2,2-Difluoroethyl Acetate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 03 September 2018 to 14 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations, i.e. 10, 18, 32, 56 and 100mg/L (nominal).
- Sampling method: 30 mL samples were taken from each test vessel at the beginning of the test, before and after each renewal and at the end of the test.
- Sample storage conditions before analysis: The samples were stored in refrigerator (2-8°C) after pretreatment if they were not analysed on the day of collection.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
* Range-finding test:
Approximately 1g of the test substance was dissolved in 1L test water to prepare a stock solution with a nominal concentration of 1g/L. The test solutions were prepared by dilution of the stock solution with test water. The stock solution used in range-finding test was renewed daily. Three nominal concentrations of 1, 10 and 100mg/L were tested.
* Definitive test:
Approximately 2g test substance was dissolved in 2L test water to prepare a stock solution with a nominal concentration of 1g/L. The test solutions were prepared by dilution of the stock solution with test water. The stock solution used in definitive test was renewed daily. Five nominal concentrations of 10, 18, 32, 56 and 100mg/L were tested.
- Controls: Test water only.
- Test concentration separation factor: ca. 1.8 in the definitive test.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

TEST ORGANISM
- Common name: Chinese Rare Minnow.
- Source: Bred in Bioassay and Safety Assessment Lab, Shanghai Academy of Public Measurement (ID: GR-SAPM-170110).
- Length: 2.5±0.1cm (Mean ± SD at the end of the test).
- Weight: 0.28±0.02g (Mean ± SD at the end of the test).
No further data available.

ACCLIMATION
- Acclimation period: 31 days.
- Acclimation conditions (same as test):
* Water: Tap water dechlorinated by activated carbon and sterilized by UV lamp.
* Temperature: 21-25℃ and constant within a range of 2°C.
* pH: 6.0-8.5.
* Dissolved oxygen concentration: > 80% of air saturation value.
* Photoperiod: Light/Dark = 12 hours/12 hours.
- Type of food during acclimation: fish were fed with brine shrimp flake (O.S.I®, USA).
- Feeding frequency during acclimation: Daily until 24 hours before the test was started.
- Health during acclimation: No diseases were observed during the acclimation period. No mortality was observed within the 7 days before the start of the test.

FEEDING DURING TEST: no.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

No data available.

Test temperature:

22.1 to 23.1°C in the test solutions.

pH:

7.78 to 8.35 in the test solutions.

Dissolved oxygen:

63.5% to 98.8% of air saturation value.

Salinity:

Not concerned.

Conductivity:

No data available.

Nominal and measured concentrations:

-Nominal concentrations: control, 10, 18, 32, 56 and 100 mg/L.
-Measured concentration on Day 0 (start of experiment): ND, 9.65, 19.1, 33.0, 57.0 and 105 mg/L.
-Measured concentration on Day 1 (end of the renewal period): ND, 9.51, 17.7, 32.2, 54.0 and 105 mg/L.
-Measured concentration on Day 1 (new test solution): ND, 10.0, 18.3, 32.4, 55.7 mg/L and NM.
-Measured concentration on Day 2 (end of the renewal period): ND, 10.0, 18.5, 31.4, 54.8 mg/L and NM.
-Measured concentration on Day 2 (new test solution): ND, 9.64, 18.8, 32.9 mg/L, NM and NM.
-Measured concentration on Day 3 (end of the renewal period): ND, 8.40, 17.7, 31.6 mg/L.
-Measured concentration on Day 3 (new test solution): ND, 9.53, 19.2 and 34.4 mg/L, NM and NM.
-Measured concentration on Day 4 (end of the test): ND, 7.77, 18.3, 32.5 mg/L, NM and NM.
-Geometric mean measured concentrations: ND, 9.28, 18.4, 32.5, 55.4 and 105mg/L.
(ND: Not Detected, NM: Not Measured)

