2,2-Difluoroethyl Acetate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 jan. 2016 to 22 apr. 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of activated sludge: Chemicals Evaluation and Research Institute, Japan (Date of receipt: December 25, 2015).
- Concentration of sludge: 3800 mg/L as Mixed liquor suspended solids (MLSS).
No further data available.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Measured continuously for 28 days (measured with a closed-system oxygen consumption measuring apparatus, OM-3100A, Ohkura Electric Co. (ID code: J)).

Parameter followed for biodegradation estimation:

DOC removal

Remarks:

On day 28 (measured with a TOC analyzer, multi N/C 2100S, Analytik Jena Japan Co., Ltd).

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

On day 28. Residual amount of the test substance (measured with GC/MS).

Parameter followed for biodegradation estimation:

other: On day 28. Amount of transformation product 2,2-difluoroethanol (measured with GC/MS).

Parameter followed for biodegradation estimation:

other: On day 28. Amount of transformation products difluoroacetic acid and acetic acid (measured with HPLC).

Details on study design:

TEST CONDITIONS
- Composition of medium: The basal salt medium (BSM) was purified water containing 0.3% each of liquids A, B, C and D. The pH value of the basal medium was adjusted to 7.0 ± 0.04 with 0.33 mol/L H3PO4 or 1 mol/L NaOH. Compositions of liquids A, B, C and D (Japanese Industrial Standards (JIS) K0102-2008, 21) were as follows:
Liquid A: In ultra-pure water, 21.75 g of K2HPO4, 8.5 g of KH2PO4, 44.6 g of Na2HPO4･12H2O and 1.7 g of NH4Cl were dissolved and filled up to 1 L.
Liquid B: In ultra-pure water, 22.5 g of MgSO4･7H2O was dissolved and filled up to 1 L.
Liquid C: In ultra-pure water, 27.5 g of CaCl2 was dissolved and filled up to 1 L.
Liquid D: Four hundred mL of ultra-pure water was added to 0.10 g of FeCl3･6H2O.
- Additional substrate: no.
- Solubilising agent (type and concentration if used): no.
- Test temperature: 25 ± 1°C.
- pH: At the end of BOD measurement, the pH values were 8.0 for the [Water + test substance] and 6.7, 6.8 and 6.8 for the [Sludge + test substance]-1, 2 and 3, respectively.
- pH adjusted: no.
- Aeration of dilution water: no.
- Suspended solids concentration: 30 mg/L.
- Continuous darkness: yes.
- Other: Continuous stirring with magnetic stirrer.

TEST SYSTEM
- Culturing apparatus: Closed system oxygen consumption measuring apparatus OM-3100A, Ohkura Electric Co. (ID code: J).
- Number of culture flasks/concentration: 3 bottles were used for the [Sludge + test substance] treatment, while 1 bottle was used for the other treatments described below under "CONTROL AND BLANK SYSTEM".
- Measuring equipments: Different parameters were followed for biodegradation estimation using different equipments:
* BOD measurements: Closed system oxygen consumption measuring apparatus OM-3100A, Ohkura Electric Co. (ID code: J).
* DOC measurements: TOC analyzer multi N/C 2100S, Analytik Jena Japan Co., Ltd (Conditions: Furnace temperature - 800°C / Injection volume - 300 µL / Repeated number - n=3, adopt mean value). The details of the DOC measurements are described hereafter. At the end of the 28-day test, the test solutions were pretreated by centrifugation for 15 minutes at 40000 m/s (4750 rpm). The obtained supernatant was processed by the TOC analyser. A calibration curve was prepared as detailed hereafter. Total Carbon (TC) and Inorganic Carbon (IC) standard solutions were injected into the TOC analyzer. The peak area was plotted against the concentration to obtain the linear regression, Y = a + bX. Amount of DOC in each bottle was calculated from the linear regression
* Amounts of residual test substance and transformation products: Measuring equipments are already detailed in the field above entitled "Details on analytical methods".
- Test performed in closed vessels.
- Details of trap for CO2: Soda lime, for carbon dioxide absorption, Kanto Chemical Co., Inc.

