Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-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:

04 - 08 Sep 2017

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)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD no. 23, ENV/JM/MONO(2000)6

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany (03.01.2017)

Analytical monitoring:

yes

Remarks:

GC-MS

Details on sampling:

- Concentrations: Control, 1.25, 2.50, 5.00, 10.0 and 20.0 mg/L (nominal) at 0 h (fresh media) and after 48 h (old media)
- Sampling method: At the start of the exposure (0 h), samples were taken after preparation of the test item concentrations and analyzed. At the end of exposure (48 h), samples of the old media were taken directly from the test vessels.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis (on an autosampler), if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test concentrations were prepared by preparation of a stock solution with a nominal concentration of 20 mg/L test item and four further dilution levels in a geometric series with a dilution factor of 2. The stock solution was prepared with dilution water 1 d before test start by stirring at approximately 1100 rpm for 24 h at room temperature. The stock solution was removed by siphoning from the approximate center of the water body after 1 h separation time.
- Controls: Dilution water without test item incubated under the same conditions as the test.
- Evidence of undissolved material: All concentration levels were visually clear throughout the exposure period.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Clone 5
- Age of daphnids at test start: < 24 h from a healthy stock
- Breeding conditions: In 2 - 3 L glass vessels with approximately 1.8 L culture medium (Elendt M4), at 20 ± 2 °C, in an incubator, 16 h illumination, light intensity of max 1500 lx
- Feeding during breeding: At least 5 times per week ad libitum with a mix of unicellular green algae (P. subcapitata and D. subspicatus at a cell density of > 10^6 cells/mL).
- Feeding during test: No feeding

ACCLIMATION
Not necessary because the composition of the dilution water was equivalent to the culture medium.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Juvenile daphnids were removed from the culture vessels at the latest 24 h before the start of the exposure and discarded. The juveniles born within the following period of max. 24 h preceding the exposure were used for the test. No first brood progeny was used for the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

Closed system without headspace to reduce the lost of test item

Hardness:

171 mg CaCO3/L (dilution water at 0 h)

Test temperature:

21.0 °C (dilution water at 0 h)

pH:

8.61 (dilution water at 0 h)
8.61 (control at 0 h), 7.36 - 7.53 (treatments at 0 h)
7.39 (control at 48 h), 7.51 - 7.60 (treatments at 48 h)

Dissolved oxygen:

9.10 mg O2/L (dilution water at 0 h)
9.10 mg O2/L (control at 0 h), 7.89 - 8.52 mg O2/L (treatments at 0 h)
8.39 mg O2/L (control at 48 h), 7.70 - 8.20 mg O2/L (treatments at 48 h)

Conductivity:

434 µS/cm (dilution water at 0 h)

Nominal and measured concentrations:

Control, 1.25, 2.50, 5.00, 10.0, and 20.0 mg/L (nominal)
< LOQ, 1.08, 2.12, 4.52, 8,39, and 17.1 mg/L (measured at 0 h)
< LOQ, 1.01, 1.92, 4.11, 8.15, and 17.1 mg/L (measured at 48 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw caps, filled with approximately 130 mL test volume, leaving no headspace
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Same composition as culture medium. The culture medium (Elendt M4), was prepared according to OECD 202, Annex 3 (2004) with a modified total water hardness of 160 - 180 mg CaCO3/L.
- Conductivity: 434 µS/cm (0 h)
- Culture medium different from test medium: Culture medium same as test medium.
- Intervals of water quality measurement: In dilution water, the pH, dissolved oxygen concentration, temperature, conductivity and total hardness were measured at the start of exposure (0 h). In test media, the pH value and dissolved oxygen concentration were measured in one additional replicate without daphnids at the start of exposure (0 h). At the end of exposure (48 h), the water quality parameters of the old media were measured in one appropriate replicate per concentration level and control, containing daphnids.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark cycle
- Light intensity: Diffuse light, light intensity max 1500 lx

EFFECT PARAMETERS MEASURED:
- Immobilization: After 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: Control, 1, 10, and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes. After 48 h, 100% mortality was recorded at the 100 mg/L treatment level and 80% at the 10 mg/L treatment level.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

8.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 4.52 - 17.1 mg/L

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All concentration levels were visually clear throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes, results were within the valid range of 0.6 - 2.1 mg/L laid down by the OECD guideline 202 (2004). The most recent test of the reference item dated from 03 - 04 Aug 2017.
- EC50 (24 h): 1.90 mg/L potassium dichromate (95% confidence limits: 1.43 - 2.69 mg/L)

Reported statistics and error estimates:

The EC10 and EC100 values were empirically derived from the observation data.
The EC50 values were calculated by sigmoidal dose-response regression. The respective 95% confidence limits were empirically derived from the observation data. All calculations were carried out from the best-fit values with the software GraphPad Prism.

