4-(2-butenylidene)-3,5,5-trimethylcyclohex-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:

04 - 06 Jul 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: Guidance document on aquatic testing of difficult substances and mixtures. OECD series on testing and assessment no. 23

Version / remarks:

2000

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: All loading levels and the control at the start (0 h) and end of the test (48 h).
- Sampling method: At the start of exposure (0 h), samples were taken from freshly prepared WAFs. At the end of the exposure (48 h), samples were taken directly from the test vessels.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Five water accommodated fractions (WAFs) were prepared by adding an appropriate volume of the test item (considering the test item density of 0.9962) into 2000 mL dilution water in brown glass flasks, leaving a headspace of approximately 100 - 200 mL, and shaking for 24 h at 20 rpm at room temperature. After a separation phase of 1 h after shaking, the aqueous phase (WAF) was removed from the approximate center of the water body by siphoning.
- Controls: Dilution water without test item incubated under the same conditions as the test group.
- Evidence of undissolved material: All WAFs were visually clear throughout the exposure phase.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Clone 5
- Culture: In 2 - 3 L glass vessels with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination, light intensity of max 1500 lx
- Food type: Unicellular green algae (e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus)
- Amount: > 10^6 cells/mL
- Frequency: At least 5 times per week ad libitum
- Age of daphnids at the start of exposure: < 24 h
- Feeding during test: No

ACCLIMATION
Not necessary

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

Hardness:

174 mg CaCO3/L

Test temperature:

20.8 °C

pH:

Control: 7.47 (0 h), 6.75 - 7.15 (48 h)
Treatments: 7.88 - 7.89 (0 h), 7.23 - 7.50 (48 h)

Dissolved oxygen:

Control: 9.10 mg O2/L (0 h), 7.93 - 8.04 mg O2/L (48 h)
Treatments: 8.12 - 8.74 mg O2/L, 7.71 - 8.59 mg O2/L (48 h)

Conductivity:

434 µS/cm

Nominal and measured concentrations:

Control, 6.25, 12.5, 25.0, 50.0, and 100 mg/L (nominal)
< LOQ, 1.79, 4.90, 9.32, 17.7, and 31.5 mg/L (measured, 0 h)
< LOQ, 1.85, 4.91, 9.88, 17.7, and 39.1 mg/L (measured, 48 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sealed glass flasks (4.5 cm (ID) x 9.5 cm (H)) with screw caps, filled with a test volume of approximately 130 mL, without headspace.
- Type: Closed
- 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: Elendt M4, according to OECD 202
- Culture medium different from test medium: Culture medium same as test medium (dilution water)
- Intervals of water quality measurement: The water parameters (pH, dissolved oxygen, temperature, conductivity and total hardness) of the dilution water was measured prior to the start of exposure (0 h). At the start of exposure (0 h), the water quality parameters of the fresh test media (dilution water + test item) were measured in one additional replicate (without daphnids) per loading level and control. At the end of the exposure (48 h), the water qualtiy parameters were measured in all replicates (containing daphnids) per loading level and control.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark cycle
- Light intensity: Diffuse light, 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 loading level)
- Results used to determine the conditions for the definitive study: Yes, 100% immobilization at a nominal loading level of 100 mg/L after 48 h.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

15.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 14.2 - 18.0 mg/L

Details on results:

- Other biological observations: No
- Mortality of control: 0%
- Any observations that might cause a difference between measured and nominal values: All WAFs 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, the results fell within the valid range.
- Relevant effect levels: EC50 (24 h) = 1.31 mg/L
- Other: The reference item is tested monthly. The most recent test dated from 11 - 12 Jul 2017.

Reported statistics and error estimates:

The EL10 and EL50 values and the confidence intervals after 24 and 48 h were calculated by sigmoidal dose-response regression. The loading-effect relationships are shown graphically. The EC50 value for the reference item was calculated by sigmoidal dose-response regression. The respective 95% confidence limits for the EC50 were calculated from the standard error and the t-distribution. All calculations were carried out with GraphPad Prism5.

VALIDITY CRITERIA

 

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

 

Table 1: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the control containing the solubilising agent, not more that 10% of the daphnids should have been immobilized.	0% immobilization in the control.	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-hour old media at the end of the exposure intervals was ≥ 7.71 mg/L in the test vessels of all loading levels and the control.	Yes

 

 

ANLAYTICAL RESULTS

 

The measured concentrations for the test item at the start of the exposure were in the range of 1.79 to 31.5 mg/L and in the old media in the range of 1.85 to 39.1 mg/L.

 

Table 2. Measured concentrations of test item during the definitive test. Determination by a group evaluation of four major peaks of the test item via GC-MS.

		0 hours	48 hours
Nominal test item loading [mg/L]	Nominal sum of four major peak of the test item [mg/L]	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
100	51.3	31.5	39.1	124
50.0	25.7	17.7	17.7	100
25.0	12.8	9.32	9.88	106
12.5	6.41	4.90	4.91	100
6.25	3.21	1.79	1.85	103
Control		< LOQ	< LOQ

 Meas.conc.      = measured concentration of the four major peaks, dilution and enrichment factors taken into account

%                         = percent of the initial measured concentration of the four major peaks

LOQ                     = Limit of quantification (0.05 mg/L test item)

 

BIOLOGICAL RESULTS

 

Table 3. Immobilization Rates after 24 and 48 hours of exposure in the definitive test (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal loading level of the test item [mg/L]	Immobilization [%]
	24 h					48 h
	1	2	3	4	MV	1	2	3	4	MV
100	100	100	100	100	100	100	100	100	100	100
50.0	60	100	100	100	90	100	100	100	100	100
25.0	60	60	60	60	60	100	100	100	60	90
12.5	20	20	20	20	20	40	20	20	20	25
6.25	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:

Based on the outcome of the Immobilization Test, the EL50 (48 h) is 15.7 mg/L (nominal, OECD 202, D. magna).

Description of key information

EL50 (48 h) = 15.7 mg/L (nominal loading rate, OECD 202, D. magna)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

15.7 mg/L

Additional information

There is one study available, in which the toxicity of the substance to aquatic invertebrates was assessed according to the OECD guideline 202 and GLP.

The substance is a UVCB and five aqueous water accommodated fractions (WAFs) with nominal loading rates ranging from 3.25 to 100 mg/L in a geometric series with a separation factor of 2 were prepared by shaking each loading level for 24 h (20 rpm) and removing the aqueous phase (WAF) by siphoning from the center of the flask after a 1 h separation phase. Daphnia magna were exposed to these WAFs in a closed system without headspace for 48 h under static conditions. The concentrations of the test item in the test vessels were verified by GC-MS analysis of the four main constituents of the substance at the beginning (0 h) and end of exposure (48 h).

The measured concentrations were in the range of 1.79 to 31.5 mg/L in the fresh media (0 h) and ranged from 1.85 to 39.1 mg/L in the old media (48 h), indicating that the substance was stable for the duration of the test. After 48 h, immobilization was observed and an EL50 (48 h) of 15.7 mg/L (nominal loading rate) was determined.