Vinyl 2-ethylhexanoate

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

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Specific details on test material used for the study:

Identification: vinyl 2-ethylhexanoate
CAS Number: 94-04-2
EC Number: 202-297-4
Batch: PM9CEH01
Purity: 99.6%
Physical state / Appearance: Clear colorless liquid
Expiry date: 04 January 2022
Storage conditions: Room temperature in the dark over silica gel

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and each surviving test group for immediate quantitative analysis. Samples of the fresh test preparations were taken from the bulk test preparations on Days 0, 6, 13 and 20 and of the expired test preparations from the pooled replicates on Days 1, 7, 14* and 21. Duplicate samples were taken and stored frozen for further analysis if necessary.

Test organisms (species):

Daphnia magna

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Details on test conditions:

Experimental Design and Study Conduct
Preliminary Media Preparation Trial
Preliminary solubility work conducted (Labcorp Study Number 8461247) indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 14 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions (see Annex 3).

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
The test item solutions were prepared in triplicate for the first three preparations, this was then reduced to one replicate. Nominal amounts of test item (56.5 mg) were separately dispensed to 565 mL of test water in completely filled glass test vessels which were sealed immediately. The flasks were shaken at approximately 150 rpm at a temperature of 24 °C for 24 hours. After the shaking period the contents of the replicate flasks (if prepared) were pooled and any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL was discarded in order to pre-condition the filter) to give a 100% v/v saturated solutions of the test item. A series of dilutions was made from this saturated solution to give further test solutions of 0.10, 1.0 and 10% v/v saturated solution.
Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
Due to the potentially volatile nature of the test item, testing was conducted in completely filled, sealed test vessels in order to minimize possible losses due to volatilization.
In the range-finding test, for each concentration a single daphnid was placed in 120 mL vessels completely filled with test preparation and sealed. For each test and control group five replicate test vessels were prepared. The water temperature was maintained at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 10 days.
The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were renewed daily. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
Each daphnid received approximately 5 to 20 μL of an algal suspension (Raphidocelis subcapitata) daily, supplemented by the addition of approximately 20 μL of GEMMA Micro 300 fish food suspension on Days 0, 1, 2 and 3. 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.
A sample of each test concentration was taken for chemical analysis on Days 0 and 1 in order to determine the stability of the test item under test conditions. All samples were analyzed on the day of sampling. Duplicate samples were taken and stored frozen for further analysis if necessary.

Definitive Test
Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 0.45, 1.0, 2.2, 4.8 and 10% v/v saturated solution for a period of 21 days. The test solutions were renewed daily throughout the test.

Experimental Preparation
A nominal amount of test item (56.5 mg) was separately dispensed to 565 mL of test water in completely filled glass test vessels which were sealed immediately. The flasks were shaken at approximately 150 rpm at a temperature of 24 °C for 24 hours. After the shaking period, any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL was discarded in order to pre-condition the filter) to give a 100% v/v saturated solutions of the test item. This saturated solution was then further diluted to give the 10, 4.8 and 2.2% v/v saturated solutions of the test item. The 10% v/v saturated solution was also further diluted to give the 1.0 and 0.45 % v/v saturated solutions of the test item.
Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 1, 6, 7, 13, 14, 20 and 21 (see Annex 4).

Exposure Conditions
For each concentration a single daphnid was placed in a 120 mL vessel completely filled with test preparation and sealed. For each test and control group 10 replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (not exceeding1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed daily throughout the test. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
Each daphnid received approximately 5 to 20 μL of an algal suspension (Raphidocelis subcapitata) daily, supplemented by the addition of approximately 20 μL of GEMMA Micro 300 fish food suspension on Days 0, 1, 2 and 3. 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.

Assessments
Test Organism Observations
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.
Young daphnids were considered to be dead if no sign of movement was apparent during examination. Adult daphnids which were unable to swim within approximately 15 seconds after gentle agitation (i.e. immobile), were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
At the end of the test, the length of each surviving parent animal was determined.

Water Quality Criteria
Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Fisherbrand digital thermometer. Measurements were made on one replicate for each test concentration. The temperature was also measured every hour in a surrogate vessel using a Testo temperature logger. The water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week.
The light intensity was also recorded daily throughout the test and was measured by a RS ILM 1332A Light meter.

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

ca. 0.26 mg/L

95% CI:

>= 0.18 - <= 0.35

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

Definitive Test
Based on the results of a preliminary range-finding test, test concentrations of 0.45, 1.0, 2.2, 4.8 and 10% v/v saturated solution were selected for the definitive test.

