Sodium (2-butoxyethoxy)acetate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

17th July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

TOXI-COOP ZRT., Arácsi út 97., 8230 Balatonfüred, Hungary

Oxygen conditions:

anaerobic

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 06 April 2018 (seven days before the main 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 with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed (5.218 g wet weight), dried and the ratio of wet sludge to dry weight (0.4927 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (5 g dry weight that was equivalent to 59.953 g wet sludge) was suspended in mineral medium (Section 5.4; 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 7 days) until use.
The pH of the activated sludge inoculum after preparation was 7.42, just before use the pH was: 7.39. A pH adjustment of activated sludge inoculum was not performed.
- Pretreatment: Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium ) for 7 days (from April 06 to 13, 2018) 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 0.1 mL of the 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 ~10^8/L; therefore, before the test the inoculum was further diluted 50000 x with mineral medium to reach the necessary ~10^4 – 10^6 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improves the precision of the method. The inoculum was not pre-adapted to the test chemical
- Concentration of sludge: 5 g/L (on dry weight basis).
- Initial cell concentration: 10^4 – 10^6 cells/L

Duration of test (contact time):

28 d

Initial conc.:

4 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Standard mineral medium (OECD 301)
- Additional substrate: no
- Test temperature: 20.8 - 21.3 °C
- pH: 7.39
- pH adjusted: no
- Aeration of dilution water: no
- Suspended solids concentration: 5g per litre (on dry weight basis).
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Winkler bottles (300 mL, coded) with special neck and glass stoppers.
- Number of culture flasks/concentration: 2
- Measuring equipment: O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]

SAMPLING
- Sampling frequency: on days 0, 2, 5, 7, 12, 14, 21 and 28
- Sampling method: Oxygen measurements
- Sample storage before analysis: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculum and mineral medium
- Positive control: reference item, inoculum and mineral medium
- Toxicity control: test item, reference item, inoculum and mineral medium

Reference substance:

benzoic acid, sodium salt

Test performance:

The study was considered as valid since oxygen depletion in the inoculum control was 1.22 mg O2/L on average, and did not exceed the validity criteria of 1.5 mg O2/L after 28 days. The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any time. The lowest value was 0.51 mg O2/L, which was measured on the 28th day in the toxicity control. The difference of duplicate values for the degradation at the plateau or at the end of the test was not greater than 20 %. At the whole biodegradation processes (test item, procedure control, and toxicity control groups) the highest difference (20 %) between duplicate values for degradation was calculated in the test item group, it was observed on the 14th day. The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. The percentage degradation of the reference item was 71.4 % on the 14th day. All validity criteria were met as required by the test guideline OECD 301.

Key result

Parameter:

% degradation (O2 consumption)

Value:

71.8

Sampling time:

28 d

Details on results:

Under the test conditions the percentage biodegradation of the test item reached a mean of 71.8 % after 28 days based on its measured COD. In this test the test item biodegradation curve reached its plateau about on the 12th day, from that day 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 sodium (2-butoxyethoxy) acetate reached a mean of 58.1 % (based on its COD) on the 7th day of the test and 68.0 % biodegradation was noticed on the 12th day of the test. The 10-day window criterion for ready biodegradation was unequivocally fulfilled, 10 % biodegradation of the test item (derived from the biodegradation curve) was reached before the 1st day of the test and the biodegradation reached the 60 % level between the 7th and 8th days of the test. The pass level for biodegradability, 60 % removal of COD, was reached in about 7 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 considered to be ready biodegradable. The test item does not contain N. For this reason, total oxidised Nitrogen (nitrate and nitrite) concentrations were not measured after each oxygen measurement.

Table 1: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration [mg/L]	Bottle No.	Percent of biodegradation after n days of exposure
			2	5	7	12	14	21	28
Test item	4.0	1a	45.7	56.3	58.6	71.6	76.3	74.6	75.0
		1b	47.5	52.4	57.6	64.5	62.6	65.9	68.5
		mean	46.6	54.3	58.1	68.0	69.5	70.3	71.8
Reference item	3.0	2a	48.3	53.8	59.3	65.4	69.1	72.4	74.2
		2b	50.5	63.8	64.3	72.2	73.7	73.0	73.0
		mean	49.4	58.8	61.8	68.8	71.4	72.7	73.6
Toxicity control	Test item: 4.0 Reference item: 3.0	4a	37.1	57.0	62.6	64.3	64.1	64.0	64.0
		4b	36.6	59.9	62.7	63.5	63.3	63.9	63.8
		mean	36.9	58.4	62.6	63.9	63.7	63.9	63.9

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item is considered to be ready biodegradable in the Closed Bottle Test according to OECD guideline 301 D.

