Vinyl 2-ethylhexanoate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity 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 test solutions:

On Days 0 to 2 and 4 to 32 a nominal amount of test item (2300 mg) was dispersed in test water and the volume adjusted to 23 L with the aid of a magnetic stirrer at approximately 250 rpm for 48 hours (aspirator was completely filled and sealed with a bung to try and reduce test item losses). After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 L used to pre-condition the filter was discarded) to give a 100% v/v saturated solution.
A series of dilutions was made from the 100% v/v saturated solution to give the required test concentrations of 0.56, 1.8, 5.7, 18 and 58% v/v saturated solution.
Each of the test concentrations were inverted several times (Days 0 and 2 and from Day 3 to Day 14) to ensure adequate mixing and homogeneity. From Day 15 onwards the test preparations were stirred via magnetic stirrer for Approximately 5 minutes at 300 rpm to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test item.

Test organisms (species):

Pimephales promelas

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Details on test conditions:

The test was carried out using newly fertilized eggs of fathead minnows (Pimephales promelas). The adult fathead minnows that produced the eggs for the test were obtained from Osage Catfisheries Inc. Missouri, USA and maintained in-house since 24 June 2021 and maintained in dechlorinated tap water (see Annex 2) with an activated carbon and biological filtration system.
The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. In the 7 days preceding the start of the test, the water temperature was controlled at approximately 25 C with a dissolved oxygen content of greater than or equal to 7.6 mg O2/L. The breeding stock fish were fed daily with frozen brine shrimp and/or ZM small granular fish feed.
Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized. Fertilized eggs were collected from the breeding tanks on 29 September 2021 and used for the definitive test. The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
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 media preparation trials were conducted in order to determine the solubility of the test item under test conditions (see Annex 3).

Range-finding Test
The results obtained from a media preparation trial conducted (Study Number: 8461328) indicated that a dissolved test item concentration of approximately 13 mg/L could be obtained using a saturated solution method of preparation.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing newly fertilized fathead minnow eggs to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 15 days.
A nominal amount of test item (2000 mg) was dispersed in 20 L of test water with the aid of a propeller stirrer at approximately 1500 rpm for approximately 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Whatman Polycap filter (first approximate 2 L used to pre-condition the filter was discarded) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10, 1.0 and 0.10% v/v saturated solution.
Each of the test concentrations were inverted several times to ensure adequate mixing and homogeneity.
The test was conducted in 1 L glass vessels each containing approximately 400 mL of test preparation from Day 0 to 6, increasing to approximately 800 mL from Day 7 to 15. Two replicate flasks were used for each control and test concentration.
Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25 °C with a maximum deviation of ± 1.5 °C between test chambers or between successive days with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 15 days under semi-static test conditions. The test preparations were renewed daily throughout the test with the exception of Day 3 where there was no water change to avoid causing premature hatching of the eggs.
The control group was maintained under identical conditions but not exposed to the test item.
The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily.
The larvae were fed freshly hatched special grade brine shrimp nauplii from Day 7 to the end of the test.
Samples of the fresh test preparations were taken on Days 0 and 14 and of the expired test preparations on Days 1 and 15. All samples were taken for immediate analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.

Initial Experiments
Several initial experiments were begun; however, these were terminated as either the hatching success rate of the control group failed the validity criteria, the analytical results showed that no test item was present within the test system or the fish within the test (including controls) showed kinked spines which was un related to treatment with the test item.

Definitive Test
Based on the results of the range-finding test, newly fertilized fathead minnow eggs (four replicates of 20 eggs per group) were exposed to solutions of the test item at nominal concentrations of 0.56, 1.8, 5.7, 18 and 58% v/v saturated solution for a period of 33 days under semi-static test conditions.