Verification of Test Concentrations
Analysis of the fresh test preparations on Days 0, 6, 13 and 20 showed measured test concentrations to range from 0.037 to 1.1 mg/L. A decline in measured test concentration of the aged test preparations on Days 1, 7, 14* and 21 was observed to be between 0.0060 and 0.73 mg/L. A duplicate control sample from Day 13 was submitted for analysis as a small amount of test item was found in the original sample (0.0041 mg/L). No contamination was found in the duplicate sample. As not all test concentrations were maintained within ± 20% of the measured initial test concentration it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.023, 0.052, 0.10, 0.32 and 0.71 mg/L.

Test Organism Observations
The observations for each test and control group are summarized in Table 2. The summary of daily observations is given in Table 3 and total cumulative production of live young is given in Table 4. The mean number of offspring per parent Daphnia (for each test group) alive at the end of the test is shown in Figure 1. Data for each individual replicate vessel are given in Appendix 2 to Appendix 7.

Validation Criteria
The following validation criteria were achieved during the test:
Required Actual
Control mortality ≤20 %0%
Mean number of live young per surviving adult (control group) ≥60 after 21 days143
Coefficient of variation for control group ≤ 25% 6.9%
Ephippia produced 0 0
Dissolved oxygen >3 mg O2/L≥7.7 mg O2/L
pH (control group) 6 to 9 Variation ≤1.57.4 to 8.3 0.9

Lethal Effects on the Parental Generation (P1)
Low levels of mortality (10%) were observed at the test concentrations of 0.023, 0.10 and 0.32 mg/L. Statistical analysis of the mortality data using Fisher’s Exact Binomial Test with Bonferroni Correction Test Procedure showed that the observed mortalities in the 0.023, 0.10 and 0.32 mg/L test groups were not significantly different (P≥0.05) when compared to the control. No mortalities occurred in the Control, 0.052 and 0.71 mg/L test groups throughout the test (see Appendix 1).
The following ECx, NOEC and LOEC (immobilization) values based on geometric mean measured test concentrations were estimated by inspection of the data or calculated by Fisher’s Exact Binomial Test with Bonferroni Correction Test Procedure incorporating Qualitative Trend analysis by Contrasts at 21 days:

Endpoint Concentration mg/L
(95% confidence limits)
Immobilization
EC10 >0.71 (not determined)
EC20 >0.71 (not determined)
EC50 >0.71 (not determined)
No Observed Effect Concentration 0.71
Lowest Observed Effect Concentration Not determined

Sub-lethal Effects on the Parental Generation (P1)
No sub-lethal effects of exposure were observed (including prior to Daphnia being found dead) in the control and at the test concentrations of 0.023, 0.052 and 0.32 mg/L; however, one Daphnia was observed to be pale in the 0.10 mg/L test group on Days 17 and 18.
At 0.71 mg/L, all parental generation Daphnia were pale on Days 6 and 7 and from Day 15 until Day 21.
The body length of surviving adults at Day 21 are given in Table 5.
After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P≥0.05) between the control and the 0.023 mg/L test group; however, a significant difference (P<0.05) was determined between the control and the 0.052, 0.10, 0.32 and 0.71 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test item (see Appendix 1). The relationship between body length and concentration at 21 days is given in Figure 2.
The following ECx, NOEC and LOEC values (body length) based on geometric mean measured test concentrations were calculated using the 3-Parameter Normal Cumulative Distribution Function and the Williams Multiple Sequential t-test incorporating Shapiro-Wilk's test on normal distribution, Levene’s test on variance homogeneity and Trend analysis by Contrasts at 21 days:

Endpoint Concentration mg/L
(95% confidence limits)
Body Length
EC10 0.26 (0.18 – 0.35)
EC20 0.64 (0.43 – 0.94)
EC50 >0.71 (not determined)
No Observed Effect Concentration 0.023
Lowest Observed Effect Concentration 0.052

Effects on Reproduction
After 21 days there were no statistically significant differences (P≥0.05) between the control and the 0.023, 0.052, 0.10 and 0.32 mg/L test groups. The 0.71 mg/L test group showed a statistically significant difference (P<0.05) from the control after 21 days in terms of producing fewer numbers of live young per adult (see Appendix 1). The relationship between percentage reduction in offspring and concentration at 21 days is given in Figure 3.
The following ECx, NOEC and LOEC (reproduction) values based on geometric mean measured test concentrations were calculated by Probit analysis using Linear-maximum likelihood regression and Williams Multiple Sequential t-test incorporating Shapiro-Wilk's test on normal distribution, Levene’s test on variance homogeneity and Trend analysis by Contrasts at 21 days:

