(R)-6-(isopropyl)-3-methylcyclohex-2-en-1-one

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

Study period:

05 - 07 Sep 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Only 3 concentrations tested, EC50 was calculated from EC0/EC100.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Directive 92/69/EEC (O.J. No. L383A, 29.12.92)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 25, 50, and 100 mg/L test item (nominal) at 0 and 48 h

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared to give the desired series of test concentrations. To achieve this, 120.2 mg of the test substance were added to 1 L of dilution water, treated with a dispersing device (ultra turrax) for 60 s and afterwards stirred for 24 h on a magnetic stirrer. Finally, undissolved particles of the test substance were removed by filtration.
- Controls: Yes
- Evidence of undissolved material: Undissovled particles of the test substance were removed by filtration.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Age of parental stock (mean and range, SD): A population of parthenogenetic females of synchronized age structure had been maintained since more than 15 y in the test facility under constant temperature conditions (20 ± 1 °C) and at a 16 h light/8 h dark photoperiod (< 1000 lux).
- Feeding during test: No
- Food type daphnia culture: Unicellular green algae (Scenedesmus subspicatus)
- Frequency: ad libitum

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The neonates are separated from their parent daphnia by filtration prior to the acute test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

15.1 °dH = 269.5 mg/L CaCO3 (dilution water)

Test temperature:

19.8 - 20 °C (48 h)

pH:

7.7 - 7.8 (48 h)

Dissolved oxygen:

8.2 - 8.6 mg/L (48 h)

Nominal and measured concentrations:

nominal: control, 25, 50, and 100 mg/L
measured: 94.9 - 98.8% of nominal (0 h) and 93.6 - 96.2% of nominal (48 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Material, size, headspace, fill volume: Material: glass; Size: 50 mL; Fill volume: 20 mL
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard reconstituted water (M4 medium), according to the recommendations of Bundegesundheitsamt Berlin, Germany.
- Culture medium different from test medium: No, the M4 medium was used for both the maintenance of the test animals as well as for the preparation of stock and test solutions of the test substance.
- Intervals of water quality measurement: After 48 h

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark
- Light intensity: < 1000 lux

EFFECT PARAMETERS MEASURED:
Immobilisation: after 24 and 48 h

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 24.1 - < 94.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

24.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

94.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Reported statistics and error estimates:

The EC50 was calculated in retrospect using the statistics program ToxRatPro Version 3.2.1 (Probit analysis and ANOVA).

ANALYTICAL RESULTS

The results are expressed in terms of nominal concentrations (at 24 h) and in terms of mean measured concentrations (at 48 h). Measured concentrations ranged from 94.9 - 98.8% of nominal values at 0 h, and from 93.6 - 96.2% of nominal values at 48 h, respectively.

BIOLOGICAL RESULTS

Since the EC50 value is not reported, the EC0 (48 h) = 24.1 mg/L and EC100 (48 h) = 94.3 mg/L can be used to calculate the geometric mean (47.7 mg/L), which can serve as an approximation of the EC50 (48 h) (Table 1).

Table 1. Analysis of the immobilization rates.

Time	EC0	EC100	Geometric mean EC0/EC100
[h]	[mg/L]	[mg/L]	[mg/L]
24	25	> 100	--
48	24.1	94.3	47.7

 

CALCULATION OF EC50

The EC50 was calculated in retrospect using the statistics program ToxRatPro Version 3.2.1 (Probit analysis and ANOVA) and the available raw data reported in the study. The program yielded the following effect concentration: EC50 (48 h) = 43.42 mg/L. However, it was not possible to perform the ANOVA and the results should be considered cautiously.

Validity criteria fulfilled:

not specified

Description of key information

24.1 mg/L < EC50 (48 h) < 94.3 mg/L (arithmetic mean measured, EU Method C.2, D. magna)

Additional information

There is one GLP study available investigating the acute toxicity of the test substance to aquatic invertebrates according to EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC, Part C, Method 2 (1992).

In a static test, Daphnia magna was exposed to the nominal test concentrations 25, 50, and 100 mg/L of test substance dissolved in water for 48 h. The test substance concentration was analytically verified at 0 h and 48 h by measuring total organic carbon (TOC) content.

The measured concentrations ranged from 94.9 – 98.8% of nominal values at 0 h and from 93.6 – 96.2% of nominal values at 48 h, respectively. Therefore, the test substance concentration was within ± 20% of the nominal concentration both at the beginning and end of the test, indicating that the test substance concentration was stable throughout the duration of the test. The results at 48 h are expressed in terms of mean measured concentrations.

The highest test concentration resulting in 0% immobilization after 48 h was 24.1 mg/L (EC0, 48 h) and the lowest test concentration resulting in 100% immobilization was 94.3 mg/L (EC100, 48 h). The EC0 (48 h) and EC100 (48 h) were used to calculate the geometric mean value (47.7 mg/L), which can serve as an approximation of the EC50 (48 h), according to the OECD guideline 202. The EC50 (48 h) value was calculated in retrospect using ToxRatPro Version 3.2.1. The calculated EC50 (48 h) was 43.42 mg/L. However, it is not possible to perform the ANOVA and therefore the value should be considered cautiously.