Experimental Preparation
On Days 0 to 2 and 4 to 32 a nominal amount of test item (2300 mg) was dispersed in test water and the volume adjusted to 23 L with the aid of a magnetic stirrer at approximately 250 rpm for 48 hours (aspirator was completely filled and sealed with a bung to try and reduce test item losses). After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 L used to pre-condition the filter was discarded) to give a 100% v/v saturated solution.
A series of dilutions was made from the 100% v/v saturated solution to give the required test concentrations of 0.56, 1.8, 5.7, 18 and 58% v/v saturated solution.
Each of the test concentrations were inverted several times (Days 0 and 2 and from Day 3 to Day 14) to ensure adequate mixing and homogeneity. From Day 15 onwards the test preparations were stirred via magnetic stirrer for Approximately 5 minutes at 300 rpm to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test item.

Exposure Conditions
In the definitive test 1 L glass vessels were used from Day 0 to Day 14 and from Day 15 to the end of the test, 5 L glass vessels were used. The vessels were completely filled with the required test item formulation. Four replicate vessels were used for each control and test concentration.
A semi-static test regime was employed in the test involving a renewal of the test preparations daily, with the exception of Day 3 where there was no water change to avoid causing premature hatching of the eggs.
Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25 °C with a maximum deviation of ± 1.5 °C between test chambers or between successive days with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 33 days.
The test vessels were aerated via narrow bore glass tubes from Day 8 onwards. The eggs and larvae were not individually identified.
The larvae were fed freshly hatched brine shrimp nauplii from Day 7 to the end of the test.

Assessments
Test Organism Observations
The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily. The criteria of death for eggs were marked loss of translucency and change in coloration leading to a white opaque appearance. The criteria of death for larvae and juvenile fish were one or more of the following: immobility, absence of respiratory movement, absence of heart beat, white opaque coloration and lack of reaction to mechanical stimulus.
At the end of the test, the length and wet weight of each surviving fish was determined.

Key result

Duration:

33 d

Dose descriptor:

EC10

Effect conc.:

ca. 0.03 mg/L

95% CI:

>= 0.021 - 0.039

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Details on results:

Definitive Test
Verification of Test Concentrations
Analysis of the fresh test preparations on Days 0*, 6, 13, 20, 27 and 32 (see Annex 4) showed measured test concentrations to range from 0.017 to 2.3 mg/L. A decline in measured test concentration of the aged test preparations on Day 1 showed measured test concentrations to range from 0.011 to 1.05 mg/L, all other aged preparation did not show any test item present (non -detected) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The Day 0 and Day 1 control samples showed signs of test item present (0.0052 and 0.0055 mg/L respectively)†.
The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration Geometric Mean Measured
(% v/v Saturated Solution) Test Concentration (mg/L)
0.56 0.0095
1.8 0.020
5.7 0.047
18 0.47
58 1.6
* = Duplicate samples were taken and submitted for analysis as the original synthetic control used by analytical chemistry was contaminated and was shown to have had a significant impact on the data and as such the original samples could not be used as they were considered unfit for purpose.
† = Considered to be due to post sample contamination as no reduction in the test item concentration was
apparent from Day 0 to Day 1 as per all other test groups and as such it is highly unlikely that this contamination came directly from within the control tank.

Number of Eggs Hatching
The numbers of dead eggs observed during the definitive test are given in Table 11 and the relationship between percentage not hatched and concentration at Day 6 is given in Figure 1.
The start of egg hatching was observed on Day 5 of the test and completion of hatching was observed on Day 7 at 0.56, 1.8 and 5.7 % v/v. None of the eggs at test concentrations of 18 and 58 % v/v survived until hatching.

Hatching Concentration 95% confidence limits
(mg/L) (mg/L)
EC10 0.030 0.021 to 0.039
EC20 0.050 0.038 to 0.064
EC50 0.11 0.083 to 0.15

Statistical analysis of the hatching data was carried out for the control and all test concentrations (see Appendix 1). There were no statistically significant differences between the control, 0.0095 and 0.020 mg/L test concentration (P0.05), however all other test concentrations were significantly different (P<0.05) and therefore, the NOEC based on the number of eggs hatching was 0.020 mg/L. Correspondingly the LOEC based on the number of eggs hatching was 0.047 mg/L.
Post-hatch Survival
The number of dead larvae and hatched (live) larvae observed during the definitive test are given in Table 12 and Table 13 respectively. The relationship between percentage post-hatch survival and concentration at Day 33 (28 days post-hatch) is given in Figure 2. The number of dead larvae were observed to be low throughout the duration of the test with no concentration dependent effects being observed.

Post-hatch Survival Concentration 95% confidence limits
(mg/L) (mg/L)*
LC10 >0.047 Not Determined
LC20 >0.047 Not Determined
LC50 >0.047 Not Determined
* = It was not possible to calculate 95% confidence limits for the ECx values as the data generated did not fit the models available for the calculation of confidence limits.

Statistical analysis of the post-hatch survival data was carried out for the control, 0.0095, 0.020 and 0.047 mg/L test groups (see Appendix 1). There were no statistically significant differences (P0.05), between the control, 0.0095, 0.020 and 0.047 mg/L test groups and therefore the NOEC based on post-hatch survival was 0.047 mg/L. Correspondingly the LOEC based on post-hatch survival could not be determined.
The percentage of eggs hatched and post-hatch survival are given in Table 14.

Length and Weight Data
The body length and wet weight data for all fish alive at the end of the test are given in Table 15 and are presented graphically in Figure 3 and Figure 4.

Length Concentration 95% confidence limits
(mg/L) (mg/L)*
EC10 >0.047 Not Determined
EC20 >0.047 Not Determined
EC50 >0.047 Not Determined

Statistical analysis of the fish body length data was carried out for the control, 0.0095, 0.020 and 0.047 mg/L test groups (see Appendix 1). There were no statistically significant differences between the control, 0.0095 and 0.020 mg/L test concentration, however, the test concentration of 0.047 mg/L was significantly different and therefore, the NOEC based on body length was 0.020 mg/L. Correspondingly the LOEC based on body length was 0.047 mg/L.

Weight Concentration 95% confidence limits
(mg/L) (mg/L)*
EC10 0.041 0.022 to 0.075
EC20 >0.047 Not Determined
EC50 >0.047 Not Determined
* = It was not possible to calculate 95% confidence limits for the LCx values as the data generated did not fit the models available for the calculation of confidence limits.

Statistical analysis of the fish wet weight data was carried out for the control, 0.0095, 0.020 and 0.047 mg/L test groups (see Appendix 1). There were no statistically significant differences between the control and 0.0095 mg/L test concentration, however, the test concentration of 0.020 and 0.047 mg/L were significantly different and therefore the NOEC based on wet weight was 0.0095 mg/L. Correspondingly the LOEC based on wet weight was 0.020 mg/L.

Observations
A single fish in Replicate R1 of the 0.047 mg/L test group was observed to have a kinked spine between Days 28 and 30.
There were no sub-lethal effects observed in the control, 0.0095 and 0.020 mg/L test groups throughout the test.

Validation Criteria
The following validity criteria were achieved during the test:
Required Achieved
1) Dissolved oxygen Equal to or greater 83% ASV
than 60% ASV
2) Water temperature
Between test chambers ± 1.5 oC ± 0.7 oC
Between successive days ± 1.5 oC ± 1.0 oC
3) Hatching success rate* > 70% 93%
4) Post-hatch survival† > 75% 99%

Water Quality Criteria
The temperature measurements recorded in a surrogate vessel by a Testo temperature logger are presented in Figure 5 and were shown to have been maintained at between 24.8 and 25.2 C. The temperature recorded in each test vessel before and after each media renewal ranged from 24 to 25 C. The dissolved oxygen concentration was maintained at least at 6.9 mg/L (equivalent to 83% ASV) and the pH ranged from 7.2 to 8.4. Throughout the test the light intensity was observed to be in the range 647 to 809 Lux.
The water hardness values were observed to range from 132 to 160 mg/L as CaCO3 at the start of the test and from 130 to 144 mg/L as CaCO3 at termination of the test (see Table 16).

Observations on Test Item Solubility
After dosing all control and test concentrations were observed to be clear, colorless solutions by visual inspection.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of fathead minnow (Pimephales promelas) to the test item gave the following results based on the nominal test concentrations:

Hatching Concentration 95% confidence limits
(mg/L) (mg/L)
EC10 0.030 0.021 to 0.039
EC20 0.050 0.038 to 0.064
EC50 0.11 0.083 to 0.15

Post-hatch Survival Concentration 95% confidence limits
(mg/L) (mg/L)*
LC10 >0.047 Not Determined
LC20 >0.047 Not Determined
LC50 >0.047 Not Determined

Length Concentration 95% confidence limits
(mg/L) (mg/L)*
EC10 >0.047 Not Determined
EC20 >0.047 Not Determined
EC50 >0.047 Not Determined

Weight Concentration 95% confidence limits
(mg/L) (mg/L)*
EC10 0.041 0.022 to 0.075
EC20 >0.047 Not Determined
EC50 >0.047 Not Determined
* = It was not possible to calculate 95% confidence limits for the LCx values as the data generated did not fit the models available for the calculation of confidence limits.

Executive summary:

Introduction
A study was performed to assess the effects of the test item on the early life stages of the fathead minnow (Pimephales promelas). The method followed that described in the OECD Guidelines for Testing of Chemicals (2013) No 210, "Fish, Early-Life Stage Toxicity Test”.

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 indicated that a dissolved test item concentration of approximately 18 mg/L was obtained from a saturated solution method of preparation.
Based on the results of a preliminary range-finding test, newly fertilized fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to solutions of the test item at nominal concentrations of 0.56, 1.8, 5.7, 18 and 58% v/v saturated solution for a period of 33 days at a temperature of approximately 24 to 25 °C under semi-static test conditions. The test solutions were renewed daily throughout the test with the exception of Day 3 where there was no water change to avoid causing premature hatching of the eggs. The test item solution was prepared by stirring an excess (100 mg/L) of test item in test water using a magnetic stirrer for approximately 48 hours (the test vessel used was completely filled and sealed with a bung to try and reduce losses of the test item). After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 L used to pre-condition the filter was discarded) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the required test groups of 0.56, 1.8, 5.7, 18 and 58% v/v saturated solution.
The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post-hatch). At test termination the length and wet weight of the surviving fish were measured.

Results
Analysis of the fresh test preparations on Days 0, 6, 13, 20, 27 and 32 showed measured test concentrations to range from 0.017 to 2.3 mg/L. A decline in measured test concentration of the aged test preparations on Day 1 showed measured test concentrations to range from 0.011 to 1.05 mg/L, all other aged preparations did not show any test item present (non-detected) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured concentrations were determined to be 0.0095, 0.020, 0.047, 0.47 and 1.6 mg/L. The Day 0 and Day 1 control samples showed signs of test item present (0.0052 and 0.0055 mg/L respectively)*.

* = Considered to be due to post sample contamination as no reduction in the test item concentration was
apparent from Day 0 to Day 1 as per all other test groups and as such it is highly unlikely that this contamination came directly from within the control tank.

Exposure of fathead minnow (Pimephales promelas) to the test item gave the following results based on the nominal test concentrations:

Hatching Concentration 95% confidence limits
(mg/L) (mg/L)
EC10 0.030 0.021 to 0.039
EC20 0.050 0.038 to 0.064
EC50 0.11 0.083 to 0.15

Post-hatch Survival Concentration 95% confidence limits
(mg/L) (mg/L)*
LC10 >0.047 Not Determined
LC20 >0.047 Not Determined
LC50 >0.047 Not Determined

Length Concentration 95% confidence limits
(mg/L) (mg/L)*
EC10 >0.047 Not Determined
EC20 >0.047 Not Determined
EC50 >0.047 Not Determined

Weight Concentration 95% confidence limits
(mg/L) (mg/L)*
EC10 0.041 0.022 to 0.075
EC20 >0.047 Not Determined
EC50 >0.047 Not Determined
* = It was not possible to calculate 95% confidence limits for the LCx values as the data generated did not fit the models available for the calculation of confidence limits.

† = It was not possible to calculate 95% confidence limits for the ECx/LCx values as the data generated did not fit the models available for the calculation of confidence limits.

Description of key information

The EC10 of freshwater fish is 0.020 mg.L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC10

Effect concentration:

0.02 mg/L