Endpoint Concentration mg/L
(95% confidence limits)
Reproduction
EC10 0.31 (0.28 – 0.34)
EC20 0.40 (0.36 – 0.42)
EC50 0.63 (0.59 – 0.67)
No Observed Effect Concentration 0.32
Lowest Observed Effect Concentration 0.71

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 the 0.023, 0.052 and 0.32 mg/L test groups were in the same general condition as the young produced by the controls over the duration of the test. However, one brood was observed to be small at 0.10 mg/L and eight daphnids produced one brood each which were small at 0.71 mg/L mg/L.
Young were first produced in the control test group on Day 8 of the test.
There were no unhatched eggs or dead young observed in the control or any of the treatment groups throughout the test.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

Endpoint Concentration mg/L
(95% confidence limits)
Immobilization
EC10 >0.71 (not determined)
EC20 >0.71 (not determined)
EC50 >0.71 (not determined)
No Observed Effect Concentration 0.71
Lowest Observed Effect Concentration Not determined

Endpoint Concentration mg/L
(95% confidence limits)
Body Length
EC10 0.26 (0.18 – 0.35)
EC20 0.64 (0.43 – 0.94)
EC50 >0.71 (not determined)
No Observed Effect Concentration 0.023
Lowest Observed Effect Concentration 0.052

Endpoint Concentration mg/L
(95% confidence limits)
Reproduction
EC10 0.31 (0.28 – 0.34)
EC20 0.40 (0.36 – 0.42)
EC50 0.63 (0.59 – 0.67)
No Observed Effect Concentration 0.32
Lowest Observed Effect Concentration 0.71

Executive summary:

Introduction
A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Methods
Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.
A preliminary media preparation trial (Labcorp Study Number: 8461247) indicated that a dissolved test item concentration of approximately 14 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.
Due to the potentially volatile nature of the test item, testing was conducted in completely filled and sealed test vessels in order to minimize possible losses due to volatilization.
Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 0.45, 1.0, 2.2, 4.8 and 10% v/v saturated solution for a period of 21 days. The test solutions were renewed daily throughout the test. The test item solutions were prepared by shaking in excess (100 mg/L) of the test item in test water at approximately 150 rpm, at a temperature of 24 °C for 24 hours. After the shaking period any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL was discarded in order to pre-condition the filter) to give a 100% v/v saturated solutions of the test item. This saturated solution was then further diluted to give the 10, 4.8 and 2.2% v/v saturated solutions of the test item. The 10% v/v saturated solution was also further diluted to give the 1.0 and 0.45 % v/v saturated solutions of the test item.
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, supplemented for the first 4 days of the test with a GEMMA Micro 300 fish food suspension.

Results
Analysis of the fresh test preparations on Days 0, 6, 13 and 20 showed measured test concentrations to range from 0.037 to 1.1 mg/L. A decline in measured test concentration of the aged test preparations on Days 1, 7, 14* and 21 was observed to be between 0.0060 and 0.73 mg/L. As not all test concentrations were maintained within ± 20% of the measured initial test concentration it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.023, 0.052, 0.10, 0.32 and 0.71 mg/L.
Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

Endpoint Concentration mg/L
(95% confidence limits)
Immobilization
EC10 >0.71 (not determined)
EC20 >0.71 (not determined)
EC50 >0.71 (not determined)
No Observed Effect Concentration 0.71
Lowest Observed Effect Concentration Not determined

Endpoint Concentration mg/L
(95% confidence limits)
Body Length
EC10 0.26 (0.18 – 0.35)
EC20 0.64 (0.43 – 0.94)
EC50 >0.71 (not determined)
No Observed Effect Concentration 0.023
Lowest Observed Effect Concentration 0.052

Endpoint Concentration mg/L
(95% confidence limits)
Reproduction
EC10 0.31 (0.28 – 0.34)
EC20 0.40 (0.36 – 0.42)
EC50 0.63 (0.59 – 0.67)
No Observed Effect Concentration 0.32
Lowest Observed Effect Concentration 0.71

* = Due to a sampling error the Day 14 (old) concentration could not be determined for the 4.8 % v/v saturated solution samples and as such could not be used in order to determine the geometric mean concentration.
† = It was not possible to calculate 95% confidence limits for the ECx value as the data generated did not fit the models available for the calculation of the confidence limits

Description of key information

The chronic EC10 for freshwater daphnia is 0.26 mg/L. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

0.26 mg/L