6,6-dimethoxy-2,5,5-trimethylhex-2-ene

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item: HR 02/197007
Batch Number: 00003
CAS No.: 67674-46-8
Active Ingredients: 6,6-Dimethoxy-2,5,5-trimethyl-2-hexene
Appearance: Colourless to pale yellowish, clear liquid
Water Solubility: Insoluble
Molecular weight: 186.30 g/mol
Molecular formula: C11H22O2

Analytical monitoring:

yes

Details on sampling:

The test item was analytically verified via DOC analysis. DOC was measured in all concentration levels and control after 0h (new media) and 48h (old media).

Details on test solutions:

Stock solution: The solution (100 mg/l were weighed out) was prepared with dilution water one day prior to application
Dispersion treatment: THe stock solution was stirred (400 rpm) for 24 h. Finally undissolved particles of the test item were removed by centrigugation (20 min, 3000 rpm)
Test Dilutions: 1:1 - 1:2 - 1:4 - 1:8 - 1:16
Control: Dilution water without test item tested under the same conditions as the test groups

Test organisms (species):

Daphnia magna

Details on test organisms:

Test System: Daphnia magna STRAUS (Clone 5)
Reason for selection: Daphnia magna STRAUS belongs, in accordance with the study guideline, to the suitable Daphnia types and are bred in the test facility.
Origin: Institut fur Wasser-, Boden- und Lufthygiene des Bundes- gesundheitsamtes, Corrensplatz, D-14195 Berlin, FRG
Breeder: Dr. U. Noack-Laboratorium
Culture: In 203 L glass vessels with approximately 1.8 L culture medium, at 21 °C (temperature of 20 - 25 °C were tolerated), in an incubator, 16 h illumination, illumination strength max. 20 μmolm2s-1 (corresponding to 1200 lx)
Culture medium: Culture medium Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
Feeding: 5 x weekly ad libitum with a mix of Scenedesmus subspicatus and Chlorella vulgaris, with an algae cell density of >10^6 cells.mL.
Number of study organisms per concentration and control: 20 animals, divided into 4 (2*) parallel samples, each with 5 (10*) animals
Number of animals per test vessel: 5 (10*)
Parallel samples per concentration: 4 (2*)
Age of test organisms: 2- 24 h old

* The information in brackets refer to the test of the reference item

Test type:

static

Total exposure duration:

48 h

Hardness:

Total hardness of 160 to 180 mg CaCO3/L

Test temperature:

Prior to test start, temperature of the dilution water were measured. At the beginning of the test, water parameters were measured in one additional replicate per concentration and control groups. After 48 hours the water parameters in the old media were measured in all replicates per concentration and control. The temperature was recorded throughout the test with a thermohygropgraph.

pH:

Prior to test start, pH value of the dilution water were measured. At the beginning of the test, water parameters were measured in one additional replicate per concentration and control groups. After 48 hours the water parameters in the old media were measured in all replicates per concentration and control.

Dissolved oxygen:

Prior to test start, dissolved oxygen of the dilution water were measured. At the beginning of the test, water parameters were measured in one additional replicate per concentration and control groups. After 48 hours the water parameters in the old media were measured in all replicates per concentration and control.

Nominal and measured concentrations:

The real test concentrations were calculated based on DOC analysis as:
62.4 - 26.9 - 14.0 - 7.0 - 3.3 mg/l

Details on test conditions:

A preliminary range finding test was conducted under static conditions with dilution levels of the test item ranging from 1:1 to 1:100, set up in a geometrical series with a factor of 10.
The definitive study was performed with 5 dilution levels ranging from 1:1 to 1:16 with a dilution factor of 2 under static conditions t enable the determination of immobilization after 24 hours and 48 hours. For the stock solution 100 mg/l were weighed out. The stock solution was stirred for 24 h. Thereafter the suspension was centrifuged (20 min, 300 rpm) to remove finally undissolved particles of the test item.

2 to 24 h old daphnids were used for the preliminary and definitive test. They had been obtained by removing the mother animals twice within 22 h. The daphnids had been acclimatized to the dilution water 2 h before the test started under test conditions.

20 daphnids divided into 4 (2*) groups with 5 (10*) animals per test vessel were used. Control replicates without test item were tested under the same conditions as the test groups. All test beakers were placed in an incubator (light.dark cycle 16/8 for 48 h at 20 +/- 1 °C).
In the preliminary and in the definitive test the immobilisation of the daphnids was observed after 24 and 48 hours of exposure.
The reference item, Potassium Dichromate p.a, was tested in five concentrations within the concentration range 0.59 to 5.8 mg/l.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate p.a (100% purity)

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

34.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

50.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 44.7 - 57.5

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 62.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

EC100 values were deduced directly from the dose-response relationship after 24 and 48 h. There was no mathematical calculation. The concentration effect relationship is shown graphically in a probability network.
EC10 and EC50 values were calculated via probit analysis. Probits according to WEBER (1986).
Calculation of the confidence intervals was carried out using standard procedures according to Breitig & Tumpling (1982).

Results with reference substance (positive control):

The EC50 value of the reference item at 1.4 mg/L after 24 h was within the prescribed concentration range of 1.0 - 2.5 mg/l according to AQS, DIN Guideline 38412 L 30.

DOC - Analysis

Dilution*	DOC (mg/L) 0 h	DOC (mg/L) 48h	DOC (mg/L) mean value	DOC (mg/L) mean value, corrected against DOC of control	Real Concentration (mg/L)**
1:1	45.97	42.80	44.39	42.94	62.4
1:2	19.11	20.73	19.92	18.47	26.9
1:4	11.36	10.80	11.08	9.63	14.0
1:8	6.49	6.08	6.29	4.84	7.0
1:16	3.82	3.61	3.72	2.27	3.3
Control	1.6	1.29	1.45	---	---

* Dilutions of the saturated solution

** calculated corresponding to a TOC of 0.6876 mg/mg test item

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study the 48h-EC50 was 50.7 mg/l based on measured concentrations (95% confidence interval between 5.9 and 12 mg/l).

Description of key information

The acute immobilisation of the test item to Daphnia Magna was determined. Under the conditions of the study the 48h-EC50 was 50.7 mg/l based on measured concentrations (95% confidence interval between 5.9 and 12 mg/l).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

50.7 mg/L

Additional information

The study was conducted under static conditions over a duration of 48 hours. Twenty test organisms were exposed to each test concentration and control. A saturated solution was prepared and diluted as follows: 1:1 - 1:2 - 1:4 - 1:8 - 1:16. The percentage immobility in the test item groups after 48h was respectively 70, 5, 0, 0 and 0%.

Samples for analytical confirmation of exposure concentrations were taken at the start (0h) and the end (48h) of exposure period. The test item was analytically verified via DOC analysis. The results showed that the concentrations remained stable over the 48 hour period. The mean value of the 0h and 24h results was corrected against the DOC of the control and used to calculate real test concentrations, which were 62.4 - 26.9 - 14.0 - 7.0 - 3.3 mg/L. The ECx values are based on these real concentrations.