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

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:

between 12 May 2006 and 31 July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, 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:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: April 2005 Date of Signature: November 2005

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
The following nominal concentrations of 2-(2-Vinyloxyethoxy) ethyl acrylate were tested: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control was tested in parallel (test water without test item).

- Sampling method:
The test design included three replicates per test concentration and six replicates of the control. Volumes of 15 mL algal suspension for each replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks.

The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath at a temperature of 24 °C (Table 8), and continuously illuminated at a measured light intensity of about 8200 Lux (mean value), range: 6760 to 9060 Lux (minimum and maximum value of measurements at nine places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed about 35 cm above the test flasks. The test vessels were
labeled with the RCC study number and all necessary additional information to ensure unmistakable identification. The test duration was 72 hours.

- Sample storage conditions before analysis:
The samples were stored deep-frozen and protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
The following nominal concentrations of 2-(2-Vinyloxyethoxy) ethyl acrylate were tested: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. The test medium of the highest nominal test item concentration of 100 mg/L was prepared by dissolving 60.5 mg of the test item completely in 600 mL of test water. For this, ultrasonic treatment for 10 minutes and intense stirring for 10 minutes at room temperature was applied. In a series of dilution steps this intensively mixed test medium was diluted with test water to prepare the test media with the lower test item concentrations. The test media were prepared just before the start of the test (= addition of algae).

- Eluate:
Not applicable

- Controls:
For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test. The latest result of the positive control test in 2005 (72-h EC50 for the growth rate: 0.78 mg/L, RCC Study No. 858774) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2005: 72-h EC50: 0.44–1.16 mg/L).

Additionally, a control was tested in parallel (test water without test item). The test design included three replicates per test concentration and six replicates of the control.

- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
none

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae

- Strain: Strain No. 86.81 SAG

- Source: Liquid cultures of Scenedesmus subspicatus were obtained from the Institute for Plant Physiology, University of Gottingen, D-37073 Gottingen, Germany.

- Age of inoculum :
The cells used in the test were taken from an exponentially growing pre-culture that was aet up four days prior to the start of the test.

- Method of cultivation:
The algae are cultivated in RCC's laboratories under standardised conditions according to the test guidelines. The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines.

ACCLIMATION

- Acclimation period:
Not recorded.

- Culturing media and conditions:
The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:
Macro-nutrients:

NaHCO3 50.0 mg/L
CaCl2 × 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 × 7 H2O 15.0 mg/L
MgCl2 × 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L

Trace elements:
Na2EDTA × 2 H2O 100.0 μg/L
FeCl3 × 6 H2O 80.0 μg/L
MnCl2 × 4 H2O 415.0 μg/L
H3BO3 185.0 μg/L
Na2MoO4 × 2 H2O 7.0 μg/L
ZnCl2 3.0 μg/L
CoCl2 × 6 H2O 1.5 μg/L
CuCl2 × 2 H2O 0.01 μg/L

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/l) was added to the prepared culture medium prior to use.

- Any deformed or abnormal cells observed:
None recorded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Calculated water hardness of the test water was 0.24 mmol/L (=24 mg/L as CaCO3)

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.

pH:

The pH values of the test media was measured and recorded in each test concentration and the control at the start of the test and after 24,48 and 72 hours in the volumes incubated for the analytical samples.

Dissolved oxygen:

Not recorded.

Salinity:

Not applicable, as freshwater study

Nominal and measured concentrations:

The following nominal concentrations of 2-(2-Vinyloxyethoxy) ethyl acrylate were tested: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 250ml
- Aeration: No aeration

- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable

- Renewal rate of test solution (frequency/flow rate): Not applicable

- Initial cells density: The test was started (0 hours) by inoculation of 10,000 algal cells per mL of test medium. These cells were taken from an exponentially growing pre-culture that was set up four days prior to the test under the same conditions as in the test. One day before the start of the test, the pre-culture was diluted threefold to keep the algae in exponential growth.

The test design included three replicates per test concentration and six replicates of the control. Volumes of 15 mL algal suspension for each replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks.