SAMPLING
- Sampling frequency: The BOD was measured continuously for 28 days. DOC and amounts of residual test substance and transformation products were measured at the end of the 28-day test period.
- Sampling method: There was no sampling for BOD measurement because the test was directly conducted in the oxygen consumption measuring apparatus. For the measurements of DOC and amounts of residual test substance and transformation products, on day 28, after the end of the BOD measurement, 12 mL of test solution was sampled in each vessel.

CONTROL AND BLANK SYSTEM
On top of the [Sludge + test substance + BSM] treatment (3 replicates, 30 mg/L activated sludge + 100 mg/L test substance), the following control and blank treatments were run:
- Inoculum blank: [Sludge + BSM] (1 replicate, 30 mg/L activated sludge).
- Abiotic sterile control: [Water + test substance] (1 replicate, 100 mg/L test substance).
- Toxicity control: no.
- Other: 1 activity control: [Sludge + aniline as reference substance + BSM] (1 replicate, 30 mg/L activated sludge + 100 mg/L aniline).

STATISTICAL METHODS: Equations for calculation of degradability:

1) Degradability based on BOD
Degradability (%) = (BODs – BODb) / ThOD × 100
Where:
BODs: Oxygen consumption (mg) in [Sludge + test substance] or [Sludge + aniline]
BODb: Oxygen consumption (mg) in [Inoculum blank]
ThOD: Theoretical oxygen demand (mg) of the test substance or aniline
Calculation of theoretical oxygen demand (ThOD)
Test substance: 30.9 mgO2/30.0 mg
The test substance is assumed to be mineralized as shown below:
C4H6F2O2 + 4O2 → 4CO2 + 2H2O + 2HF
Aniline: 72.2 mgO2/30.0 mg
Aniline was assumed to be mineralized as shown below:
C6H7N + 7O2 → 6CO2 + 2H2O + NH3

2) Degradability based on DOC
Degradability (%) = [1 – (DOCs – DOCb) / DOCc] × 100
Where:
DOCs: DOC (mg) in [Sludge + test substance]
DOCb: DOC (mg) in [Inoculum blank]
DOCc: DOC (mg) in [Water + test substance]
In this study, disappearance rate was calculated because transformation product was observed in the [Water + test substance].
Disappearance rate (%) = (Theoretical value – DOC) / Theoretical value × 100
Where
DOC: DOCs – DOCb ( [Sludge + test substance] )
DOCc ( [Water + test substance] )

3) Degradability based on the residual amount of the test substance
Degradability (%) = (1 – Cs / Cc) × 100
Where
Cs: amount (mg) in [Sludge + test substance]
Cc: amount (mg) in [Water + test substance]
In this study, disappearance rate was calculated instead of degradability because transformation product was observed in the [Water + test substance].
Disappearance rate (%) = (Initial amount – Residual amount) / Initial amount × 100

Reference substance:

aniline

Preliminary study:

No preliminary study.

Test performance:

There was no specific factor which might have affected the reliability of the test results.

Key result

Parameter:

% degradation (O2 consumption)

Value:

52

St. dev.:

3.1

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Value:

53

St. dev.:

0.6

Sampling time:

28 d

Remarks on result:

other: Since transformation products were formed in the abiotic control [i.e. Water + test substance], this percentage reflects a disappearance rate instead of biodegradability.

Parameter:

% degradation (test mat. analysis)

Value:

> 99

Sampling time:

28 d

Remarks on result:

other: Since the test substance was transformed in the abiotic control [i.e. Water + test substance], this percentage reflects a disappearance rate instead of biodegradability.

Details on results:

DEGRADABILITY BASED ON BOD MEASUREMENTS
After 28 days, the degradability percentages were calculated to be 51%, 49% and 55% for the three replicates of the [Sludge + test substance] treatment.

DEGRADABILITY BASED ON DOC MEASUREMENTS
As transformation products were formed in the abiotic control treatment [i.e. Water + test substance], a disappearance rate (%) was calculated instead of a biodegradability percentage. The disappearance rates were 59% for the abiotic control treatment [i.e. Water + test substance] and 53%, 53% and 52% for the three replicates of the [Sludge + test substance] treatment.

