Reaction mass of (4R)-isopropenyl-1-methylcyclohexene and (4S)-isopropenyl-1-methylcyclohexene and 1-isopropyl-4-methylcyclohexa-1,3-diene and 1-isopropyl-4-methylcyclohexa-1,4-diene and 4-isopropylidene-1-methylcyclohexene

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-02-11 to 2016-03-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

05 march 2015

Analytical monitoring:

yes

Remarks:

Dissolved [02]

Details on sampling:

- Concentrations: 2 mg/L (nominal)
- Sampling method: Determinations of the concentration of dissolved oxygen using an oxygen electrode in duplicate vessels taken from each group (except the procedure control) were made at day 0, 7, 14, 21 and 28, and only at the start of the test and after 7 and 14 days of incubation for the procedure control.

Vehicle:

yes

Remarks:

silica gel

Details on test solutions:

Approximately 30 mg of test item (actual measured amount: 34.2 mg) was added to 10 g of silica gel present in a 100-mL glass flask. The flask was closed with a screw cap and the content was mixed vigorously by hand shaking. Then 0.2 g of silica gel dosed with test item was added to the respective test vessels in order to obtain a nominal concentration of 2 mg test item.L-1 that were thereafter completely filled with the inoculated mineral medium (300 mL of test medium) and closed.
No small bubble was observed in the test vessels. The test was carried out without adjustment of the pH.

Test organisms (species):

other: River water inoculum

Details on inoculum:

- Origin: River water was sampled from “L’Adour”, situated downstream of a plant treating domestic wastewater (Aureilhan, France).
- Pre-conditioning: The river water was aerated for about 7 days before use as inoculum at the test temperature to reduce endogenous respiration.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

28 d

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

No data.

Test temperature:

Temperatures were situated between 21.1 and 22.1°C throughout the test (average value: 21.8°C), and complied with the requirements as laid down in the study plan (22°C ± 2°C, constant within 1°C).

pH:

No data.

Dissolved oxygen:

Prameter used for analysis.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

2 mg.L-1 (nominal)

Details on test conditions:

TEST CONDITIONS
- Test water: Mineral medium, Reconstituted water, as prescribed by the OECD Guideline No. 301D
- Composition of medium: Mineral medium (8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.40 mg Na2HPO4•2H2O, 0.50 mg NH4Cl; 36.40 mg CaCl2•2H2O, 22.50 mg MgSO4•7H2O and 0.25 mg FeCl3•6H2O.)
- River water was aerated for about 7 days before use as inoculum at the test temperature to reduce endogenous respiration.
- Test temperature: 21.1-22.1 °C throughout the test (average value: 21.8 °C)
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: All-glass sealed with ground glass stoppers BOD (Biological Oxygen Demand) bottles of approximately 300 mL capacity.
- Inoculum: A concentration of inoculum of approximately 10^5 cells/L was used.
- Number of culture flasks/concentration: 10 bottles containing test item, inoculum, mineral medium and silica gel (test suspension)

SAMPLING
- Sampling frequency: Determinations of the concentration of dissolved oxygen using an oxygen electrode in duplicate vessels taken from each group (except the procedure control) were made at day 0, 7, 14, 21 and 28, and only at the start of the test and after 7 and 14 days of incubation for the procedure control.
- Temperature: Measured continuously in the growth chamber.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 10 bottles containing inoculum, mineral medium and silica gel (inoculum blank + with silica gel); 10 bottles containing inoculum and mineral medium (inoculum blank without silica gel)
- Procedure control: 6 bottles containing reference substance, inoculum and mineral medium (procedure control)
- Toxicity control: No

Reference substance (positive control):

yes

Remarks:

benzoic acid, sodium salt

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biodegradability of test item

Remarks on result:

other: Observed results

Details on results:

Toxicity effect of the substance on aquatic microorganisms has been derived from data on its ready biodegradability.
Test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was biodegraded by 87% at day 28 in the Closed Bottle Test. The pass level of 60% was reached after less than 7 days (approximately 5 days) upon achieving 10% biodegradation.
Therefore, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE is considered as readily biodegradable based on the biodegradation percentage of 87% at day 28 and fulfilling the 10-day time window requirement (ca. less than 7 days).

Based on these results, it can be considered that the substance has no inhibition nor toxic effect on aquatic microorganisms.

Results with reference substance (positive control):

The calculated ThOD of the test item and the reference substance sodium benzoate were calculated from their empirical formulae to be:ThOD = 1.67 mgO2.mg-1 (test item calculated ThOD = 3.29 mgO2.mg-1).

Reported statistics and error estimates:

Not applicable.

The validity of the test was demonstrated by an endogenous respiration < 1.5 mgO2.L-1 and by a biodegradation of the reference substance of 73% of its ThOD after 14 days of incubation. Moreover, the differences of the replicate values at day 28 were less than 20% and oxygen concentrations were > 0.5 mg.L-1 in all bottles during the test period.

Substances are considered to be readily biodegradable in the Closed Bottle test if oxygen consumption is equal to or greater than 60% of the ThOD value in the ten-day window within the 28 day period of the test (the 10-day window begins when the degree of biodegradation has reached 10% of the ThOD).

Validity criteria fulfilled:

not applicable

Conclusions:

Toxicity of REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE has been derived from data obtained during a laboratory study on ready biodegradability under closed bottle following OECD Guideline 301 D.
The substance is readily biodegradable, and based on these results, it is considered that the substance has no ecotoxic effect on aquatic microorganisms at the tested nominal concentration of 2 mg/L.
NOEC of REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE is considered to be equal or higher than 2 mg/L.

Executive summary:

The biotic degradation of test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was assessed by performing a ready biodegradability test. The method followed was designed to be compliant with ISO 10634 and OECD Guideline for Testing of Chemicals No. 301 D, "Ready biodegradability – Closed Bottle Test", referenced as Method C.4-E of Commission Regulation No. 440/2008.

Results obtained during this study showed that the substance is readily biodegradable and that it can be considered as not having ecotoxic effect on aquatic microorganisms at the tested nominal concentration of 2 mg /L.

NOEC of REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE is considered to be equal or higher than 2 mg/L.

Description of key information

The biotic degradation of test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was assessed by performing a ready biodegradability test. The method followed was designed to be compliant with ISO 10634 and OECD Guideline for Testing of Chemicals No. 301 D, "Ready biodegradability – Closed Bottle Test", referenced as Method C.4-E of Commission Regulation No. 440/2008.

Results obtained during this study showed that the substance is well degraded and is considered as readily biodegradable.

It is proposed to consider the test concentration of 2 mg/L as NOEC value for the registered substance, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE, in accordance with recommandation of the guidance on Information requirements and Chemical Safety Assessment Chapter R.7b.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

2 mg/L