Ethyl octanoate

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 2017-02-03 to 2017-02-23

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: 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. Additionally, samples for each test item concentration and the control were prepared with and without algae for the determination of the adsorption to algae and glass at the end of the exposure.
- 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 (on an autosampler)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Two saturated solutions with a nominal loading of 100 mg/L test item were prepared once with demineralized water 72 ± 1 hour prior to the start of the exposure.
1) The test item was placed on to the surface of the demineralized water (pH value of 6.15 to 6.21) with a pipette. A
2) Slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) with a magnetic stirrer at 30 °C for 72 ± 1 hour.
3) Slow stirring procedure at room temperature for one hour.
4) The saturated solution was taken from the bottom and used for testing.
5) Check of Tyndall effect (result was negative)
6) Test concentrations were acquired by further dilution of the saturated solution.
- Test concentrations: 2.56 - 6.40 - 16.0 - 40.0 - 100% of the saturated solution, corresponding to initial measured test item concentration of 0.735, 1.9, 4.91, 12.3 and 30.4 mg/L.
- Control: Six replicates (without test item) were incubated under the same conditions as the test concentrations

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: HINDAK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 hours per day

ACCLIMATION
- Acclimation period: three days
- Culturing media and conditions (same as test or not): Nutrient medium Z, same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

mean temperature: 22.5°C

pH:

7.25 to 7.48 at test start and 7.22 to 8.33 at test end

Nominal and measured concentrations:

Nominal concentrations: 2.56 - 6.40 - 16.0 - 40.0 - 100% of the saturated solution
Initial measured concentrations: 0.735 - 1.90 - 4.91 - 12.3 - 30.4 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks
- Type: Closed
- Material, size, headspace, fill volume: volume: 59 mL, with aluminium tops with PTFE seals, fill volume: 59 mL
- Initial cells density: 6839 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, threefold concentrated AAP medium according to the guidelines OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24 hours/day light
- Light intensity and quality: range of the measured values: 4672 to 6043 lux, mean value: 5479 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Chlorophyll a-fluorescence
- Other: Microscopic evaluation: at the start and the end of the incubation period; Physico-chemical Properties: The pH-values at the start of exposure were measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: yes
- Test concentrations: 0.1, 1, 10 and 100% saturated solution (nominal loading level of 100 mg/L)
- Results used to determine the conditions for the definitive study: The saturated solution and the dilution levels of 0.1, 1 and 10% of the saturated solution were visually clear throughout the exposure period. Growth rate inhibitions were 0%, 0%, 7% and 100% for 0.1, 1, 10 and 100% saturated solution, respectively.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.57 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: confidence limits: 5.24 to 6.99 mg/L)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.53 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Confidence limits: 2.75 to 4.24 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.83 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.735 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

- Microscopic evaluation: revealed no morphological abnormalities.
- Physicochemical Data: The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test media were clear throughout the exposure phase.
- Chemical analysis of test item concentrations: The measured concentrations after 24 hours were in the range of 80 to 92% of the initially measured values. The measured test item concentration after 48 hours were between < LOQ to 0.2% of the initially measured values. At the end of the exposure (72 hours) all concentration levels were < LOQ. For the adsorption to glass and the adsorption to algae samples no signals above the lowest calibration standard were observed.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: most recent test: growth rate 0.435 mg/L (95% CI: 0.414-0.458), yield 0.230 mg/L (95% CI: 0.179-0.322)

Results of non-GLP range finding test:

Saturated solution (%)	% inhibition at test end (72h)
	growth rate	yield
Control	0	0
0.100	0	0
1.00	0	26
10.0	7	100
100	100	58.3

Mean cell Densities:

Initial measured concentration (mg/L)	0h	24h	48h	72h
30.4	6839	n.a.	n.a.	n.a.
12.3	6839	4244	n.a.	n.a.
4.91	6839	7930	9516	130154
1.9	6839	10068	43765	421073
0.735	6839	12012	171573	572961
Control	6839	25937	173706	579979

n.a. not applicable

Mean growth rates and yields after 72 hours:

