[2-(cyclohexyloxy)ethyl]benzene

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:

08 July 2016 - 16 August 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:

Samples for possible analysis were taken from all test concentrations and the control at the start and the end of both 24-hour renewal periods.
The glass wool used for the preparation of the saturated solution (SS) was retained for possible analysis of the residue.

Volume 2.0 mL from the approximate centre of the test vessels
Storage Samples were stored in a freezer (<= -15°C) until analysis.

At the end of both refreshment periods, samples were taken from one of the replicates of each concentration.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: saturated solution
The batch of Phenafleur tested was a clear colourless liquid with a purity of 98.51%. The test item was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixture was left to stabilize for approximately 1 hour whereafter the clear and colourless Saturated Solution (SS) was siphoned out through glass wool. The SS was used as the highest test concentration for the combined limit range-finding test and as a stock solution for the final test. All lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Controls: ISO-medium without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

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 a bout 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg CaCO3/L

Test temperature:

20-21°C

pH:

in fresh solutions: 7.6
in used solutions: 7.6-7.7

Dissolved oxygen:

in fresh solutions: 8.9-9.2
in used solutions: 8.4-9.1

Nominal and measured concentrations:

Based on the results of the combined limit/range-finding test, EC50 was expected to range between concentrations obtained in 1.0 and 10% of a saturated solution (SS) at 100 mg/L.
Therefore the following test concentrations were assigned to the definitive test:
Test concentrations: 0.46, 1.0, 2.2, 4.6, 10 and 22% of an SS prepared at a loading rate of 100 mg/L
Mean measured test concentrations:
* first renewal period: 0.097, 0.21, 0.47, 0.97, 2.5 and 5.6 mg/L (all concentrations remained stable, i.e. 81-101% of initial)
* second renewal period: 0.095, 0.21, 0.47, 0.98, 2.4 and 5.2 mg/L (all concentrations remained stable, i.e. 100-102% of initial)

EC50 values were expressed in terms of mean initial measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, air-tight closed
- Fill-volume: 60 mL
- Aeration: no
- Renewal rate of test solution (frequency): after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Other: intgroduction of daphnids within 35 minutes after preparation of the test solutions

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium (prepared in RO water)
- Culture medium different from test medium: yes (M7)
- Intervals of water quality measurement: pH and dissolved oxygen at the start and the end of both renewal periods, for all concentrations and the control. Temperature continuously.

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mobility at 24 and 48 hours

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100% of an SS prepared at a loading rate of 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.:

0.39 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 0.34-0.44 mg/L

Details on results:

- Behavioural abnormalities: none reported
- Other biological observations: none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: no

Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study (pH: 6.0-8.5, not varying by more than 1.5 unit; temperature: 18-22°C, constant within 2°C; oxygen: >=3 mg/L at the end of the test).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 48-h EC50
- Dose-response test: yes (0.10-1.8 mg potassium dichromate/L)
- 48-h EC50: 0.39 (95% CI: 0.33-0.44)
- Other: The historical ranges for the 48h-EC50 lie between 0.28 and 0.90 mg/L. The observed 48h-EC50 corresponds with this range.

Reported statistics and error estimates:

The 48h-EC50-value was calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (mean initial) using the maximum likelihood estimation method.

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

An overview of the statistics is attached under 'Illustration'.

 Table 1: Concentrations of the test item in test medium - final test

 

Time of sampling^1 [hours]	Percentage of SS^2 [%]	Analysed concentration [mg/L]	Relative to initial [%]
0	0	n.d.
	0.46	0.0972
	1.0	0.212
	2.2	0.469
	4.6	0.974
	10	2.46
	22	5.61
24 (old)	0	n.d.	n.a.
	0.46	0.0810	83
	1.0	0.208	98
	2.2	0.381	81
	4.6	0.943	97
	10	2.42	98
	22	5.68	101
24 (new)	0	n.d.
	0.46	0.0949
	1.0	0.211
	2.2	0.467
	4.6	0.975
	10	2.42
	22	5.17
48 (old)	0	n.d.	n.a.
	0.46	0.0971	102
	1.0	0.212	100
	2.2	0.473	101
	4.6	0.991	102
	10	2.41	100
	22	5.20	101

¹      Samples were stored in the freezer (≤ -15°C) until the day of analysis.

²      Percentage of a saturated solution prepared at a loading rate of 100 mg/L.

n.d. Not detected.

n.a. Not applicable.

