Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

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:

05-03-2001 to 28-03-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test, the test item was dispersed in ethanol due to limited water solubility.
Different solutions were prepared by dissolution of test item in ethanol in order to add always the same volume (i.e. 5 mL) of ethanol in each cylinder.
The following solutions were prepared:
A 1 : solution of 1000 mg of test item in 100 mL ethanol.
A2: solution of 316 mg of test item in 100 mL ethanol.
A3: preparation of a 100 mg of test item in 100 mL ethanol solution by dilution of the A 1 solution.
A4: preparation of a 31.6 mg of test item in 100 mL ethanol solution by dilution of the A2 solution.
A5: preparation of a 10 mg of test item in 100 mL ethanol solution by dilution of the A3 solution.
Test vessel S1: at time "15 minutes", in another 500-mL cylinder, 16 mL of the synthetic sewage feed + 5 mL of test substance stock solution A 1 were made up to 300 mL with water. 200 mL of sludge were added and the mixture was aerated. Final concentration: 100 mg/L.
Test vessel S2: at time "30 minutes", in another 500-mL cylinder, 16 mL of the synthetic sewage feed + 5 mL of test substance stock solution A2 were made up to 300 mL with water. 200 mL of sludge were added and the mixture was aerated. Final concentration: 31.6 mg/L.
Test vessel S3: at time "45 minutes", in another 500-mL cylinder, 16 mL of the synthetic sewage feed + 5 mL of test substance stock solution A3 were made up to 300 mL with water. 200 mL of sludge were added and the mixture was aerated. Final concentration: 10 mg/L.
Test vessel S4: at time "60 minutes", in another 500-mL cylinder, 16 mL of the synthetic sewage feed + 5 mL of test substance stock solution A4 were made up to 300 mL with water. 200 mL of sludge were added and the mixture was aerated. Final concentration: 3.16 mg/L.
Test vessel S5: at time "75 minutes", in another 500-mL cylinder, 16 mL of the synthetic sewage feed + 5 mL of test substance stock solution A5 were made up to 300 mL with water. 200 mL of sludge were added and the mixture was aerated. Final concentration: 1.0 mg/L.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls:
For blank control:
Control C1: at time "0 minutes", in a 500-mL scaled cylinder, 16 mL of the synthetic sewage feed and 5 mL ethanol was made up to 300 mL with water. 200 mL of sludge were added and the mixture was aerated continuously for 3 hours by bubbling air into it.
Control C2: at time "120 minutes", in a 500-mL scaled cylinder, 16 mL of the synthetic sewage feed and 5 mL ethanol was made up to 300 mL with water. 200 mL of sludge were added and the mixture was aerated continuously for 3 hours by bubbling air into it.
For positive control – reference item:
Test R1 : at time "90 minutes", in another 500-mL cylinder, 16 mL of the synthetic sewage feed + 5 mL of 3,5-dichlorophenol stock solution + 5 mL ethanol were made up to 300 mL with water. 200 mL of sludge were added and the mixture was aerated.
Test R1 : at time "105 minutes", in another 500-mL cylinder, 16 mL of the synthetic sewage feed +30 mL of 3,5-dichlorophenol stock solution + 5 mL ethanol were made up to 300 mL with water. 200 mL of sludge were added and the mixture was aerated
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Ethanol
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: A mixed population of activated sewage sludge microorganisms was obtained from the aeration stage of the sewage treatment plant at Geneva, Peney-Dessous, Switzerlan (full details available in the full study report), which treats predominantly domestic sewage wastewater.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at temperature of 21°C and was used on the day of collection. The pH of the sample was 7 to 8.
- Pretreatment: Not applicable.
- Initial biomass concentration: The suspended solids concentration in the inoculum was determined to be 6.44 g (dry weight) / L prior to use. The final suspended solids concentration was 2.57 g/L (per ISO 8192-1986)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

Respiration was measured at 30 minutes and 3 hours using a dissolved oxygen meter BOD probe

Post exposure observation period:

At time "3 hours", the pH of the first cylinder C1 was measured (pH-indicating paper strips). The content was used to fill up the BOD flask which was closed with the O2-electrode and the measurement of the oxygen consumption was started for ca. 10 minutes. The same procedure was repeated every 15 minutes for each cylinder in the order they had been prepared, so that the contact time is three hours for each of vessel.

