(E)-1-(2,4,4-trimethyl-2-cyclohexen-1-yl)-2-buten-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:

21 - 23 Nov 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

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany (03 Jan 2017)

Analytical monitoring:

yes

Remarks:

GC-MS

Details on sampling:

- Concentrations: All concentration levels and the control in fresh (0 and 24 h) and aged media (24 and 48 h)
- Sampling method: Fresh media were sampled after preparation of the test item concentration. Aged media were sampled 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 analysis (on an autosampler), if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 3.0 mg/L stock solution was prepared with dilution water (culture medium) 1 d prior to the start of the exposure (at -24 h) and 1 d prior to the renewal of the test solutions (at 0 h). The stock solution was prepared in a tightly closed glass flask with nearly no headspace and was mixed by constant stirring with a magnetic stirrer at approximately 1100 rpm for 24 h at room temperature. The 5 test item concentrations in a geometric series with a separation factor of 2.2 were prepared by dilution of the stock solution with dilution water.
- Controls: Dilution water without test item
- Evidence of undissolved material: All tested 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 parental stock at test start: < 24 h. 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.
- Culture conditions: In 2-3 L glass vessels filled with approx. 1.8 L culture medium (Elendt M4) at 20 ± 2 °C in an incubator, 16 h illumination with a light intensity of max. 1500 lx
- Feeding of culture stocks: At least 5 times per week ad libitum with a mix of unicellular green algae (e.g. P. subcapitata and D. subspicatus) with an algae cell density of > 10^6 cells/mL.
- Feeding during test: No feeding during the test.

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

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

160 - 180 mg CaCO3/L (dilution water with modified total water hardness)

Test temperature:

Dilution water: 20.0 °C (0 h), 20.4 °C (24 h)

pH:

0 h: 7.69 (control), 7.70 - 7.81 (treatments)
48 h: 7.39 (control), 7.48 - 7.55 (treatments)

Dissolved oxygen:

0 h: 9.02 mg/L (control), 8.45 - 8.94 mg/L (treatments)
48 h: 8.34 mg/L (control), 7.79 - 8.28 mg/L (treatments)

Conductivity:

0 h: 450 µS/cm (dilution water)
48 h: 497 µS/cm (dilution water)

Nominal and measured concentrations:

Control, 0.128, 0.282, 0.620, 1.36, and 3.00 mg/L (nominal)
< LOQ, 0.134, 0.277, 0.580, 1.31, and 2.89 mg/L (geometric mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw, filled to the top with test solution (approximately 130 mL).
- Renewal rate of test solution: 24 h
- 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: Culture medium Elendt M4, according to guideline
- pH: 8.2 ± 0.8
- Culture medium different from test medium: Culture medium same as test medium.
- Intervals of water quality measurement: Water quality was measured in dilution water and test media. In dilution water, pH, dissolved oxygen, temperature, conductivity and total hardness were measured at test start (at 0 h) and at renewal (24 h). In test media, pH and dissolved oxygen were measured at the start and at renewal (0 and 24 h) in one additional replicate without daphnids per concentration level and control.

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

EFFECT PARAMETERS MEASURED:
- Immobilization: Recorded after 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: Control, 0.5, 5, and 50 mg/L
- Results used to determine the conditions for the definitive study: After 24 h, 30% mortality in 0.5 mg/L treatment and 100% mortality in 5 and 50 mg/L treatments. After 48 h, 55% mortality in 0.5 mg/L treatment.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

95% confidence interval 1.51 - 2.86 mg/L

Details on results:

- Behavioural abnormalities: No
- Other biological observations: No immobility or any adverse effects were observed at the concentration levels 0.128 and 0.282 mg/L and in the control.
- Mortality of control: 0%
- Any observations that might cause a difference between measured and nominal values: All tested 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 defined by the OECD guideline.
- EC50 (24 h): 2.10 mg/L potassium dichromate, 95% confidence interval 1.92 - 2.43 mg/L
- Other: The cited reference test dates from 02 - 03 Nov 2017

Reported statistics and error estimates:

The EC10 and EC50 values were calculated by sigmoidal dose-response regression using GraphPad Prism. The 95% confidence limits were calculated from the standard error and the t-distribution.

