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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 26 2004 - June 22 2004

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:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: November 18-22 2002, Date of Signature: June 22 2004

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Same as tested concentration.

- Sampling method:
Not recorded.
The stability of the frozen sample was tested during non-GLP pre-experiments.

- 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 (especially for difficult test substances)

The test media were prepared as follows: A concentrated stock solution of nominal 100 mg/L was freshly prepared just before the start of the test (=introduction of the fish) and prior to each test medium renewal. For this, 100 mg (range: 100.0 - 100.9 mg) of the test item were dissolved completly in 1000 mL of test water using ultrasonic treatment for 5 minutes and stirring for 10 minutes at room temperature. In a series of subsequant dilution steps, adequate volumes of this intensively stirred stock solution were added to the test water in the aquaria and were intensively mixed to prepare the test media with the test concentrations as stated.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name:
Zebra fish

- Strain:
Not recorded

- Source:
Zoohaus Schaub, CH-4410 Liestal, Switzerland.

- Age at study initiation (mean and range, SD):

- Length at study initiation:
The mean body length of the fish was 3.1 +/- 0.2 cm

- Weight at study initiation:
The mean body wet weight was 0.25 +/- 0.05g (Mean +/- SD)

- Method of breeding:
Not applicable

- Feeding during test
Not fed during test


ACCLIMATION
- Acclimation period:
One week

- Acclimation conditions (same as test or not):
Same as test - test water and temperature

- Type and amount of food:
Commercial fish diet (Tetra Min (R) Hauptfutter, supplied by TETRA-Werke, D-49304 Melle, Germany

- Feeding frequency:
During holding and acclimation until one day before the start of the test.

- Health during acclimation (any mortality observed):
During holding and acclimation the mortality rate in the test fish batch was lower than 5% and all fish were healthy.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not recorded.

Hardness:

2.5 mmol/L (=250mg/L) as CaCO3

Test temperature:

The water temperature, pH-values and oxygen concentrations were measured in each test concentration and the control at the start of the test and once every day during the test in the freshly prepared and old test media. At the same dates the appearance of the test media was recoded.

21-22 deg C during the test period (please see Table 5)

pH:

The water temperature, pH-values and oxygen concentrations were measured in each test concentration and the control at the start of the test and once every day during the test in the freshly prepared and old test media. At the same dates the appearance of the test media was recoded.
7.8 +/- 0.1 (Please see Table 3)
Alkalinity : 0.8 mmol/L (of the test water)

Dissolved oxygen:

The water temperature, pH-values and oxygen concentrations were measured in each test concentration and the control at the start of the test and once every day during the test in the freshly prepared and old test media. At the same dates the appearance of the test media was recoded.

8.5 +/- 0.2 (Please see Table 4)

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal
0.46, 1.0, 2.2, 4.6 and 10 mg/L.
The test concentrations of the test item in the test media were analytically determined.
Additionally, a control was tested in parallel (test water without test item).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: Glass aquarium with 5 litres test material
- Aeration: The test water was aerated prior to the preparation of the test media until oxygen saturation was reached.

- Type of flow-through:
Not recorded

- Renewal rate of test solution:
The test media were renewed daily to keep the concentrations of the test substance in the test media as constant as possible during the test period of 96 hours.
During this semi-static test, each day the surviving test fish were placed into a clean aquarium with freshly prepared test medium of the corresponding test concentration.

- No. of organisms per vessel:
7

- No. of vessels per concentration:
1

- No. of vessels per control:
1

- No. of vessels per vehicle control:
Not applicable

- Biomass loading rate:
The loading rate was 0.35g fish wet weight per litre test medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reconstituted water: analytical grade salts were dissolved in deionized water to obtain the correct nominal concentrations (as listed below).

- Total organic carbon:
Not recorded

- Particulate matter:
Not recorded

- Metals:
MgSO4 x 7H2) : 0.5 mmol/L (=123 mg/L)
NaHCO3 : 0.75 mmol/L (=65 mg/L)
KCl : 0.075 mmol/L (=5.8 mg/L)

- Pesticides:
Not recorded

- Chlorine:
CaCl2 x 2H2O : 2.0 mmol/L (=294 mg/L)

- Alkalinity:
0.8 mmol/L

- Ca/mg ratio:
4:1 (based on molarity)

- Conductivity:
Not recorded

- Culture medium different from test medium:
No

- Intervals of water quality measurement:
Daily


OTHER TEST CONDITIONS
- Adjustment of pH:
Not recorded

- Photoperiod:
16 hour light to 8 hour dark photoperiod with a 30 minute transition period

- Light intensity:
Light intensity during the light period was approximately within the range of 90 to 120 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The test fish were observed after approximately 3, 24, 48, 72 and 96 hours test duration for mortality and visible abnormalities.

