Butyl ethyl ether

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-03 to 2017-11-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

23 March 2006

Deviations:

yes

Remarks:

sealed test vessels of 120 mL serum glass bottles, addition of NaHCO3 to the OECD standard medium (300 mg/L)

Qualifier:

according to guideline

Guideline:

other: ISO 14442

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lenzing AG, 60930023
- Expiration date of the lot/batch: 30 September 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at room temperature in a dark and dry place in tightly closed receptacles
- Stability under test conditions: Stability of reference item, prepared in 50% Methanol/Medium (v/v), was established during validation.
- Solubility and stability of the test substance in the solvent/vehicle: Up to a nominal loading rate of 1000 mg/L no undissolved material was observed after shaking.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: direct addition of test substance with immediate sealing of the flask and shaking for approximately 24 hours
- Preliminary purification step (if any): no

Analytical monitoring:

yes

Details on sampling:

Substance specific analysis was performed at the test site of biochemA GmbH.
- Concentrations (Nominal loading rate): 0.0 (Sample code: NC - negative control); 69.1 (Sample code: A); 432.0 (Sample code: C); 2700.0 (Sample code: E); (Sample code: B) mg/L
- Sampling method: Samples of 2.0 mL were taken from the additional vessels of treatment NC, A, C and E at 0 h, 24 h, 48 h and 72 h in duplicate and stabilized with 2 mL Methanol. At 72 h additionally samples of NC, A, C and E from test vessels with algae were taken.
- Sample storage conditions before analysis: The samples were stored in the freezer at ≤ -18 °C until hand-over to the analytical laboratory.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct addition of test substance with immediate sealing of the flask and shaking for approximately 24 hours was used to obtain maximum solubility and to reduce loss of test substance through volatilization.
The pipetting scheme shown in "Table 1: Preparation of the test treatment and control" (refer to "Any other information on materials and methods incl. tables) describes the preparation of the test vessels and controls.
The volume of algal inoculum was added to the water saturated fraction in one batch. For homogeneous distribution on replicates the solutions were mixed by a magnetic stirrer. All test vessels were filled up to the brim to avoid headspace while sealing with crimp caps. The controls were treated the same way. The additional vessels for chemical analysis without algae were treated the same way.
Modified algal medium was added instead of algal inoculum suspension.
- Differential loading (nominal concentrations): 0.0 (Sample code: NC), 69.1 (Sample code: A), 172.8 (Sample code: B), 432.0 (Sample code: C), 1080.0 (Sample code: D), 2700.0 (Sample code: E) mg/L.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Up to a nominal loading rate of 1000 mg/L no undissolved material was observed after shaking.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Culture Collection of Algae at the University of Goettingen
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: Twice a week, the stock suspension is diluted into fresh Holm-Hansen medium under axenic conditions to keep it in exponential growth.

ACCLIMATION
- Acclimation period: Four days before starting the test, 0.86 mL of the algae stock suspension was diluted into 49.14 mL algal growth medium (according to OECD 201) to obtain a starting concentration of 0.5 x 10^5 algal cells per mL. They were incubated at 22.8 – 23.2°C and 82.9 μE/m² s +/- 3.8% (PAR). After 4 days, the cell concentration was determined using the Coulter Counter Z2.
- Culturing media and conditions (same as test or not): algal growth medium according to OECD 201
- Any deformed or abnormal cells observed: not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.6 - 23.3 °C.

pH:

approximately 8.45 (at 0 h) and 8.63 (after 72 h)
See also Table 2: "pH-values measured at the beginning and at the end of the test" in "Any other informations on materials and methods".

Nominal and measured concentrations:

Nominal loading rate in the test vessel [mg/L]: 0, 69.1, 172.8, 432, 1080, 2700
mean measured test concentration [mg/L]: 0, 52.7, 255.0, 1650.0

See also Table 3: Results of the accompanying chemical analysis" in "Any other informations on materials and methods".

Details on test conditions:

TEST SYSTEM
- Test vessel: sealed test vessels of 120 mL serum glass bottles
- Type (delete if not applicable): closed with crimp caps (Alucrimp Cap with inlaid Septum of Silicone/PTFE)
- Material, size, headspace, fill volume: All test vessels were filled up to the brim to avoid headspace while sealing with crimp caps.
- Aeration: not reported
- Initial cells density: concentration of 0.5 x 10^5 algal cells/mL
- Control end cells density: 4.039 x 10^5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: Modified algal medium was added instead of algal inoculum suspension. Addition of NaHCO3 to the OECD standard medium (300 mg/L).

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: continuous lightning
- Light intensity and quality: 64.0 μE/m² s +/- 1.2 %


EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Chlorophyll measurement: The algal growth was determined by measuring the chlorophyll fluorescence as a measure for algal biomass. At the start of the test and after 24, 48 and 72 hours, 200 μL samples were taken out of each test vessel and chlorophyll fluorescence was measured in a 96-well microplate for each sample using a fluorescence micro-plate reader (excitation wave length: 465 nm, emission wave length: 670 nm). The measured chlorophyll fluorescence is converted into cell counts per mL by means of a correlation factor. This correlation factor is determined twice a year during quality check by measuring different cell densities with micro-plate fluorescence reader and coulter counter and subsequent calculation of a calibration curve. The algal count was determined for a cell size between 3 and 9 μm using a Coulter Counter Z2.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: Yes: Direct addition of the test substance into the test medium without sealing resulted in high loss of substance due to the volatile property of the test substance. It was also not possible to dissolve the test material in the test solution without shaking for several hours. The test solution with a nominal loading rate of 4000 mg/L resulted in a considerable amount of non-dissolved test material, visible as droplets on the surface of the test medium. The clear water saturated fraction obtained from the 1000 mg/L loading rate had strong evaporation of the test substance after incubation in the standard test vessels.
Therefore direct addition of test substance with immediate sealing of the flask and shaking for approximately 24 hours was used to obtain maximum solubility and to reduce loss of test substance through volatilization. Up to a nominal loading rate of 1000 mg/L no undissolved material was observed after shaking.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

309.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 260.7-367.1 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

190.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 165.8-219.7 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

148.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 127.6-171.9 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 69.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 69.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

127.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 120.8-134.7 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 59.3-64.8 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

42.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 40.5-44.5 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 69.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 69.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

175.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL: 124.1-247.8 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

88.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 66.3-118.2 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 45.1-85.4 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 52.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 52.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

79.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 47.9-128.1 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

46.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 32.7-65.1 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

34.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 24.3-49.6 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 52.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 52.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities

Results with reference substance (positive control):

- Results with reference substance valid?: Yes
- ErC50: (Growth Rate) 0.92 mg/L (95%-CI: 0.84 - 1.00mg/L)
- EyC50: (Yield) 0.61 mg/L (95%-CI: 0.51 - 0.72 mg/L)

Reported statistics and error estimates:

With statistical software ToxRat Professional 3.2.1.

Table 3: Results of the accompanying chemical analysis.

	NC	A	C	E
Nominal test concentration [mg/L]	0	69.1	432.0	2700.0
Measured concentration [mg/L]
0 h	< LOQ*	59.7	301.0	1880.0
24 h	< LOQ	52.2	252.0	1430.0
48 h	< LOQ	50.2	246.0	1700.0
72 h	< LOQ	48.8	221.0	1590.0
72 h**	< LOQ	44.9	222.0	1290.0
Mean measured test concentration (0-72 h)***		52.7	255.0	1650.0
Relative standard deviation (%)		10.4	12.8	13.9
Recovery rate (%)		76.3	59.0	61.1
*LOQ: Limit of Quantification: 20 μg/mL
**test solution with algae
***mean values were calculated without the test solution with algae

Validity criteria fulfilled:

yes

Conclusions:

For the static algae growth inhibition test with Raphidocelis subcapitata, applying nominal concentrations of 0.0 (control), 69.1, 172.8, 432.0, 1080.0, 2700.0 mg/L butyl ethyl ether, the following effect levels - based on the growth rate - are calculated:
- 72 h EC50 (measured): 175.2 mg/L (95 % C.I. 124.1 – 247.8 mg/L);
- 72 h EC20 (measured): 88.6 mg/L (95 % C.I. 66.3 – 118.2 mg/L);
- 72 h EC10 (measured): 62.0 mg/L (95 % C.I. 45.1 – 85.4 mg/L)
- 72 h NOEC (measured): < 52.7 mg/L and
- 72 h LOEC (measured): ≤ 52.7 mg/L.

Executive summary:

In a 72 hour algae growth inhibition test according to OECD 201 (2006), cultures of Raphidocelis subcapitata (Strain No. 61.81 SAG) were exposed to butyl ethyl ether at nominal concentrations of 0.0 (control), 69.1, 172.8, 432.0, 1080.0 and 2700.0 mg/L and measured concentrations of 0.0, 52.7, 255.0 and 1650.0 mg/L (geometric mean) under static conditions for 72 hours.

Substance-specific chemical analysis was based on the detection of the test item.

In the control, the test item was not detected. The initial measured test item concentrations in the test item treatments were 59.0 – 76.3 % of the nominal test concentrations.

Therefore EC values were given additionally as measured effective concentrations.

There were no compound related phytotoxic effects.  

This toxicity study is classified as acceptable and satisfies the guideline requirements for an algae growth inhibition toxicity study.  

 

Results Synopsis

As the measured test item concentrations are not within ± 20 % of the nominal concentrations, according to OECD 201 (2004) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations. 

Test Organism: Raphidocelis subcapitata (Strain No. 61.81 SAG)

Test Type: static

-         72 h EC50 (measured): 175.2 mg/L (95 % C.I. 124.1 – 247.8 mg/L);

-         72 h EC20 (measured): 88.6 mg/L (95 % C.I. 66.3 – 118.2 mg/L);

-         72 h EC10 (measured): 62.0 mg/L (95 % C.I. 45.1 – 85.4 mg/L)

-         72 h NOEC: < 52.7 mg/L and

-         72 h LOEC: ≤ 52.7 mg/L.  

Endpoint Effected: Growth rate

Description of key information

In a 72 hour algae growth inhibition test cultures of Raphidocelis subcapitata (Strain No. 61.81 SAG) were exposed to butyl ethyl ether at nominal concentrations of 0.0 (control), 69.1, 172.8, 432.0, 1080.0 and 2700.0 mg/L and measured concentrations of 0.0, 52.7, 255.0 and 1650.0 mg/L (geometric mean) under static conditions in accordance with the OECD 201 (2006), OECD 23 (2000) and under GLP.

As the measured test item concentrations are not within ± 20 % of the nominal concentrations, according to OECD 201 (2006) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations. 

The following effect levels - based on the growth rate - are calculated:

- 72 h EC50 (measured): 175.2 mg/L (95 % C.I. 124.1 – 247.8 mg/L);

- 72 h EC20 (measured): 88.6 mg/L (95 % C.I. 66.3 – 118.2 mg/L);

- 72 h EC10 (measured): 62.0 mg/L (95 % C.I. 45.1 – 85.4 mg/L);

- 72 h NOEC: < 52.7 mg/L and

- 72 h LOEC: ≤ 52.7 mg/L.  

Key value for chemical safety assessment

EC50 for freshwater algae:

175.2 mg/L

EC10 or NOEC for freshwater algae:

62 mg/L