2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between April 07, 2006 to July 19, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols/ Current OECD Guidelines and to GLP standards, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: April 2005 Date of signature: November 2005

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method:
Definitive Test:
For the purpose of the definitive test, the test material was dispersed directly in water.
An amount of test material (500 mg) was dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 15 minutes. Intense stirring for 24 hours. After 24 hours synthetic sewage (16 ml), activated sewage sludge (200 ml) were added

- Synthetic waste water: The synthetic waste water was made as shown below:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl filled up to a final volume of 1 liter
0.4 g CaCl2 · 2H2O with deionized water
0.2 g MgSO4 · 7H2O
2.8 g K2HPO4

This was filled up to a final volume of 1 l with deionised water

At the start of the test (after the stirring period of 24 hours), synthetic wastewater and activated sludge inoculum were added. The inoculum had a sludge concentration of 3.3 g/L dry weight (corresponding to about 1.3 g dry material per litre test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, secondly to the test solutions of the reference item, thirdly to the test solutions of the test item, and finally to the second control.

- Differential loading:
Not applicable.

- Controls:
Two controls containing only tap water, synthetic wastewater and inoculum were tested in parallel to the five test item concentrations under identical test conditions.

The reference item 3,5-dichlorophenol was tested in parallel under identical test conditions, and functioned as a positive control. The nominal concentrations tested were 5, 16 and 50 mg/L. A stock solution of 3,5-dichlorophenol was prepared according to the test guidelines: 0.5 g of 3,5-dichlorophenol was dissolved in 10 mL 1 M NaOH and diluted to about 30 mL with purified water. Excess NaOH was neutralized with 0.5 mol/L H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to one litre with purified water. The pH was 7.8. The final concentration of 3,5-dichlorophenol amounted to 500 mg/L. Aliquots of this 3,5-dichlorophenol stock solution were mixed with synthetic wastewater and tap water in the test vessels to obtain the desired concentrations.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable.

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Not applicable

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Hardness:

not recorded

Test temperature:

The temperature in the test media, measured in one control, was 20 °C at the start and 19 °C at the end of the incubation period.

pH:

The pH of the solution was 7.8

Dissolved oxygen:

The dissolved oxygen concentrations was determined after the addition of synthetic wastewater and activated sludge in all test media and the controls at the start and at the end of the 3-hour incubation period.

The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period, and just before measurement of the respiration rates, the dissolved oxygen concentrations was at least 7.4 mg/L.

Salinity:

Not applicable.

Nominal and measured concentrations:

The final nominal test concentrations were 10, 32, 100, 320 and 1000 mg/L. Test item amounts of 4.93, 16.51, 48.00, 155.20 and 520.76 mg were directly weighed by means of an analytical balance and transferred to the test vessels with 284 mL of tap water.

The nominal concentartions for the reference item 3,5-dichlorophenol were 5, 16 and 50 mg/L.

Details on test conditions:

TEST SYSTEM
Preparation of test system: Test item amounts of 4.93, 16.51, 48.00, 155.20 and 520.76 mg were directly weighed by means of an analytical balance and transferred to the test vessels with 284 mL of tap water. The test item was mixed into the tap water by ultrasonic treatment for 15 minutes and intense stirring for 24 hours at room temperature in the dark, to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used. After the 24-hour stirring period, 16 mL of synthetic wastewater and 200 mL of the activated sludge inoculum were added (see table 1 in methods section).

OTHER TEST CONDITIONS
- Adjustment of pH:
The pH values of the test preparations at the start and end of the exposure period are given in Table 4

- Photoperiod:
3 hours.

- Light intensity:
Normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
In order to calculate the inhibitory effect of the test and reference materials the respiration rate was expressed as a percentage of the two control respiration rates. The inhibitory effect of the test item, or of the reference item on the respiration rate (oxygen consumption per minute) is expressed as a percentage of the mean respiration rate of the two controls measured at the start and at the end of the test series (vessels 1 and 10).

% inhibition = [ 1 – (2 RS/RC1 + RC2) ] x 100

where
RS = oxygen consumption rate for test or reference sample
RC1 + RC2 = oxygen consumption rates for controls 1 and 2

The NOEC (3-hour EC15), the 3-hour EC20, EC50 and EC80 of the test and reference item (and their 95%-confidence limits) were (as far as possible) calculated by Probit Analysis. All test results were based on nominal concentrations of the test and reference item.

TEST CONCENTRATIONS

- Test concentrations:

The final nominal test concentrations were 10, 32, 100, 320 and 1000 mg/L. Test item amounts of 4.93, 16.51, 48.00, 155.20 and 520.76 mg were directly weighed by means of an analytical balance and transferred to the test vessels with 284 mL of tap water.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

741 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL = 286 mg/l - upper limit could not be determined

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

82 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 7.0 – 209 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

124 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 20 – 331 mg/L

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The influence of the test item 2-(2-Vinyloxyethoxy) ethyl acrylate on the respiration rate of activated sludge is presented in Table 2 (results section).
All test concentrations in the range 10 to 1000 mg/L were clear solutions. Up to and including the test concentration of 32 mg/L, the test item 2-(2-Vinyloxyethoxy) ethyl acrylate had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. At the next higher test item concentrations of 100, 320 and 1000 mg/L, significant inhibitory effects of 15, 33 and 60% were noted, respectively.

