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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 12 April 2010 and 30 June 2010.

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 guidlines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 15/09/09 Date of signature: 26/11/09

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.11, 0.23, 0.35, 1.1 and 2.3
- Sampling method: Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 2, 5, 7, 9, 12, 14, 16 and 19 and of the expired test preparations on Days 2, 5, 7, 9, 12, 14, 16, 19 and 21.

Sodium chloride (15 g/50 ml w/v) was added to a volume of test sample which was then extracted direct in the bottle with ethyl acetate to give final theoretical concentrations of 1.1 to 12 mg/I.

- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

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

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 12480 Duplex Water Softener) giving water with a total hardeness of approximately 140 mg/l as CaC03. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

For the purpose of the definitive test the test item was dissolved directly in dechlorinated tap water.

An amount of test item (100 mg) was dissolved in dechlorinated tap water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. From this a dilution was prepared in dechlorinated tap water to give a further stock solution of 10 mg/l. Aliquots (22, 46, 70, 220 and 460 ml) of the 10 mg/l stock solution were each separately diluted in a final volume of 2 litres of dechlorinated tap water to give the nominal test concentrations of 0.11, 0.23, 0.35, 1.1 and 2.3 mg/l test concentrations respectively. Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: 1st instar Daphnia magna derived from in house cultures
- Source: derived from in house cultures
- Age of parental stock (mean and range, SD): not stated in report
- Feeding during test: yes
- Food type: algae (Chlorella sp)
- Amount: Each daphnid received approximately 5 µI of a unicellular algal culture (ChIarella sp.), daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
- Frequency: daily

ACCLIMATION
- Acclimation period: at least 5 days
- Acclimation conditions (same as test or not): the test water used for the test was the same as that used to maintain the stock animals
- Type and amount of food: algae (Chlorella sp)
- Feeding frequency: ach daphnid received approximately 5 µI of a unicellular algal culture (ChIarella sp.), daily.
- Health during acclimation (any mortality observed): not reported

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

The water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week.
The water hardness was observed to be in the range 128 to 152 mgll as CaC03 in the control and the highest surviving test group throughout the test. v

Test temperature:

The temperature were recorded before and after each test media renewal. The flasks were maintained at between 20 and 22 degrees C. the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. A single temperature
during the definitive test was measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.

pH:

pH was recorded before and after each test media renewal. During the study there were no treatment related differences for pH.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded before and after each test media renewal. The dissolved oxygen content was measured using a dissolved oxygen meter. During the study there were no treatment related differences for dissolved oxygen concentration.

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

Salinity:

Not applicable as fresh water was used for the study.

Nominal and measured concentrations:

Based on the data supplied by the Sponsor the following nominal test concentrations were initially assigned to the test: 0.55, 1.8, 5.5, 18 and 55 mg/l.The test conducted using these nominal concentrations showed immobilisation at all test concentrations by Day 14 of the test indicating a prolonged toxic effect at the concentrations used. In agreement with the Sponsor the nominal test concentrations to be used in the definitive test were revised to 0.11, 0.23, 0.35, 1.1 and 2.3 mg/l.

Measured concentrations:
Analysis of the freshly prepared media showed measured concentrations to range from 102%> to 157% of nominal. Analysis of the old media showed measured concentrations to range from B20/0 to 1440/0 of nominal. Some of these results were outside the 80% to 120% acceptance limits. Duplicate samples, stored frozen prior to analysis, were analysed and showed variable results significantly lower than the initial analyses. This was considered to be possibly due to instability of the test item during the freezing and/or thawing process. It was considered that the results from the duplicate samples did not accurately reflect the concentrations of the test item in the samples and therefore were not considered in any calculation of the results.

Details on test conditions:

TEST SYSTEM
- Test vessel: 150ml flasks
- Type (delete if not applicable): covered with a plastic lid to reduce evaporation
- Material, size, headspace, fill volume: fill volume: 100ml
- Aeration: not aerated
- Type of flow-through (e.g. peristaltic or proportional diluter): not reported
- Renewal rate of test solution (frequency/flow rate): The test preparations were renewed 3 times per week on Days 0, 2, 5, 7, 9, 12, 14, 16 and 19. The adult Daphnia were transferred to fresh media by wide-bore pipette.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 0
- Biomass loading rate: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon: Please see attachment 1 (appendix 2) in the attachments section
- Particulate matter: Please see attachment 1 (appendix 2) in the attachments section
- Metals: Please see attachment 1 (appendix 2) in the attachments section
- Pesticides: Please see attachment 1 (appendix 2) in the attachments section
- Chlorine: the distilled water was dechlorinated by passage through an activated carbon filter (Purite 500).
- Alkalinity: Please see attachment 1 (appendix 2) in the attachments section
- Ca/mg ratio: Please see attachment 1 (appendix 2) in the attachments section
- Conductivity: Please see attachment 1 (appendix 2) in the attachments section
- Salinity: not applicable as freshwater used


OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 hours light and 8 hours darkness, with 20 minute duck periods imbetween
- Light intensity: 507 to 531 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19 (old and fresh media) and 21 (old media).

On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) Daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.

The parental (P1) generation were tested for immobilisation, it was deemed unappropriate to test the young daphnids for this due to the large numbers produced. At the end of the test, the length of each surviving parent animal was determined.


VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY
Not applicable

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation and reproduction

Remarks on result:

other: 95%CL 2.5 - 14 mg/L

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.42 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.26 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation and reproduction

Details on results:

- Mortality of parent animals: 0
- No. of offspring produced per day per female: see table 9
- Body length and weight of parent animals: After 21 days the length of each surviving adult was determined, the results of which are given in Appendix 4. The results showed that there were no statistically significant differences (P> 0.05) between the control and the 0.13 and 0.26 mg/l test groups in terms of length of the daphnids after 21 days exposure to the test item. The daphnids in the 0.42, 1.4 and 2.6 mg/l test concentrations were significant smaller compared to the control group.
- Type and number of morphological abnormalities: none
- Type and number of behavioural abnormalities: none
- Number of males and females (parental)
- Time to first brood release or time to hatch: see table 9
- Egg development time: see table 9
- Effect concentrations exceeding solubility of substance in test medium: None as the test item was shown to be stable in the test medium.

Reported statistics and error estimates:

Evaluation of data:
The EC50 (immobilisation) values up to Day 21 of the test were estimated by inspection of the data. The EC50 (immobilisation) value and associated confidence limits at 21 days were calculated by the maximum-likelihood logic method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999). Logit analysis is used where two or more partial responses to exposure are shown.
The observed mortalities in the parental (P1) generation of the 0.23, 0.35 and 1.1 mg/l test groups were compared to the control group using the corrected chi-squared statistic (Breslow and Day 1980).

The EC50 (reproduction) value and associated confidence limits after 21 days were calculated by the maximum-likelihood logit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).

For the estimation of the "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) the numbers of live young produced per adult over the duration of the test for the control and each test group were compared 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) (see Appendix 4). Results from the control and each test group Daphnia length data, determined for the surviving daphnids on termination of the test, were compared 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) (see Appendix 4). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001). For more information see appendix 4 in the attachments section.

Observations on Test Item Solubility:

The freshly prepared test media were observed to be clear, colourless solutions whereas the old test media were observed to be green tinged due to the presence of algal cells used as feed for the daphnids.

Effects on the Filial Generation (F1):

Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.

Young were first produced in the control test group on Day 8 of the test. Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

For results tables, please see attachment 3 in the attachments section.

Validity criteria fulfilled:

yes

Remarks:

The validation criterion were achieved throughout the test:

Conclusions:

Exposure of Daphnia magna to the test item resulted in significant mortalities at the timeweighted mean measured test concentration of 2.6 mg/l resulting in 44% mortalities by Day 21.

The 21-Day EC50 (immobilisation) value, based on time-weighted mean measured test concentrations, for the parental Daphnia generation (P1) was calculated to be 4.3 mg/l with 95% confidence Iirnits of 2.5 - 14 mg/l

The 21-Day EC50 (reproduction) based on time-weighted mean measured test concentrations was 4.3 mg/l with 95% confidence limits of 2.5 - 14 mg/l.

The "Lowest Observed Effect Concentration" based on the length of the surviving adults was considered to be 0.42 mg/l. The "Lowest Observed Effect Concentrations" based on immobilisation and reproduction were considered to be 2.6 and 1.4 mg/l respectively.

The "No Observed Effect Concentration" (NOEC) was 0.26 mg/I as there were no significant mortalities (immobilisation) observed in the parental generation (P1), there were no significant differences (P > 0.05) in terms of the number of live young produced per adult and no significant differences (P > 0.05) in terms of adult daphnia length when compared to the control after 21 days.

Executive summary:

Introduction:

A study was performed to assess the effect of the test item on the reproduction of Daphnia magna over a 21-day period. The method followed that

described in the GECD Guidelines for Testing of Chemicals No 211 (1998) "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Regulation (EC) No 440/2008.

