[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl acetate

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:

17 April 2016 - 21 July 2016

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:

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 items and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control
- Sampling method:
Frequency: At the start and the end of both 24-hour renewal periods
Volume: 4.0 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis: Not applicable, samples were analysed on the day of sampling

At the end of each renewal 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
- Method: water accommodated fraction (WAF)

The batch of Cedryl Acetate tested was a clear pale yellow to yellow liquid with a purity of 53.6% and the test item was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.

All test concentrations were prepared separately. Loading rates ranging from 1.0 to 100 mg/L were magnetically stirred in closed vessels for two days to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for 1-1½ hours whereafter the WAF was siphoned out through glass wool and used for testing.
The final test solutions were all clear and colourless.

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.
- Validity of batch: 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.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

no

Hardness:

180 mg CaCO3/L

Test temperature:

20-21°C

pH:

7.6-7.9

Dissolved oxygen:

in fresh solutions (t=0, t=24): 8.4-9.0 mg O2/L
in used solutions (t=24, t=48): 8.1-8.5 mg O2/L

Nominal and measured concentrations:

Based on the results of the combined limit/range-finding test, the EC50 was expected to be in the range of a WAF prepared at loading rates of 10 mg/L, thus the following test concentrations were assigned to the definitive test:
Final test 2 - WAFs prepared separately at loading rates of 2.2, 4.6, 10, 22 and 46 mg/L
Final test 2 - mean measured concentrations: 0.2, 0.35, 0.63, 1.4 and 1.7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, air-tight closed; fill-volume: 60 mL
- Aeration: None
- Renewal rate of test solution: every 24 hours
- 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.

RANGE-FINDING STUDY
- Test concentrations: WAFs prepared separately at loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate (July 2016)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 0.20 - 0.63 mg/L

Details on results:

Recovery: In final test 2, measured concentrations at the end of the renewal periods ranged from 69-111% relative to initial analysed concentrations, except for the 4.6 mg/L and 22 mg/L concentrations prepared for the second renewal period, where the concentrations were above and much below, relative to the initial analysed concentrations (129 and 26%, respectively). (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):

The 48h-EC50 was within the expected range of 0.30 to 1.0 mg/L, to be specific between 0.32 and 0.56 mg/L. 100% effect was observed at 1.0 mg/L.

A range-finding test and two final test were performed.

Final test 1 was rejected because analytical results were not acceptable at 48 hours, thus only data from Final test 2 (same test set-up as Final test 1) are presented.

Table 1: Number of introduced daphnids and incidence of immobility in Final test 2

Time (h)	Replicate	Cedryl Acetate, WAF at x mg/L
		Control (-)	2.2 (0.20)	4.6 (0.35)	10 (0.63)	22 (1.4)	46 (1.7)
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	2	4	4 [2]	5
	B	0	0	0 [2]	4 [2]	5	5
	C	0	0	0	4 [5]	5 [2]	5
	D	0	0	1	5 [1]	5	5 [5]
	Total immobilised	0	0	3	17	19	20
	Effect %	0	0	15	85	95	100
48	A	0	0	2 [2]	5	5	5
	B	0	0	2	5	5	5
	C	0	0	3 [3]	5	5	5
	D	0	0	4	5	5	5
	Total immobilised	0	0	11	20	20	20
	Effect %	0	0	55	100	100	100

[ ]: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

( ): Mean concentration (mg/L)

(-): Not detected

Table 2: Mean concentrations

Cedryl Acetate, WAF prepared at x mg/L	Mean measured concentration (mg/L)		Mean concentration (mg/L)
	t = 0 – 24 h	t = 24 – 48 h	t = 0 – 48 h
2.2	0.230	0.175	0.20
4.6	0.489	0.209	0.35
10	0.691	0.576	0.63
22	0.814	1.90	1.4
46	2.80	0.699	1.7

Table 3: Measured concentrations

Time of sampling	Loading rate^1	Concentration analysed	Relative to initial
[hours]	[mg/L]	[mg/L]	[%]
0	0	n.d.
	2.2	0.277
	4.6	0.569
	10	0.805
	22	0.943
	46	3.19
24 (old)	0	n.d.	n.a.
	2.2	0.191	69
	4.6	0.420	74
	10	0.593	74
	22	0.703	75
	46	2.46	77
24 (fresh)	0	n.d.
	2.2	0.179
	4.6	0.184
	10	0.575
	22	3.69
	46	0.665
48 (old)	0	n.d.	n.a.
	2.2	0.172	96
	4.6	0.237	129
	10	0.577	100
	22	0.975	26
	46	0.735	111

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

n.d. Not detected.

n.a. Not applicable.

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 determined for Daphnia magna was 0.33 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 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 2.2, 4.6, 10, 22 and 46 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 at the end of the renewal periods ranged from 69-111% relative to initial analysed concentrations, except for the 4.6 mg/L and 22 mg/L concentrations prepared for the second renewal period, where the concentrations were above and much below, relative to the initial analysed concentrations (129 and 26%, respectively). Based on these results, the mean measured concentrations were calculated. The range tested based on mean concentrations was 0.2, 0.35, 0.63, 1.4 and 1.7 mg/L.

The 48h-EC50 value determined for Daphnia magna was 0.33 mg/L based on mean measured concentrations.

Description of key information

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 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 2.2, 4.6, 10, 22 and 46 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. 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 at the end of the renewal periods ranged from 69-111% relative to initial analysed concentrations, except for the 4.6 mg/L and 22 mg/L concentrations prepared for the second renewal period, where the concentrations were above and much below, relative to the initial analysed concentrations (129 and 26%, respectively).

Based on these results, the mean measured concentrations were calculated. The range tested based on mean concentrations was 0.2, 0.35, 0.63, 1.4 and 1.7 mg/L.

The 48h-EC50 value determined for Daphnia magna was 0.33 mg/L based on mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.33 mg/L