DEGRADABILITY BASED ON RESIDUAL TEST SUBSTANCE AMOUNT MEASUREMENTS
Since the test substance was transformed in the abiotic control treatment [i.e. Water + test substance], a disappearance rate (%) was calculated instead of a biodegradability percentage. The disappearance rates were calculated to be >99% for the three replicates of the [Sludge + test substance] treatment and 89% for the abiotic control treatment [i.e. Water + test substance].

FORMATION OF TRANSFORMATION PRODUCTS
* 2,2-Difluoroethanol: Formation rates were 50, 56, 58 and 62 % for the abiotic control treatment [i.e. Water + test substance] and the three replicates of the [Sludge + test substance] treatment, respectively.
* Acetic acid: Formation rates were 1, <1, <1 and <1 % for the abiotic control treatment [i.e. Water + test substance] and the three replicates of the [Sludge + test substance] treatment, respectively.
* Difluoroacetic acid: Formation rates were <1, 33, 31 and 29 % for the abiotic control treatment [i.e. Water + test substance] and the three replicates of the [Sludge + test substance] treatment, respectively.

DISCUSSION
According to the mean degradability percentage based on BOD after 28 days (52%; key result for ready biodegradability), the test substance was concluded not to be readily biodegradable.
Based on measurements of the amounts of residual test substance and transformation products, the test substance disappeared completely in the [Sludge + test substance] treatment, and transformation products 2,2-difluoroethanol and difluoroacetic acid were formed with average formation rate of 59% and 31%, respectively. The total formation rate of transformation products (2,2-difluoroethanol and difluoroacetic acid) was 90%; however, as the test substance is volatile, the rest was supposed to have been volatilized as the test substance or 2,2-difluoroethanol during the 28-day exposure.
The test substance would be hydrolyzed into 2,2-difluoroethanol and acetic acid. Acetic acid was considered to have been completely mineralized because the acetic acid is known to be readily biodegradable. ThOD of the presumed acetic acid is calculated to be 15.5 mg. BOD measured in the [Sludge + test substance] treatment was 15.7, 15.1 and 16.9 mg, which might correspond to the value for mineralization of acetic acid and partial oxidation of 2,2-difluoroethanol to difluoroacetic acid.
Meanwhile, in the abiotic control treatment [i.e. water + test substance], residual rate of the test substance accounted for 11% and the 2,2-difluoroethanol and acetic acid production rate accounted for 50% and 1%, respectively. As the test substance is hydrolyzable, it was considered that 2,2-difluoroethanol and acetic acid will be formed on hydrolysis in test solution. Although 89% of the test substance disappeared, only a trace amount of acetic acid was detected. Acetic acid was considered not to degrade in the [water + test substance], but to volatilize from the water and to be trapped in soda lime. The total balance of the test substance and 2,2-difluoroethanol was 61% and lower than expected. The undetected component was considered to have been volatized during the exposure.

Results with reference substance:

The degradability of aniline based on the BOD after 7 and 14 days were calculated to be 91% and 94%, respectively, thus meeding the validity crieria of >40% after 7 days and >65% after 14 days.

Table 1: BOD measurement

Bottle No.	Sample description	BOD (mg)
		day 7	day 14	day 21	day 28
1	Water + test substance (Abiotic control)	0.0	0.0	0.0	0.0
2	Sludge + test substance -1 (Test suspension -1)	14.1	17.4	18.9	20.0
3	Sludge + test substance -2 (Test suspension -2)	14.0	17.1	18.3	19.4
4	Sludge + test substance -3 (Test suspension -3)	14.7	18.0	19.9	21.2
5	Sludge + aniline (Activity control)	66.9	69.4	70.7	71.8
6	Inoculum blank (Control blank)	1.3	1.7	2.1	4.3

 