During the test, the measured concentrations of the test substance in each group analysed by GC-FID varied less than 20% during each renewal period (range of variation was between -18.5% and 1.09% over the four renewal periods). Concentrations measured in all the freshly prepared solutions varied within ±20% of their arithmetic mean (range of variation as compared to arithmetic mean was between -2.92% and 3.69%). Finally, it has to be noticed that geometric mean measured concentrations did not deviate by more that 20% from nominal concentrations in any of the treatments.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessels.
- Type (delete if not applicable): closed with seals.
- Material, size, headspace, fill volume: full volume of six litres without headspace.
- Aeration: no.
- Renewal rate of test solution (frequency): every 24h.
- No. of organisms per vessel: 7 fish/concentration.
- No. of vessels per concentration (replicates): 1 vessel/concentration.
- No. of vessels per control (replicates): 1 vessel/concentration.
- Biomass loading rate: 0.33 g fish/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water dechlorinated by activated carbon and sterilized by UV lamp.
- Intervals of water quality measurement: pH, dissolved oxygen concentration and temperature were measured in the test vessels at the beginning of the test, before and after each renewal and at the end of the test.
No further data available.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: Light/Dark=12 hours/12 hours.
- Light intensity: no data available.

EFFECT PARAMETERS MEASURED
Test fish were observed at 3, 24, 48, 72 and 96 hours after the start of exposure for mortality and abnormality.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8.
- Range finding study: yes.
* Test concentrations: 1, 10 and 100mg/L.
* Results used to determine the conditions for the definitive study: no mortality at 1 and 10 mg/L, 100% of mortality at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (No reference substance was directly tested in the context of the present study. However, potassium dichromate is routinely tested once a year in the testing laboratory)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

36.3 mg/L

95% CI:

30 - 43.8

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Abnormalities: Lost of equilibrium was observed in some treatments (see in the table below in the field "Sublethal observations / clinical signs").
- Mortality of control: no.
- Other adverse effects control: no.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: no.

Results with reference substance (positive control):

- Results with reference substance are valid (within the ±2SD range of the QC chart of the testing laboratory, i.e. 82.2-310mg/L).
- LC50: 257mg/L (95% confidence limits: 207-309mg/L).

Reported statistics and error estimates:

The 24h-and 96h-LC50 with 95% confidence limits were calculated using the method of Spearman-Karber, the 48h- and 72h-LC50 were directly deduced from the graph. The data analysis was carried out with software TOXCALC-Toxicity Data Analysis Software (v5.0.32).

Sublethal observations / clinical signs:

Mortalities and abnormalities in definitive test (7 fish in each vessel):

Group		Duration (h)	Mortalities	Cumulative mortalities	Abnormalities
Blank control		0	0	0	none
		3	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	0	0	none
Treatment groups (geometric mean measured concentration, mg/L)	9.28	0	0	0	none
		3	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	0	0	none
	18.4	0	0	0	none
		3	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	0	0	none
	32. 5	0	0	0	none
		3	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	Two lost equilibrium
		96	2	2	Two lost equilibrium
	55.4	0	0	0	none
		3	0	0	none
		24	6	6	One lost equilibrium
		48	1	7	/
		72	/	7	/
		96	/	7	/
	105	0	0	0	none
		3	0	0	Three lost equilibrium
		24	7	7	/
		48	/	7	/
		72	/	7	/
		96	/	7	/

 

 

The 24h, 48h, 72h and 96h LC₅₀ values as well as 95% confidence limits:

LC50 (mg/L)		95% Confidence Limits (mg/L)
		Lower	Upper
24h	46.1	39.5	53.9
48h	42.4	-	-
72h	42.4	-	-
96h	36.3	30.0	43.8

"-" : not available.

Validity criteria fulfilled:

yes

Remarks:

No mortality in controls, dissolved oxygen concentrations > 60 % air saturation value throughout the test (minimum value during the test: 63.5%).