- Control end cells density:

- No. of organisms per vessel:
initial cell density of approximately 1 x 10E4 cells/ml.

- No. of vessels per concentration (replicates):
Three replicate flasks per concentration.

- No. of vessels per control (replicates):
Six replicate flasks.


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM

The test medium of the highest nominal test item concentration of 100 mg/L was prepared by dissolving 60.5 mg of the test item completely in 600 mL of test water. For this, ultrasonic treatment for 10 minutes and intense stirring for 10 minutes at room temperature was applied. In a series of dilution steps this intensively mixed test medium was diluted with test water to prepare the test media with the lower test item concentrations. The test media were prepared just before the start of the test (= addition of algae).

The actual concentrations of the test item in the test media were analytically determined

OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH: No adjustments recorded

- Photoperiod: Not stated

- Light intensity and quality: light intensity of about 8,200 Lux (mean value) and the range was calculated to be between 6760 to 9060 Lux.

EXPOSURE CONDITIONS :
The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath at a temperature of 24 °C (Table 8), and continuously illuminated at a measured light intensity of about 8200 Lux (mean value), range: 6760 to 9060 Lux (minimum and maximum value of measurements at nine places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed about 35 cm above the test flasks. The test vessels were labeled with the RCC study number and all necessary additional information to ensure unmistakable identification.


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Small volumes of the test media and the control (1.0 mL) were taken out of all test flasks after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter®, Model ZM), with at least two measurements per sample.

In addition, after 72 hours exposure, a sample was taken from the control and from a test concentration with reduced algal growth (nominal 10 mg/L). The shape and size of the algal cells were examined microscopically in these samples. This test concentration (nominal 10 mg/L was chosen since, at the two highest nominal concentrations of 32 and 100 mg/L, the algal cell density was too low for a reliable microscopic examination.

For the determination of the LOEC and NOEC, the calculated mean biomass and the mean growth rate at the test concentrations were tested for significant differences when compared to the control values by a Dunnett-test

- Chlorophyll measurement: Not recorded

TEST CONCENTRATIONS
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 20, 40, 80 and 160 mg/l.

- Range finding study:
The test concentrations were based on the results of a range-finding test (without GLP). The enlarged spacing factor of 3.2 between the test concentrations was chosen because, according to the results of the range-finding test, the concentration-effect relationship was rather flat and thus, a large concentration range had to be tested.

POSITIVE CONTROL:
For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test. The latest result of the positive control test in 2005 (72-h EC50 for the growth rate: 0.78 mg/L, RCC Study No. 858774) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2005: 72-h EC50: 0.44–1.16 mg/L).


VALIDATION:

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL 0 - 3.6%

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL 1.8–4.6%

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL 0.24–14

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL: 7.9–14%

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

18 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL 7.8–60

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 23–62

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.78 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.78 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

RESULTS AND DISCUSSION
At the start of the test, the measured test item concentrations in the analyzed test media ranged from 92 to 102% of the nominal values (see analytical results and Table 2 in the attached analytical phase report). This shows the correct preparation of the test media.

At the test concentrations of nominal 10 to 100 mg/L the test item was sufficiently stable during the test period of 72 hours (85 to 96% of the nominal test concentrations at test end). At nominal 1.0 to 3.2 mg/L, the test item concentrations decreased to 46 to 70% of nominal at the end of the 72 hour test period. The losses could be due to an adsorption of the test item onto the algae and/or glass surfaces.

The mean measured test concentrations (calculated as the geometric mean over all measurements per test concentration) varied in the range of 78 to 98% of the nominal values. Therefore, the reported biological results are based on the mean measured test item concentrations. The tabulated values represent rounded results obtained by calculation using the exact raw data.


