2-{2-[(2-ethylhexyl)oxy]ethoxy}ethyl prop-2-enoate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 13 August 2015 and 03 September 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 1= Reliable without restriction o GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-{2-[(2-ethylhexyl)oxy]ethoxy}ethyl prop-2-enoate

Cas Number:

117646-83-0

Molecular formula:

C15H28O4

IUPAC Name:

2-{2-[(2-ethylhexyl)oxy]ethoxy}ethyl prop-2-enoate

Test material form:

other: liquid

Details on test material:

Identification:
M120

Physical state/Appearance:
Clear colorless liquid

Batch:
NC-5635-01

Purity:
99.4%

Expiry date:
5 December 2015

Storage conditions:
Room temperature in the dark

Test organisms

Test organisms (species):

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 4.4 - 6.6 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.47 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Any other information on results incl. tables

Range-finding Test

The results showed no effect on growth at the test concentrations of 0.10 and 1.0% v/v saturated solution. However, growth was observed to be reduced at 10 and 100% v/v saturated solution.

 

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.

 

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.56 to 32 mg/L. A concentration dependant decline in measured concentration was observed at 72 hours in the range of 0.13 to 34 mg/L. The decline in measured test concentration was considered to be due to adsorption to algal cells. Overall the decline in measured concentrations followed a concentration dependent pattern with greater losses being observed at the lower test concentrations. This effect was considered to be due to there being greater numbers of algal cells in the lower concentrations and hence greater surface area for adsorption to occur

 

 

DefinitiveTest

Verification of Test Concentrations

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.53 to 28 mg/L. A concentration dependant decline in measured test concentration was observed at 72 hours to between 0.043 and 31 mg/L (8% to 111% of the  0-Hour measured test concentrations) indicating that the test item was adsorbing to the algal cells present. Greater losses of test item were observed at the lowest test concentrations employed where there were larger numbers of algal cells present. 

 

Analysis of test samples at 72 hours which had been prepared with the omission of algal cells showed measured test concentrations to range from 0.42 to 37 mg/L (78% to 134% of the 0-Hour measured concentrations) thereby confirming that the losses seen in the test samples which contained algal cells was due to adsorption of the test item to the algal cells present rather than instability of the test item over the test duration. 

 

It was therefore considered appropriate to calculate the results based on the measured concentrations obtained from the test samples prepared with the omission of algal cells as these samples gave a more accurate representation of the concentration of test item to which the algae were exposed. Given that two values fell slightly outside of the acceptable 80-120% of nominal range (78% and 134%), the results were calculated using the geometric mean measured test concentrations. The geometric mean measured test concentrations were determined to be:

 

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a % of the 0-Hour Measured Test Concentration
1.0	0.47	88
3.2	2.5	94
10	5.3	93
32	15	109
100	32	115

 

 

Growth Data

From the data given in the two tables below, it is clear that the growth rate (r) and yield (y) ofPseudokirchneriella subcapitata(CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

 

Inhibition of Growth Rate

E_rC₁₀(0 - 72 h)           : 2.0 mg/L

E_rC₂₀(0 - 72 h)           : 2.9 mg/L

E_rC₅₀(0 - 72 h)           : 5.4 mg/L; 95% confidence limits 4.4 – 6.6 mg/L

 

where E_rC_xis the test concentration that reduced growth rate by x%.

 

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control and 0.47 mg/L test concentration (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.47 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 2.5 mg/L.

 

 

Inhibition of Yield

E_yC₁₀(0 - 72 h)          : 1.2 mg/L
E_yC₂₀(0 - 72 h)          : 1.6 mg/L
E_yC₅₀(0 - 72 h)          : 2.5 mg/L; 95% confidence limits 2.1 – 2.9 mg/L

 

Where:

 

E_yC_xis the test concentration that reduced yield by x%.

 

Statistical analysis of the yield data was carried out as in Section 4.2.2.1. There were no statistically significant differences between the control and 0.47 mg/L test concentration (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.47 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 2.5 mg/L.

 

 

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 156 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

 

Mean cell density of control at 0 hours         :          3.98 x 10³cells per mL

Mean cell density of control at 72 hours       :          6.21 x 10⁵cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 16% and hence satisfied the validation criterion given in the OECD 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 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

 

 

Observationson Cultures

Due to a technical oversight, microscopic examination of the control and test cultures was not conducted at the end of the definitive test. This was considered to have had no adverse effect on the outcome of the test.

 

Water QualityCriteria

Temperature was maintained at 24 ± 1 ºC throughout the test.

