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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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:

The batch of Kephalis tested was a pale yellow liquid with a purity of 85.2% (sum of two peaks) and not completely soluble in test medium at the loading rate initially prepared.

In accordance with OECD No 23 for poorly soluble substances, a saturated solution (SS) was prepared at a loading rate of 100 mg/l applying a 2-day period of magnetic stirring followed by a 1-hour settlement period. Thereafter, the aqueous SS was siphoned off and used as the highest concentration. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

Due to light sensitivity of Kephalis all handlings were performed under dimmed light conditions, moreover, amber coloured glassware or glassware wrapped in aluminium foil was used during preparation.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.

Source: In-house laboratory culture with a known history.

Reason for selection: This system has been selected as an internationally accepted invertebrate species.

Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old. 6.3.

Breeding:
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.

Maximum age of the cultures: 4 weeks

Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.

Temperature of medium: 18-22°C

Feeding: Daily, a suspension of fresh water algae.

Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Test temperature:

18-22°C, constant within 2°C

pH:

pH: 6.0-8.5

Dissolved oxygen:

oxygen: ≥3 mg/l at the end of the test

Nominal and measured concentrations:

Kephalis: Solutions containing 1.0, 2.2, 4.6, 10, 22 and 46% of the SS prepared at a loading rate of 100 mg/l, which corresponds to initial measured concentrations of 0.6, 1.4, 2.8, 5.9, 12 and 28 mg/L.
Controls: Test medium without test substance or other additives

Details on test conditions:

Test duration: 48 hours
Test type: Static
Test vessels: 60 ml, all-glass, closed airtight, headspace reduced to minimum
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 60 ml of test solution
Light: Study was performed in dark
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 1 hour after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

8.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: (95% confidence interval between 5.9 and 12 mg/l).

Details on results:

Samples taken from all concentrations were analysed. The actual concentration increased with the applied dose from 0.60 mg/l at the lowest concentration to 28 mg/l in the highest concentration. Measured concentrations remained stable during the test (97-107% of initial). Therefore, the effects parameters were expressed in terms of initially measured concentrations.

Results with reference substance (positive control):

The study procedures described in this report were based on the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004 and the ISO International Standard 6341.
The reference test was carried out to check the sensitivity of the test system as used by WIL Research Europe. Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/l and to a control. Twenty daphnids were exposed per concentration.
The reference substance, potassium dichromate (K2Cr2O7, art. 1.04864, batch no. K44879664) was obtained from Merck, Darmstadt, Germany.
The 24h-EC50 was 0.51 mg/l with a 95% confidence interval between 0.46 and 0.57 mg/l.
The 48h-EC50 was 0.32 mg/l with a 95% confidence interval between 0.29 and 0.37 mg/l.
The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/l. Hence, the sensitivity of the daphnia was within the range determined with the historical data collected at WIL Research Europe.
The raw data from this study are kept in the WIL Research Europe archives. The test described above was performed under GLP with a QA-check.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study the 48h-EC50 was 8.4 mg/l based on initially measured concentrations (95% confidence interval between 5.9 and 12 mg/l).

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on initial measured test concentrations gave a 48-Hour EC50 value of 8.4 mg/l (95% confidence interval between 5.9 and 12 mg/L). The study was performed according to GLP and OECD Guideline 202

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

8.4 mg/L

Additional information

Based on the results of a range-finding test, the definitive test was conducted under static conditions for 48 hours at test concentrations of 1.0, 2.2, 4.6, 10, 22 and 46% of a saturated solution prepared at a loading rate of 100 mg/L. Samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. Because Kephalis was light sensitive and volatile, the exposure took place under dimmed light conditions and in airtight closed vessels with headspace reduced to minimum.

Samples taken from all concentrations were analysed. The actual concentration increased with the applied dose from 0.60 mg/l at the lowest concentration to 28 mg/l in the highest concentration. Measured concentrations remained stable during the test (97-107% of initial). Therefore, the effects parameters were expressed in terms of initially measured concentrations, which were respectively 0.6, 1.4, 2.8, 5.9, 12 and 28 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid.