Methyl 2,3-dibromopropionate

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:

From 2018-12-18 to 2019-03-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 98%
Batch No.: 800327680

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Treatment: Freshly obtained sludge was kept under continuous aeration until further treatment. Before use, sludge was coarsely sieved (1 mm). After treatment concentration of suspended solids (SS) was determined to be 3.7 g/L in concentrated sludge as used for the test. Magnetically stirred sludge was used as inoculum at an amount of 3 mL per litre of mineral medium, leading to a SS concentration of 11 mg/L.
- Concentration of sludge: 11 mg/L

Duration of test (contact time):

28 d

Initial conc.:

61.5 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST SYSTEM
Reason for selection: The test has been accepted internationally for determining 'ready' biodegradability of test items under aerobic conditions.

Test Procedure and Conditions
-Test duration: 28 days for inoculum blank and test item (last CO2 measurement on day 29). 14 days for procedure and toxicity control (last CO2 measurement on day 15). During the test period, test media were aerated and stirred continuously.
-Test vessels: 2 litre brown coloured glass bottles.
-Milli- RO water: Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.
-Stock solutions of mineral components: A) 8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.50 g NH4Cl, dissolved in Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2; B) 22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre; C) 36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 litre; D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 litre.
-Mineral medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.
-Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from air.
-Synthetic air (CO2 < 1 ppm) : A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. Synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
-Illumination: Test media were excluded from light.
Preparation of Bottles
-Pre-incubation medium: The day before start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
-Type and number of bottles: Test suspension: containing test item and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles). Procedure control: containing reference item and inoculum (1 bottle). Toxicity control: containing test item, reference item and inoculum (1 bottle).
-Preparation: At the start of the test (day 0), test and reference item were added to bottles containing microbial organisms and mineral components. Volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.
Determination of CO2
-Experimental CO2 production: CO2 produced in each test bottle reacted with barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol ampoule), Merck, Darmstadt, Germany).
-Theoretical CO2 production: Theoretical CO2 production was calculated from the molecular formula.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

28 d

Remarks on result:

other: The mean biodegradation of two parallel samples

Details on results:

Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of Methyl 2,3-dibromopropionate (2% and 0%, based on ThCO2).
In the toxicity control, more than 25% biodegradation occurred within 14 days (42%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.

Results with reference substance:

Reference item was biodegraded by 88% within 14 days

Comparison of Biodegradation of the Test Item in Bottles A and B

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A and B	∆ A-B^1)
2	0	0	0	0
5	0	0	0	0
8	2	0	1	2
12	2	0	1	2
15	2	0	1	2
19	2	0	1	2
23	2	0	1	2
29^2)	2	0	1	2
29^2)	2	0	1	2
29^2)	2	0	1	2
^1): Absolute difference in biodegradation between bottles A and B ^2): Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

The objective of the study was to evaluate test item for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludgein compliance with OECD guideline No. 301 B, 1992.

The test item was tested in duplicate at a target concentration of 61.5 mg/L, corresponding to 12 mg TOC/L. Organic carbon content was based on the molecular formula. Theoretical CO2 production (ThCO2) of the test item was calculated to be 0.72 mg CO2/mg.

The study consisted of six bottles: 2 inoculum blanks (no test item), 2 test bottles (test item), 1 procedure control (sodium acetate) and 1 toxicity control (test item plus sodium acetate).

Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to 2 litre test bottles containing medium with microbial organisms and mineral components. To this end, small watch glasses were used to transfer weighed amounts of test item to respective test bottles. Test solutions were continuously stirred during the test to ensure optimal contact between test item and test organisms. Test duration was 28 days for inoculum blank and test item (last CO2 measurement on day 29) and 14 days for procedure and toxicity control (last CO2 measurement on day 15).

Relative biodegradation values calculated from measurements performed during the test period revealed nobiologically relevantbiodegradation of the test item (2% and 0%, based on ThCO2).

In the toxicity control, the test item was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion,the test item was designated as not readily biodegradable.

Description of key information

In a study to OECD Guideline 301 B, relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of the test item (2% and 0%, based on ThCO2). The test substance is therefore not considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater