Bis(2-ethylhexyl) 2-(4-hydroxy-3,5-dimethoxybenzylidene)malonate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-10-20 to 2004-11-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

adopted 04 April 1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 1988

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

not performed

Test solutions

Vehicle:

no

Details on test solutions:

Due to the low water solubility of the test item in test water, adequate amounts of the test item were directly weighed into each test flask.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Type: activated sludge, micro organisms from a domestic waste water treatment plant.
Source: Sewage plant Groß-Zimmern, Germany

The sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and again centrifuged. The latter procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge (g) to its dry weight (g) determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 2.63 g dry material per litre, were made up with tap water. The pH of the activated sludge was 7.4.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

21-22°C

pH:

7.4 - 7.6

Dissolved oxygen:

6.2-7.6 mg/L

Nominal and measured concentrations:

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: open
- Fill volume: 500 mL
- Aeration: yes, with compressed air (0.613 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

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 x min)) was determined from the most linear part of the respiration curve, and were determined in the range between approximately 8.5 - 2.7 mg O2/L.

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

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

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

Applicant's summary and conclusion

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.

Results

The 3-hour EC 50 for the positive control 3,5-Dichlorophenol, which was tested in the same way as the test item, was found to be 5.1 mg/L and is within the range of 5 - 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge. In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited by 2.6 % at the two highest nominal concentrations of 1000 and 320 mg/L, respectively. At the nominal concentrations of 100 mg/L down to 10 mg/L test item, no inhibition was measured. Conclusion It can be concluded that the 3-hour EC 20 and 3-hour EC 50 will be more than 1000 mg test item per litre.

Conclusion

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