4-(isopropyl)cyclohexyl propionate

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:

10 March 2017 - 03 May 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:

13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

30 May 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

Version / remarks:

14 December 2000

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Vapour pressure: 5.35 Pa at 24°C
- Solubility in water: 13.3 mg/L

Analytical monitoring:

yes

Details on sampling:

Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency: at t=0 h and t=24 h from the freshly prepared solutions; at t=24 h and t=48 h from the 24-hours old solutions
Volume: 4.0 mL from the approximate centre of the test vessels
Storage: Not applicable, samples were analysed on the day of sampling.

At the end of each refreshment period, the replicates were not pooled before sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Correction for purity: no correction for purity was made.
- Method: Direct application to the test medium.
The substance was a clear, colourless liquid and not completely soluble in test medium at the loading rate initially prepared. Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of approximately one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: test medium without test item or other additives.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, 1820
- Source: in-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock: 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: < 24 hours, from parental daphnids of more than two weeks old.
- Feeding of test organisms during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started 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 at 18-22 °C. Cultures were max. 4 weeks old. After 7 days of cultivation half of the medium was renewed twice a week.
- Feeding: daily, a suspension of fresh water algae.

Test type:

semi-static

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L

Test temperature:

20°C

pH:

Start of the test: 8.0-8.1
End of the test: 7.6-7.8

Dissolved oxygen:

Start of the test: 8.5-8.9 mg/L
End of the test: 8.1-9.0 mg/L

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56 and 100% of a saturated solution prepared at 100 mg/L
Average exposure concentrations: 1.2, 2.1, 3.7, 7.1 and 11 mg/L. For measured concentrations per sampling point see table 1 in 'Any other information on results incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, air-tight closed vessels with headspace reduced to minimum
- Fill volume: 60 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, adjusted ISO medium
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Culture medium different from test medium: yes, M7 was used as culture medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 14 hours photoperiod daily (the incubator containing the test vessels was incorrectly set to a 14 light:10 dark cycle during the final test instead of 16 light:8 dark. However, since the validity criteria were met, this deviation had no effect on the obtained test results).


EFFECT PARAMETERS MEASURED: mobility at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen at the beginning, after 24 hours and at the end of the test, in all concentrations and the control. Temperature of the medium: continuously in a temperature control vessel.

COMBINED LIMIT/RANGE-FINDING STUDY:
- Test concentrations: 1.0, 10 and 100% of SS prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: yes, the results of a combined limit/range-finding test showed an immobility effect of 85% at t=24 and 100% at t=48 in the highest test concentration (100% of a SS prepared at 100 mg/L). Therefore, the final effect concentration was expected to be between 10% and 100% of a SS prepared at 100 mg/L. Based on this expectation, the final test concentrations were chosen.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (February 2017)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95%-CI: 4.2-5.5 mg/L

Details on results:

- For details on incidence of immobility see table 2 in 'Any other information on results incl. tables'.
- In the highest two concentrations, several daphnids were trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording mobility. Microscopic observations showed that daphnids exposed to 7.1 mg/L had no test item attached to their bodies after 48 hours of exposure.
- No other abnormalities were observed in the control group or any of the test concentrations.
- The single immobile daphnia observed in the lowest test concentration was considered to be biologically not relevant. However, it interfered with determining a suitable model for the estimation of effect concentrations. Considering that no responses were observed at the subsequent test group, it was decided to remove the lowest test group from the statistical analysis. The remaining responses recorded in this test allowed for reliable determination of an EC50.

- Analyses of the samples taken from the old solutions at t=24 h and t=48 h were within 82-96% of the initial measured concentrations in the fresh solutions, indicating that the exposure concentrations remained stable during the 24 hours exposure periods. Nonetheless, the effect concentrations were calculated as the arithmetic means of the geometric mean measured concentrations, which can be considered as worst case.

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

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 48h-EC50: 0.48 mg/L, 95%-confidence interval: 0.43-0.53 mg/L
- Other: results fell within the historical range.

Reported statistics and error estimates:

The 24 and 48h-EC50-value were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations using the maximum likelihood estimation method. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Table 1 Measured concentrations

Test item %SS at 100 mg/L	Measured concentration (mg/L)				Average exposure concentration (mg/L)
	t=0h (fresh)	t=24h (old)	t=24h (fresh)	t=48h (old)
10	1.25	1.17	1.18	1.05	1.2
18	2.27	2.17	2.10	1.92	2.1
32	4.42	3.73	3.53	3.34	3.7
56	8.47	7.30	6.71	5.84	7.1
100	12.8	11.4	11.5	9.45	11

Table 2 Incidence of immobility (final test)

Time (h)	Replicate	Average exposure concentration (mg/L)
		Control	1.2	2.1	3.7	7.1	11
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	4	4
	Total introduced	20	20	20	20	19	19
24	A	0	1	0	0	0	5
	B	0	0	0	0	1 (2)	3 (3)
	C	0	0	0	0	2 (3)	5 (4)
	D	0	0	0	0	3	4 (4)
	Total immobilised	0	1	0	0	6	17
	Effect %	0	5	0	0	32	89
48	A	0	1	0	0	4	5
	B	0	0	0	0	5	5 (2)
	C	0	0	0	2	5	5 (1)
	D	0	0	0	1	4	4
	Total immobilised	0	1	0	3	18	19
	Effect %	0	5	0	15	95	100

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

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 (e.g., ≥8.1 mg/L).

Conclusions:

The 48h-EC50 of the substance towards Daphnia magna was 4.8 mg/L based on mean measured exposure 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 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: 10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 100 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 were taken from all concentrations at the start and the end of both 24-hour renewal periods and analysed with a validated GC-FID method.

Measured concentrations ranged from 82% to 96% of initial measured concentrations at the start of the 24 -hour renewal periods indicating that all test concentrations remained stable during the renewal periods. Based on these results, the mean measured concentrations were calculated to be 1.2, 2.1, 3.7, 7.1 and 11 mg/L. All acceptability criteria were met and the study was considered valid.

The 48h-EC50 value to Daphnia magna was 4.8 mg/L based on mean measured exposure concentrations.

Description of key information

A study was performed to assess the acute toxicity of the substance toDaphnia magna. The study was conducted in accordance with OECD Guideline 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: 10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 100 mg/L.For each concentration and a control group, twentyDaphnia magna(less than 24 hours old) were exposed for 48 hoursin air-tight closed vessels.The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours.Samples were taken from all concentrations atthe start and the end of both 24-hour renewal periods andanalysed with a validated GC-FID method.

Measured concentrations ranged from 82% to 96% of initial measured concentrations at the start of the 24 -hour renewal periods indicating that all test concentrations remained stable during the renewal periods.Based on these results, the mean measured concentrations were calculated to be 1.2, 2.1, 3.7, 7.1 and 11 mg/L.All acceptability criteria were met and the study was considered valid.

The 48h-EC50 value toDaphnia magnawas 4.8 mg/L based on mean measured exposure concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.8 mg/L