8-[4-(3,4-Difluorophenyl)-3-cyclohexene-1-yl)-1,4-dioxaspiro[4.5]decane

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

Study period:

March 1, 2003 - April 25, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 4, 1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

EEC, 1992
Annex to Commission Directive 92/69/EEC of 31st July 1992 adapting to technical progress for the seventeenth time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packing and labeling of dangerous substances
(Official Journal No. L 383 A, December 29th, 1992)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

8-[4-(3,4-difluorophenyl)cyclohex-3-en-1-yl]-1,4-dioxaspiro[4.5]decane

EC Number:

604-591-1

Cas Number:

147622-83-1

Molecular formula:

C20H24F2O2

IUPAC Name:

8-[4-(3,4-difluorophenyl)cyclohex-3-en-1-yl]-1,4-dioxaspiro[4.5]decane

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test medium (reconstituted water and test material) was freshly prepared. Therefore, the calibrated flask with test material and vehicle, reconstituted water, was treated in an ultrasonic device for 1 hour. Subsequently the preparation was stirred with a magnetic stirrer for further 23 hours. After 24 hours the formulation was given through a nutsch filter (pore size >10 - <16 µm) and a single use syringe filter (pore size 0.2 µm). The filtrate was used for the study.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

The solubility in water was examined in the “Institut für Zentrale Dienste-Analytik, Merck KGaA “ (SCHÖNIG H. J., 2003). The solubility of the test material Art. 272318 could not be determined but was < 0.024 mg/L in the aqueous vehicle using a HPLC method. Further refinement of the analytical method would be extremely cost- and time- consuming. Thus, no further analysis were performed during this study.

Test solutions

Vehicle:

yes

Details on test solutions:

Preparation of the Test Item:
The test medium (reconstituted water and test material) was freshly prepared. Therefore, the calibrated flask with test material and vehicle, reconstituted water, was treated in an ultrasonic device for 1 hour. Subsequently the preparation was stirred with a magnetic stirrer for further 23 hours. After 24 hours the formulation was given through a nutsch filter (pore size >10 - <16 µm) and a single use syringe filter (pore size 0.2 µm). The filtrate was used for the study.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna Straus
Culture conditions: The Daphnia magna were kept in reconstituted water in glass-vessels. The study was located in an air-conditioned room in the Institute of Toxicology. Lighting was controlled by a timer to provide a 16 hours light - 8 hours dark regime.
Temperature and humidity in the experimental room was measured using a thermo-hygrograph.
At the start of the experimental phase 5 Daphnia magna were introduced in each test vessel (25 ml, all-glass) containing 10 ml reconstituted water with or without test material. The vessels were open. The test medium was freshly prepared before the introduction of the young Daphnia magna. The Daphnia magna were not fed, and the test medium was not aerated during the test.
Feeding: The parental daphnids are fed ad libitum with unicellular green algae Desmodesmus subspicatus
Age: Offspring less than 24 hours old were used for the study.
Acclimation period: same as test

Feeding during test: None

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Test temperature:

20 - 21°C

pH:

7.92 - 7.97

Dissolved oxygen:

94.7 - 96.8%

Nominal and measured concentrations:

Nominal 100 mg/L

Details on test conditions:

EXPOSURE:
At the start of the experimental phase, 5 Daphnia magna were placed into 10 ml of reconstituted water (control group) or test medium (test material group). The Daphnia magna were not fed, and the control medium and test medium were not aerated during the test.

The test was performed as a static test in open vessels.

The duration of exposure was 48 hours. During the exposure period, the mobility of the daphnids was assessed daily, i.e. after 24 and 48 hours.

NO. OF DAPHNIDS:
Control Group: 20 daphnids
100 mg/L: 20 daphnids

CONCENTRATION(S)
In a pre-test no immobilization was observed at a concentration of 100 mg/L under open static conditions. Therefore, the solution of a nominal test item concentration of 100 mg/L was tested in the present study.

For the control, reconstituted water (ELENDT M4 medium) was used.

VEHICLE
Reconstituted water (ELENDT M4 medium) was used as vehicle.

Macro nutrients (mg/L)
CaCI2 •2H20 293.80
MgSO4•7H20 123.30
NaHCO3 64.80
KCI 5.80
Na2SiO3•9H20 10.00
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Trace elements (mg/L)
B 0.5000
Fe 0.2000
Mn 0.1000
Li, Rb and Sr 0.0500
Mo 0.0250
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003

Macro nutrients (mg/L)
Na2EDTA • 2H20 2.50

Vitamins (µg/L):
Thiamine 75.00
B12 1.00
Biotin 0.75

- pH: 7.97
- O2-Concentration: 96.8%
- Culture medium different from test medium: no
- Intervals of water quality measurement: 48 h


OTHER TEST CONDITIONS
- Photoperiod: 16h light / 8h dark

References:

ELENDT, B.-P. Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus.
Protoplasma 154, 25-33, 1990

Reference substance (positive control):

no

Remarks:

No positive control used in this study. The accuracy and reliability of the test method is demonstrated periodically as recommended by guidelines with potassium dichromate.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The 48 hour EC50 for the test material to Daphnia magna could not be quantified due to the absence of toxicity at the highest test concentration achievable in water. An aqueous filtrate of 0.1 g/L nominally revealed no aquatic toxicity in the test system.


The 48h EC50 exceeded the water solubility of <0.024 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

- Behavioural abnormalities: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Any other information on results incl. tables

Study Design

The objective of this study was to determine the acute toxicity of the test material in an open static test using Daphnia magna.

For this purpose, juvenile Daphnia magna were exposed to an aqueous test material solution over 48 hours, under defined conditions. The study comprised of four vessels per concentration containing five Daphnia magna each, i.e., 20 Daphnia per concentration (test medium group).

Daphnia magna were exposed to a test material concentration of nominal 100 mg/L (limit-test) in an open static system.

Results

Daphnia magna exposed to an aqueous preparation of the nominal concentration of 100 mg were not affected.

The analysis of the saturated aqueous preparation revealed that the water solubility of the test item was < 0.024 mg/L and could not be determined. At the start and at the end of the study, the test material concentration in the aqueous medium could not be quantified, since it was below the level of detection.

For the test material, the following nominal EC₅₀ values and no effect concentration for Daphnia magna were determined:

24 h EC₅₀      > 100 mg/L

48 h EC₅₀      > 100 mg/L

NOEC >100 mg/L

Conclusion

A saturated aqueous solution of the test item revealed no aquatic toxicity in this test system. The test item has a very poor solubility.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour EC50 for the test material to Daphnia magna could not be quantified due to the absence of toxicity at the highest test concentration achievable in water. An aqueous filtrate of 0.1 g/L nominally revealed no aquatic toxicity in the test system.

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 202. The 48 hour EC50 for the test material to Daphnia magna could not be quantified due to the absence of toxicity at the highest test concentration achievable in water. An aqueous filtrate of 0.1 g/L nominally revealed no aquatic toxicity in the test system.