Test temperature:

23 °C

pH:

3 hours: control: 7 to 8 and test groups: 7 to 8 and reference item: 7 to 8

Dissolved oxygen:

Initial: control: 4.0 – 4.4 mgO2/L and test groups: 3.6 – 5.6 mgO2/L and reference item: 4.8 -7.1 mgO2/L
3 hours: control: 2.7 – 2.3 mgO2/L and test groups: 1.9 – 2.6 mgO2/L and reference item: 2.2 – 2.8 mgO2/L

Nominal and measured concentrations:

Definitive test: nominal: 0 (control), 1, 3.16, 10, 31.6 and 100 mg/L
Reference item was completed: nominal: 5 and 30 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL glass flasks
- Type (delete if not applicable): Open, vessel continuously aerated with seal.
- Aeration: Yes, with stirring on magnetic stirrers but without foaming.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): None.
- No. of vessels per concentration (replicates): Definitive test: single
- No. of vessels per control (replicates): control: duplicate ; duplicate (reference item)
- Nitrification inhibitor used (delete if not applicable): Not applicable.
- Biomass loading rate: See table.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Test water used for both the range-finding and definitive tests was laboratory tap water. Water quality characteristics is reported in the full study report.
- Culture medium different from test medium: Not applicable.
- Intervals of water quality measurement: Temperature, pH and dissolved oxygen values were determined in all test media and controls; prior to and at the end of the 3 hour incubation period.

OTHER TEST CONDITIONS
- Adjustment of pH: No. pH at end of test was in range 7 to 8 in controls and treatment groups ; 7 to 8 in the reference item groups
- Light intensity: The test was conducted under normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Biological Oxygen Demand. Monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase.

TEST CONCENTRATIONS
- Test concentrations: Definitive test: nominal: 0 (control), 1, 3.16, 10, 31.6 and 100 mg/L
Reference item was completed: nominal: 5 and 30 mg/L
- Range finding study: No, the definitive test served as a range-finding limit test
- Results used to determine the conditions for the definitive study: Not applicable.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not applicable.
- Effect concentrations exceeding solubility of substance in test medium: Yes. However, an dispersant was used to increase test item concentration.
- Adsorption (e.g. of test material to the walls of the test container): Not reported.
- Blank controls oxygen uptake rate: 16.8 to 17.1 mgO2/L/min
- Coefficient of variation of oxygen uptake rate in control replicates: The oxygen consumption rates of the two controls differed by 1.7% (guideline, recommended maximum variation: 15%).

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Relevant effect levels: the EC50 for 3,5-dichlorophenol was: between 5 mg/L (22.9% inhibition) and 30 mg/L (97.4% inhibition). This was within the expected range: 5 to 30 mg/L. Full information is provided in the full study report.

Table 1.0 – Definitive test results

Vessel	pH	Iniital O2 concentration (mg/L)	Final O2 concentration (mg/L)	Duration (min)	Respiration Rate (mg/L/min)	% inhibition
C1	7-8	4.4	2.7	0.1	16.8
C2	7-8	4.0	2.3	0.1	17.1
S1	7-8	4.8	2.2	0.2	13.2	21.9
S2	7-8	5.6	2.6	10	15.3	9.5
S3	7-8	5.2	2.2	0.2	15.0	11.4
S4	7-8	4.8	1.9	0.2	14.5	14.3
S5	7-8	3.6	2.3	0.1	13.6	20.0
R1	7-8	4.8	2.2	0.2	13.1	22.9
R2	7-8	7.1	2.8	0.2	0.44	97.4

 

The EC50 could not be calculated as no significant inhibition was observed below 100 mg/L nominal test item concentration.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 3 hour EC50 for total inhibition was > 100 mg/L based on nominal test item concentrations prepared by dispersion of the test item in ethanol dispersant then the test system.

Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Tests conducted over a range of nominal test concentrations of 0 (control), 1, 3.16, 10, 31.6 and 100 mg/L test item pre-dispersed into ethanol dispersant, activated sewage sludge was exposed in a definitive limit test to the test item for a period of 3 hours at 23°C with the addition of synthetic sewage as a respiratory substrate examining total respiration under static conditions. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The variation of oxygen uptake in the inoculum control vessels was 1.7%. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 between 5 and 30 mg/L. This confirmed the suitability of the activated sludge and method used. All validity criteria were considered to have been satisfied. Under the conditions of this study, the test item total respiration EC50 was > 100 mg/L based on nominal test item concentrations.

Description of key information

ASRIT: EC50 : > 100 mg/L (nominal), 23°C, OECD TG 209, 2005

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

Additional information

Key study : OECD TG 209, 2005 : The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Tests conducted over a range of nominal test concentrations of 0 (control), 1, 3.16, 10, 31.6 and 100 mg/L test item pre-dispersed into ethanol dispersant, activated sewage sludge was exposed in a definitive limit test to the test item for a period of 3 hours at 23°C with the addition of synthetic sewage as a respiratory substrate examining total respiration under static conditions. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The variation of oxygen uptake in the inoculum control vessels was 1.7%. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 between 5 and 30 mg/L. This confirmed the suitability of the activated sludge and method used. All validity criteria were considered to have been satisfied. Under the conditions of this study, the test item total respiration EC50 was > 100 mg/L based on nominal test item concentrations.