4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Kephalis
Appearance: Pale yellow liquid
Batch: PE00097828
Purity/Composition: 85.2% (sum of two peaks)
Test substance storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 14 March 2015 (expiry date)
Chemical name (IUPAC): 4-(1-ethoxyvinyl)-3,3,5,5,-tetramethylcyclohexanone (main component)
CAS Number: 36306-87-3
EC Number: 252-961-2
Molecular formula: C14H24O2
Molecular weight: 224.4 g/mol

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Kephalis: Solutions prepared at 1.0, 3.2, 10, 32 and 100% SS prepared at a loading rate.
Controls: Test medium without test substance or other additives
Replicates:
3 replicates of each test concentration.
6 replicates of the control.
1 or 2 replicates of each test concentration without algae.
1 replicate of each test concentration for sampling purposes at 24 hours of exposure.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture/test medium: adjusted M2; according to the OECD 201 Guideline, formulated using Milli-RO water

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

21-24°C, constant within 2°C

pH:

pH 6.0-9.0

Nominal and measured concentrations:

Time Weighted Average (TWA) Exposure concentrations were calculated and determined to be 0.21, 1.5, 4.7, 13 and 48 mg/L for the solutions containing 1.0, 3.2, 10, 32 and 100% SS, respectively.

Details on test conditions:

Test vessels: 120 ml, all-glass, completely filled with test solution. Sealed to minimise possible losses of test item due to volatilization.
Medium: adjusted M2
Cell density: An initial cell density of 1 x 104 cells/ml.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 82 to 85 μE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval ranging from 24 to 27 mg/l

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval from 7.8 to 10 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Remarks on result:

other: 95% confidence interval ranging from 9.3 to 12 mg/l

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Results with reference substance (positive control):

The study procedures described in this report were based on the OECD guideline No. 201, Adopted March 23, 2006; Annex 5 corrected 28 July 2011.
This reference test was carried out to check the sensitivity of the test system used by WIL Research Europe to Potassium dichromate (Merck, Art. 1.04864, Batch K44879664). Closed test vessels were used as to mimic testing a volatile test article and results were compared with standard ‘open’ systems.
Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and to a control. The initial cell density was 1.0 x 104 cells/ml.

The following results were obtained:
The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/l with a 95% confidence interval ranging from 1.2 to 1.5 mg/l. The historical ranges for growth rate inhibition using standard ‘open’ systems range between 0.82 and 2.3 mg/l. Hence, the 72h-ERC50 for the present test in closed systems is within the historical range.
The EC50 for yield inhibition (72h-EYC50) was 0.50 mg/l with a 95% confidence interval ranging from 0.47 to 0.53 mg/l. The historical ranges for yield inhibition using standard ‘open’ systems lie between 0.43 and 1.1 mg/l. Hence, the 72h-EYC50 for the present test in closed systems was within range.
It was concluded that the sensitivity of algae exposed in closed test systems towards Potassium dichromate was not significantly different from the sensitivity of algae exposed in standard ‘open’ reference studies during the past years.
The protocol, raw data and report of this study are kept in the WIL Research Europe archives. The test described above was performed under GLP conditions with a QA-check.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Kephalis reduced growth rate of this fresh water algae species significantly at a TWA concentration of 13 mg/l and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 25 mg/l with a 95% confidence interval ranging from 24 to 27 mg/l.
The EC50 for yield inhibition (72h-EYC50) was 10 mg/l with a 95% confidence interval ranging from 9.3 to 12 mg/l.
The EC10 for growth rate inhibition (72h-ERC50) was 9.0 mg/l with a 95% confidence interval ranging from 7.8 to 10 mg/l.
The EC10 for yield inhibition (72h-EYC50) was 3.1 mg/l with a 95% confidence interval ranging from 2.4 to 3.8 mg/l.
The 72h-NOEC for growth rate inhibition was 4.7 mg/l.
The 72h-NOEC for yield inhibition was 1.5 mg/l.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate are used for classification purposes, which were determined in this study to be 25mg/L and 9.0 mg/L respectively.

Description of key information

The effects of the test item on the growth of the green alga, Pseudokirchneriella subcapitata, were determined during a 72-hour growth inhibition test. The study was performed according to GLP and OECD Guideline 201.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate, which were determined in this study to be 25mg/L and 9.0 mg/L respectively, are used for classification purposes and have been selected as the key values for chemical safety assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

25 mg/L

EC10 or NOEC for freshwater algae:

9 mg/L

Additional information

Based on the results of a range-finding test, the definitive test was conducted at test concentrations of 1.0, 3.2, 10, 22 and 100% of a saturated solution prepared at a loading rate of 100 mg/L. Due to light sensitivity of Kephalis all solutions were protected from light during preparation. The test item was known to be volatile and hence testing was conducted in sealed test vessels in order to minimize possible losses due to volatilization. In order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure. The measured concentrations increased with the applied dose at the start of the test and were at the level of 70-95% of initial at the end of the test. Based on these results, the Time Weighted Average (TWA) Exposure concentrations were calculated and determined to be 0.21, 1.5, 4.7, 13 and 48 mg/L for the solutions containing 1.0, 3.2, 10, 32 and 100% SS, respectively.

The study met the acceptability criteria prescribed by the protocol and was considered valid.