methyl 2-hydroxybut-3-enoate

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:

2021-07-01 to 2021-07-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 17th July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on June 25, 2021 (six days before the test).
- Preparation of inoculum for exposure: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution by shaking and was again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed (5.688 g wet weight), dried and the ratio of wet sludge to dry weight (0.5394 g dry weight) was determined. Based on this ratio, calculated amount of wet sludge (5 g dry weight that was equivalent to 52.725 g wet sludge) was suspended in mineral medium (ad. 1000 mL) to yield a concentration equivalent to about 5 g per litre (on dry weight basis). The prepared activated sludge inoculum was aerated under test conditions (for 6 days) until use. The pH of the activated sludge inoculum after preparation was 7.49, just before use the pH was: 7.28. A pH adjustment of activated sludge inoculum was not performed.
- Storage conditions: The prepared activated sludge was continuously aerated (2 L/minute) at the test temperature of 20.0 – 20.8 °C, for about 6 days.

PRE-CONDITIONING
Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium1 ) for 6 days (from June 25 to July 01, 2021) at test temperature (the actual temperature: 20.0 – 20.8 °C). Before use the cell count of inoculum was checked as follows: the viability of the cultured sludge was determined by plating of 0.1 mL undiluted sample and different, 10-1, 10-2, 10-3 and 10-4 dilutions of cultures on nutrient agar plates. Plates were incubated at 37 °C for 24 hours. The viable cell number of the cultures was determined by these plating experiments by manual colony counting. The approximately cell count of aerated inoculum was ~ 108 - 109 /L; therefore, before the test the inoculum was further diluted 100 000x with mineral medium to reach the necessary ~104 – 106 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning (see above) improves the precision of the method. The inoculum was not pre-adapted to the test chemical.
- Storage length: 6 days
- Concentration of sludge: 5 g per litre (on dry weight basis).
- Water filtered: no (centrifuged)

Duration of test (contact time):

28 d

Initial conc.:

3.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
In purified, deionized water analytical grade salts were added to prepare the following stock solutions:
A) Solution: KH2PO4 8.50 g K2HPO4 21.75 g Na2HPO4 x 12H2O 67.16 g2 NH4Cl 0.50 g Water ad 1000 mL
B) Solution: CaCl2 x 2 H2O 36.40 g Water ad 1000 mL
C) Solution: MgSO4 x 7 H2O 22.50 g Water ad 1000 mL
D) Solution: FeCl3 x 6 H2O 0.25 g Water ad 1000 mL (The “D” stock solution was prepared on the day of the mineral medium preparation and was not further stored).
The mineral medium was prepared in the following ratio: 1 mL of the stock solutions A - D) were combined per 1000 mL total volume, filled with water (purified deionized) . The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was checked and found 8.10 mg/L. The pH of the mineral medium was 7.46.
- Additional substrate: no
- Test temperature: 20.0 - 20.6 °C
- pH: 7.46
- pH adjusted: no
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: incubator
- Number of culture flasks/concentration: 16 (+2 reserve) bottles containing the test item and inoculum
- Method used to create aerobic conditions: prepared activated sludge was continuously aerated (2 L/minute) at the test temperature of 20.0 – 20.8 °C, for about 6 days
- Measuring equipment: O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]
- Test performed in closed vessels due to significant volatility of test substance: yes
- Test performed in open system: no

SAMPLING
- Sampling frequency: Oxygen measurements were performed in all duplicate bottles in all groups on days 0, 2, 5, 7, 12, 14, 21 and 28
- Sampling method: electrode

CONTROL AND BLANK SYSTEM
- Inoculum blank: 16 (+2 reserve) bottles containing only inoculum (inoculum control)
- Procedure control: 16 (+2 reserve) bottles containing the sodium benzoate and inoculum
- Toxicity control: 16 (+2 reserve) bottles containing the test item, sodium benzoate and inoculum

Reference substance:

benzoic acid, sodium salt

Preliminary study:

The test item solubility, behavior, and toxicity were tested in a 14-day pre-experiment. The
test design was the same as described at the main experiment. In the preliminary experiment
the test item was investigated at the concentration of 4 mg/L. In this toxicity test (containing
the test and reference items) the biodegradation degree was higher than 25 % (the actual
value: 38 %). The test item can be assumed non-inhibitory at ≤ 4 mg/L concentration.
The pre-experiments were not performed in compliance with the GLP-Regulations and are
excluded from the Statement of Compliance in the final report, but the raw data of these
tests will be archived under the study code of the present study.