 

Table 2: Exposure concentrations

 

Phenafleur %SS at 100 mg/L	Measured concentration (mg/L)		Mean initial concentration (mg/L)
	t=0h (fresh)	t=24h (fresh)
0.46	0.0972	0.0949	0.096
1.0	0.212	0.211	0.21
2.2	0.469	0.467	0.47
4.6	0.974	0.975	0.97
10	2.46	2.42	2.4
22	5.61	5.17	5.4

 

 

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

 

Time (h)	Replicate	Mean initial concentration Phenafleur (mg/L)
		Control	0.096	0.21	0.47	0.97	2.4	5.4
0	A	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20	20
24	A	0	0	0	0	4	5	5
	B	0	0	0	0	5	5	5
	C	0	0	0	0	5	5	5
	D	0	0	0	0	4	5	5
	Total immobilised	0	0	0	0	18	20	20
	Effect %	0	0	0	0	90	100	100
48	A	0	0	0	3	5	5	5
	B	0	0	0	4	5	5	5
	C	0	0	0	5	5	5	5
	D	0	1	0	4	5	5	5
	Total immobilised	0	1	0	16	20	20	20
	Effect %	0	5	0	80	100	100	100

 

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 in Daphnia magna was 0.39 mg/L (95% confidence interval between 0.34 and 0.44 mg/L) based on mean initial 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. An initial range-finding test performed at 1, 10 and 100 mg/l, showed toxicity starting at 10 mg/l after 24 h and starting at 1 mg/l after 48 h. Based on these results, the following nominal concentrations were tested in the definitive test: 0.46, 1.0, 2.2, 4.6, 10 and 22% of a saturated solution prepared at a loading rate of 100 mg/L using dilution. Dilution can be done because the substance is a mono-constituent and the toxicity is below 1 mg/l (OECD guidance 23, 2000). 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 HPLC-UV method. nalysis of the samples taken at the start of the first renewal period showed measured concentrations of 0.097, 0.21, 0.47, 0.97, 2.5 and 5.6 mg/L in 0.46, 1.0, 2.2, 4.6, 10 and 22% of the SS at 100 mg/L, respectively. All concentrations remained stable (81-101% of initial) during the first 24-hour renewal period. Analysis of the samples taken at the start of the second renewal period showed measured concentrations of 0.095, 0.21, 0.47, 0.98, 2.4 and 5.2 mg/L. All concentrations remained stable (100-102% of initial) during the second 24-hour renewal period. Given this result, the EC50 values were expressed in terms of mean initial exposure concentrations.The mean initial measured concentrations corresponding with 0.46, 1.0, 2.2, 4.6, 10 and 22% of a saturated solution prepared at a loading rate of 100 mg/L were 0.096, 0.21, 0.47, 0.97, 2.4 and 5.4 mg/L. The 48h-EC50 value in Daphnia magna was 0.39 mg/L based on mean initial 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 and GLP under semi-static conditions. An initial range-finding test performed at 1, 10 and 100 mg/l, showed toxicity starting at 10 mg/l after 24 h and starting at 1 mg/l after 48 h. Based on these results, the following nominal concentrations were tested in the definitive test: 0.46, 1.0, 2.2, 4.6, 10 and 22% of a saturated solution prepared at a loading rate of 100 mg/L using dilution. Dilution can be done because the substance is a mono-constituent and the toxicity is below 1 mg/l (OECD guidance 23, 2000). 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 HPLC-UV method. nalysis of the samples taken at the start of the first renewal period showed measured concentrations of 0.097, 0.21, 0.47, 0.97, 2.5 and 5.6 mg/L in 0.46, 1.0, 2.2, 4.6, 10 and 22% of the SS at 100 mg/L, respectively. All concentrations remained stable (81-101% of initial) during the first 24-hour renewal period. Analysis of the samples taken at the start of the second renewal period showed measured concentrations of 0.095, 0.21, 0.47, 0.98, 2.4 and 5.2 mg/L. All concentrations remained stable (100-102% of initial) during the second 24-hour renewal period. Given this result, the EC50 values were expressed in terms of mean initial exposure concentrations.The mean initial measured concentrations corresponding with 0.46, 1.0, 2.2, 4.6, 10 and 22% of a saturated solution prepared at a loading rate of 100 mg/L were 0.096, 0.21, 0.47, 0.97, 2.4 and 5.4 mg/L. The 48h-EC50 value in Daphnia magna was 0.39 mg/L based on mean initial measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.39 mg/L