[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

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

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 (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewarbeaufsichtsamt Hildesheim

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test item was pipetted directly in the laboratory bottles. The bottles were closed immediately.
- Differential loading: yes
- Controls: yes

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: no
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant of 31137 Hildesheim, Germany (16 Mar 2017)
- Preparation of inoculum for exposure: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10%. The sludge was used within 24 h after sampling.
- Initial biomass concentration: 2.74 g/L, corresponding to 1.37 g/L in the test vessel (500 mL test solution containing 250 mL activated sludge)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.5 °C

pH:

7.58 (activated sludge)
7.26 (synthetic waste water)

Dissolved oxygen:

above 60 - 70% saturation

Nominal and measured concentrations:

nominal: control, 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: closed
- Material, size, headspace, fill volume: 1000 mL laboratory bottles with screw cap, ISO 4796, 500 mL fill-volume, 500 mL headspace
- Aeration: Shaking of closed bottles at 150 rpm to keep the dissolved oxygen concentration above 60 - 70% saturation
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): No
- Sludge concentration (weight of dry solids per volume): 2.74 g/L
- Nutrients provided for bacteria: Synthetic waste water according to OECD Guideline 209
- Biomass loading rate: 2.74 g/L, corresponding to 1.37 g/L in the test vessel (500 mL test solution containing 250 mL activated sludge)

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

OTHER TEST CONDITIONS
- Adjustment of pH: not necessary
- Details on termination of incubation: After 3 h incubation, the oxygen concentration was determined after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes.

EFFECT PARAMETERS MEASURED: oxygen consumption as a proxy for respiration rate after aeration time of 3 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.1
Range finding study
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Based on the results fof the non-GLP range-finding test the main test was performed.

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

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:

EC10

Effect conc.:

16.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: Confidential limits 95%

Remarks:

13.6 - 18.8 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

24.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: Confidential limits 95%

Remarks:

22.8 - 27.3 mg/L

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Blank controls oxygen uptake rate: yes
- Coefficient of variation of oxygen uptake rate in control replicates: 8.7%
- Others: Biological results are summarized within the tables in section "Any other information on results incl. tables".

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h): 96.9 mg/L (95% CI: 92.9 - 101 mg/L)

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and Dunnett’s test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha=0.05. The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively, using software GraphPadPrism. Calculations of the confidence intervals for the EC-values were carried out using standard procedures.

Biological results of the range-finding test:

In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification. Two replicates 1000 mg/L were measured with addition of Allylthiourea ATU. No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed. The results indicate that the respiration rates in the closed laboratory bottles are a little lower than in open Erlenmeyer flasks. Nevertheless, the respiration rates in closed bottles (test volume 500 mL and 500 mL headspace) are in the valid and necessary range for this test. The closed bottles prevent the volatile test item from leaving the test vessel while the vigorously shaking at 150 rpm maintains aeration and exchange with the gas phase.

Biological results main test:

Table 1: Oxygen Uptake Rates and Inhibition of the Respiration of the Control and Test Item Concentrations

Test Concentration [mg/L]	Repl. [%]	Oxygen Uptake	Inhibition Rate R [mg O2/L-h]	Mean Inhibition [%]
Control	1	33.2
	2	29.2
	3	29.6
	4	27.6
	5	26.4
	6	26.8
10	1	28.0	3
	2	27.6	4
	3	27.6	4	5
	4	26.4	8
	5	26.8	7
32	1	19.6	32
	2	20.8	28
	3	20.4	29	28
	4	21.6	25
	5	20.8	28
100	1	13.6	53
	2	15.6	46
	3	15.6	46	47
	4	16.0	44
	5	15.2	47
320	1	15.2	47
	2	16.0	44
	3	16.0	44	45
	4	16.4	43
	5	14.8	49
1000	1	14.0	51
	2	15.2	47
	3	13.2	54	51
	4	15.2	47
	5	13.2	54

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 8.7% Suspended solids in the test vessel: 1.37 g/L.

Table 2: Specific respiration rates of the control replicates

	Repl.	Specific Respiration Rate Rs [mg O2/g-h]
	1	24.2
	2	21.3
	3	21.6
Control	4	20.1
	5	19.3
	6	19.6
Mean value	21.0

Repl. = Replicate

Suspended solids in the test vessel: 1.37 g/L

Validity criteria fulfilled:

yes

Conclusions:

The NOEC (3 h) of Menthyl methyl ether is 10 mg test item/L. The EC10 (3 h) is 16.6 mg test item /L and the EC50 (3 h) is > 1000 mg test item/L.

Description of key information

NOEC (3 h) = 10 mg/L (activated sludge, OECD 209)

EC10 (3 h) = 16.6 mg/L (activated sludge, OECD 209)

EC50 (3 h) > 1000 mg/L (activated sludge, OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

16.6 mg/L

Additional information

One experimental study was conducted in accordance with OECD Guideline 209 and EU Methode C.11 under GLP conditions. The activated sludge was exposed to the test item concentrations of 10, 32, 100, 320 and 1000 mg/L. The respiration rate of each mixture was determined after aeration periods of 3 hours (no nitrification set up was performed). The EC50 (3 h) is higher than 1000 mg/L based on statistical evaluation. The EC10 (3 h) is observed at 16.6 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed. No inhibition of the degradation process in sewage treatment plants is expected.