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EC number: 211-112-6 | CAS number: 629-82-3
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Toxicity to aquatic plants other than algae
Administrative data
Link to relevant study record(s)
Description of key information
In order to overcome potential short comings with the analytical monitoring of the test concentration in previous tests, it was decided to repeat the algae test OECD 201 with a special focus on recovery rates of the test item in the exposure vessels.
In sum 6 different pretest were performed in order to verify the intended exposure concentrations in the range of close to 2 µg/L which corresponds to the water solubility in the algae media (1.68 -1.83 µg/L).
Despite of all reasonable efforts, it was not possible to keep the intended concentration in the exposure vessels under static conditions over a period of 72 hours.
However, to predict effects on plant organisms, it was decided to switch to a semi-static testing regime with Lemna (Lemna gibba, OECD 221) with a daily water exchange of the test medium and a close analytical monitoring of the intended test concentration.
In a new OECD 221 study according to GLP, Dioctyl ether was found not to inhibit the growth of the monocotyledonous aquatic plant Lemna gibba after 7-day exposure under semi-static conditions at a functional water solubility limit of 6 μg/L (nominally). The following effect values (geometric mean measured test item concentrations) were determined: The EC50-values for both, inhibition of the specific growth rate and inhibition of yield, based on frond numbers (ErC50) and dry weight (ErdwC50) were > 0.942 μg/L. The NOEC-values for all endpoints were 0.942 μg/L.
Key value for chemical safety assessment
- EC50 for freshwater plants:
- 0.942 µg/L
- EC10 or NOEC for freshwater plants:
- 0.942 µg/L
Additional information
Pre-Testing:
1 Summary
Several preliminary range finding tests in an open system with headspace and the 6th preliminary test in a closed system without headspace under static conditions over a period of 72 hours were conducted at the test facility. The cell density was determined after 0 and 72 hours, the inhibition of growth rate and yield was calculated. Specific analysis of the test media was carried via GC-MS. Details on the range of analysed samples and results of all tests are given below.
- 1st Preliminary Test: regular algae test without analytical determination (saturated solution)
- 2nd Preliminary Test: regular algae test with analytical determination (saturated solution)
- 3rd Preliminary Test: regular algae test with solvent
- 4th Preliminary Test: Test with solvent and coating phase
- 5th Preliminary Test: Test with solvent and multiple coating phases and adsorption to glass
- 6th Preliminary Test: Test with solvent and multiple coating phases and adsorption to glass, additional samples with algae (closed system, without headspace)
The effects of saturated solutions of Dioctyl ether on the growth rate of the green alga Pseudokirchneriella subcapitata were negligible or very low. Solutions of Dioctyl ether, prepared with different procedures, were not stable for a period of 72 hours, i.e. the exposure time in an alga toxicity test.
2 Preliminary Range Finding Tests
2.1 1st Range Finding Test (non-GLP)
Preliminary Test 0 to 72 hours, 3 dilution levels
Application from saturated solution (loading rate: 100 mg/L)
Solvent none
Coating phase none
Analytical determination none
Replicates 4 for the control, 2 per concentration level
Test method static procedure, open system
Study date May 28 to 31, 2019
The saturated solution were prepared 72 hours prior to the start of exposure with dilution water (TG OECD 201 medium). The test item was placed onto the surface of the dilution water with a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 72 ± 1 hours. After stirring the saturated solution was taken from the bottom of the glass flask. The Tyndall effect was negative. The results are presented in Table 1.
Table 1: Results of the 1st Range Finding Test (0 - 72 hours)
Saturated Solution [%] |
Growth Rate Inhibition [%] |
Yield Inhibition [%] |
100 |
-2 |
-13 |
10.0 |
0 |
1 |
1.00 |
0 |
-1 |
Negative values = growth stimulation
2.2 2nd Range Finding Test (non-GLP)
Preliminary Test 0 to 72 hours, 2 dilution levels
Application from saturated solution (loading rate: 200 mg/L)
Solvent none
Coating phase none
Analytical determination at the start and at the end of the exposure
Replicates 4 for the control, 2 per concentration level
Test method static procedure, open system
Study date July 22 to 25, 2019
The saturated solutions were prepared 72 hours prior to the start of exposure with dilution water (TG OECD 201 medium). The test item was placed onto the surface of the dilution water with a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 72 ± 1 hours. After stirring the saturated solution was taken from the bottom of the glass flask. The Tyndall effect was negative. The results are presented in Table 2 and Table 3.