EVALUATION OF THE EC10 AND EC50 VALUE AFTER 48 HOURS BASED ON THE INITIAL TEST ITEM CONCENTRATION MEASURED

Equation: Sigmoidal dose-response, variable slope
Y = Bottom + (Top-Bottom) / (1+10^((logEC50-X)*HillSlope))

Fitting Results:
Bottom = -1.34E-09, Top = 100.0, LogEC50 = 0.9193, HillSlope = 39.35; EC10 [mg/L] = 7.853; EC50 [mg/L] = 8.304
Goodness of fit:
Degrees of freedom: 16; R square: 0.977; Absolute sum of squares: 800.0; Stand. deviation of residuals Sy.x: 7.071

VALIDITY CRITERIA

The validity criteria of the test guideline 202 were fulfilled (Table 1).

Table 1: Validity criteria for OECD 202.

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.	In the control, no daphnids were immobilized or showed any signs of disease or stress.	Yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	The dissolved O2 concentration in the 48 h old media at test end was ≥ 7.70 mg/L in all concentration levels and in the control.	Yes

 

RESULTS

Immobilization results are presented in Table 2.

 

Table 2: Immobilization rates after 24 and 48 h of exposure in the definitive test.

Measured initial test item concentration [mg/L]	Immobilization [%]
	24 h					48 h
	replicates					replicates
	1	2	3	4	MV	1	2	3	4	MV
17.1	80	80	100	100	90	100	100	100	100	100
8.39	40	20	20	20	25	60	40	60	80	60
4.52	0	0	0	0	0	0	0	0	0	0
2.12	0	0	0	0	0	0	0	0	0	0
1.08	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

The guideline study resulted in an EC50 (48 h) of 8.30 mg/L for aquatic invertebrates (OECD 202, D. magna).

Executive summary:

The short-term toxicity of the test item to aquatic invertebrates was assessed according to OECD guideline 202 and GLP. In a static test using closed test vessels without headspace, Daphnia magna was exposed to five nominal test item concentrations ranging from 1.25 to 20.0 mg/L in a geometric series with a dilution factor of 2. Test item concentrations in all treatment levels were analytically verified in fresh media at the beginning of the test (0 h) and in aged media at the end of the test (48 h) by GC-MS. The measured test item concentrations in fresh media at 0 h ranged from 1.08 to 17.1 mg/L. The measured test item concentrations in aged media at 48 h ranged from 91 – 100% of the measured initial concentrations in the fresh media, indicating that test item concentrations were stable throughout the duration of the test. Therefore, all effect concentrations were based on the measured initial concentrations of the test item. After 48 h immobilization was observed and an EC50 (48 h) of 8.30 mg/L was obtained.

Description of key information

EC50 (48 h) = 8.30 mg/L (initial measured test item concentration, OECD 202, D. magna)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

8.3 mg/L

Additional information

There is one study available, in which the short-term toxicity of the test item to aquatic invertebrates was assessed according to OECD guideline 202 and GLP.

In a static test using closed test vessels without headspace, Daphnia magna was exposed to five nominal test item concentrations ranging from 1.25 to 20.0 mg/L in a geometric series with a dilution factor of 2. Test item concentrations in all treatment levels were analytically verified in fresh media at the beginning of the test (0 h) and in aged media at the end of the test (48 h) by GC-MS.

The measured test item concentrations in fresh media at 0 h ranged from 1.08 to 17.1 mg/L. The measured test item concentrations in aged media at 48 h ranged from 91 – 100% of the measured initial concentrations in the fresh media, indicating that test item concentrations were stable throughout the duration of the test. Therefore, all effect concentrations were based on the measured initial concentrations of the test item.

After 48 h immobilization was observed and an EC50 (48 h) of 8.30 mg/L was obtained.