(E)-4-Ethoxy-1,1,1-trifluoro-3-buten-2-one (stabilized with BHT)

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Jan.-02 Mar. 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted according to an internationally recognised method, and under GLP. The substance is adequately characterised with its purity. A Klimisch score of 2 was however assigned due to a deviation from the validity criteria recommended in the current version of OECD 301/OECD 301D guideline. Indeed,all the current validity criteria are fulfilled, except one which was not in place in the 1992 version of the guideline that was used at the time of the study (see the field "Overall remarks" for further information). This deviation was tolerated especially considering that the final conclusion is a worst-case scenario, i.e. ETFBO was considered as not readily biodegradable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

1992

Deviations:

yes

Remarks:

One minor deviation from OECD 301D guideline was that ammonium chloride was omitted from the medium to prevent nitrification.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: About 0.5L secondary (biological) activated sludge was obtained from the RWZI Horstermeer in Nederhorst den Berg, The Netherlands on 26 January 2006. The RWZI Horstermeer is an activated sludge plant treating predominantly domestic waste water.
- Preparation of inoculum for exposure: The sludge was preconditioned to reduce the endogenous respiration rate. This was done by aerating the sludge (50 mg dry weight per litre mineral salt solution) at 20 +/- 1 °C for one week.
- Concentration of sludge: After this preconditioning, the sludge was diluted to a concentration in the test bottles of 2 mg/L dry weight.
No further data.

Duration of test (contact time):

28 d

Initial conc.:

5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of the mineral salt medium (= closed bottle medium): 1 mL of each one of the following solutions was added to 1 L purified water prepared by Solvay Pharmaceuticals according to the methods of the European Pharmacopeia and the USP Purified water:
* Solution 1 containing KH2PO4 at 8.5 g/L, K2HPO4 at 21.8 g/L, Na2HPO4.12H2O at 67.0 g/L.
* Solution 2 containing MgSO4.7H2O at 22.5 g/L.
* Solution 3 containing CaCl2.2H2O at 36.4 g/L.
* Solution 4 containing FeCl3.6H2O at 0.25 g/L.
One minor deviation from the OECD guideline is that ammonium chloride was omitted from the medium to prevent nitrification.
- Test temperature: 20 to 20.7 °C (temperature recorded once every hour).
- pH: 6.8 to 7.2 (pH measured on day 0 and day 28 in two bottles of each group).
- pH adjusted: no.
- Continuous darkness: yes.
No further data.

TEST SYSTEM
- Culturing apparatus: 280 mL BOD bottles (Wertheim, cat. no. 270202).
- Number of culture flasks/concentration: 10.
- Measuring equipment: Oxygen meter, HACH, model HQ10.
- Test performed in closed vessels: yes, the bottles were completely filled with closed bottle medium (to avoid a headspace) and closed with glass stoppers sealed with silicon paste.
No further data.

SAMPLING
- Sampling frequency: Sampling immediately after filling the bottles (days 0) and after 7, 14, 21 and 28 days.
- Sampling method: At each sampling time, two bottles of each group were used for oxygen measurements.
No further data.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes.
- Abiotic sterile control: no.
- Positive control: yes.
- Toxicity control: yes.
(See the compositions of the experimental bottles in the field "Any other information on materials and methods incl. tables")

STATISTICAL METHODS:
No statistics seem to have been performed (no information in the report).

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

49

Sampling time:

28 d

Details on results:

Test substance:
- 40% biodegradation after 7 days.
- 47% biodegradation after 14 days.
- 49% biodegradation after 21 days.
- 49% biodegradation after 28 days.
ETFBO was significantly degraded, but the threshold of 60% biodegradation was not reached and therefore the 10-day window criterion was not assessed; thus preventing to consider ETFBO as readily biodegradable.

Positive control:
- 62% biodegradation after 7 days.
- 78% biodegradation after 14 days.
- 92% biodegradation after 21 days.
- 67% biodegradation after 28 days.
It seems surprising that the biodegradation percentage was lower after 28 days (67%) than after 14 days (78%) and 21 days (92%). Because different bottles were "sacrified" for oxygen content measurement at each time intervals, it is possible that biodegradation had proceeded at different extents in the different bottles. Anyway, the threshold of 60% for the study to be considered as valid was achieved in any case.

Toxicity control: Biodegradation percentages were not provided in the report for the toxicity control group. It was however reported that ETFBO was not considered as toxic to the micro-organisms because sodium acetate was biodegraded in the presence of ETFBO for more than 25%.