Nominal test concentration (mg/L) Mean measured test item concentration (average over all measurements per test concentration) (mg/L)
0.32 not analyzed
1.0 0.78 (78% of nominal)
3.2 2.7 (83% of nominal)
10 8.8 (88% of nominal)
32 31 (96% of nominal)
100 98 (98% of nominal)

The influence of the test item 2-(2-Vinyloxyethoxy) ethyl acrylate on the growth of Scenedesmus subspicatus is shown in Tables 1–5 and Figure 1. The test item had a statistically significant inhibitory effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after the test period of 72 hours at concentrations of 2.7 mg/L (nominal 3.2 mg/L) and above (results of Dunnett-tests, one-sided, α = 0.05, Tables 4 and 5). Thus, the test concentration of 2.7 mg/L was determined as the 72-hour LOEC (lowest concentration tested with toxic effects).

The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined to be 0.78 mg/L, since up to and including this test concentration both the mean biomass and the mean growth rate of the algae were not statistically significantly lower than in the control (Tables 4 and 5).

The EC values as well as the NOEC and LOEC were calculated for both parameters, the algal biomass (b) and the growth rate (r), after 72 hours test duration, based on mean measured concentrations:
Parameter (0-72 h) Biomass b (mg/L) Growth rate r (mg/L)
EC50 5.0 10
95%-confidence limits 0.24–14 7.9–14
EC10 1.4 3.2
95%-confidence limits 0–3.6 1.8–4.6
EC90 18 33
95%-confidence limits 7.8–60 23–62
NOEC 0.78 0.78
LOEC 2.7 2.7

The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing in test medium with reduced algal growth (nominal 10 mg/L) and the algal cells in the control. There were no obvious effects on the shape and size of the algal cells growing in test media containing the test item at up to and including this nominal concentration.

At the start of the test, the pH values in the test media and the control ranged from 7.9 to 8.1 (Table 7). The pH increased during the test period and was in the range of 8.4 to 10.6 at test end, when measured in the volumes incubated for the analytical samples. The increase of the pH during the testwas obviously caused by the CO2-consumption of the algae due to their rapid growth or their high densities (although the test media have been intensively stirred). The pH measured in the actual test itself was in the range if 8.4 to 8.9 at test end. The differences in pH between the flasks with the analytical samples and the actual test are obviously caused by differences in the incubation flasks and the test medium volume (for analytical purpose volumes up to 100 mL per sample were needed).

Results with reference substance (positive control):

In the control the cell density increased from a nominal N = 1 × 104 cells/mL at the start of the test (0 hours) to N = 64 × 104 cells/mL (mean value) after 72 hours (Table 1). Thus, the algal growth in the control was sufficiently high under the conditions of the test. The validity criterion of increase of cell density by at least a factor of 16 over 72 hours was fulfilled. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period (Table 6).

Reported statistics and error estimates:

Calculation of LC10, LC50, LC90, NOEC and LOEC.

Dunnett's test.

For tables (1 -8) please see attachment 2.

Validity criteria fulfilled:

yes

Conclusions:

Parameter (0 – 72 hours) Biomass b (mg/L) Growth rate r (mg/L)
EC 50 – 95% confidence limits 5.0 (0.24 – 14) 10 (7.9 – 14)
EC 10 – 95% confidence limits 1.4 (0 – 3.6) 3.2 (1.8 – 4.6)
EC 90 – 95% confidence limits 18 (7.8 – 60) 33 (23 – 62)
NOEC 0.78 0.78
LOEC 2.7 2.7

Executive summary:

The influence of the test item 2 -2(Vinyloxyethoxyether ethyl acrylate on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72 hour static test according to the EU commission Directive 92/69/EEC C.3, 1992 and the OECD Guideline No. 201, 1984.

The nominal test concentrations were 0.32,1.0, 3.2, 10, 32 and 100 mg/L in parallel with a control.

At the start of the test, the measured test item concentrations in the analyzed test media ranged from 92 to 102% of the nominal values. At the test concentrations of nominal 10 to 100 mg/L the test item was sufficiently stable during the test period of 72 hours (85 to 96% of the nominal test concentrations at test end). At nominal 1.0 to 3.2 mg/L, the test item concentrations decreased to 46 to 70% of nominal at the end of the 72 hour test period. The losses could be due to an adsorption of the test item onto the algae and/or glass surfaces.

The mean measured test concentrations (calculated as the geometric mean over all measurements per test concentration) varied in the range 78% to 98% of the nominal values. Therefore, the reported biological results are based on the mean measured test item concentrations.