Table 2: Results of DOC measurement on day 28

Bottle No.	Sample description	Added amount mg	Theoretical value mgC	DOC in bottle mgC	DOCs - DOCb mgC	Ratio to theoretical value %	Disappearance rate %
1	Water + test substance (Abiotic control)	30.0	11.6	4.8	－	41	59
2	Sludge + test substance-1 (Test suspension -1)	30.0	11.6	6.5	5.4	47	53
3	Sludge + test substance-2 (Test suspension -2)	30.0	11.6	6.5	5.4	47	53
4	Sludge + test substance-3 (Test suspension -3)	30.0	11.6	6.7	5.6	48	52
5	Sludge + aniline (Activity control)	－	－	－	－	－	－
6	Inoculum blank (Control blank)	－	－	1.1	－	－	－
Average (Bottle 2, 3, 4)						47	53

Where

DOCs: DOC (mg) in [Sludge + test substance] treatment
DOCb: DOC (mg) in [Inoculum blank] treatment

 

Table 3: Measured values of BOD, DOC, test substance and transformation products on day 28

		Activated sludge + test substance			Water + test substance	Theoretical value
		No.1	No.2	No.3
		Bottle 2	Bottle 3	Bottle 4	Bottle 1
BOD^*1	mg	15.7	15.1	16.9	0.0	30.9
DOC*1	mg	5.4	5.4	5.6	4.8	11.6
Test substance (GC/MS)	mg	<0.2	<0.2	<0.2	3.3	30.0
	%①	<1	<1	<1	11	－
2,2-Difluoroethanol (GC/MS)	mg	11.2	11.6	12.4	9.9	19.8
	%②	56	58	62	50	－
Acetic acid (HPLC)	mg	<0.1	<0.1	<0.1	0.1	14.5
	%③	<1	<1	<1	1	－
Difluoroacetic acid (HPLC)	mg	7.7	7.2	6.8	<0.1	23.2
	%④	33	31	29	<1	－
Total(①＋②＋④) *2	%	89	89	91	61	－

 *1 : Value of [sludge + test substance] treatment is corrected with BOD or DOC value of [Control blank] treatment.

 *2: Total balance was calculated with the residual test substance, 2,2-Difluoroethanol and Difluoroacetic acid.

 

Table 4: Degradability (%) on day 28

		Activated sludge + test substance			Average
		No.1	No.2	No.3
		Bottle 2	Bottle 3	Bottle 4
BOD	%	51	49	55	52
DOC^*3	%	53	53	52	53
Test substance^*3	%	>99	>99	>99	>99

*3: A disappearance rate instead of a biodegradability percentage was calculated because the test substance was shown to be transformed in abiotic control.

 

Table 5: Total balance on day 28

Bottle No.	Sample description	A	B	C	D	E
		Residual test substance rate %	Formation rate (2,2-Difluoroethanol) %	Formation rate (Acetic acid) %	Formation rate (Difluoroacetic acid) %	Total balance %
1	Water + test substance (Abiotic control)	11	50	1	<1	61
2	Sludge + test substance-1 (Test suspension -1)	<1	56	<1	33	89
3	Sludge + test substance-2 (Test suspension -2)	<1	58	<1	31	89
4	Sludge + test substance-3 (Test suspension -3)	<1	62	<1	29	91
5	Sludge + aniline (Activity control)	-	-	-	-	-
6	Inoculum blank (Control blank)	-	-	-	-	-
Average (Bottle 2, 3, 4)		<1	59	<1	31	90

 Equation :  E=A+B+D

Total balance was calculated with the residual test substance, 2,2-Difluoroethanol and Difluoroacetic acid.

 

Validity

The test is considered valid because the following validity criteria were met:

• The degradability of reference substance based on the BOD should exceed 40% after 7 days and 65% after 14 days=> 91% after 7 days and 94% after 14 days in this study


• The difference between the maximum and minimum values of the degradability of the test substance after 28 days should be less than 20% => 6% in this study


• The BOD in the [Inoculum blank] treatment should be less than 18 mg O2 in 28 days => 4.3 mg O2 in this study

Validity criteria fulfilled:

yes

Remarks:

See above in the field "Any other information on results incl. tables".

Interpretation of results:

not readily biodegradable

Conclusions:

2,2-Difluoroethyl acetate was found to be not readily biodegradable and to completely disappear to form 2,2-difluoroethanol and difluoroacetic acid.