Conclusions:

The 96-hour LC50 of 2,2-Difluoroethyl acetate to Gobiocypris rarus was determined to be 36.3 mg/L.

Executive summary:

The acute toxicity of 2,2-Difluoroethyl acetate to Gobiocypris rarus was investigated in a 96-hours semi-static test according to the OECD guideline 203 under GLP compliance.

Groups of seven fish were exposed to a control treatment and the test chemical at various nominal concentrations: 10, 18, 32, 56 and 100 mg/L, with a daily renewal. The corresponding actual concentrations were measured using GC/FID as being equal to 9.28, 18.4, 32.5, 55.4 and 105 mg/L (geometric mean measured concentrations), respectively. Test fish were observed at 3, 24, 48, 72 and 96 hours after the start of exposure for mortality and abnormality.

After 96 hours of exposure, no mortality were recorded in the control and up to and including the mean measured concentration of 18.4 mg/L. At the mean measured concentration of 32.5 mg/L, 2 out of 7 fish were found to be dead and at 55.4 and 105 mg/L, 100% of the fish were found to be dead. The 96-h LC50 was determined to be 36.3 mg/L; meaning 2,2-Difluoroethyl acetate is acutely harmful to fish.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 04 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 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 18, 32, 56 and 100 mg/L nominal.
- Sampling method and collection: Samples of the test solutions were taken at the beginning and the end of two different renewal periods. These two sets of test solutions, one at the beginning of exposure (0 hour) and before renewal at 24 hours and the other after renewal at 72 hours and at the end of exposure (96 hours), were analyzed.
- 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 (580 mg) was dissolved in test water and the volume adjusted to 5800 mL 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 each renewal period. The stock solution and its dilution were stirring for 5 min 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):

Oryzias latipes

Details on test organisms:

TEST ORGANISM
- Common name: Medaka.
- Source: Kitamura-yourijou (388 Gunchiku 12-bancho, Yatsushiro-city, Kumamoto, Japan).
- Age: Less than a half year after hatching.
- Length of control fish at the end of exposure: 2.06 cm (1.82 – 2.33 cm), n=10.
- Weight of control fish at the end of exposure: 0.058 g (0.034 – 0.088 g), n=10.

ACCLIMATION
- Acclimation period: November 30, 2015 –January 4, 2016.
- Acclimation conditions:
Water: Dechlorinated water.
Procedure: Flow-through (Biomass loading: 1 g fish/L/day or less).
Temperature: 24 ± 1°C.
Dissolved oxygen concentration (D.O.): 80% or more of the saturation.
pH: 6.0 – 8.5.
Light: Fluorescent light, 16-hour light (1000 lux or less) / 8-hour dark.
- Type/amount of food and feeding frequency during acclimation:
Feed: TetraMin, TetraWerke.
Feeding amount: 2% of the body weight/day (approx.) (until 24 hours before exposure) .
- Medication during acclimation: Not applied.
- Health during acclimation (any mortality observed): Less than 5% mortality during the 7 days before the test.

FEEDING DURING TEST: no.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

54 mg CaCO3/L (for the dilution water)

Test temperature:

23.4 to 23.6°C.

pH:

7.1 to 7.7.

Dissolved oxygen:

6.7 to 8.8 mg/L.

Salinity:

Not concerned.

Conductivity:

15 mS/m (for the dilution water).

Nominal and measured concentrations:

-Nominal concentrations: control, 10, 18, 32, 56 and 100 mg/L.
-Measured concentration on Day 0 (start of experiment): <0.1, 9.42, 17.5, 31.5, 49.0, 100 mg/L.
-Measured concentration on Day 1 (end of the renewal period): <0.1, 9.36, 20.3, 29.9, 54.5, 92.2 mg/L.
-Measured concentration on Day 3 (new test solution): <0.1, 9.86, 17.6, 36.4 mg/L, NM*, NM.
-Measured concentration on Day 4 (end of the renewal period): <0.1, 8.26, 16.9, 32.2 mg/L, NM, NM.
* NM = Not measured because all fish were dead in these treatments.
-Time-weighted mean measured concentrations: <0.1, 9.21, 18.1, 32.5, 51.7, 96.0 mg/L being almost equivalent to the nominal concentrations.
The proportions of the time-weighted mean measured concentrations to the nominal concentrations were 83-114%.