Nominal test concentration (mg/L)	Mean measured test item concentration (average over all measurements per test concentration) (mg/L)
0.32	Not analysed
1	0.78
3.2	2.7
10	8.8
32	31
100	98

The test item had a statistically significant inhibitary effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after the test period of 72 hours at concentrations of 2.7 mg/L and above (results of Dunnett-tests, one sided α = 0.05). Thus, the test concentration of 2.7 mg/L was determined as as the 72 -hour LOEC (lowest concentration tested with toxic effects after a test period of 72 hours) was determined to be 0.78 mg/L, since up to and including this test concentration both the mean and the biomass and the mean growth rate of the algae were not statistically significantly lower than in the control.

The EC values as well as the NOEC and LOEC were calculated for both parameters, the algal biomass (b) and the growth rate (r), after 72 hours test duration, based on mean measured test concentrations:

Parameter (0 – 72 hours)	Biomass b (mg/L)	Growth rate r (mg/L)
EC 50 – 95% confidence limits	5.0 (0.24 – 14)	10 (7.9 – 14)
EC 10 – 95% confidence limits	1.4 (0 – 3.6)	3.2 (1.8 – 4.6)
EC 90 – 95% confidence limits	18 (7.8 – 60)	33 (23 – 62)
NOEC	0.78	0.78
LOEC	2.7	2.7

Description of key information

The 72 hr EC50 value for growth rate (based on mean measured test concentrations) was 10 mg/L. The 72 hr NOEC value for growth rate (based on mean measured test concentrations) was 0.78 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

10 mg/L

EC10 or NOEC for freshwater algae:

0.78 mg/L

Additional information

The influence of the test item 2 -2(Vinyloxyethoxyether ethyl acrylate on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72 hour static test according to the EU commission Directive 92/69/EEC C.3, 1992 and the OECD Guideline No. 201, 1984.

The nominal test concentrations were 0.32,1.0, 3.2, 10, 32 and 100 mg/L in parallel with a control.

At the start of the test, the measured test item concentrations in the analyzed test media ranged from 92 to 102% of the nominal values. At the test concentrations of nominal 10 to 100 mg/L the test item was sufficiently stable during the test period of 72 hours (85 to 96% of the nominal test concentrations at test end). At nominal 1.0 to 3.2 mg/L, the test item concentrations decreased to 46 to 70% of nominal at the end of the 72 hour test period. The losses could be due to an adsorption of the test item onto the algae and/or glass surfaces.

The mean measured test concentrations (calculated as the geometric mean over all measurements per test concentration) varied in the range 78% to 98% of the nominal values. Therefore, the reported biological results are based on the mean measured test item concentrations.

Nominal test concentration (mg/L)	Mean measured test item concentration (average over all measurements per test concentration) (mg/L)
0.32	Not analysed
1	0.78
3.2	2.7
10	8.8
32	31
100	98

The test item had a statistically significant inhibitary effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after the test period of 72 hours at concentrations of 2.7 mg/L and above (results of Dunnett-tests, one sided α = 0.05). Thus, the test concentration of 2.7 mg/L was determined as the 72 -hour LOEC (lowest concentration tested with toxic effects after a test period of 72 hours) and the NOEC was determined to be 0.78 mg/L, since up to and including this test concentration both the mean and the biomass and the mean growth rate of the algae were not statistically significantly lower than in the control.

The EC values as well as the NOEC and LOEC were calculated for both parameters, the algal biomass (b) and the growth rate (r), after 72 hours test duration, based on mean measured test concentrations:

Parameter (0 – 72 hours)	Biomass b (mg/L)	Growth rate r (mg/L)
EC 50 – 95% confidence limits	5.0 (0.24 – 14)	10 (7.9 – 14)
EC 10 – 95% confidence limits	1.4 (0 – 3.6)	3.2 (1.8 – 4.6)
EC 90 – 95% confidence limits	18 (7.8 – 60)	33 (23 – 62)
NOEC	0.78	0.78
LOEC	2.7	2.7