Executive summary:

The ready biodegradation potential of 2,2-Difluoroethyl acetate was investigated in a study performed according to OECD test guideline 301C (Modified MITI Test I) under GLP compliance. Activated sludge obtained from the Chemicals Evaluation and Research Institute (Japan) was used as the inoculum. Four different treatment groups were used:

- Inoculum blank: sludge + basal salt medium.

- Abiotic control group: test substance at 100 mg/L + purified water.

- Test substance group: sludge + test substance at 100 mg/L + basal salt medium.

-  Positive control group: sludge + reference substance (aniline) at 100 mg/L + basal salt medium.

Three experimental bottles were used for the test substance group and one bottle for the other groups; each with an end-volume of 300 mL. Bottles were incubated at 25 ± 1 °C for 28 days with continuous magnetic stirring.

The Biochemical Oxygen Demand (BOD) was measured continuously for 28 days using a closed-system oxygen consumption measuring apparatus. At the end of the 28-day period, the Dissolved Oxygen Carbon (DOC) was measured using a TOC analyzer, and the amounts of residual test substance and three transformation products (2,2-difluoroethanol, difluoroacetic acid and acetic acid) were measured using different analytical methods (gas chromatograph/mass spectrometer (GC/MS) for DFEA and 2,2-difluoro ethanol, reversed phase high performance liquid chromatograph (HPLC) for difluoroacetic acid and acetic acid).

Based on BOD measurements, 94 % and 52 % biodegradation after 28 days were obtained for the reference substance (aniline) and the test substance, respectively. The results obtained with the reference substance thus confirmed the suitability of the test system. The results obtained with 2,2-Difluoroethyl acetate showed it is not readily biodegradable (i.e. % biodegradation < 60 %).

DOC and residual test substance measurements in the test substance group revealed that 2,2-Difluoroethyl acetate disappeared. Based on GC/MS analyses, 2,2-Difluoroethyl acetate disappearance rate was above 99 %, while the average formation rate of the transformation products 2,2-difluoroethanol and difluoroacetic acid were 59 and 31 % based on GC/MS and HPLC analyses, respectively. Acetic acid is another expected product in the transformation pathway, but it was not detected by HPLC in this study likely because it is readily biodegradable. The total formation rate of transformation products (2,2-difluoroethanol and difluoroacetic acid) was thus 90% (i.e. 59 % + 31 %); the rest was supposed to have been volatilized as the test substance or 2,2-difluoroethanol during the 28-day exposure.

In the abiotic control group, a 11 % residual rate was determined for 2,2-Difluoroethyl acetate. The average formation rate of the transformation products 2,2-difluoroethanol and acetic acid were 50 and 1 %, respectively. The total balance of 2,2-Difluoroethyl acetate and transformation products was thus 62% (i.e. 11 % + 50 % + 1 %). The rest was supposed to have been volatized during the exposure.

All validity criteria were met.

2,2-Difluoroethyl acetate was found to be not readily biodegradable and to completely disappear to form 2,2-difluoroethanol and difluoroacetic acid.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 january 2016 to 30 may 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Version / remarks:

1981

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge, surface soil and surface water were sampled from ten sites distributed in four districts throughout Nanjing city, such as Chengdong, Chengbei, Jiangxinzhou and Jiangning. 1 L of the sludge, soil and water were collected and mixed thoroughly together. After removing floating matter, the mixture was allowed to stand and then the supernatant was filtrated through filter paper. After that the filtrate was adjusted to pH 7.0 with sodium hydroxide or phosphoric acid. Finally an appropriate volume of the filtrate was transferred to a fill-and-draw activated sludge vessel and aerated for about 23.5 h.
- Laboratory culture: Thirty minutes after stopping the aeration, about one third of the whole volume of supernatant was discarded. Then an equal volume of synthetic sludge (a solution at pH 7.0 containing 0.1% each of glucose, peptone and potassium orthophosphate) was added into the settled material which was then aerated again. This procedure was repeated once per day during one month.
- Preparation of inoculum for exposure: Before use the mixture was allowed to stand, and the supernatant was removed. A small quantity of sludge was taken to be centrifuged (2500 r/minx10 min) and then weighed. Then the sludge was dried in the oven and weighed again in order to calculate the content of dry sludge was 13.4%. At last 15.0 g of centrifuged sludge was diluted 0.5 L with basal culture.
- Concentration of sludge: 4000 mg/L (dry basis).