Details on test conditions:

TEST SYSTEM
- Test vessel: 5-L glass vessel covered with a Teflon sheet.
- Aeration: not aerated.
- Renewal rate of test solution (frequency): every 24-hour.
- No. of organisms per vessel: 10 fish/concentration.
- No. of vessels per concentration (replicates): 1 vessel/concentration.
- No. of vessels per control (replicates): 1 vessel/concentration.
- Biomass loading rate: 1 g fish/L or less.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water for rearing organisms (dechlorinated water).
- Total organic carbon: not detected.
- Particulate matter: not detected.
- Metals: not detected.
- Pesticides: not detected.
- Chlorine: not detected.
- Alkalinity: 35 mg CaCO3/L.
- Ca/mg ratio: 15/3.9.
- 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 each renewal and at the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: Fluorescent light, 16-hour light / 8-hour dark.
- Light intensity: 1000 lux or less.

EFFECT PARAMETERS MEASURED
Test fish were observed at 3, 24, 48, 72 and 96 hours after the start of exposure for mortality, critical and abnormal behaviour and appearance.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78.
- Range finding study: yes.
Test concentrations: 1.0, 10, 100 mg/L (nominal).
Results used to determine the conditions for the definitive study: no mortality at 1 and 10 mg/L, 100% mortality at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

CuSO4(II)·5H2O, guaranteed reagent

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

39 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Mortality of control: no.
- Other adverse effects control: no.
- Abnormal responses: See in the field below "Sublethal observations / clinical signs".
- 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):

- Results with reference substance is valid.
- 96h-LC50: 0.51 mg/L (95% confidence limits: 0.34 – 0.76 mg/L). Date: December 1, 2015. This value was consistent with the average value found in this laboratory (0.74 ± 0.28 mg/L).

Reported statistics and error estimates:

See below in the table of results.

Sublethal observations / clinical signs:

Observation of Fish:

Control
Symptoms	3 hr		24 h		48h		72h		96h
	No	Detail	No	Detail	No	Detail	No	Detail	No	Detail
Death (cumulative)	0		0		0		0		0
Critical	0		0		0		0		0
Abnormal	0		0		0		0		0
Normal	10		10		10		10		10
Total	10		10		10		10		10
Conc. 1: nominal: 10 mg/L  - mean measured: 9.21 mg/L
Symptoms	3 hr		24 h		48h		72h		96h
	No	Detail	No	Detail	No	Detail	No	Detail	No	Detail
Death (cumulative)	0		0		0		0		0
Critical	0		0		0		0		0
Abnormal	0		0		0		0		0
Normal	10		10		10		10		10
Total	10		10		10		10		10
Conc. 2: nominal: 18 mg/L  - mean measured: 18.1 mg/L
Symptoms	3 hr		24 h		48h		72h		96h
	No	Detail	No	Detail	No	Detail	No	Detail	No	Detail
Death (cumulative)	0		0		0		0		0
Critical	0		0		0		0		0
Abnormal	0		0		0		0		0
Normal	10		10		10		10		10
Total	10		10		10		10		10
Conc. 3: nominal: 32 mg/L  - mean measured: 32.5 mg/L
Symptoms	3 hr		24 h		48h		72h		96h
	No	Detail	No	Detail	No	Detail	No	Detail	No	Detail
Death (cumulative)	0		0		0		0		1
Critical	0		0		0		0		0
Abnormal	0		0		0		0		9	AR 9
Normal	10		10		10		10		0
Total	10		10		10		10		10
Conc. 4: nominal: 56 mg/L  - mean measured: 51.7 mg/L
Symptoms	3 hr		24 h		48h		72h		96h
	No	Detail	No	Detail	No	Detail	No	Detail	No	Detail
Death (cumulative)	0		7		9		10		10
Critical	0		0		1	IS 1	0		0
Abnormal	0		3	AS 1 AR 2	0		0		0
Normal	10		0		0		0		0
Total	10		10		10		10		10
Conc. 5: nominal: 100 mg/L  - mean measured: 96.0 mg/L
Symptoms	3 hr		24 h		48h		72h		96h
	No	Detail	No	Detail	No	Detail	No	Detail	No	Detail
Death (cumulative)	0		10		10		10		10
Critical	7	IS 7	0		0		0		0
Abnormal	3	AS 3	0		0		0		0
Normal	0		0		0		0		0
Total	10		10		10		10		10