Executive summary:

The ready biodegradability of the test item was determined in the Closed Bottle Test according to OECD guideline 301 D, EU method C.4 E and OPPTS 835.3110. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. The biodegradation was followed by 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 chosen test item concentration of 4.0 mg/L investigated in the main test was based on the testing laboratory’s experience with this test item originated from other studies applying bacterial test system and on the calculated ThODNH3 of 1.61 mg O2/mg sodium (2-butoxyethoxy) acetate. However, for correct calculations the chemical oxygen demand (COD) of the sodium (2-butoxyethoxy) acetate (1.24 mg O2/ mg) was measured at the start of the main experiment.

Under the test conditions ready biodegradation of test item was noticed. The percentage biodegradation of sodium (2-butoxyethoxy) acetate reached a mean of 69.5 % on the 14th day of the test and 71.8 % after 28 days based on its COD. The 10-day window criterion for ready biodegradation was unequivocally fulfilled, 10 % biodegradation of the test item (derived from the biodegradation curve) was reached before the 1st day of the test and the biodegradation reached the 60 % level between the 7th and 8th days of the test.

The test item is considered to be ready biodegradable, since fulfilled the pass level for ready biodegradability that is the removal of 60 % COD in a 10-day window.

The test item does not contain N. For this reason, total oxidised Nitrogen (nitrate and nitrite) concentrations were not measured after the oxygen measurements. Errors due to not considering nitrification in the assessment by oxygen uptake of the biodegradability of test substances not containing N are marginal (not greater than 5 %), even if oxidation of the ammonium N in the medium occurs erratically as between test and blank vessels.

The reference item sodium benzoate was sufficiently degraded to a mean of 71.4 % after 14 days, and to a mean of 73.6 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

In the toxicity control containing both, the test item and the reference item, a mean of 63.7 % biodegradation was noted within 14 days and 63.9 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).

Description of key information

The test item is considered to be ready biodegradable in a Closed Bottle Test according to OECD guideline 301 D.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

The ready biodegradability of the test item was determined in the Closed Bottle Test according to OECD guideline 301 D, EU method C.4 E and OPPTS 835.3110. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. The biodegradation was followed by 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 chosen test item concentration of 4.0 mg/L investigated in the main test was based on the testing laboratory’s experience with this test item originated from other studies applying bacterial test system and on the calculated ThODNH3 of 1.61 mg O2/mg sodium (2-butoxyethoxy) acetate. However, for correct calculations the chemical oxygen demand (COD) of the sodium (2-butoxyethoxy) acetate (1.24 mg O2/ mg) was measured at the start of the main experiment.

Under the test conditions ready biodegradation of test item was noticed. The percentage biodegradation of sodium (2-butoxyethoxy) acetate reached a mean of 69.5 % on the 14th day of the test and 71.8 % after 28 days based on its COD. The 10-day window criterion for ready biodegradation was unequivocally fulfilled, 10 % biodegradation of the test item (derived from the biodegradation curve) was reached before the 1st day of the test and the biodegradation reached the 60 % level between the 7th and 8th days of the test.

The test item is considered to be ready biodegradable, since fulfilled the pass level for ready biodegradability that is the removal of 60 % COD in a 10-day window.

The test item does not contain N. For this reason, total oxidised Nitrogen (nitrate and nitrite) concentrations were not measured after the oxygen measurements. Errors due to not considering nitrification in the assessment by oxygen uptake of the biodegradability of test substances not containing N are marginal (not greater than 5 %), even if oxidation of the ammonium N in the medium occurs erratically as between test and blank vessels.

The reference item sodium benzoate was sufficiently degraded to a mean of 71.4 % after 14 days, and to a mean of 73.6 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

In the toxicity control containing both, the test item and the reference item, a mean of 63.7 % biodegradation was noted within 14 days and 63.9 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).