The calculated 3-hour EC-values and their 95% confidence limits were as follows:

calculated 3-hour EC15):
82 mg/L (95% confidence limits: 7.0 – 209 mg/L)
EC20: 124 mg/L (95% confidence limits: 20 – 331 mg/L)
EC50: 741 mg/L (95% confidence limits*: 286 mg/L – n.d.*)
EC80: >1000 mg/L
*: The upper confidence limit could not be calculated (>1000 mg/L).

The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 5% (guideline-recommended maximum variation of 15%).

Results with reference substance (positive control):

- Results with reference substance valid?
Yes.

- Relevant effect levels:
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 14 mg/L (the 95% confidence limits were not calculable). The 3-hour EC50 is within the guideline-recommended range of 5 – 30 mg/L, confirming suitability of the activated sludge used.

Reported statistics and error estimates:

None.

Table 2: Influence of 2-(2-Vinyloxyethoxy) ethyl acrylate and 3,5-dichlorophenol on the Oxygen consumption of activated sludge

Vessel no.	Nominal concentration of test chemical	Oxygen consumption rate	Inhibition		pH values			Oxygen concentration
					start*	end*	start*	end*
1 10	0 0	1.430 1.357			7.4 7.4	8.1 8.2	8.3 8.2	7.4 8.6
Mean deviation (%)		1.393 5.1
	Test item
5 6 7 8 9	10 32 100 320 1000	1.308 1.357 1.187 0.940 0.557	6.1 2.6 14.8 32.5 60.0		7.3 7.3 7.3 7.3 7.4	8.2 8.2 8.2 8.2 8.2	8.1 8.4 8.4 8.2 8.2	8.6 8.6 8.5 8.9 8.9
	Reference item
2 3 4	5 16 50	1.211 0.545 0.140	13.1 60.9 90.0		7.2 7.3 7.3	8.2 8.2 8.2	8.6 8.3 8.7	8.8 8.8 8.6

* Start and end of the 3-hour incubation period

Validity criteria fulfilled:

yes

Conclusions:

The 3-hour EC50 of VEEA was 741 mg/l, the NOEC was calculated as 82 mg/l.

Executive summary:

The inhibitory effect of the test item 2-(2-Vinyloxyethoxy) ethyl acrylate on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11, and the OECD Guideline for Testing of Chemicals, No. 209.

The following nominal concentrations of 2-(2-Vinyloxyethoxy) ethyl acrylate were tested: 10, 32, 100, 320 and 1000 mg/L.

In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16 and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

Up to and including the test concentration of 32 mg/L, the test item 2-(2-Vinyloxyethoxy) ethyl acrylate had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. At the next higher test item concentrations of 100, 320 and 1000 mg/L, significant inhibitory effects of 15, 33 and 60% were noted, respectively.

The calculated 3-hour EC-values and their 95% confidence limits were as follows:

NOEC (determined as the calculated 3-hour EC15): 82 mg/L (95% confidence limits: 7.0 – 209 mg/L)

EC20: 124 mg/L (95% confidence limits: 20 – 331 mg/L)

EC50: 741 mg/L (95% confidence limits: 286 mg/L – n.d.*)

EC80: >1000 mg/L

*: The upper confidence limit could not be calculated (>1000 mg/L).

Description of key information

The 3-hour EC50 of VEEA was 741 mg/l, the NOEC was calculated as 82 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:

741 mg/L

EC10 or NOEC for microorganisms:

82 mg/L

Additional information

The inhibitory effect of the test item 2-(2-Vinyloxyethoxy) ethyl acrylate on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11, and the OECD Guideline for Testing of Chemicals, No. 209.

The following nominal concentrations of 2-(2-Vinyloxyethoxy) ethyl acrylate were tested: 10, 32, 100, 320 and 1000 mg/L.

In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16 and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

Up to and including the test concentration of 32 mg/L, the test item 2-(2-Vinyloxyethoxy) ethyl acrylate had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. At the next higher test item concentrations of 100, 320 and 1000 mg/L, significant inhibitory effects of 15, 33 and 60% were noted, respectively.

The calculated 3-hour EC-values and their 95% confidence limits were as follows:

NOEC (determined as the calculated 3-hour EC15): 82 mg/L (95% confidence limits: 7.0 – 209 mg/L)

EC20: 124 mg/L (95% confidence limits: 20 – 331 mg/L)

EC50: 741 mg/L (95% confidence limits: 286 mg/L – n.d.*)

EC80: >1000 mg/L

*: The upper confidence limit could not be calculated (>1000 mg/L).