Table 2: Results of the 2nd Range Finding Test (0 - 72 hours)
Saturated Solution [%] |
Growth Rate Inhibition [%] |
Yield Inhibition [%] |
100 |
8 |
30 |
10.0 |
4 |
14 |
Table 3: Measured Exposure Concentrations during the 2nd non-GLP Preliminary Range Finding Test
Determination of the test item via GC-MS
Sampling |
0 hours Start of the exposure |
72 hours End of the exposure |
Saturated solution |
Dioctyl ether |
|
Meas. conc. |
Meas. conc. |
|
100 |
1.83 |
< LCL |
10.0 |
0.152 |
< LCL |
Control |
< LCL |
< LCL |
LCL = Lowest Calibration Level (0.05 µg test item/L)
Meas. Conc. = measured concentration of the test item
2.3 3rd Range Finding Test (non-GLP)
Preliminary Test 0 to 72 hours, 2 concentration levels
Application from stock solution (3.00 µg/L)
Solvent acetone (0.05 mL/L)
Coating phase none
Analytical determination at the start, after 24 and 48 hours and at the end of the exposure
Replicates 4 for the control, 2 per concentration level
Test method static procedure, open system
Study date September 03 to 06, 2019
For the preparation of the test concentrations of the test item Dioctyl ether, appropriate stock solutions were prepared in Acetone. The stock solutions were prepared in measuring flasks (20 and 10 mL capacity) by weighing out appropriate amounts of the test item and transferring it into the measuring glass flasks. The stock solutions were agitated. The measuring glass flasks containing the stock solutions prepared with Acetone were closed and opened only for taking aliquots for preparation of the test concentrations.
The test concentrations were prepared by adding 0.05 mL/L of the appropriate stock solutions to the dilution water. These solutions were agitated and used for testing.
The results are presented in Table 4 and Table 5.
Table 4: Results of the 3rd Range Finding Test (0 - 72 hours)
Nominal test item concentration [µg/L] |
Growth Rate Inhibition [%] |
Yield Inhibition [%] |
3.00 |
1 |
6 |
0.300 |
2 |
13 |
Solvent control |
1 |
5 |
Table 5: Measured Exposure Concentrations during the 3rd non-GLP Preliminary Range Finding Test
Determination of the test item via GC-MS
Sampling date |
0 hours Start of the exposure |
24 hours (with algae) |
48 hours (with algae) |
72 hours End of the exposure |
|
Nominal test item concentration [µg/L] |
Dioctyl ether |
||||
Meas. Conc. [µg/L] |
Meas. Conc. [µg/L] |
Meas. Conc. [µg/L] |
Meas. Conc. [µg/L] |
||
3.00 |
1.68 |
0.0555 |
< LCL |
< LCL |
|
0.300 |
0.281 |
< LCL |
< LCL |
< LCL |
|
LCL = Lowest Calibration Level (0.05 µg test item/L)
Meas. Conc. = measured concentration of the test item
2.4 4th Range Finding Test (non-GLP)
Preliminary Test 0 to 72 hours, one concentration level and solvent control
Application from stock solution (3.00 µg/L)
Solvent acetone (0.05 mL/L)
Coating phase 24 hours
Analytical determination at the start and after 24 hours of exposure
Replicates 4 for the control, 2 per concentration level
Test method static procedure, open system
Study date October 15 to 18, 2019
For the preparation of the test concentrations of the test item Dioctyl ether, a stock solution was prepared in Acetone. The stock solution was prepared in a measuring flask (20 mL capacity) by weighing out appropriate amounts of the test item and transferring it into the measuring glass flask. The stock solution was agitated. The measuring glass flask containing the stock solution prepared with Acetone was closed and opened only for taking aliquots for preparation of the test concentration. The test concentration was prepared by adding 0.05 mL/L of the appropriate stock solutions to the dilution water. These solutions were agitated and used for testing.
A coating phase (saturation of the test container) was carried out. The test containers were pre-treated with the appropriate test solution for at least 24 hours under test conditions. Before the start of the exposure, the test containers were emptied and refilled with freshly prepared test solution. Additionally, all test vessels and pipette tips used in the test were pre-treated with the appropriate test solution for at least 1 hour before the start of the exposure.
The results are presented in Table 6 and Table 7.