Initial measured concentration (mg/L)	growth rate (d-1)	inhibition of growth rate (%)	yield (cells/ml)	inhibition of yield (%)
30.4	n.a.	100	n.a.	100
12.3	n.a.	100	n.a.	100
4.91	0.981	34	123315	78
1.9	1.37	7	414234	28
0.735	1.48	0	566122	1
Control	1.48		573140

Measured Concentrations and Percentage of the Initial Concentrations of test item:

Sample time	0 h	24 h		48 h		72 h
Dilution Level of the Saturated Solution [%]	Meas. conc. [µg/L]	Meas. conc. [µg/L]	[%]	Meas. conc. [µg/L]	[%]	Meas. conc. [µg/L]
100	30.4	27.9	92	< LOQ		< LOQ
40.0	12.3	10.2	83	< LOQ		< LOQ
16.0	4.91	3.90	80	0.0101	0.2	< LOQ
6.40	1.9	1.55	81	< LOQ		< LOQ
2.56	0.735	0.675	92	< LOQ		< LOQ
Control	< LOQ	< LOQ		< LOQ		< LOQ

Meas. Conc. = Measured concentration of the test item (dilution factor taken into account)

%= Percentage of the initial concentration of the test item

LOQ= Limit of quantification (0.01 mg test item /L)

Validity criteria fulfilled:

yes

Conclusions:

In this study, 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 initial measured test item concentration): ErC50 = 5.57 mg/L and EyC50 = 2.83 mg/L; ErC10 = 3.53 mg/L and EyC10 = 1.20 mg/L, respectively and the NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.735 mg/L.

Executive summary:

The toxicity of test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The aim of the study was the determination of NOEC, LOEC, EC10, EC20- and EC50-values based on growth rate and yield over a period of 72 hours.

The study was carried out in closed bottles without headspace in order to avoid losses of test item to the headspace. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. The saturated solution and four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2.5: 2.56-6.40 -16.0-40.0 - 100% of the saturated solution, corresponding to the initial measured test item concentrations of 0.735 - 1.90 - 4.91 -12.3 - 30.4 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 measured initial concentrations of test item were 0.735 - 1.90 - 4.91 - 12.3 -30.4 mg/L. The measured concentrations after 24 hours were in the range of 80 to 92% of the initially measured values. The measured concentration after 48 hours were between < LOQ to 0.2% of the initially measured values. At the end of the exposure (72 hours) all concentration levels were < LOQ. The measured concentrations of the adsorption to glass sample and the adsorption to algae samples were < LOQ.

In this study, 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 initial measured test item concentration): ErC50 = 5.57 mg/L and EyC50 = 2.83 mg/L;  ErC10 =  3.53 mg/L and EyC10 =  1.20 mg/L, respectively and the NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.735 mg/L.

Description of key information

ErC50 (72 h) = 5.57 mg/L (Pseudokirchneriella subcapitata ; OECD 201)

ErC10 (72 h) = 3.53 mg/L (Pseudokirchneriella subcapitata ; OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

5.57 mg/L

EC10 or NOEC for freshwater algae:

3.53 mg/L

Additional information

The toxicity of test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The study was carried out under static conditions in closed bottles without headspace in order to avoid losses of test item to the headspace. A saturated solution and four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2.5: 2.56-6.40 -16.0-40.0 - 100% of the saturated solution, corresponding to the initial measured test item concentrations of 0.735 - 1.90 - 4.91 -12.3 - 30.4 mg/L. 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 initial measured test item concentration): ErC50 = 5.57 mg/L and EyC50 = 2.83 mg/L;  ErC10 =  3.53 mg/L and EyC10 =  1.20 mg/L, respectively and the NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.735 mg/L.