditions.

Conclusion

The 3-hour EC₂₀and EC₅₀are clearly higher than 1000 mg/L under the present test conditions.

Description of key information

The 3-hour EC₂₀and EC₅₀ are greater than 1000 mg/L under the test conditions.

Additional information

Purpose

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions.

Study design

The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations (10, 32, 100, 320 and 1000 mg/L) of the test item after an incubation period of 3 hours. This study was conducted according to Good Laboratory Practice (GLP) and the method applied followed the OECD Guideline for Testing of Chemicals, Section 2, No.209: "Activated Sludge, Respiration Inhibition Test", adopted April 04, 1984.

3,5-Dichlorophenol was used as a positive control.

 

Results

The following nominal concentrations of the positive control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 32, 10 and 3.2 mg/L. In comparison to the controls the respiration rate of the activated sludge was moderately inhibited by 15.3 % at the lowest nominal concentration of 3.2 mg/L. At the nominal concentrations of 10 and 32 mg test item/L, the respiration rate was inhibited by 49.2% and 79.7 %, respectively.

All validity parameters were fulfilled in this study:

- Inoculum control: The respiration rates of the two controls did not differ by more than 15%.

- Reference item: The 3h EC50 of the refernce item 3,5-Dichlorophenol was determined to be 10.8 mg/L and is thus in the range of 5 to 30 mg/L for the used activated sludge batch.

- Dissolved oxygen: The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 7.3 mg O2/L and thus >6.5 mg/L

In comparison to the inoculum controls the respiration rate of the activated sludge was slightly enhanced by 5.1% and 1.7% at the two lowest nominal test concentrations of 10 and 32 mg/L, respectively. At the next higher concentrations of nominal 100 and 320 mg/L, the respiration rate was moderately enhanced by 22%: At the highest nominal test concentration of 1000 mg/L an activation of activated sludge respiration of 32% was measured. Concentrations exceeding 1000 mg/L nominal were not tested.

Based on measured inhibition rates, the 3-hour EC₂₀and ECs0 could not be quantified because up to the highest nominal test concentration of 1000 mg/L no inhibition was noted after three hours incubation. Nevertheless, the 3 -hour EC₂₀and EC₅₀are clearly higher than 1000 mg/L under the present test conditions.

Conclusion

The 3-hour EC₂₀and EC₅₀are clearly higher than 1000 mg/L under the present test conditions.