Ethyl hexanoate

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:

2015-07-27 to 2016-08-20

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:

2011

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the control were analytically verified in replicates prepared after 0 hours (start of exposure), 24 hours, 48 hours and 72 hours (end of exposure).
- Sample storage conditions before analysis: All samples were stored in a refrigerator at 6 ± 2 °C until sample preparation.Prepared samples were stored at ambient temperature in an auto sampler rack prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Considering the density of the test substance (0.8705) appropriate volumes of the test item were pipetted directly into measuring flasks, containing the respective volume of the dilution water. The test solutions were stirred for at least 15 minutes at approximately 1100 rpm at room temperature. Thereafter, the readily prepared test solutions were transferred to the test vessels which were tightly closed, without headspace. The test concentrations were selected based on the results of a non- GLP preliminary range finding test.
- Controls: negative control (Six replicates (without test item) were exposed under the same conditions as the test concentrations); positive control: Potassium dichromate

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green freshwater algae
- Strain: Pseudokirchneriella subcapitata HINDÄK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar and cultivated in nutrient medium Z according to Lüttge et al. (1994) under a light intensity of 2567 - 5130 lux, corresponding to 35 - 70 µE • m-2 * s-1 for 24 hours per day at a temperature of 21 - 24 °C.

ACCLIMATION
- Acclimation period: 4 days in AAP medium.
- Culturing media and conditions (same as test or not): Same as test.
- Any deformed or abnormal cells observed: No.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.5 - 22°C

pH:

at 0 hours: 7.47 - 7.57
at 72 hours: 7.63 - 8.39

Nominal and measured concentrations:

Nominal: 0 (control), 2.65, 4.76, 8.57, 15.4, 27.8, 50 mg test substance/L
Measured (mean): < LOQ (control), 0.250, 0.977, 2.32, 5.23, 10.1, 33.8 mg test substance/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks (volume 59 mL) with aluminum tops with PTFE seals.
- Type (delete if not applicable): Closed, without headspace.
- Material, size, headspace, fill volume: 59 mL
- Aeration: No.
- Renewal rate of test solution (frequency/flow rate): No.
- Initial cells density: Approximately 5 x 10^3 - 10^4 cells/mL, Actual: 6305 cells/mL.
- Control end cells density: 494842 cells/mL.
- No. of vessels per concentration (replicates): 3 (Separate replicates for each measuring time were prepared.)
- No. of vessels per control (replicates): 6 (Separate replicates for each measuring time were prepared.)

GROWTH MEDIUM
- Standard medium used: Yes.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 3 x AAP medium with compounds additionally added to enable sufficient growth under conditions without headspace.
- Ca/Mg ratio: 1/3
- Culture medium different from test medium: Yes.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes.
- Adjustment of pH: No data.
- Photoperiod: 24 hours light
- Light intensity and quality: 5475 (range: 4751 - 6107) µE*m-2*s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: The cells were checked by microscopic evaluation for unusual cell shapes, color differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: None.
- Range finding study: Yes.
- Test concentrations: 1.00, 10.0 and 100 mg/L of the test item were used in the range-finding study.
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.23 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.:

9.97 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield inhibition

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield inhibition

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield inhibition

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- EC50: 0.796 ± 0.509 mg/L (growth rate inhibition), 0.419 + 0.275 mg/L (yield inhibition)

Table 1: Cell Densities

Time weighted average mean measured test item concentration (mg/L)	Cell density [cells/mL]
	0 hours	24 hours	48 hours	72 hours
33.8	6305	16055	17677	n.a.
10.1	6305	23885	44063	213618
5.23	6305	28170	112540	603844
2.32	6305	33371	168414	667022
0.977	6305	36767	184836	575123
0.250	6305	14858	191568	545185
Control	6305	38799	192542	494842

 

Table 2: Evaluation after 72 hours

Time weighted average mean measured test item concentration (mg/L)	Growth rate [1/d]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
33.8	(+) n.a.	100	(+) n.a.	100
10.1	(+) 1.17	19	(+) 207313	58
5.23	(-) 1.52	-4	(-) 597539	-22
2.32	(-) 1.55	-7	(-) 660717	-35
0.977	(-) 1.50	-3	(-) 568818	-16
0.250	(-) 1.49	-2	(-) 538880	-10
Control	1.45		488537

 

Table 3: Measured Concentrations and Percentage of the Nominal Concentration of the test item in Fresh Medium (0 hours) and Old Medium (72 hours)

Sampling date	0 hours without algae		24 hours with algae		48 hours with algae		72 hours with algae		Geometric Mean (Time weighed average)
Nominal loading level of the test item [mg/L]	Meas. cone. [mg/L]	%	Meas. cone. [mg/L]	%	Meas. cone. [mg/L]	%	Meas. cone. [mg/L]	%	mg/L
50	44.7	89	40.7	81	45.7	91	9.59	19	33.8
27.8	22.6	81	23.0	83	18.9	68	< LOQ	-	10.1
15.4	12.5	81	12.7	82	6.36	74	< LOQ	-	5.23
8.57	7.07	82	7.38	86	1.28	8	< LOQ	-	2.32
4.76	4.26	89	3.61	76	< LOQ	-	< LOQ	-	0.977
2.65	0.185	7	0.170	6	0.180	7	< LOQ	-	0.250
Control	< LOQ		< LOQ		< LOQ		< LOQ

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

% = Percent of the nominal concentration of the test item

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

Validity criteria fulfilled:

yes

Conclusions:

An Algae test with Pseudokirchneriella subcapitata according to OECD 201 and in compliance with GLP was conducted with Ethyl hexanoate. The following values were estimated:
ErC50 (72 h): 11.8 mg/L based on growth rate.
ErC10 (72h) = 9.41 mg/L based on growth rate.
EyC50 (72 h): 9.97 mg/L based on yield inhibition.
EyC10 (72h) = 9.29 mg/L based on yield inhibition.
NOEC (72h) = 5.23 mg/L based on growth rate and yield inhibition.
(based on geometric mean measured concentrations)

Description of key information

ErC50 (72 h): 11.8 mg/L

ErC10 (72h) = 9.41 mg/L

EyC50 (72 h): 9.97 mg/L

EyC10 (72h) = 9.29 mg/L

NOEC (72h) = 5.23 mg/L based on growth rate and yield inhibition

(based on geometric mean measured concentrations)

Key value for chemical safety assessment

EC50 for freshwater algae:

11.8 mg/L

EC10 or NOEC for freshwater algae:

9.41 mg/L

Additional information

The toxicity of the test item to algae and cyanobacteria was determined in a static test performed according to OECD 201 and GLP. Cultures of Pseudokirchneriella subcapitata were exposed to nominal test concentrations of 2.65, 4.76, 8.57, 15.4, 27.8 and 50.0 mg/L, equivalent to geometric mean measured concentrations of 0.250, 0.977, 2.32, 5.23, 10.1 and 33.8 mg/L. The test concentrations were analysed by GC-FID determinations daily. After 72 h 10% inhibition of growth rate was observed at 9.41 mg/L, 10% inhibition of yield was recorded at 9.29 mg/L. The ErC50 (72 h) was calculated at 11.8 mg/L (measured), the EyC50 (72 h) at 9.97 mg/L (measured). The NOEC values for both endpoints was estimated as 5.23 mg/L.