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Remarks:

(nominal)

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Measured daily during the 28-day test (BOD).

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

Measured at the end of the 28-day test.

Details on study design:

TEST CONDITIONS
- Composition of medium: The Basal Salt Medium (BSM) was prepared by adding 3 mL, of each of the following stock solutions prepared in pre-aerated deionized water to 1 litre of deionized water. 10 L BSM was prepared.
Stock solution A
KH2PO4 (potassium dihydrogen phosphate): 8.50 g/L.
K2HPO4 (dipotassium hydrogen phosphate): 21.75 g/L.
Na2HPO4.12H2O (disodium phosphate dodecahydrate): 44.60 g/L.
NH4Cl (ammonium chloride): 1.70 g/L.
The pH of this solution was 7.32.
Stock solution B: CaCl2 (calcium chloride): 27.50 g/L.
Stock solution C: MgSO4.7H20 (magnesium sulphate heptahydrate): 22.50 g/L.
Stock solution D: FeCl3.6H20 (iron (III) chloride hexahydrate): 0.25 g/L.
- Test temperature: 25 ± 2 °C.
- pH: 7.26-7.49.
- Suspended solids concentration: concentration of activated sludge: 100 mg/L.
- Continuous darkness: yes.
- Other: Vigorous stirring with mechanical stirrer.

TEST SYSTEM
- Number of culture flasks/concentration: 3 bottles for the test substance treatment, 1 bottle for the other treatments described below under "CONTROL AND BLANK SYSTEM".
- Measuring equipment: Different parameters were followed for biodegradation estimation using different equipments:
* BOD measurements: BOD meter (OM3100, Ohkura, Japan) and BOD bottles (500 mL).
* Amounts of residual test substance: Measuring equipment is already detailed in the field above entitled "Details on analytical methods".
- Test performed in closed vessels.

SAMPLING
- Sampling frequency: The BOD was measured continuously for 28 days. Amounts of residual test substance were measured at the end of the 28-day test period..
- Sampling method: There was no sampling for BOD measurement because the test was directly conducted in the BOD meter. For the measurements of amounts of residual test substance, 10 mL samples were taken from "abiotic" bottle, "test" bottles, and "blank" bottle.

CONTROL AND BLANK SYSTEM
On top of the test substance treatment (3 replicates, 100 mg/L activated sludge + 100 mg/L test substance + BSM), the following control and blank treatments were run:
- Inoculum blank: 1 replicate, 100 mg/L activated sludge + BSM.
- Abiotic sterile control: 1 replicate, 100 mg/L test substance + BSM.
- Toxicity control: no.
- Other: Positive control: 1 replicate, 100 mg/L activated sludge + 100 mg/L sodium benzoate + BSM.

STATISTICAL METHODS / CALCULATIONS
The biodegradability percentage was calculated from the BOD and ThOD values according to the following equation:
% degradation = BOD / ThOD x 100
Where:
BOD (Biochemical Oxygen Demand) = [T(n) - C(n)] / mg (test substance or reference substance)/L
Where:
T(n) = mg O2/L content in the test substance or reference substance bottles on day n.
C(n) = mean mg O2/L content in the inoculum control bottles on day n.
ThOD (Theoretical Oxygen Demand) = 1.03 and 1.67 mg O2/mg for the test substance and reference substance, respectively.

No further data available.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

No

Key result

Parameter:

% degradation (O2 consumption)

Value:

25.8

St. dev.:

2.3

Sampling time:

28 d

Parameter:

% degradation (test mat. analysis)

Value:

32.1

St. dev.:

1.4

Sampling time:

28 d

Details on results:

Based on the study results, 2,2-Difluoroethyl acetate was considered to be inherently biodegradable (i.e. inherent degradation rate ≥ 20% after 28 days).