Dead fish were removed at least once daily and discarded.

THE LC50 and the 95% confidence interval at the observation date after 96 hours could not be calculated by Probi Analysis or Moving Average Interpolation due to the steep concentration-effect relationship. Instead, the 96 hours LC50-value was determined as the geometric mean value of the two consecutive test concentrations with 0 and 100% mortality, and the 95% confidence intervals for the LC50 as the test concentrations with 0 and 100% mortality. The LC50 at the observation date after 72 hours was estimated by linear interpolation.

The NOEC, LOEC, LC0 and LC100 were determined directly from the raw data.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0.46, 1.0, 2.2, 4.6 and 10 mg/L.
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: Not recorded

- Results used to determine the conditions for the definitive study: Not recorded

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

6.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence interval: 4.6 - 10 mg/L

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The biological results (visible abnormalities observed at the test fish, mortalities and the LC50-values) are listed in Table 3. In the control and at the test concentrations up to and including 2.2 mg/L all fish survived until the end of the test and no visible abnormalities were observed at some test fish, but all test fish survived until the end of the test at this test concentration. At the highest tst concentration of 10 mg/L visible abnormalities were observed at the test fish three hours after test start, and at the end of the test all fish were dead.

Thus, the 96-hour NOEC (highest concentration tested withou observable toxic effects after the exposure period of 96 hours) and the 96-hour LC0 of the test substance to zebra fish were determined to be 2.2 mg/L. The 96-hour LOEC (lowest concentration tested with observable toxic effects) was 4.6 mg/L.

The 96-hour LC50 of the tested substance was calculated to be 6.8 mg/L with a 95% confidence interval from 4.6 to 10 mg/L. The 96-hour LC100 was 10 mg/L/ Thus, the concentration effect relationship of the test item was rather steep.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test medium renewal periods (Table 2).

The pH values in the test media and the control ranged from 7.7 to 7.9 (Table 3). The oxygen concentration was always 8.0 mg/L or higher (Table 4), and thus higher than 60% oxygen saturation. The water temperature ranged from 21 to 22 deg C (Table 5).

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

None reported.

Sublethal observations / clinical signs:

Table 1: Mortality and visible abnormalities observed at the test fish

Abbreviations of visible abnormalities for which fish were observed during the study:

AK: Strongly extended gills                            OB: Fish mainly at the water surface

AP: Apathy                                                    SA: Mucus secretion

BA: Distended abdomen                                SR: Fish lying on side or back on the bottom

BO: Fish mainly at the bottom of the aquarium SV: Strong ventilation

GA: Exophthalmus                                          TS: Tumbling during swimming

KR: Convulsions                                             VF: Changed body colour

Number of fish tested at each concentration and the control: 7

Nominal test item concentration (mg/L)	Number of affected fish*/number of dead fish visible abnormalities
	Observation time
	3 hours	24 hours	48 hours	72 hours	96 hours
Control	0/0	0/0	0/0	0/0	0/0
0.46	0/0	0/0	0/0	0/0	0/0
1.0	0/0	0/0	0/0	0/0	0/0
2.2	0/0	0/0	0/0	0/0	0/0
4.6	1/0 TS	2/0 TS, BO	7/0 TS, BO	7/6 TS, SR	07-Jul
LC50	>10	>10	>10	7.8	6.8
95% CL	-	-	-	nd	4.6 - 10

* - number of dead fish plus number of fish with visible abnormalities

95% CL - 95% confidence interval of the LC50

nd - could not be determined

Table 2: Appearance of the freshly prepared and old test media

Abbreviations:

0: no remarkable observations, clear test medium

1: homogeneous dispersion in the water, turbidity observable

2: noticeable turbility caused by test item

3: noticeable colouration caused by the test item

4: inhomogeneous dispersion of the test item

5: precipitation of the test item

6: test item at the surface

7: test item lying at the bottom of the aquarium

Nominal test item concentration (mg/L)	Exposure time
	0 h	24 h		48 h		72 h		96 h
	new	old	new	old	new	old	new	old
0.46	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0
2.2	0	0	0	0	0	0	0	0
4.6	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0

