Reaction mass of ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate and ethyl 2,6,6-trimethylcyclohexa-2,4-diene-1-carboxylate and ethyl 6,6-dimethyl-2-methylenecyclohex-3-enecarboxylate

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:

19 October 2020 - 04 March 2021

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 20 to 21 November 2018 and 07 June 2019; Date of decision: 21 August 2019

Analytical monitoring:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture
- Name and location of sewage treatment plant where inoculum was collected:
Activated sludge was obtained from a water treatment plant treating predominantly domestic sewage (Bois-de Bay, 1242 Satigny, Switzerland). The sludge was kept aerobic until being used on the next day

Test type:

static

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 ± 2°C

Nominal and measured concentrations:

Nominal concentration: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500-ml scaled cylinders
- Type: closed
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 1.5 g/L
- Nutrients provided for bacteria: Synthetic sewage feed

EFFECT PARAMETERS MEASURED: oxygen consumption at 3 hours

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

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

The EC50 (3 hours) of 3,5-dichlorophenol is between 2 and 25 mg/L.

Table 6.1.7/1 % inhibition by test item

Recipient	pH	O2(mg/L) T0 min	O2(mg/L) T5.0 min	Δ O2(mg/L)	Temps (min)	Rate	% Inhibition	Concentration finale (mg/L)
C1	8.41	6.80	4.30	2.50	5.0	0.50	-	-
C2	8.46	6.70	4.30	2.40	5.0	0.48	-	-
C3	8.50	7.75	5.25	2.50	5.0	0.50	-	-
S1	8.40	6.80	4.35	2.45	5.0	0.49	11.7	100.16
S2	8.47	7.80	5.35	2.45	5.0	0.49	11.7	99.84
S3	8.44	7.30	4.80	2.50	5.0	0.50	9.9	100.10
R1	8.45	-	-	-	-	-	-	2.14
R2	8.42	6.75	4.30	2.45	5.0	0.49	11.7	2.12
R3	8.47	7.50	5.00	2.50	5.0	0.50	9.9	2.08
R4	8.51	8.30	7.40	0.90	5.0	0.18	67.6	25.14
R5	8.55	8.45	7.55	0.90	5.0	0.18	67.6	24.92
R6	8.47	8.25	7.30	0.95	5.0	0.19	65.8	24.92
C4	8.51	6.65	3.70	2.95	5.0	0.59	-	-
C5	8.56	8.15	5.15	3.00	5.0	0.60	-	-
C6	8.58	8.10	4.80	3.30	5.0	0.66	-	-

 

The data of reference number 1 (R1) is not exploitable due to a measurement error, as Reference 2 (R2) and Reference 3 (R3) are in very good agreement the results of this study are not impacted.

- The variation coefficient of the controls was 13.0%. This is within the validity range (30%).

- the EC₅₀(3 hours) of 3,5-dichlorophenol is between 2 and 25 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the EC50(3 hours) of test item > 100 mg/L and NOEC (3 hours) of test item is 100 mg/L.

Executive summary:

An activated sludge respiration inhibition test was performed with test item according to OECD Guideline 209, ISO 8192-1986 and EU method C.11, Activated Sludge Respiration Inhibition Test. In a limit test, a concentration of 100 mg/L (nominal) was evaluated.

 

The percentage values of inhibition in the three test replicates of 11.7%, 11.7% and 9.9% are well within the 30% error (validity criteria) of the test, and therefore we can consider that at the tested concentration (nominal 100 mg/L) no significant inhibition with test item was observed.

 

Therefore, EC₅₀(3 hours) of test item > 100 mg/L; NOEC (3 hours) of test item is 100 mg/L.

Description of key information

An activated sludge respiration inhibition test was performed with test item according to OECD Guideline 209, ISO 8192-1986 and EU method C.11, Activated Sludge Respiration Inhibition Test. In a limit test, a concentration of 100 mg/L (nominal) was evaluated. The percentage values of inhibition in the three test replicates of 11.7%, 11.7% and 9.9% are well within the 30% error (validity criteria) of the test, and therefore it can be considered that at the tested concentration (nominal 100 mg/L) no significant inhibition with Ethyl Safranate was observed.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L