Reaction mass of 1-(2,6,6-trimethylcyclohex-2-en-1-yl)hepta-1,6-dien-3-one and 1-(2,6,6-trimethylcyclohex-1-en-1-yl)hepta-1,6-dien-3-one

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a guideline study

Justification for type of information:

The acute Daphnia toxicity of Hexalon is based on read across from Galbascone. The documentation is presented in the Endpoint summary of Aquatic toxicity.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.4 mg/L

Basis for effect:

mobility

Remarks on result:

other: read-across from Galbascone

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 August - 15 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Hexalon.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

(2004)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test concentrations and the control according to the schedule below.

Frequency: At the start and the end of both 24-hour renewal periods.
Volume: 3.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.

At the end of each refreshment period, samples were taken from one replicate of each test concentration and the control.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

The batch of GALBASCONE tested was a clear colourless liquid and not completely soluble in test medium at loading rates above 3.2 mg/L. No correction was made for the composition of the test item.

All test solutions were prepared separately applying one day of magnetic stirring in a closed vessel followed by a stabilization period of one day in order to reach the maximum solubility of the test item in the test medium. The resulting clear and colourless solutions (concentrations up to and including 3.2 mg/L) and the aqueous mixtures (concentrations above 3.2 mg/L) were siphoned off. The final test solutions were all referred to as Water Accommodated Fractions (WAFs). Note that during the preparation of test solutions for the range-finding test the test item was not completely dissolved at a loading rate of 1.0 mg/L after stirring and stabilization.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus, 1820)
- Source: In-house laboratory culture with a known history, at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions. Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age at study initiation: young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
- Method of breeding: start of each batch with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium (M7) in an all-glass culture vessel. Renewal of the cultures: after 7 days of cultivation half of the medium twice a week.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

180 mg CaCO3/L

Test temperature:

20 °C

pH:

7.6 - 8.1

Dissolved oxygen:

8.5 - 9.3 mg O2/L

Nominal and measured concentrations:

Based on the results of the combined limit/range-finding test, EC50 was expected to range between WAFs prepared at loading rates of 1.0 and 10 mg/L, thus the following test concentrations were assigned to the definitive test:
Test concentrations: WAFs separately prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L
Mean measured test concentrations: 1.4, 1.7, 3.1, 5.6 and 11 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, air-tight closed; fill-volume: 60 mL
- Aeration: None
- 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: Adjusted ISO medium
- Culture medium different from test medium: Yes, M7

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h daily

EFFECT PARAMETERS MEASURED: mobility at 24 and 48 hours ; pH and dissolved oxygen at the start and the end of both renewal periods, for all concentrations and the control. Temperature continuously.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (September 2015)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 3.1 - 5.6 mg/L

Details on results:

Recovery: Measured concentrations ranged from 84 -111% of initial loading rates, except for the highest and the lowest concentration prepared for the first renewal period, where the concentrations were above the initial loading rates (127 and 170%, respectively)
Relative to initial [%] ranged from 90 - 109% (see table 3 below)

Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Results with reference substance (positive control):

48h-EC50: 0.39 mg/L.

Table 1: Measured concentrations

Time of sampling [hours]	Loading rate^1 [mg/l]	Concentration analysed [mg/l]	Relative to initial [%]	Relative to initial loading rate^2 [%]
0	0	n.d.
	1.0	1.70		170
	1.8	1.73		96
	3.2	3.49		109
	5.6	6.21		111
	10	12.7		127
24	0	n.d.
(Old)	1.0	1.81	107
	1.8	1.89	109
	3.2	3.35	96
	5.6	6.04	97
	10	12.3	96
24	0	n.d.
(Fresh)	1.0	1.08		108
	1.8	1.51		84
	3.2	2.78		87
	5.6	4.92		88
	10	10.7		107
48	0	n.d.
(Old)	1.0	0.970	90
	1.8	1.53	101
	3.2	2.81	101
	5.6	5.16	105
	10	10.1	95

1         A water accommodated fraction (WAF) prepared at the loading rate.

2         Calculated by reviewer

n.d.   Not detected

Table 2: Mean concentrations

GALBASCONE WAF prepared at x mg/L	Mean concentration (mg/L)		Mean concentration 0-48 h (mg/L)
	t=0-24 h	t=24-48 h
1.0	1.8	1.0	1.4
1.8	1.8	1.5	1.7
3.2	3.4	2.8	3.1
5.6	6.1	5.0	5.6
10	12	10	11

Table 3: Number of introduced daphnids and incidence of immobilityin the final test

Mean conc. GALBASCONE (mg/L)	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			number	Total %	number	Total %
Control	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
1.4	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
1.7	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
3.1	A  B C D	5 5 5 5	1 1 1 0	15	0 0 0 0	0
5.6	A  B C D	5 5 5 5	0 1 2 2	25	4 5 5 5	95
11	A  B C D	5 5 5 5	5 5 5 5	100	5 4 5 5	95

Vessel number A, B, C, D = replicate 1, 2, 3, 4

Validity criteria fulfilled:

yes

Remarks:

1) In the control, no daphnids became immobilised or showed other signs of disease or stress. 2) The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

Conclusions:

The 48h-EC50 value to Daphnia magna was 4.2 mg/L based on mean measured concentrations.

Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP under semi-static conditions.

Based on the results of a combined limit/range-finding test, the following nominal concentrations were tested in the definitive test: WAFs prepared separately at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L.

For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours in air-tight closed vessels. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24-hour renewal periods were analysed with a validated GC-FID method.

Measured concentrations ranged from 84 -111% of initial loading rates, except for the highest and the lowest concentration prepared for the first renewal period, where the concentrations were above the initial loading rates (127 and 170%, respectively). All test concentrations remained stable during the 24 -hour renewal periods (90 -109% of initial). Based on these results, the mean measured concentrations were calculated. The range tested based on mean measured concentrations was 1.4, 1.7, 3.1, 5.6 and 11 mg/L.

The 48h-EC50 value to Daphnia magna was 4.2 mg/L based on mean measured concentrations.

Description of key information

The short-term toxicity to aquatic invertebrates is assessed based on read-across from the close structural analogue Galbascone (CAS# 56973 -85 -4) and the EC50 value determined at 3.4 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.4 mg/L

Additional information

The acute Daphnia toxicity for Hexalon is based on read across from Galbascone (CAS# 56973 -85 -4). The read across justification is presented in the Endpoint summary on Aquatic toxicity. The executive summary of the test with Galbascone is presented below in the present section.

Galbascone and its acute toxicity for Daphnia

For Galbascone, a study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP under semi-static conditions. Based on the results of a combined limit/range-finding test, the following nominal concentrations were tested in the definitive test: WAFs prepared separately at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L. For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours in air-tight closed vessels. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24-hour renewal periods were analysed with a validated GC-FID method. Measured concentrations ranged from 84 -111% of initial loading rates, except for the highest and the lowest concentration prepared for the first renewal period, where the concentrations were above the initial loading rates (127 and 170%, respectively). All test concentrations remained stable during the 24 -hour renewal periods (90 -109% of initial). Based on these results, the mean measured concentrations were calculated. The range tested based on mean measured concentrations was 1.4, 1.7, 3.1, 5.6 and 11 mg/L. The 48h-EC50 value to Daphnia magna was 4.2 mg/L based on mean measured concentrations.