Results with reference substance:

The level of biodegradation of the reference substance sodium benzoate was 84.3% after 7 days and 89.8% after 14 days, thus meeting the validity criteria of > 40% and > 65% after 7 and 14 days, respectively.

Table 1: Results of BOD measurements

Time (d)	BOD (mg/L)
	"abiotic control"	"test"			"positive control"	"blank control"
	Bottle 1	Bottle 2	Bottle 3	Bottle 4	Bottle 5	Bottle 6
0	0	0	0	0	0	0
1	0	0	0	0	72.2	0
2	0	0	0	0	126	0
3	0	0	0	0	132	0
4	0	0	0	0	134	0
5	0	0	0	0	137	0
6	0	0	0	0	138	0
7	0	0	0	0	141	0
8	0	0.12	0.12	0.42	143	0
9	0	1.35	1.44	1.78	145	0
10	0	1.97	2.10	2.56	146	0
11	0	2.86	3.06	3.48	147	0
12	0	3.95	4.22	4.50	149	0.32
13	0	5.30	5.66	5.75	150	1.04
14	0	6.39	6.82	6.61	152	1.81
15	0	7.17	7.66	7.94	153	2.68
16	0	8.96	9.57	9.29	155	3.80
17	0	10.5	11.3	11.0	157	5.51
18	0	11.8	12.6	12.4	158	6.64
19	0	12.7	13.6	12.9	159	7.43
20	0	13.6	14.5	14.1	160	8.04
21	0	14.2	15.1	14.5	160	8.56
22	0	15.0	16.0	15.4	161	9.05
23	0	15.9	17.0	16.3	161	9.84
24	0	17.1	18.2	17.6	162	10.7
25	0	18.2	19.5	18.4	163	11.6
26	0	18.8	20.1	19.3	163	12.2
27	0	19.5	20.8	20.1	164	12.7
28	0	20.9	22.3	21.7	165	13.7

 

Table 2: Biodegradation as BOD

Time (d)	Biodegradation after deduction of the % due to abiotic process (%)
	"test"				"procedure control"
	Bottle 2	Bottle 3	Bottle 4	Mean	Bottle 5
0	0	0	0	0	0
1	0	0	0	0	43.2
2	0	0	0	0	75.5
3	0	0	0	0	78.9
4	0	0	0	0	80.2
5	0	0	0	0	81.8
6	0	0	0	0	82.8
7	0	0	0	0	84.3
8	0.40	0.38	1.35	0.71	85.9
9	4.37	4.66	5.77	4.93	86.8
10	6.36	6.79	8.27	7.14	87.3
11	9.27	9.89	11.3	10.1	88.3
12	11.8	12.6	13.5	12.6	89.0
13	13.8	14.9	15.3	14.7	89.4
14	14.8	16.2	15.5	15.5	89.8
15	14.6	16.1	17.0	15.9	90.0
16	16.7	18.7	17.8	17.7	90.3
17	16.3	18.6	17.8	17.6	90.4
18	16.6	19.1	18.5	18.1	90.6
19	17.2	19.9	17.5	18.2	90.7
20	17.9	20.9	19.6	19.5	90.8
21	18.1	21.2	19.2	19.5	90.7
22	19.1	22.3	20.7	20.7	90.7
23	19.6	23.1	20.8	21.2	90.7
24	20.4	24.2	22.1	22.2	90.6
25	21.5	25.5	22.1	23.0	90.6
26	21.4	25.5	22.8	23.2	90.5
27	21.9	26.2	23.8	23.9	90.4
28	23.4	27.9	26.0	25.8	90.5

 

Table 3: Residual results and degradation of the test substance by GC/MS

Sample	Time (d)	Bottle	Concentration (mg/L)	Degradation (%)
"abiotic control"	28	1	34.2	-
"blank"	28	6	ND	-
"test"	28	2	24.8	27.5
		3	22.6	33.9
		4	22.3	34.8
		Average	23.2	32.1

ND: Not detected, since below the detection limit of instrument.