Table 3: pH-values in the freshly prepared and old test media and in the control

Nominal test item concentration (mg/L)	Exposure time
	0 h	24 h		48 h		72 h		96 h
	new	old	new	old	new	old	new	old
control	7.7	7.8	7.8	7.8	7.9	7.8	7.8	7.8
0.46	7.8	7.8	7.8	7.9	7.9	7.8	7.8	7.8
1.0	7.8	7.8	7.8	7.9	7.9	7.8	7.8	7.8
2.2	7.8	7.8	7.8	7.9	7.9	7.9	7.9	7.8
4.6	7.8	7.9	7.8	7.9	7.9	7.9	7.9	7.8
10	7.8	7.9	7.9	7.9	7.9	7.9	7.9	7.9

Table 4: Oxygen concentrations (mg/L) in the freshly prepared and old media and in the control

Nominal test item concentration (mg/L)	Exposure time
	0 h	24 h		48 h		72 h		96 h
	new	old	new	old	new	old	new	old
control	8.5	8.0	8.5	8.2	8.2	8.1	8.4	8.5
0.46	8.4	8.5	8.6	8.3	8.3	8.4	8.5	8.5
1.0	8.4	8.5	8.6	8.3	8.3	8.4	8.5	8.5
2.2	8.4	8.5	8.6	8.4	8.4	8.5	8.5	8.6
4.6	8.3	8.5	8.7	8.4	8.3	8.5	8.5	8.6
10	8.3	8.5	8.7	8.4	8.4	8.5	8.5	8.6

Table 5: Temperature (deg C) in the freshly prepared and old test media and in the control

Nominal test item concentration (mg/L)	Exposure time
	0 h	24 h		48 h		72 h		96 h
	new	old	new	old	new	old	new	old
control	21	22	21	21	22	21	21	21
0.46	21	21	21	21	21	21	21	21
1.0	21	21	21	21	21	21	21	21
2.2	21	21	21	21	21	21	21	21
4.6	21	21	21	21	21	21	21	21
10	21	21	21	21	21	21	21	21

Validity criteria fulfilled:

yes

Conclusions:

The biological test results after 96 hours test duration:
- 96 -hour LC50: 6.8 mg/L
(95% confidence interal: 4.6 - 10 mg/L)
- 96 -hour NOEC: 2.2 mg/L

Executive summary:

The acute toxcity of the test item 2-(2'-Vinyloxyethoxy)ethyl acrylate to zebra fish (Brachydanio rerio) was determined in a 96 -hour semi-static test with a daily test medium renewal according to the EU Commission Directive 92/69/EEC, Part C.1 (1992) and the OECD Guideline for Testing of Chemicals No. 203 (1992).

The nominal test item concentrations tested were 0.46, 1.0, 4.6 and 10 mg/L, and in parallel a control.

In the analysed test medium samples from the start of the test medium renewal periods the measured test item concentrations ranged between 97 and 111% of the nominal values. This shows the correct dosage of the test item. At the end of the test medium renewal periods 91 - 101% of the nominal values were found. Thus, the test item concentrations were sufficiently constant during the test medium renewal periods of one day. Therefore, the reported biological results are related to the nominal test item concentrations.

The biological test results after 96 hours test duration:

- 96 -hour LC50: 6.8 mg/L

(95% confidence interal: 4.6 - 10 mg/L)

- 96 -hour LC0: 4.6 mg/L

- 96 -hour LC100: 10 mg/L

- 96 -hour NOEC: 2.2 mg/L

- 96 -hour LOEC: 4.6 mg/L

Description of key information

The 96-hour LC50 of the tested substance was calculated to be 6.8 mg/L.

The 96-hour NOEC was  determined to be 2.2 mg/L.  

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

6.8 mg/L

Additional information

The acute toxicity of the test item 2-(2'-Vinyloxyethoxy)ethyl acrylate to zebra fish (Brachydanio rerio) was determined in a 96 -hour semi-static test with a daily test medium renewal according to the EU Commission Directive 92/69/EEC, Part C.1 (1992) and the OECD Guideline for Testing of Chemicals No. 203 (1992).

The nominal test item concentrations tested were 0.46, 1.0, 4.6 and 10 mg/L, and in parallel a control.

In the analysed test medium samples from the start of the test medium renewal periods the measured test item concentrations ranged between 97 and 111% of the nominal values. This shows the correct dosage of the test item. At the end of the test medium renewal periods 91 - 101% of the nominal values were found. Thus, the test item concentrations were sufficiently constant during the test medium renewal periods of one day. Therefore, the reported biological results are related to the nominal test item concentrations.

The biological test results after 96 hours test duration:

- 96 -hour LC50: 6.8 mg/L

(95% confidence interval: 4.6 - 10 mg/L)

- 96 -hour LC0: 4.6 mg/L

- 96 -hour LC100: 10 mg/L

- 96 -hour NOEC: 2.2 mg/L

- 96 -hour LOEC: 4.6 mg/L