Table 6: Results of the 4th Range Finding Test (0 - 72 hours)
Nominal test item concentration [µg/L] |
Growth Rate Inhibition [%] |
Yield Inhibition [%] |
3.00 |
3 |
14 |
Solvent control |
2 |
11 |
Table 7: Measured Exposure Concentrations during the 4th non-GLP Preliminary Range Finding Test
Determination of the test item via GC-MS
Sampling date |
0 hours Start of the exposure |
24 hours
|
|
|
Nominal test item concentration [µg/L] |
Dioctyl ether |
|||
Meas. Conc. [µg/L] |
Meas. Conc. [µg/L] |
|||
3.00 with algae |
2.36 |
< LCL |
||
3.00 without algae |
2.33 |
< LCL |
||
LCL = Lowest Calibration Level (0.05 µg test item/L)
Meas. Conc. = measured concentration of the test item
2.5 5th Range Finding Test (non-GLP)
Preliminary Test 3, 6 and 9 hours, one concentration level
Application from stock solution (3.00 µg/L)
Solvent acetone (0.05 mL/L)
Coating phase multiple coating phases
Analytical determination 3, 6 and 9 hours, adsorption to glass
Replicates 3 replicates
Test method static procedure, open system
Study date October 15 to 18, 2019
Study date January 07, 2020
For the preparation of the test concentration of the test item Dioctyl ether, a stock solution was prepared in Acetone. The stock solution was prepared in a measuring flask (20 mL capacity) by weighing appropriate amounts of the test item and transferring them into the measuring glass flask. The stock solution was agitated. The measuring glass flask containing the stock solution prepared with Acetone was closed and opened only for taking aliquots for preparation of the test concentration. The test concentration was prepared by adding 0.05 mL/L of the appropriate stock solutions to the dilution water (OECD medium). These solutions were agitated and used for testing. Additionally, all test vessels and pipette tips used in the test were pre-treated with the appropriate test solution for at least 3 hours before the start of the exposure.
Three test vessels were used for the test. A coating phase (saturation of the test container) was carried out. The test containers were pre-treated with the appropriate test solution for 3 hours under test conditions. The test solutions from all test vessels were analyzed. Then the containers were emptied. The adsorption of the first replicate was analyzed after 3 hours of coating. The replicates 2 and 3 were refilled with test solution and coated for further 3 hours. The replicate 3 was refilled with test solution and coated for further 3 hours. The duration of the coating phase was 3 hours for replicate 1, 6 hours for replicate 2 and 9 hours for replicate 3. Results are given in the table below. The test item concentration in the glass adsorption samples was below the detection limit.
Table 8: Measured Aqueous Concentrations during the 5th Preliminary Test without Algae
Determination of the test item via GC-MS
Sampling date |
3 hours |
6 hours |
9 hours |
|
Nominal test item [µg/L] |
Rep. |
Dioctyl ether |
||
Meas. Conc. [µg/L] |
Meas. Conc. [µg/L] |
Meas. Conc. [µg/L] |
||
3.00 |
1 |
0.810 |
- |
- |
2 |
0.669 |
0.725 |
- |
|
3 |
0.624 |
0.693 |
0.767 |
|
Control |
- |
< LOQ |
< LOQ |
< LOQ |
LOQ = Limit of quantification (0.25 µg test item/L)
Meas. Conc. = measured concentration of the test item
Rep. = Replicate
2.6 6th Range Finding Test (non-GLP)
Preliminary Test 3, 6 and 9 hours, one concentration level
Application from stock solution (3.00 µg/L)
Solvent acetone (0.05 mL/L)
Coating phase multiples coating phases
Analytical determination 3, 6 and 9 hours, adsorption to glass
Replicates 3 replicates
Test method static procedure, closed system
Study date February 04 to 07, 2020
Preparation of the solutions:
For the preparation of the test concentration of the test item Dioctyl ether, a stock solution was prepared in Acetone. The stock solution was prepared in a measuring flask (20 mL capacity) by weighing an appropriate amount of the test item and transferring it into the measuring glass flask. The stock solution was agitated. The measuring glass flask containing the stock solution prepared with Acetone was closed and opened only for taking aliquots for preparation of the test concentration. The test concentration was prepared by adding 0.05 mL/L of the appropriate stock solutions to the dilution water (OECD medium). These solutions were agitated and used for testing. Additionally, all test vessels and pipette tips used in the test were pre-treated with the appropriate test solution for at least 3 hours before the start of the exposure.
