2,2-Difluoroethyl Acetate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 January 2016 to 08 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10; 18; 32; 56; and 100 mg/L nominal.
- Sampling method: Two sets of samples were taken for analysis. One set consisted of new samples of test solutions at the time of preparation and another set consisted of old samples of the same test solutions immediately prior to renewal or at the end of exposure. At the time of preparation, samples were collected from the middle layer of the vessel for measuring the test conditions of each test group. At the time immediately prior to renewal and at the end of exposure, samples were collected from the middle layer of one vessel of each test group.
- Sample storage conditions before analysis: no data available.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A nominal amount of test item (110 mg) was dissolved in test water and the volume adjusted to 1100 L to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give the required test solutions of 10, 18, 32, 56 and 100 mg/L. The stock solution and its dilutions were prepared at the beginning of the test and before the renewal period. The stock solution and its dilution were mixing by invertion to ensure adequate mixing and homogeneity.
- Controls: Only dilution water.
- Test concentration separation factor: 1.78.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea.
- Age at study initiation: Juveniles less than 24-hour-old (female).
- Source: National Institute for Environmental Studies, Japan in 1995; then the daphnids have been maintained at the testing facility.
- Method of breeding of the parent daphnids that produced the juveniles used for the test:
* Water: Elendt M4 medium.
* Biomass loading: 1 daphnid or less/80 mL (25 daphnids or less/2 L).
* Vessel: 2-L glass beaker.
* Temperature: 20 ± 1°C.
* Dissolved oxygen concentration (D.O.): 60% or more of saturation.
* pH: 6.0–9.0.
* Light: Fluorescent light, 16-hour light (800 lux or less) / 8-hour dark.
* Renewal of water in the culture vessel: The first renewal was performed within 5 days after the start of culturing; subsequently, 3 times per week.
* Food type: Chlorella vulgaris (unicellular green algae).
* Amount and frequency: 6 mg C (organic carbon)/2 L/day.
* Offspring removal: Removed on the day offspring were produced.
* At the start of exposure, it was confirmed that the parent daphnids fulfilled the following conditions:
Week age: 2–4 weeks (Culture period: December 22, 2015 to January 6, 2016).
Mortality of parent daphnids: 20% or less during the 2-week period prior to the start of exposure.
Abnormality: Not observed.
Production of first brood: within 12 days.
- Age of parental stock (mean and range, SD): 28 days or less.
- Feeding during test: no.

ACCLIMATION
- Acclimation period: no.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

228 mg CaCO3/L before the start of exposure.

Test temperature:

19.8 °C - 19.9 °C.

pH:

7.5 – 8.1.

Dissolved oxygen:

8.0 to 8.8 mg/L, i.e.: > =3 mg/L.

Salinity:

Not relevant.

Conductivity:

No data available.

Nominal and measured concentrations:

-Nominal concentations: control, 10, 18, 32, 56 and 100 mg/L.
-Mesured concentration on Day 0 (start of experiment): <0.1, 9.44, 16.4, 26.6, 49.2, 91.0 mg/L.
-Mesured concentration on Day 1 (end of the renewal period): <0.1, 7.53, 11.0, 22.9, 46.3, 87.3 mg/L.
-Mesured concentration on Day 1 (new test solution): <0.1, 8.76, 15.5, 32.4, 48.3, 86.3 mg/L.
-Mesured concentration on Day 2 (end of the renewal period): <0.1, 7.10, 12.8, 22.6, 36.8, 67.1 mg/L.
-Time-weighted mean measured concentrations: <0.1, 8.18, 13.8, 26.0, 45.0 and 82.7 mg/L.
The proportions of the time-weighted mean measured concentrations to the nominal concentrations were 77–83%.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100-mL glass incubation bottle with ground-in stopper.
- Type: closed.
- Volume of solution: 100 mL/vessel.
- Aeration: No aeration during the test.
- Renewal rate of test solution (frequency): daily.
- No. of organisms per vessel: 5 daphnids/vessel.
- No. of vessels per concentration (replicates): 4 vessels/test group.
- No. of vessels per control (replicates): 4 vessels/test group.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Temperature, dissolved oxygen and pH were measured in the test solutions at the beginning of the test, before and after the renewal and at the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: 16-hour light/8-hour dark.
- Light intensity: 800 lux or less.

EFFECT PARAMETERS MEASURED
Number immobile and other abnormal symptoms after 24-hour and 48-hour exposure.

