2-(2-ethoxyethoxy)ethyl acetate

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:

20 Jul - 20 Aug 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No justification on differences in measured concentrations in range-finding study compared to the definitive study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from the control and the 100 mg/L test group (replicates R1 - R6 pooled) at 0 and 72 hours for quantitative analysis. All samples were stored at approximately -20 °C prior to analysis.
- Sample storage conditions before analysis: Duplicate samples were taken at 0 and 72 hours and stored at approximately -20 ºC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Range-finding study: An amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (5.5 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L. Definitive Test: An amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution. This stock solution was inoculated with 20 mL of algal suspension to give the required test concentration of 100 mg/L. The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
- Eluate: no
- Differential loading: no (range-finding study); yes (definitive study)
- Controls: yes, test medium control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

ACCLIMATION
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 ºC

pH:

7.4 - 7.8

Nominal and measured concentrations:

nominal: control, 100 mg/L
measured: < LOQ, 130 mg/L (0 h)/3.65 mg/L (72 h)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): The flasks were plugged with polyurethane foam bungs.
- Material, size, headspace, fill volume: glass, size: 250 mL, headspace: 150 mL, fill volume: 100 mL
- Aeration: constantly shaken at approximately 150 rpm for 72 h
- Initial cells density: 5E+03 cells/mL
- Control end cells density: 6.56E+05 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux (warm white lighting 380 - 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
- Other: Cell abnormalities were recorded microscopically.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth rate at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 22 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.:

> 22 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.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 22 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
- Unusual cell shape: No abnormalities were observed in the control and test vessels.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: ErC50 (0-72 h): 1.4 mg/L (95% CL: 1.2 - 1.7 mg/L); EyC50 (0-72 h): 0.59 mg/L (95% CL: 0.53 - 0.65 mg/L)
- Other: A positive control (Harlan Laboratories Ltd, Study Number: 41104038) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and 100 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences (P ≥ 0.05), between the control and 100 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 mg/L.

Analysis of the 100 mg/L test preparation at 0 hours showed a measured test concentration of 130% of nominal was obtained. A significant decline in measured test concentration was observed at 72 hours to 4% of nominal. Analysis of the 100 mg/L test preparations in the range-finding test showed that near nominal concentrations were obtained at both 0 and 72 hours. Analysis of a duplicate sample showed a measured concentration of 4% of nominal was obtained. The decline observed in the definitive test was considered to be due to degradation of the test item, predominantly by alkaline hydrolysis at the pH of the study. The rate at which this occurs may change drastically with subtle differences in pH or the presence of other catalysts for the reaction.

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Test Concentration [mg/L]		Growth Rate [cells/mL/h]		Yield [cells/mL]		Biomass Integral [cells/mL/h]
		0-72 h	% inhibition	0-72 h	% inhibition*	0-72 h	% inhibition
Control	R1	0.067	-	6.20E+05	-	1.08E+07	-
	R2	0.067		6.60E+05		1.10E+07
	R3	0.068		6.88E+05		1.18E+07
	R4	0.069		7.07E+05		1.17E+07
	R5	0.069		7.04E+05		1.18E+07
	R6	0.066		5.78E+05		9.62E+06
	Mean	0.068		6.50E+05		1.11E+07
	SD	0.001		5.58E+04		8.41E+05
100	R1	0.067	1	6.33E+05		1.14E+07	[3]
	R2	0.067	1	6.11E+05		1.06E+07	5
	R3	0.067	1	5.98E+05		1.08E+07	3
	R1	0.068	0	6.54E+05		1.12E+07	[1]
	R2	0.068	0	6.62E+05		1.14E+07	[2]
	R3	0.068	0	6.54E+05		1.15E+07	[4]
	Mean	0.068	1	6.35E+05	2	1.11E+07	0
	SD	0.001		2.59E+04		3.81E+05

* In accordance with the OECD test guideline only the mean value for yield is calculated

[Increase in growth as compared to controls]

Validation criteria

Daily Specific Growth Rates for the Control Cultures

 

	Daily specific growth rate (cells/mL/hour)
	0 – 24 hours	24 – 48 hours	48 – 72 hours
R1	0.071	0.062	0.068
R2	0.072	0.067	0.062
R3	0.067	0.070	0.069
R4	0.063	0.070	0.074
R5	0.067	0.066	0.073
R6	0.060	0.065	0.073
Mean	0.067	0.067	0.070

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation criterion given in the OECD test Guideline which states the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 72 h) was 1% and hence satisfied the validation criterion given in the OECD Test Guideline which states that this must not exceed 7%.

 

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour No Observed Effect Concentration (NOEC) was 100 mg/L based on nominal concentration / 22 mg/L based on the mean of the measured concentration.

Executive summary:

The potential effects of exposure to 2 -(2 -ethoxyeythoxy)ethyl acetate to freshwater algae has been investigated according to methods described by OECD TG 201. No growth inhibition was observed and the 72 hour No Observed Effect Concentration (NOEC) was 100 mg/L based on nominal concentration / 22 mg/L based on the mean of the measured concentration.

Description of key information

ErC50 (72 h) > 100 mg/L (nominal) for Pseudokirchneriella subcapitata (OECD 201)
NOErC (72 h) ≥ 100 mg/L (nominal) for Pseudokirchneriella subcapitata (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

One study is available investigating the effects of 2-(2-ethoxyethoxy)ethyl acetate (CAS 112-15-2) to algae (Vryenhoef, 2012). The study was performed according to OECD 201 (GLP) with the freshwater algae Pseudokirchneriella subcapitata. A preliminary range-finding study did not result in any effects on growth and yield up to a concentration of 100 mg/L. Test concentrations determined by GC/FID were within the range of 80-120% of nominal. Based on the results from the range-finding study a limit test with a nominal concentration of 100 mg/L was performed. No effects on growth rate and yield were recorded after an exposure of 72 h resulting in an ErC50 (72 h) > 100 mg/L and a NOErC (72 h) ≥ 100 mg/L (based on the nominal concentrations). The analytical determination resulted in a drop of concentration from 130% of nominal at the beginning of the study to 3% of nominal at the end of the study resulting in an ErC50 (72 h) > 22 mg/L and a NOErC (72 h) of ≥ 22 mg/L (based on the geometric mean concentrations). This decline was considered by the laboratory to be due to degradation of the test item, predominantly by alkaline hydrolysis at the pH of the study. However, this decline was not observed in the preliminary range-finding study and in the study with Daphnia magna. Moreover, hydrolysis at pH 7 and pH 8 is not considered to be relevant since DT50 values were determined to be greater than the duration of the study (pH 7: 304.78 d; pH 8: 30.48 d (Müller, 2011)). Nevertheless, even if hydrolysis occurred in this study it is justifiable to base the effect concentrations on the nominal values since this concentration was initially introduced in the study and reflects the effects of possible hydrolysis products as well as it would also occur in the aquatic environment. Differences between nominal and measured concentrations might be due to deficiencies in the analytical methods. The results of the range-finding study, showing no effects up to a concentration of 100 mg/L, support the conclusion that no toxicity of 2-(2-ethoxyethoxy)ethyl acetate to algal species is expected.