Results with reference substance:

See in the field "Details on results".

Oxygen content (mg/L) measured in the test bottles:

Sampling time (days)	Groups
	Mineral medium	Inoculum blank	Positive control	Toxicity control	Test substance
0	9.10	9.10	9.10	9.10	9.10
7	8.95	8.70	5.50	3.70	6.45
14	8.75	8.65	4.65	3.10	5.95
21	9.00	8.75	4.00	4.10	5.95
28	8.90	8.60	5.15	3.65	5.80

Each value corresponds to the mean of oxygen contents measured in both bottles sacrified at the same time interval.

Oxygen depletion (mg/L) recorded during the test period:

Sampling time (days)	Endogenous^(1)	Positive control^(2)	Toxicity control^(3)	Test substance^(4)
7	0.25	3.20	5.00	2.25
14	0.10	4.00	5.55	2.70
21	0.25	4.75	4.65	2.80
28	0.30	3.45	4.95	2.80

⁽¹⁾ Endogenous oxygen depletion was obtained by subtracting the oxygen content in the inoculum blank group from the oxygen content in the mineral medium group.

⁽²⁾ Obtained by subtracting the oxygen content in the positive control group from the oxygen content in the inoculum blank group.

⁽³⁾ Obtained by subtracting the oxygen content in the toxicity control group from the oxygen content in the inoculum blank group.

⁽⁴⁾ Obtained by subtracting the oxygen content in the test substance group from the oxygen content in the inoculum blank group.

BOD values of sodium acetate and ETFBO to be used in the biodegradation calculation (see in the field "Any other information on materials and methods incl. tables") were calculated by dividing the corresponding oxygen depletion values by the final concentration in reference substance (sodium acetate: 6.6 mg/L) and test substance (ETFBO: 5.0 mg/L), respectively.

Biodegradation percentages (%) during the test period:

Sampling time (days)	Positive control	Test substance
7	62	40
14	78	47
21	92	49
28	67	49

Validity criteria fulfilled:

not specified

Remarks:

See the discussion on the validity criteria in the field "Overall remarks".

Interpretation of results:

not readily biodegradable

Conclusions:

49% biodegradation were obtained after 28 days; implying that ETFBO has to be considered as not readily biodegradable.

Executive summary:

Ready biodegradability was investigated in a GPL-compliant study performed according to OECD test guideline 301D.

Secondary (biological) activated sludge obtained from a plant treating predominantly domestic waste water was used as the inoculum. Five different treatment groups were used:

- Mineral medium group: containing only mineral salt medium.

- Inoculum blank: containing the inoculum and mineral salt medium.

- Positive control group: containing the inoculum, the reference substance (sodium acetate) at 6.6 mg/L and mineral salt medium.

- Toxicity control group: containing the inoculum, the reference substance (sodium acetate) at 6.6 mg/L, the test substance (ETFBO) at 5 mg/L and mineral salt medium.

- Test substance group: containing the inoculum, the test substance (ETFBO) at 5 mg/L and mineral salt medium.

Ten experimental bottles were used per group; each with an endvolume of 280 mL. Bottles were incubated in the dark in a thermostated room at 20 +/- 1 °C. Immediately after filling (day 0) and after 7, 14, 21 and 28 days, two experimental bottles of each group were “sacrified” in order to measure oxygen content. The Biochemical Oxygen Demand (BOD) was calculated from these oxygen contents. And biodegradation was calculated as the ratio of the BOD and the theoretical oxygen demand (ThOD).

The reference substance, sodium acetate, was demonstrated to be biodegraded for more than 60% at each sampling time. As observed in the toxicity control group, sodium acetate was also biodegraded in the presence of the test substance, ETFBO; and this, for more than 25 % implying that ETFBO was not toxic to the micro-organisms. Finally, 49 % biodegradation were recorded after 28 days for ETFBO. The threshold of 60% biodegradation was not reached and therefore the 10-day window criterion was not assessed; implying that ETFBO has to be considered as not readily biodegradable.

Description of key information

49% biodegradation were obtained after 28 days in a Closed Bottle Test (OECD 301D); implying that the registered substance has to be considered as not readily biodegradable.

Additional information

An experimental study, conducted according to OECD guideline and GLP, is available. Despite a limitation due to the use of a version of OECD 301D guideline presenting different validity criteria as compared to the ones currently in place, the study is considered as reliable with restriction, and the result is retained as key data.