Methods:

Based on data supplied by the Sponsor, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test item over a range of time-weighted mean measured test concentrations of 0.13, 0.26, 0.42, 1.4 and 2.6 mg/I for a period of 21 days. The test solutions were renewed 3 times per week. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with an algal suspension.

Results.

Analysis of the freshly prepared media showed measured concentrations to range from 102% to 157% of nominal. Analysis of the old media showed measured concentrations to range from 82% to 144% of nominal. Some of these results were outside the 80% to 120% acceptance limits. Duplicate samples, stored frozen prior to analysis, were analysed and showed variable results significantly lower than the initial analyses. This was considered to be possibly due to instability of the test item during the freezing and/or thawing process. It was considered that the results from the duplicate samples did not accurately reflect the concentrations of the test item in the samples and therefore were not considered in any calculation of the results.

A general trend for a decline in measured concentration over each dosing period was observed and therefore it was considered justifiable to base the results on time-weighted mean measured test concentrations to give a "worst case" analysis of the data. These were calculated to be 0.13, 0.26, 0.42, 1.4 and 2.6 mg/I.

The 21-Day EC50 (immobilisation) value based on the time-weighted mean measured test concentrations, for the parental Daphnia generation (P1) was calculated to be 4.3 mg/I with 95% confidence limits of 2.5 - 14 mg/I.

The 21-Day EC50 (reproduction) value based on the time-weighted mean measured test concentrations was calculated to be 4.3 mg/l with 95% confidence limits of 2.5 - 14 mg/l.

The "Lowest Observed Effect Concentration" was considered to be 0.42 mg/l on the basis that at this test concentration, although no significant mortalities (immobilisation) were observed in the parental generation (P1) and that there were no significant differences (P2 > 0.05) between the control and this test group in terms of numbers of live young produced per adult by Day 21, the surviving adults were significantly smaller in length compared to the control group.

The "No Observed Effect Concentration" was considered to be 0.26 mg/l on the basis that at this test concentration there were no significant mortalities (immobilisation) observed in the parental generation (P1) and there were no significant differences (P2 > 0.05) in terms of the number of live young produced per adult when compared to the control group after 21 days.

Description of key information

The "No Observed Effect Concentration" was considered to be 0.26 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.26 mg/L

Additional information

Introduction:

A study was performed to assess the effect of the test item on the reproduction of Daphnia magna over a 21-day period. The method followed that

described in the GECD Guidelines for Testing of Chemicals No 211 (1998) "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Regulation (EC) No 440/2008.

Methods:

Based on data supplied by the Sponsor, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test item over a range of time-weighted mean measured test concentrations of 0.13, 0.26, 0.42, 1.4 and 2.6 mg/I for a period of 21 days. The test solutions were renewed 3 times per week. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with an algal suspension.

Results.

Analysis of the freshly prepared media showed measured concentrations to range from 102% to 157% of nominal. Analysis of the old media showed measured concentrations to range from 82% to 144% of nominal. Some of these results were outside the 80% to 120% acceptance limits. Duplicate samples, stored frozen prior to analysis, were analysed and showed variable results significantly lower than the initial analyses. This was considered to be possibly due to instability of the test item during the freezing and/or thawing process. It was considered that the results from the duplicate samples did not accurately reflect the concentrations of the test item in the samples and therefore were not considered in any calculation of the results.

A general trend for a decline in measured concentration over each dosing period was observed and therefore it was considered justifiable to base the results on time-weighted mean measured test concentrations to give a "worst case" analysis of the data. These were calculated to be 0.13, 0.26, 0.42, 1.4 and 2.6 mg/I.

The 21-Day EC50 (immobilisation) value based on the time-weighted mean measured test concentrations, for the parental Daphnia generation (P1) was calculated to be 4.3 mg/I with 95% confidence limits of 2.5 - 14 mg/I.

The 21-Day EC50 (reproduction) value based on the time-weighted mean measured test concentrations was calculated to be 4.3 mg/l with 95% confidence limits of 2.5 - 14 mg/l.

The "Lowest Observed Effect Concentration" was considered to be 0.42 mg/l on the basis that at this test concentration, although no significant mortalities (immobilisation) were observed in the parental generation (P1) and that there were no significant differences (P2 > 0.05) between the control and this test group in terms of numbers of live young produced per adult by Day 21, the surviving adults were significantly smaller in length compared to the control group.

The "No Observed Effect Concentration" was considered to be 0.26 mg/l on the basis that at this test concentration there were no significant mortalities (immobilisation) observed in the parental generation (P1) and there were no significant differences (P2 > 0.05) in terms of the number of live young produced per adult when compared to the control group after 21 days.