Validity criteria fulfilled:

yes

Remarks:

The biodegradation of the reference substance sodium benzoate was 84.3% after 7 days (> 40%), and 89.8% after 14 days (> 65%), and the recovery rate of residual amount of the test compound in the "abiotic" test was found to be more than 10% after 28 days.

Interpretation of results:

inherently biodegradable

Conclusions:

The inherent biodegradation percentages of 2,2-Difluoroethyl acetate were 25.8% and 32.1% after 28 days based on BOD and residual test substance measurements, respectively. Based on these results, 2,2-Difluoroethyl acetate is considered to be inherently biodegradable.

Executive summary:

The inherent biodegradation potential of 2,2-Difluoroethyl acetate was investigated in a study performed according to OECD test guideline 302C (Modified MITI Test II) under GLP compliance. A mixed inoculum obtained from activated sludge, surface soil and surface water sampled from ten sites near Nanjing city was used. Four different treatment groups were tested:

   - Inoculum blank: inoculum + basal salt medium.

   - Abiotic control group: test substance at 30 mg/L + basal salt medium.

   - Test substance group: inoculum + test substance at 30 mg/L + basal salt medium.

    - Positive control group: inoculum + reference substance (sodium benzoate) at 100 mg/L + basal salt medium.

Three experimental bottles were used for the test substance group and one bottle for the other groups; each with an end-volume of 300 mL. Bottles were incubated at 25 ± 2 °C for 28 days under darkness with continuous magnetic stirring.

The Biochemical Oxygen Demand (BOD) was measured daily for 28 days. At the end of the 28-day period, the amounts of residual test substance was also measured using GC/MS.

Based on BOD measurements, 89.8 % biodegradation were obtained after 14 days for the reference substance (sodium benzoate), which thus confirmed the suitability of the test system. For the test substance, 25.8 % and 32.1 % biodegradation were obtained after 28 days based on BOD and residual test substance measurements, respectively, showing that 2,2-Difluoroethyl acetate is inherently biodegradable (i.e. % biodegradation ≥ 20 %).

In the abiotic control group, the residual amount of 2,2-Difluoroethyl acetate was more than 10 % after 28 days.

All validity criteria were met.

2,2-Difluoroethyl acetate was found to be inherently biodegradable. Indeed, according to ECHA Guidance R.7b (v4.0, June 2017), biodegradation above 20% may be regarded as evidence of inherent, primary, biodegradability and suggests that stable degradation products are likely to be formed as it was actually evidenced in the ready biodegradability (see the other endpoint study record in the present IUCLID section ) and hydrolysis (see in IUCLID section 5.1.2) studies.

Description of key information

Ready biodegradability: 2,2-Difluoroethyl acetate was found to be not readily biodegradable considering that 52 % biodegradation were achieved after 28 days based on BOD measurements. Based on measurements of the amounts of residual test substance and of transformation products, 2,2-Difluoroethyl acetate was demonstrated to completely disappear to form 2,2-difluoroethanol and difluoroacetic acid under the conditions of the ready biodegradation study.

 

Inherent biodegradability: The inherent biodegradation percentages of 2,2-Difluoroethyl acetate were 25.8% and 32.1% after 28 days based on BOD and residual test substance measurements, respectively. According to ECHA Guidance R.7b (v4.0, June 2017), biodegradation above 20% may be regarded as evidence of inherent, primary, biodegradability and suggests that stable degradation products are likely to be formed as it was actually evidenced in the ready biodegradability (see just above) and hydrolysis (see in IUCLID section 5.1.2) studies.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Type of water:

freshwater

Additional information

Two experimental studies performed under GLP compliance and in accordance with OECD test guidelines 301C and 302C were assigned a Klimisch score of 1 and flagged as key studies for ready and inherent biodegradation endpoints, respectively. 2,2-Difluoroethyl acetate was found to completely disappear to form 2,2-difluoroethanol and difluoroacetic acid under the conditions of the OECD 301C study in which it was concluded to be not readily biodegradable. Under the conditions of the OECD 302C study, inherent primary degradability of 2,2-Difluoroethyl acetate was evidenced.