2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

rel-(1R,2R,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

Cas Number:

125673-86-1

Molecular formula:

C15H26O

IUPAC Name:

rel-(1R,2R,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

Constituent 2

Reference substance name:

rel-(1R,2S,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

Molecular formula:

C15H26O

IUPAC Name:

rel-(1R,2S,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

impurity 1

Reference substance name:

4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

EC Number:

250-657-4

EC Name:

4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

Cas Number:

31499-72-6

Molecular formula:

C13H22O

IUPAC Name:

4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-one

Test material form:

liquid

Specific details on test material used for the study:

Name: Boisiris
Givaudan Substance Code: 2786903
Batch No.: VE00461936
Physical appearance: Liquid
Color: Colourless to pale yellow
Storage conditions: In a dry, well ventilated dark location at ambient temperature/10–30 °C

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations.

Control and test solutions were sampled in duplicate. The duplicate samples were kept separately as a reserve. The volume of each sample was recorded.

After sampling and before shipment, all samples were stored in amber glass bottles in the dark at a temperature of ≤ -18°C. A record was kept for each sample.

Samples were taken at the start of exposure in the fresh test solution (0 hour), in the aged test solution before renewal (24h), in the fresh renewal solution (24h) and in the aged renewal test solution (48h).

Samples of untreated test medium were provided to the analytical laboratory in order to determine the recovery of the analytical method with the specific test medium. Samples were transferred to the test site for chemical analysis under the required storage conditions. The dates of transfer of the samples from ECT to the laboratory for chemical analysis were recorded in the raw data.

Analysis was performed according to an analytical method validated by the test site for chemical analysis. Further details describing the analytical procedures are contained in the analytical phase report. The analytical work was reported by the principal investigator, and is included in the final report of this study.

Test solutions

Vehicle:

no

Details on test solutions:

As the test item is poorly water-soluble, the following approach was used taking into account aspects of OECD 2000, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Series on Testing and Assessment Number 23. ENV/JM/MONO(2000)6.

A saturated stock solution was prepared at a nominal loading rate of 100 mg/L as described below by adding 402 mg of the test item to nominal 4000 mL of the test medium. This stock solution (S1) was slowly stirred for 22.5 hours at ambient temperature in the dark. Thereafter this stock solution was left to settle for one hour. The stock solution was visually examined for undissolved/particulate matter in the free water column.

As no particles were visible, a 200 mL portion of the free water column of the stock solution was drained from the stock vessel (S1) into a glass bottle for pre-conditioning. Afterwards, this 200 mL-portion was also used to pre-rinse the flasks to hold the test concentrations (C5 to C1). This initial volume was discarded after rinsing the bottle and flasks. Thereafter the remaining volume of the free water column of S1 was transferred to the pre-conditioned bottle. This undiluted free water column of S1 was termed S2 (100% treatment). It was used immediately to prepare the desired test item concentrations. The test vessels were pre-conditioned once with the designated test concentration.

The stock solution S2 was prepared once to prepare the test solutions.

Thereafter the test solutions were adapted to test temperature. Then a first set of test vessels was filled with the test solutions for a 1 hour pre-conditioning of the test vessels under test conditions.

After pre-conditioning, the conditioning solutions were removed, and the first set of test vessels was re-filled with the respective test solutions and temperature-adapted before the test organisms were added to the test vessels.

For pre-conditioning a second set of test vessels was prepared from the remaining S2 solutions which was used after 24 h. These test vessels were kept in the dark at 20°C.

S2 was stored at 20±2°C in the dark for 24 h.

The test solutions for renewal were prepared from S2. The second set of vessels was pre-conditioned with the new test solutions as described above. Thereafter the conditioning solutions were discarded, and the second set of test vessels was re-filled with the test solutions. Then the test organisms were transferred from the first to the second set of test vessels after 24-h exposure.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

• Species: The test system used in this study was Daphnia magna STRAUS (clone M10), a water flea, cultured at ECT Oekotoxikologie GmbH since December 22, 2011.
• Origin: The organisms were originally supplied by KU Leuven, Belgium.
• Age of parental daphnids: less than 35 d
• Age of the daphnids at the beginning of the test: less than 24 h

The culture conditions of Daphnia magna:

Material of stock vessel: glass
Volume of medium per stock vessel: 1.8 L
Depth of medium in the stock vessel: approximately 15 cm
Number of daphnids kept as stock per culture vessel (batch size): approximately 20
Separation of adult and young daphnids: 2-5 times per week
Medium: Elendt medium M4
Renewal of medium: twice per week
Temperature: within required ranges (20 +/- 2°C)
Photoperiod (light/dark): 16/8 h
Light intensity: within required ranges (50 to 1000 lx)
Aeration: none
Food: algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
Feeding frequency: 4 times per week

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

18 to 22°C, constant within a range of 2°C

pH:

pH: 7.6 - 7.9

Nominal and measured concentrations:

Based on the results of a preliminary non-GLP range finding test, the following concentrations in a geometrical series (spacing factor: 1.8) were tested in the definitive test (nominal): 1.00, 3.16, 10.0, 31.6 and 100% of a saturated solution at a loading rate of 100 mg/L. The corresponding time-weighted mean measured concentrations over 48h were 0.0239, 0.0695, 0.305, 0.733 and 2.43 mg test item/L.

Details on test conditions:

At start of the test, after temperature adaptation of the test solutions, and at the end of the test the following parameters were measured and recorded in one replicate test vessel of the control and the highest test concentration:

• temperature,
• pH,
• dissolved oxygen content in % of air saturation value (ASV) and in mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.48 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The % immobility after 48 hours was 0, 0, 5, 0 and 85% in respectively the nominal concentrations of 1.0, 3.16, 10.0, 31.6 and 100% of saturated solution. The latter correspond to time-weighted mean measured concentrations of 0.0239, 0.0695, 0.305, 0.733, and 2.43 g/L. The test item concentrations were stable during each 24h renewal period between 58 and 78% of the concentrations measured in freshly prepared solutions.The biological endpoints were reported based on nominal (% of saturated solution at a loading rate of 100mg/L) and mean measured concentrations (mg/L). The latter are reported above as these are more appropriate for the assessment of the aquatic toxicity of Boisiris.

Results with reference substance (positive control):

The health of the Daphnia culture and the overall quality of the test organisms is monitored regularly by using the reference toxicant potassium dichromate (K2Cr2O7) in an acute test design in order to determine the sensitivity of the organisms from the culture and to demonstrate that the test conditions are appropriate.

The most recent reference test with potassium dichromate performed in a separate study (Study No. IDA1705) resulted in an EC50(24 h) of 1.29 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48h EC50 of Boisiris was determined to be 1.48 mg/L based on time-weighted mean measured concentrations.