Key result

Parameter:

% degradation (O2 consumption)

Value:

67.9

Sampling time:

28 d

Details on results:

Under the test conditions the percentage biodegradation of the test item reached a mean of 67.9 % after 28 days based on its calculated ThODNH3. In this test the test item biodegradation curve reached its plateau about the 12th day, from that day onwards only slight increases in biodegradability values (the slight changes were considered as being within the biological variability range of the applied test system) occurred. For informative reason the test was not stopped before the 28th day. The percentage biodegradation of Methyl vinyl glycolate (MVG) reached a mean of 46 % (based on its ThODNH3) on the 7th day of the test and 61.8 % on the next measurement occasion, on the 12th day of the test. The 10-day window criterion for ready biodegradation was unequivocally fulfilled, 10 % biodegradation of the test item (estimated on the basis of calculated biodegradation results and derived from the biodegradation curve) was reached after the second day of the test (between 2nd and 3rd day, start of the 10-day window) and the biodegradation reached the 60 % level before the 12th days of the test (between 11th and 12th day). The pass level for biodegradability, 60 % removal of ThODNH3, was reached in about 9 days from the attainment of 10 % biodegradation. The 10-day window calculation is based on the biological observations and the values were derived from the biodegradation curve. Based on the obtained values the test item is evaluated as readily biodegradable.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to a mean of 75.0 % after 14 days, and to a mean of 78.3 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

Table: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration	Bottle	Percent of biodegradation after n days of exposure
	[mg/L]	No.	2	5	7	12	14	21	28
Test item		1a	11.3	28.9	45.3	60.9	62.7	65.5	66.5
		1b	6.8	26.1	46.7	62.8	63.4	67.2	69.2
		mean	9	27.5	46	63	63	66.3	67.9
Reference item		2a	41.2	63.7	68.9	73.5	73.5	71.1	72.6
		2b	43	68.7	70.7	76.5	76.5	83.1	84
		mean	42.1	66.2	69.8	75	75	77.1	78.3
Toxicity control		4a	24.8	39.2	53.5	55.6	55.6	55.4	57.4
		4b	28.1	40.3	51.6	55	55	58	58.2
		mean	26.4	39.7	52.5	55.3	55.3	56.7	57.8

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

In a Closed Bottle Test according to OECD TG 301 D the test item was determined to be readily biodegradable (67.9 % based on theoretical oxygen demand (ThODNH3) meeting the 10-day window criterion. The test item revealed to be non-toxic to aquatic microorganisms.

Executive summary:

A Closed Bottle Test was carried out in accordance with OECD TG 301 D and the GLP principles. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate (at a concentration of 3.0 mg/L) was tested simultaneously under the same conditions as the test item, and functioned as a procedure control (reference control). Additionally, inoculum (containing the filtered inoculum, only) and toxicity (containing both the test item and reference item) controls were examined. The test item was investigated at the concentration of 3.5 mg/L. The chosen test item concentration was based on the performed preliminary experiment on toxicity; furthermore, on the calculated ThODNH3 value of the test item (ThODNH3: 1.52 mg test item O2/mg). All validity criteria of the test were met. Under the applied test conditions, was ready biodegradable. The percentage biodegradation of the test item reached a mean of 46 % (based on its ThODNH3) on the 7th day of the test and 61.8 % on the next measurement occasion, on the 12th day of the test. The 10-day window criterion for ready biodegradation was unequivocally fulfilled, 10 % biodegradation of the test item was reached after the second day (start point of the 10-day window) of the test (between 2nd and 3rd day) and the biodegradation reached the 60 % level before the 12th days of the test (between 11th and 12th day). The test item is evaluated to be ready biodegradable, as the pass level for ready biodegradability, the removal of 60 % theoretical oxygen demand (ThODNH3) within a 10-day window, is fulfilled. The reference item sodium benzoate was sufficiently degraded to a mean of 75.0 % after 14 days, and to a mean of 78.3 % after 28 days of incubation, based on ThODNH3. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 55.3 % biodegradation was noted within 14 days and the calculated biodegradation was 57.8 % after 28 days of incubation. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control was higher than 25 % within 14 days.

Description of key information

In a Closed Bottle Test according to OECD TG 301 D the test item was determined to be readily biodegradable (67.9 % based on theoretical oxygen demand (ThODNH3) meeting the 10-day window criterion). The test item revealed to be non-toxic to aquatic microorganisms.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

A Closed Bottle Test was carried out in accordance with OECD TG 301 D and the GLP principles. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate (at a concentration of 3.0 mg/L) was tested simultaneously under the same conditions as the test item, and functioned as a procedure control (reference control). Additionally, inoculum (containing the filtered inoculum, only) and toxicity (containing both the test item and reference item) controls were examined. The test item was investigated at the concentration of 3.5 mg/L. The chosen test item concentration was based on the performed preliminary experiment on toxicity; furthermore, on the calculated ThODNH3 value of the test item (ThODNH3: 1.52 mg test item O2/mg). All validity criteria of the test were met. Under the applied test conditions, it was ready biodegradable. The percentage biodegradation of the test item reached a mean of 46 % (based on its ThODNH3) on the 7th day of the test and 61.8 % on the next measurement occasion, on the 12th day of the test. The 10-day window criterion for ready biodegradation was unequivocally fulfilled, 10 % biodegradation of the test item was reached after the second day (start point of the 10-day window) of the test (between 2nd and 3rd day) and the biodegradation reached the 60 % level before the 12th days of the test (between 11th and 12th day). The test item is evaluated to be ready biodegradable, as the pass level for ready biodegradability, the removal of 60 % theoretical oxygen demand (ThODNH3) within a 10-day window, is fulfilled. The reference item sodium benzoate was sufficiently degraded to a mean of 75.0 % after 14 days, and to a mean of 78.3 % after 28 days of incubation, based on ThODNH3. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 55.3 % biodegradation was noted within 14 days and the calculated biodegradation was 57.8 % after 28 days of incubation. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control was higher than 25 % within 14 days.