Vinyl 2-ethylhexanoate

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

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute 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

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Details on test conditions:

Experimental Design and Study Conduct
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 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 fish were exposed to a series of nominal test concentrations of 0.1, 1.0, 10 and 100% v/v saturated solution for a period of 96 hours.
A nominal amount of test item (2200 mg) was dispersed in 22 L of culture medium with the aid of propeller stirring at approximately 1500 rpm for 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 produce a 100% v/v saturated solution. A series of dilutions were made from this saturated solution to give further test solutions of 10, 1.0 and 0.10% v/v saturated solution.
To ensure adequate mixing and homogeneity each test solution was mixed with a flat bladed stirrer for approximately 1 minute.
In the range-finding test three fish were placed in each test and control vessel and maintained in a temperature controlled room at approximately 13 °C to 14 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under semi-static test conditions. Each 25 L test and control vessel contained 18 L of test media and was covered to reduce evaporation. After 1, 3, 6, 24, 29, 48, 52½, 72, 77 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each test concentration was taken for chemical analysis at 0 and 24 hours 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 the range-finding test the following test concentrations were assigned to the definitive test: 0.56, 1.8, 5.7, 18 and 58% v/v saturated solution.

Experimental Preparation
A nominal amount of test item (2200 mg) was dispersed in 22 L of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 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 this saturated solution to give further test solutions of 0.56, 1.8, 5.7, 18 and 58% v/v saturated solution.
To ensure adequate mixing and homogeneity each test solution was stirred using a magnetic stirrer for approximately 5 minutes.
The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh media at 0, 24, 48 and 72 hours and of the old media at 24, 48, 72 and 96 hours (see Annex 4).

Exposure Conditions
Glass bowls (approximately 20 L in volume), completely filled with test media, were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then sealed to reduce evaporation and maintained at approximately 13 °C to 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels received no auxiliary aeration. The fish were not individually identified and received no food during exposure.
The control group was maintained under identical conditions but not exposed to the test item.

A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.7 mg/L

95% CI:

1.1 - 2.5

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

Sub-lethal effects of exposure were observed at test concentrations of 2.5 and 9.1 mg/L. These responses were increased pigmentation, swimming at bottom of vessel, flared gills, lethargic, swimming with head up, loss of equilibrium, coughing and trailing mucous.
Excessive feces and mucous were observed in the tank at a test concentration of 2.5 mg/L.
After approximately 1½ to 2 hours exposure all of the seven fish at 9.1 mg/L were observed to be moribund and after approximately 47 or 53 hours exposure six of the seven fish at 2.5 mg/L were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following observational time point.
There were no sub-lethal effects of exposure observed in the seven fish exposed to a test concentration of 0.067, 0.21 and 0.69 mg/L for a period of 96 hours.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Rainbow trout (Oncorhynchus mykiss) to the test item gave the following results based on the geometric mean measured test concentrations:

Time Point LC50 95% Confidence Limits NOEC LOEC
(Hours) (mg/L) (mg/L) (mg/L) (mg/L)
96 1.7 1.1 - 2.5 0.69 2.5

Executive summary:

Introduction
A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2019) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 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 indicated that a dissolved test item concentration of approximately 13 mg/L was obtained from a saturated solution method of preparation.
Following a preliminary range-finding test, fish were exposed, in groups of seven, to an aqueous solution 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 96 hours at a temperature of approximately 13 °C to 14 °C under semi-static test conditions. The test item solution was prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. 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 determined 1, 3, 6, 24, 30, 48, 53, 72, 78 and 96 hours after the start of exposure.

Results
Analysis of the fresh test preparations at 0, 24, 48 and 72 hours showed measured test concentrations to range from 0.083 to 9.8 mg/L. Measured test concentrations of the aged test preparations at 24, 48, 72 and 96 hours ranged from 0.039 to 8.5 mg/L. Given that 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 0.067, 0.21, 0.69, 2.5 and 9.1 mg/L.
Exposure of Rainbow trout (Oncorhynchus mykiss) to the test item gave the following results based on the geometric mean measured test concentrations:
Time Point     LC50    95% Confidence Limits    NOEC    LOEC
  (Hours)      (mg/L)             (mg/L)                (mg/L)  (mg/L)
      96            1.7               1.1 - 2.5                0.69       2.5

Description of key information

The LC50 for freshwater fish data is 1.7 mg/L. 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

1.7 mg/L