RANGE-FINDING STUDY
* Range-finding test No.1.
- Test concentrations: 0.10, 1.0, 10, 100 mg/L nominal.
* Range-finding test No.2
- Test concentrations: 100 mg/L nominal.
- Results used to determine the conditions for the definitive study: 20 % daphnids immobile after 48h of exposure in the 100 mg/L test group. Test concentrations for the definitive study were selected on this basis.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

63 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: no.
- Other adverse effects control: no.
- Immobilisation of control: no.
- Abnormal responses: no.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values / Effect concentrations exceeding solubility of substance in test medium: no, in all test groups, the test solutions appeared colorless, and suspended, floating and precipitated substance was not observed in the test solutions during exposure.

Results with reference substance (positive control):

Acute immobilisation tests of the reference substance (potassium dichromate, guaranteed reagent) have been conducted periodically (approximately every 6 months).
- Results with reference substance is valid.
- 48h-EC50: 0.53 mg/L (95% confidence limits: 0.48–0.59 mg/L). Exposure period: July 14, 2015 to July 16, 2015. This value was within the range found in this laboratory (minimum–maximum: 0.53–1.02 mg/L).

Reported statistics and error estimates:

Determination of EC50: Probit method.
Data-processing program: TOXDAT3_02_D.

Number of immobilised Daphnia magna (percent immobility):

Test group	Nominal concentration (mg/l)	Mean^a measured concentration (mg/l)	Cumulative number of immobilised daphnia (percent immobility)
			24 hours	48 hours
Control	--	--	0 (0)	0 (0)
Conc.1	10	8.18	0 (0)	0 (0)
Conc.2	18	13.8	0 (0)	0 (0)
Conc.3	32	26.0	0 (0)	0 (0)
Conc.4	56	45.0	0 (0)	2 (10)
Conc.5	100	82.7	2 (10)	17 (85)

a: Time-weighted mean

 

Median effect concentration (EC50):

Exposure period (hours)	EC50 (mg/L)	95-percent confidence limits (mg/l)	Statistical method
24	>82.7 ^*1	--	--
48	63.0 ^*2	54.6     -     72.9	Probit

*1: The EC50 value and its 95% confidence limits could not be determined by statistical method because the immobility of Daphnids at the maximum concentration level was less than 50%. Therefore, the EC50 value was given as an estimated concentration range.

*2: Using the data of Conc.1 - 5

 

Highest concentration with 0％ immobility (EC0) and lowest concentration with 100％ immobility (EC100):

Exposure period (hours)	EC0 (mg/L)	EC100 (mg/L)
48	26.0	--

-- : Not observed

Validity criteria fulfilled:

yes

Remarks:

Dissolved oxygen concentration > 3 mg/L (minimum 8.0 mg/L in the study); immobilised daphnids and daphnids floating on the surface of the water in the control < 10% (no immobilised daphnids in the control during the test).

Conclusions:

The 48-hour EC50 of 2,2-Difluoroethyl acetate to Daphnia magna was determined to be 63.0 mg/L.

Executive summary:

The acute toxicity of 2,2-Difluoroethyl acetate to Daphnia magna was investigated in a 48-hour semi-static test according to the OECD guideline 202 under GLP compliance.

Groups of 20 daphnids (divided in 4 replicates of 5 daphnids each) were exposed to control, and test chemical at nominal concentrations of 10, 18, 32, 56 and 100 mg/L. Test solutions were renewed daily. The corresponding actual concentrations were measured using GC/MS as being equal to 8.18, 13.8, 26.0, 45.0 and 82.7 mg/L (time-weighted mean measured concentrations), respectively. The number of immobilized daphnids in test solutions were counted after 24 and 48 hours of exposure and compared to those of the controls.

After 48 hours of exposure, no immobilized test organisms were recorded in the control and up to and including the mean measured concentration of 26 mg/L. At the mean measured concentrations of 56 and 100 mg/L, 10% and 85% of the daphnids were found to be immobile, respectively. The 48-h EC50 was determined to be 63.0 mg/L; meaning 2,2-Difluoroethyl acetate is acutely harmful to aquatic invertebrates.

Description of key information

The 48-hour EC50 of 2,2-Difluoroethyl acetate to Daphnia magna was determined to be 63.0 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

63 mg/L

Additional information

An experimental study performed under GLP compliance and in accordance with OECD test guideline 202 was flagged as a key study and assigned a Klimisch score of 1. It reports a 48-hour EC50 of 63.0 mg/L; meaning 2,2-Difluoroethyl acetate was thus acutely harmful to aquatic invertebrates.