No: Number of fish showing corresponding symptoms

IS: Impossible to swim

AS: Abnormal swimming

AR: Abnormal respiration

 

 

Median Lethal Concentrations (LC50) :

Exposure period (hr)	LC50 (mg/L)	95-Percent confidence limits (mg/L)	Statistical method
24	46.5 ^*	32.5    -    96.0	Binomial
48	43.0 ^*	32.5    -    51.7	Binomial
72	41.0 ^*	32.5    -    51.7	Binomial
96	39.0 ^*	32.5    -    51.7	Binomial

*: Using the data of Conc.1 – 5

 

 

Highest Concentration with 0% Mortality (LC0) and Lowest Concentration with 100% Mortality (LC100) :

Exposure period (hr)	LC0 (mg/L)	LC100 (mg/L)
96	18.1	51.7

 

Validity criteria fulfilled:

yes

Remarks:

No mortality in controls, dissolved oxygen concentrations > 60 % air saturation value throughout the test (minimum 6.7 mg/L O2).

Conclusions:

The 96-hour LC50 of 2,2-Difluoroethyl acetate to Oryzias latipes was determined to be 39.0 mg/L.

Executive summary:

The acute toxicity of 2,2-Difluoroethyl acetate to Oryzias latipes was investigated in a 96-hour semi-static test according to the OECD guideline 203 under GLP compliance.

Groups of ten fish were exposed to control, and test chemical at nominal concentrations of 10, 18, 32, 56, 100 mg/L. Test solutions were renewed daily. The corresponding actual concentrations were measured using GC/MS as being equal to 9.21, 18.1, 32.5, 51.7, 96.0 mg/L (time-weighted mean measured concentrations), respectively. These concentrations were almost equivalent to the nominal concentrations. Test fish were observed at 3, 24, 48, 72 and 96 hours after the start of exposure for mortality, critical and abnormal behaviour and appearance.

After 96 hours of exposure, no mortality were recorded in the control and up to and including the mean measured concentration of 18.1 mg/L. At the mean measured concentration of 32.5 mg/L, one fish was found to be dead and at 51.7 and 96 mg/L 100% of the fish were found to be dead. The 96-h LC50 was determined to be 39.0 mg/L; meaning 2,2-Difluoroethyl acetate is acutely harmful to fish.

Description of key information

The 96-hour LC50 of 2,2-Difluoroethyl acetate to Gobiocypris rarus was determined to be 36.3 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

36.3 mg/L

Additional information

Two experimental studies performed under GLP compliance and in accordance with OECD test guideline 203 were assigned a Klimisch score of 1 and used in a weight-of-evidence approach because giving very consistent 96h-LC50 values in the range from 30 to 40 mg/L in two different fish species. On this basis, 2,2-Difluoroethyl acetate was found to be acutely toxic to fish. The lowest value obtained on Gobiocypris rarus (i.e. 96h-LC50 = 36.3 mg/L) was selected as key value for chemical safety assessment.