EVALUATION OF THE EC50 48 HOURS BASED ON THE GEOMETRIC MEAN MEASURED CONCENTRATIONS OF THE TEST ITEM

Equation:
Sigmoidal dose-response, variable slope
Y = Bottom + (Top-Bottom)/(1+10^((log EC50-X)*HillSlope))

Fitting results
Bottom: 2.752, Top: 19760, logEC50: 1.224, HillSlope: 3.053, EC10: 1.255, EC50: 2.320

Goodness of Fit
Degrees of freedom: 16; R square: 0.9512; Absolute Sum of Squares: 1339; Sy.x: 9.147

VALIDITY CRITERIA

The study fulfilled the validity criteria laid down by the test guideline (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.	0% immobilization and no signs of disease or stress 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 concentrations in the 24 h old media were ≥ 7.62 mg/L in all concentration levels and the control.	Yes

 

ANALYTICAL RESULTS

The measured concentrations in fresh media (0 and 24 h) ranged from 94 to 137% of the nominal values. The measured concentration in aged media (24 and 48 h) ranged from 86 to 101% of the nominal concentrations (Table 2).

 

Table 2. Measured concentrations of the test item during the definitive test.

Nominal test item concentration [mg/L]	fresh media 0 h		old media 24 h		fresh media 24 h		old media 48 h		Geometric mean measured test item concentration [mg/L]
	meas. conc. [mg/L]	%	meas. conc. [mg/L]	%	meas. conc. [mg/L]	%	meas. conc. [mg/L]	%
3.00	3.09	103	2.83	94	2.86	95	2.79	93	2.89
1.36	1.41	104	1.23	91	1.34	98	1.26	93	1.31
0.620	0.614	99	0.560	90	0.583	94	0.563	91	0.580
0.282	0.312	111	0.272	97	0.264	94	0.261	93	0.277
0.128	0.130	102	0.110	86	0.176^1)	137	0.130	101	0.134
Control	< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item

%  = percentage of the nominal test item concentration

LOQ = limit of quantification of the analytical method (0.010 mg/L test item)

1) = reanalyzed

 

BIOLOGICAL RESULTS

The effect values were based on the geometric mean measured concentrations of the test item (Table 3).

The EC10 (48 h) was 1.26 mg/L with 95% confidence limits from 0.483 - 2.82 mg/L. The EC100 (48 h) was > 2.89 mg/L.

Table 3. Immobilization after 24 and 48 h of exposure (n = 20, divided into 4 replicates with 5 daphnids each).

Geometric mean measured test item concentration [mg/L]	immobilization [%]
	24 h					48 h
	replicates					replicates
	1	2	3	4	MV	1	2	3	4	MV
2.89	100	60	100	80	85	100	80	100	100	95
1.31	20	0	0	0	5	20	0	0	20	10
0.580	0	0	0	0	0	20	20	0	0	10
0.277	0	0	0	0	0	0	0	0	0	0
0.134	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 test for the short-term toxicity of the substance to aquatic invertebrates resulted in an EC50 (48 h) of 2.32 mg/L (geometric mean measured concentration, OECD 202, D. magna).

Executive summary:

The short-term toxicity of the substance to aquatic invertebrates was investigated according to OECD guideline 202 and GLP. In a semi-static test with a 24 h renewal rate, Daphnia magna was exposed to five, nominal concentration levels ranging from 0.128 to 1.35 mg/L in a geometric series with a separation factor of 2.2 and a control in sealed glass flasks for 48 h. The test item concentrations in the test solutions were analytically verified by GC-MS in fresh media (0 and 24 h) and aged media (24 and 48 h). The measured test item concentrations in fresh media ranged from 94 to 137% of the nominal values and from 86 to 101% in aged media. Thus, effect values were based on the geometric mean measured test item concentrations, which were 0.134, 0.277, 0.580, 1.31, and 2.89 mg/L. After 48 h, immobilization was observed in the 0.580, 1.31, and 2.89 mg/L treatments and no immobilization or any other adverse effects were recorded in the lowest treatment levels (0.134 and 0.277 mg/L) and the control. The derived EC50 (48 h) value was 2.32 mg/L.

Description of key information

EC50 (48 h) = 2.32 mg/L (geometric mean measured, OECD 202, D. magna)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.32 mg/L

Additional information

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

In a semi-static test with a 24 h renewal rate, Daphnia magna was exposed to five, nominal concentration levels ranging from 0.128 to 1.35 mg/L in a geometric series with a separation factor of 2.2 and a control in sealed glass flasks for 48 h. The test item concentrations in the test solutions were analytically verified by GC-MS in fresh media (0 and 24 h) and aged media (24 and 48 h).

The measured test item concentrations in fresh media ranged from 94 to 137% of the nominal values and from 86 to 101% in aged media. Thus, effect values were based on the geometric mean measured test item concentrations, which were 0.134, 0.277, 0.580, 1.31, and 2.89 mg/L.

After 48 h, immobilization was observed in the 0.580, 1.31, and 2.89 mg/L treatments and no immobilization or any other adverse effects were recorded in the lowest treatment levels (0.134 and 0.277 mg/L) and the control. The derived EC50 (48 h) value was 2.32 mg/L.