Sampling schedule:
Three test vessels were used for the test. A coating phase (saturation of the test container) was carried out. The test containers were pre-treated with the appropriate test solution for 3 hours under test conditions. The test solutions from all test vessels were analyzed. Then the containers were emptied. The adsorption of the first replicate was analyzed after 3 hours of coating. The replicates 2 and 3 were refilled with freshly prepared test solution and coated for further 3 hours. The replicate 3 was refilled with test solution and coated for further 3 hours. The duration of the coating phase was 3 hours for replicate 1, 6 hours for replicate 2 and 9 hours for replicate 3. Results are given in the table below. Additionally, the freshly prepared stock solutions were analyzed.
Analysis of aqueous solutions:
For the determination of the test item concentration in the
water phase, a defined quantity of the aqueous phase was transferred to
an extraction vial and was liquid extracted with cyclohexane. The
organic phase was subsequently analyzed via liquid injection with GC-MS
analytics.
Determination of glass adsorption:
For the determination of the test item amount on the glass surface of the 59 ml vials, the emptied vials were rinsed with ultra-pure water. After discarding the water, the glass wall was rinsed with 5 ml methanol and vortexed for 30 seconds. Since the methanol extract contained significant amount of water, liquid injection with GC-MS was not performed to avoid interferences with water. Thus, 100 µL of the methanol extract was transferred to a headspace vial containing 10 ml pure water and 10 % sodium chloride. The sample was transferred by the autosampler of the GC-MS system just before the start of the analytics to avoid sample losses through degradation or glass adsorption. Calibration standards were also transferred from a spiking solution in methanol to the headspace vials just before the start of the measurement. The samples were agitated and heated and analyzed via headspace-solid phase micro extraction-GC-MS/MS. The limit of quantification of this method was 5 µg/L, corresponding to a theoretical total amount of 25 ng test item in the 5 ml methanol extract. The maximum amount of the test item in 59 ml in the vials with a concentration of 3 µg/L is 177 ng. Thus, the limit of quantification for the glass adsorption samples was 14% of the total applied amount of the test item. Methanol as solvent to dilute adsorbed Dioctyl ether had been chosen because previous investigations (carried out by BASF) had proven its suitability.
Results:
The concentrations in the aqueous samples are given in Table 9. The objective of this test was to show the influence of the duration of the coating phase (3, 6 and 9 hours). All glass adsorption samples were below the detection limit. It can be stated that the test item is not lost by glass adsorption. There was no clear relation between the duration of the coating phase and the measured aqueous concentrations.
In a separate test with algae (Tab. 10), after a coating phase of {Bitte genaue Zeitdauer der Coating phase angeben} no Dioctyl ether could be measured after an exposure period of 72 hours .
Table 9: Measured Aqueous Concentrations during the 6th Preliminary Test without Algae
Determination of the test item via GC-MS
Sampling date |
3 hours |
6 hours |
9 hours |
|
Nominal test item [µg/L] |
Rep. |
Dioctyl ether |
||
Meas. Conc. [µg/L] |
Meas. Conc. [µg/L] |
Meas. Conc. [µg/L] |
||
3.00 |
1 |
1.68 |
- |
- |
2 |
1.50 |
2.13 |
- |
|
3 |
1.35 |
1.88 |
1.94 |
|
SL |
1.94 |
2.20 |
2.00 |
|
Control |
- |
< LOQ |
< LOQ |
< LOQ |
LOQ = Limit of quantification (0.5 µg test item/L)
Meas. Conc. = measured concentration of the test item
Rep. = Replicate
SL = Stock solution
Table 10: Measured Concentrations during the 6th Preliminary Range Finding Test with Algae
Determination of the test item via GC-MS
Sampling |
0 hours Start of the exposure |
72 hours End of the exposure |
||
Nominal test item concentration |
Dioctyl ether |
|||
Meas. conc. |
% |
Meas. conc. |
% |
|
3.00 |
2.81 |
94 |
< LOQ |
|
0.300 |
0.349 |
119 |
< LOQ |
|
Control |
< LOQ |
< LOQ |
% = Percent of the nominal test item concentration
LOQ = Limit of quantification (0.5 µg test item/L)
Meas. Conc. = measured concentration of the test item
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