 

The pH value of the control cultures was determined to be pH 7.8 at 0 and 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

 

 

Observations on Test Item Solubility

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 0.47 mg/L test cultures were observed to be green dispersions. The 2.5 mg/L test cultures were pale green dispersions, the 5.3 mg/L test cultures were extremely pale green dispersions whilst the 15 and 32 mg/L test cultures were clear colorless solutions.

 

 

Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities*(cells per mL)				pH
		0 h	0 h	24 h	47 h	72 h	72 h
Control	R1	7.8	4.24E+03	1.83E+04	1.18E+05	6.47E+05	7.8
	R2		4.64E+03	2.56E+04	1.08E+05	6.43E+05
	R3		3.42E+03	2.19E+04	1.07E+05	6.57E+05
	R4		3.47E+03	2.06E+04	1.22E+05	5.40E+05
	R5		3.94E+03	1.67E+04	1.16E+05	4.95E+05
	R6		4.16E+03	2.08E+04	1.23E+05	7.42E+05
	Mean		3.98E+03	2.06E+04	1.16E+05	6.21E+05
0.47	R1	7.5	3.30E+03	1.91E+04	1.14E+05	6.12E+05	7.9
	R2		3.06E+03	1.80E+04	1.14E+05	6.37E+05
	R3		3.34E+03	2.08E+04	1.17E+05	5.66E+05
	Mean		3.23E+03	1.93E+04	1.15E+05	6.05E+05
2.5	R1	7.6	4.16E+03	1.32E+04	8.24E+04	3.23E+05	7.8
	R2		2.96E+03	1.43E+04	9.35E+04	3.52E+05
	R3		3.55E+03	1.51E+04	7.81E+04	2.52E+05
	Mean		3.56E+03	1.42E+04	8.47E+04	3.09E+05
5.3	R1	7.6	3.04E+03	8.91E+03	3.90E+04	5.47E+04	7.7
	R2		3.17E+03	1.06E+04	3.35E+04	5.54E+04
	R3		3.31E+03	8.67E+03	3.11E+04	6.20E+04
	Mean		3.17E+03	9.41E+03	3.45E+04	5.74E+04
15	R1	7.6	3.25E+03	4.14E+03	9.33E+03	8.85E+03	7.7
	R2		3.76E+03	4.69E+03	7.58E+03	7.25E+03
	R3		2.96E+03	4.46E+03	7.90E+03	7.92E+03
	Mean		3.32E+03	4.43E+03	8.27E+03	8.01E+03
32	R1	7.6	2.93E+03	3.36E+03	5.68E+03	5.89E+03	7.7
	R2		3.47E+03	3.92E+03	5.26E+03	6.99E+03
	R3		3.81E+03	3.78E+03	5.18E+03	4.94E+03
	Mean		3.40E+03	3.69E+03	5.38E+03	5.94E+03

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

 

Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.068		6.42E+05
	R2	0.067		6.38E+05
	R3	0.068		6.53E+05
	R4	0.065	-	5.36E+05	-
	R5	0.064		4.91E+05
	R6	0.069		7.38E+05
	Mean	0.067		6.17E+05
	SD	0.002		8.86E+04
0.47	R1	0.067	0	6.08E+05
	R2	0.067	0	6.34E+05
	R3	0.066	1	5.62E+05
	Mean	0.067	0	6.01E+05	2
	SD	0.001		3.63E+04
2.5	R1	0.058	13	3.19E+05
	R2	0.059	12	3.49E+05
	R3	0.054	19	2.48E+05
	Mean	0.057	15	3.05E+05	50
	SD	0.003		5.16E+04
5.3	R1	0.033	51	5.17E+04
	R2	0.033	51	5.22E+04
	R3	0.035	48	5.86E+04
	Mean	0.034	50	5.42E+04	91
	SD	0.001		3.87E+03
15	R1	0.008	88	5.60E+03
	R2	0.005	93	3.49E+03
	R3	0.006	91	4.96E+03
	Mean	0.006	91	4.68E+03	99
	SD	0.002		1.08E+03
32	R1	0.002	97	2.96E+03
	R2	0.005	93	3.52E+03
	R3	0.000	100	1.14E+03
	Mean	0.002	97	2.54E+03	100
	SD	0.003		1.25E+03

*    In accordance with the OECD test guideline only thean value for yield for each test concentration is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:

Growth Rate
EC50 (mg/L) = 5.4
95% Confidence Limits (mg/L) = 4.4 - 6.6
No Observed Effect Concentration (NOEC) (mg/L) = 0.47
Lowest Observed Effect Concentration (LOEC) (mg/L) = 2.5

Yield
EC50 (mg/L) = 2.5
95% Confidence Limits (mg/L) = 2.1 - 2.9
No Observed Effect Concentration (NOEC) (mg/L) = 0.47
Lowest Observed Effect Concentration (LOEC) (mg/L) = 2.5