Ethyl isobutyrate

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:

From 2017-02-28 to 2017-03-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Test concentrations: 25.0, 50.0, 100, 200, 400 mg/L and control
- Sampling method: sampling point: 0 (start of exposure) and 72 hours (end of the exposure). Separate replicates for each test item concentration and control for the test item analysis at the beginning of the exposure were prepared with algae. The samples for the test item analysis after 72 hours were prepared with algae and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis.

Vehicle:

yes

Remarks:

dilution water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: freshly prepared with dilution water by adding an appropriate amount of the test item.
- Test concentrations: 25.0, 50.0, 100, 200, 400 mg/L
- Control: Dilution water without test item tested under the same conditions as the test groups.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: HINDAK, SAG 61.81
- Method of cultivation: Fresh stocks prepared every month on Z-Agar. Light intensity: 2567 - 5130 lux corresponding to 35 - 70 µE • m-2 • s-1 for 24 hours per day.
- Culture medium: Nutrient medium Z (Lüttge et al. 1994)

PRECULTURE
- Preculture: four days old preculture, prepared in dilution water
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

ACCLIMATION
- Acclimation period: none

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Nominal hardness of dilution water: 0.24 mmol Ca+Mg/L

Test temperature:

min.: 22.0 max.: 23.0 mean value: 22.5
measured by Hygro-thermograph (Klimatherm)

pH:

pH at beginning of the test: 8.01-8.18 (test groups), 8.25 (control)
pH at end of the test: 7.98-9.45 (test groups), 9.55 (control)
measured by pH-Meter, pH 191 (WTW)

Nominal and measured concentrations:

Nominal concentrations: 25.0, 50.0, 100, 200, 400 mg/L
Measured concentrations: 23.5, 45.7, 100, 199, 392 mg/L
Measured concentrations are within 20 % of the normal concentrations, hence, nominal concentrations can be used for reporting. More specifically, measured concentrations in the fresh media (0h) were between 91 and 100% of the nominal values, after 72h concentrations were between 81 and 97% of the nominal values.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks, with aluminum tops with PTFE seals.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: volume: 59 mL; Fill volume: 59 mL, no headspace
- Initial cells density: 6839 cells/mL
- Control end cells density: 491639 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, as recommended in OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24 hours/day light
- Light intensity and quality: mean value: 5684 lux, range of the measured values: 4842 to 6213 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: via daily Chlorophyll measurement: fluorometer, excitation at 436 nm, emission at 685 nm.
Other: microscopic evaluation of cells

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Range finding study
- Test concentrations: 1.00, 10.0 and 100 mg/L
- Results used to determine the conditions for the definitive study: The tested concentration levels were all visually clear throughout the exposure. Growth Rate Inhibition at 72 h were 0%, 2%, 7% for 1.00, 10.0 and 100 mg/L, respectively.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

238 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

107 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

55.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

48.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

40.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

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 morphological abnormalities
- Flocculation: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50 for Growth Rate Inhibition: with headspace: 0.435 mg/L (95% CI: 0.414-0.458); without headspace: 0.811 mg/L (95% CI: 0.763-0.883)

Reported statistics and error estimates:

- ECx-values with confidence intervals of growth rate inhibition and for yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.
- The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and D u n n e t t ’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Mean cell densities (over 3 replicates for test concentrations; 6 replicates for control):

Nominal concentration (mg/L)	Mean cell density (cells/mL)
	0 h	24 h	48 h	72 h
400	6839	7436	3857	8927
200	6839	12595	26416	142834
100	6839	8030	18347	104960
50	6839	19257	72112	381656
25	6839	16374	153455	488113
control	6839	28951	151017	491639

Effect on growth rate and yield (means):

Nominal concentration (mg/L)	growth rate	Inhibtion growth rate	Yield	Inhibition yield
	d^-1	%	cells/mL	%
400	+ 0.093	94	+ 2088	100
200	+ 1.01	29	+ 135995	72
100	+ 0.908	36	+ 98121	80
50	- 1.34	6	+ 374817	23
25	- 1.42	0	- 481274	1
control	1.42

Measured concentrations and precentage of nominal concentration of the test item

	0 hours fresh medium		72 hours old medium
Nominal test item concentration [mg/L]	test item
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
400	392	98	325	81
200	199	99	175	88
100	100	100	97.3	97
50.0	45.7	91	42.2	84
25.0	23.5	94	21.8	87
Control	< LOQ		< LOQ

LOQ  = Limit of quantification (0.5 mg test item /L)

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 238 mg/L and 66.0 mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 55.7 mg/L and 40.6 mg/L, respectively. The NOEC-values of growth rate and yield after 72 hours were 50.0 and 25.0 mg/L, respectively.

Executive summary:

The toxicity of test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. Five test solutions were tested in the definitive test in a geometrical series with a dilution factor of 2: 25.0 - 50.0 - 100 - 200 - 400 mg/L. Three replicates were tested for each test item concentration and six replicates for the control.
The test media were clear throughout the test period. The measured concentrations of test item in the fresh media (0 h) were between 91 and 100% of the nominal values. The measured concentrations in the media after 72 hours were between 81 and 97% of the nominal values. Reporting was done based on nominal concentrations.

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 238 (183 - 326) mg/L and 66.0 (56.2 - 84.1) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 55.7 (50.0 - 119) mg/L and 40.6 (25.0 - 50.9) mg/L, respectively. The NOEC-values of growth rate and yield after 72 hours were 50.0 and 25.0 mg/L, respectively.

Validity criteria were met throughout the study.

Description of key information

For this endpoint, one experimental study is available, which was performed according to OECD 201.

Based on nominal test item concentrations, the EC50 value for inhibition of growth rate (ErC50) after 72 hours was 238 mg/L, the EC10 for inhibition of growth rate (ErC10) was found to be 55.7 mg/L. The NOEC of growth rate after 72 hours was 50.0 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

238 mg/L

EC10 or NOEC for freshwater algae:

55.7 mg/L

Additional information

For this endpoint only one test is available. In this algae growth inhibition test with unicellular freshwater green alga Pseudokirchneriella subcapitata, the effects of the test item were determined according to OECD 201 (2011).

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. Five test solutions were tested in the definitive test in a geometrical series with a dilution factor of 2: 25.0 - 50.0 - 100 - 200 - 400 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.
The test media were clear throughout the test period. The measured concentrations of test item in the fresh media (0 h) were between 91 and 100% of the nominal values. The measured concentrations in the media after 72 hours were between 81 and 97% of the nominal values.

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 238 (183 - 326) mg/L and 66.0 (56.2 - 84.1) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 55.7 (50.0 - 119) mg/L and 40.6 (25.0 - 50.9) mg/L, respectively. The NOEC-values of growth rate and yield after 72 hours were 50.0 and 25.0 mg/L, respectively.