Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

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:

28 October 2016 - 14 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines (OECD 202) and in accordance with GLP. All relevant guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: +6 ºC ± 2 ºC, protected from light and stored under nitrogen.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: no
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: n/a

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 5.61, 17.9, 56.1, 179 and 561 µL of test item pipetted directly into Erlenmeyer test vessels containing 16 mL of synthetic waste water (prepared per OECD 209), 234 mL of demineralised water and 250 mL of innoculum to prepare nominal concnetrations of 10, 32, 100, 320 and 1000 mg/L.
- Eluate: Activated sewage sludge from the sewage plant at Hildesheim as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Differential loading: n/a
- Controls: Vessels prepared ad above but without test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: n/a
- Name and location of sewage treatment plant where inoculum was collected: Sewage plant at Hildesheim , Germany (no. 31137).
- Method of cultivation: n/a
- 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.
- Pretreatment: n/a
- Initial biomass concentration: Dry sludge contents = 2.84 g/L (1.42 g/L in the test vessels).

Test type:

static

Water media type:

other: Activated sewage sludge

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 ºC

pH:

7.5 ± 0.5

Dissolved oxygen:

60 - 70 % saturation

Nominal and measured concentrations:

Nominal: 10, 32, 100, 320 and 1000 mg/L

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate (Merck, batch # A0382290) tested at 3 concentrations within the range of 58 - 180 mg/L and a dilution factor of 1.8. Solutions prepared from a 300 g/L stock and applied to vessels as per test treatment flasks.

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

61.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

264 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
- Adsorption (e.g. of test material to the walls of the test container): Not reported
- Blank controls oxygen uptake rate: 33.2 - 37.2 mg O2/L/h
- Coefficient of variation of oxygen uptake rate in control replicates: 4.8 %

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 for inhibition was 109 mg/L (95 % confidence limits = 107 - 112 mg/L) i.e. within the accepeted range of 53 - 155 mg/L.
- Other: No

Reported statistics and error estimates:

EC10 95 % confidence limits = 49.2 - 74.2 mg/L.
EC50 95 % confidence limits = 216 - 324 mg/L.

Table 1       Oxygen uptake rates and inhibition of the respiration of the control, test item and reference item solutions

Test Concentration (mg/L)	Rep	Oxygen Uptake Rate (mg O2/L/h)	Inhibition (%)	Mean Inhibition (%)
Treatment vessels
Control	1	36.4	-	-
	2	37.2	-
	3	35.2	-
	4	33.6	-
	5	33.2	-
	6	33.6	-
10	1	34.8	0	0
	2	36.4	-4
	3	34.4	1
	4	34.0	3
	5	35.2	-1
32	1	34.0	3	2
	2	34.4	1
	3	34.0	3
	4	34.4	1
	5	33.6	4
100	1	23.2	34	22
	2	30.4	13
	3	29.6	15
	4	28.4	19
	5	25.2	28
320	1	15.6	55	54
	2	16.0	54
	3	15.6	55
	4	18.0	48
	5	15.2	56
1000	1	14.0	60	62
	2	13.2	62
	3	12.4	64
	4	12.8	63
	5	12.8	63
Reference item
58	1	27.2	22	21
	2	27.6	21
	3	27.6	21
100	1	19.2	45	45
	2	19.6	44
	3	19.2	45
180	1	9.2	74	74
	2	9.6	72
	3	8.8	75

Table 2       Specific respiration rates of the control replicates

	Rep	Specific Respiration Rate (mg O2/g/h)
Control	1	25.6
	2	26.2
	3	24.8
	4	23.7
	5	23.4
	6	23.7
Mean value		24.6

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item was 32 mg test item/L. The EC10 was 61.2 mg test item /L (95 % CI = 49.2 - 74.2 mg/L). The EC50 was 264 mg test item /L (95 % CI = 216 - 324 mg/L).

Executive summary:

OECD 209 (2017) - A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations; 10, 32, 100, 320 and 1000 mg/L. The respiration rates of the control, reference 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 mean inhibition of respiration for the test item replicates ranged from 0 % to 62 %.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀ value for the reference item was 109 mg/L, within guideline specifications.

The NOEC of the test item was 32 mg test item/L.  The EC10 was 61.2 mg test item /L (95 % CI = 49.2 - 74.2 mg/L).  The EC50 was 264 mg test item /L (95 % CI = 216 - 324 mg/L).

Description of key information

3h EC50 = 264 mg/L. 3h EC10 = 61.2 mg/L; OECD 209; Anon. (2017)

Key value for chemical safety assessment

EC50 for microorganisms:

264 mg/L

EC10 or NOEC for microorganisms:

61.2 mg/L

Additional information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item (Anon., 2017). The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations; 10, 32, 100, 320 and 1000 mg/L. The respiration rates of the control, reference 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 mean inhibition of respiration for the test item replicates ranged from 0 % to 62 %.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50 value for the reference item was 109 mg/L, within guideline specifications.

The NOEC of the test item was 32 mg test item/L.  The EC10 was 61.2 mg test item /L (95 % CI = 49.2 - 74.2 mg/L).  The EC50 was 264 mg test item /L (95 % CI = 216 - 324 mg/L).