2-(3,5-dimethylhex-3-en-2-yloxy)-2-methylpropyl cyclopropanecarboxylate

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:

From 25 June 2008 to 15 August 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

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:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 17 and 18 March 2008 / Date of signature: 23 June 2008

Specific details on test material used for the study:

Batch No. : LDP0800001
Purity : 97.0%
Date of Expiry : 01 May, 2010

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Ethanol

Details on test solutions:

SOLUTIONS OF THE TEST SUBSTANCE
The test substance is not soluble enough in water to allow the preparation of an aqueous stock solution in tap water. Therefore, different solutions were prepared by dissolution of the test substance in ethanol in order to add always the same volume (i.e. 5 mL) of ethanol in each cylinder.
The following solutions were prepared:
A1: solution of 1000.3 mg of test substance in 100 mL ethanol
A2: solution of 316.1 mg of test substance in 100 mL ethanol
A3: 2 mL of solution A1 diiluted to 20 mL with ethanol
A4: 2 mL of solution A2 diluted to 20 mL with ethanol
A5: 2 mL of solution A3 diluted to 20 mL with ethanol

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge was obtained from a water treatment plant treating predominantly domestic sewage (city of Geneva, Peney-Dessous). The sludge was kept aerobic until being used on the same day. The pH was between 7 and 8. The suspended solids content was determined by drying and weighing 50 mL of the sludge: dry weight of suspended solids: 4.794 g/L. The sludge was diluted with tap water to a concentration of 2.55 g/L. By adding 200 mL of sludge to the 500 mL test vessels, the final concentration of suspended solids was 1.02 g/L, i.e. in the range 0.8 to 1.6 g/L as recommended in ISO 8192-1986.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

no data

Test temperature:

The whole experiment was carried out at 20 +/- 1°C (temperature controlled water bath)

pH:

8

Nominal and measured concentrations:

Nominal values: 1.00, 3.16, 10.00, 31.61 and 100.03 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Several 500-mL scaled cylinders are used as test vessels.
- Aeration:
A clean compressed air supply is used to aerate every test vessel.

SYNTHETIC SEWAGE FEED
A synthetic sewage feed was made by dissolving the following amounts of substances in 1 L of tap water:
- 16.00 g peptone (from casein trypsin-digested)
- 11.00 g yeast extract powder
- 3.00 g urea
- 0.70 g NaCl
- 0.40 g CaCl2.2H2O
- 0.30 g MgSO4.7H2O
- 2.80 g K2HPO4

TEST PROCEDURE
- Control C1: at time 0, in a 500-mL sclaed 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.
- Test S1: at time 15 minutes, in another 500-mL cylinder, 16 mL of the synthetic sewage feed + 5 mL of test substance stock solution A1 were made up to 300 mL with water. 200 mL of sludge were added and the mixture was aerated. Final concentration = 100.03 mg/L.
- Test 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 = 31.61 mg/L.
- Test 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 = 10.00 mg/L.
- Test 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 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 = 1.00 mg/L.
- 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. Final concentration = 5.00 mg/L.
- Test R2: 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. Final concentration = 30.01 mg/L.
- Control TR: at time 120 minutes, the same as for control 1 was done. This flask serves only as spacer between the rather toxic R2 and the final control C2.
- Control C2: at time 120 minutes, the same as for control C1 was done.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
A dissolved oxygen measuring device, composed of a flat bottom flask (BOD type) in which an O2-electrode fits tightly and an oxygen meter connected to a strip-chart recorder, is used to measure oxygen concentration versus time.
At time 3 hours, the pH of the first cylinder C1 was measured. 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 about 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 3 hours for each of them.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: Observed inhibition of respiration at 100 mg/L test concentration relative to Controls was -0.8%.

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 on result:

other: Observed inhibition of respiration at 100 mg/L test concentration relative to Controls was -0.8%.

Details on results:

The test was repeated two times as in the first tests the oxygen consumption of the two flasks was different by more than 15% (validity criterion). Only the results of the third test are reported here.
No significant inhibition is observed for all concentrations (1 to 100 mg/L) tested. The variation in the degree of inhibition observed is not signficant. Therefore, the 3h-EC50 is higher than 100 mg/L.
See table 6.1.7/1 in "Any other information on results incl. tables".

Results with reference substance (positive control):

.- Results with reference substance valid? yes
- Relevant effect levels: The 3h-EC50 of 3,5-dichlorophenol is between 5 and 30 mg/L as % inhibition is 11.8% at 5 mg/L and 84.1% at 30 mg/L

Table 6.1.7/1: Test results

Vessel	pH	O2 (mg/L)	Time (min)	Rate	% inhibition	Final concentration (mg/L)	Log (conc)
C1 C2	8 8	4.00 4.50	5.6 6.4	0.71 0.70
R1 R2	8 8	3.50 0.90	5.6 8.0	0.63 0.11	11.8 84.1	5.00 30.01	0.69897 1.47727
S1 S2 S3 S4 S5	8 8 8 8 8	4.00 4.50 3.30 4.40 3.90	5.6 5.6 4.2 6.0 5.4	0.71 0.80 0.79 0.73 0.72	-0.8 -13.4 -10.9 -3.5 -1.9	100.03 31.61 10.00 3.16 1.00	2.00013 1.49982 1.00013 0.49982 0.00000

Validity criteria fulfilled:

yes

Remarks:

The difference between the two controls C1 and C2 was 1.6% (so within the validity range 15% max). The 3h-EC50 of 3,5-dichlorophenol is between 5 and 30 mg/L as % inhibition is 11.8% at 5 mg/L and 84.1% at 30 mg/L.

Conclusions:

No significant inhibition is observed for all concentrations (1 to 100 mg/L) tested. The variation in the degree of inhibition observed is not signficant. Therefore, the 3h-EC50 is higher than 100 mg/L, and, the 3h-EC0 is > = 100 mg/L..

Executive summary:

The activated sludge respiration inhibition test according to OECD Guideline 209 was performed with the test substance. Five concentrations in the range 1 to 100 mg/L were tested. None of them produced significant inhibition. Therefore, the 3h-EC50 is > 100 mg/L, and, the 3h-EC0 is > = 100 mg/L.

Description of key information

OECD Guideline 209, GLP, key study, validity 2:

3h-EC50 > 100 mg/L (highest tested concentration) based on nominal concentrations

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

To assess the toxicity of the registered substance to microorganisms, one experimental study is available.

This valid study was performed according to OECD Guideline 209 with GLP statement to assess the effect of the registered substance on the respiration rate of activated sludge. The test was carried out under static conditions with the test substance concentrations 1.00, 3.16, 10.00, 31.61 and 100.03 mg/L. The respiration rates of the control, reference and test substances were measured after a contact time of three hours. According to the results of this study, all validity criteria were fulfilled and no significant inhibition was observed for all tested concentrations. The variation in the degree of inhibition observed was not signficant. Therefore, the 3h-EC50 was determined to be > 100 mg/L.