3-tert-butyladipic acid

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:

2017-08-16 to 2017-09-13

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:

17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

January 1998

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Changzhou Sunlight Pharmaceutical Co., Ltd., China; 20170601
- Expiration date of the batch: June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge, microorganisms from a domestic waste water treatment plant. The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 09 August 2017 (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 and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with test water (mineral medium, see above) and then aerated under test conditions (for 7 days) until use. The pH of the activated sludge inoculum after preparation was 7.38, just before use: 7.39. A pH adjustment of activated sludge inoculum was not performed.
- Pretreatment: Pre-conditioning (09-16 August 2017) consisted of aerating (2 L/minute) activated sludge (in mineral medium5) for 7 days at the test temperature (the actual temperature: 20.0 – 21.8 °C). During the aeration the cell count of inoculum was checked as follows: the viability of the cultured sludge was determined by plating 0.1 mL of 10-1, 10-2, 10-3 and 10-4 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by these plating experiments by manual colony counting.
During the pre-conditioning the approximately cell count of aerated inoculum was in the range of ~109/L; therefore, before the test the inoculum was further diluted 100000 x with mineral medium to reach the necessary 10^4 – 10^5 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improved the precision of the test methods by reducing blank values.
The inoculum was not pre-adapted to the test chemical.
- Initial cell/biomass concentration: 10^4 – 10^5 cells/L
- Water filtered: yes
- Type and size of filter used, if any: The sludge was filtered through cotton wool

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: According to OECD guideline 301D
- Solubilising agent: No
- Test temperature:
During the preparation, aeration and incubation of the mineral medium the temperature was 20.1 - 21.3 °C.
During the incubation (28 days) of the test units the temperature range was the following: 20.0 - 20.1 °C.
- pH: 7.31
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Winkler bottles (300 mL, coded) with special neck and glass stoppers.
- Number of culture flasks/concentration:
16 (+ 2 reserve) bottles containing the test item and inoculum
16 (+ 2 reserve) bottles containing the reference item and inoculum (procedure control)
16 (+ 2 reserve) bottles containing only inoculum (inoculum control)7
16 (+ 2 reserve) bottles containing the test item, reference item and inoculum (toxicity control)
- Measuring equipment:
Large glass tank (volume: ~30 L) and
Large glass flasks (volume: at least 5 L),
Narrow necked, Winkler bottles with glass stoppers,
Funnels and coarse filter papers,
Oxygen and pH meter with appropriate O2 and pH electrode,
Aeration system, Moisture analyzer
Temperature controlled (22 ± 2 °C) environment room (or incubator) with thermometer with exclusion of light,
Balance, Centrifuge, Ultrasonic bath.

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.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. Only filtered inoculum (12 mL) was added to the aqueous test medium (ad. 6000 mL).
- Abiotic sterile control: No
- Toxicity control: Yes. Test (200 mL) and reference item (60 mL) stock solutions were mixed into the aqueous test medium (ad. 6000 mL) corresponding to the test item concentration of 3.0 mg/L [chosen based on the preliminary experiment and ThODNH4] and to 3.0 mg/L concentration of the reference item.
- Procedure Control: Sodium benzoate. Based on the theoretical oxygen demand (ThODNH4) of sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), at the start of the test a stock solution of sodium benzoate (60 mL) was mixed into the aqueous test medium (ad. 6000 mL, corresponding to 3.0 mg/L reference item, respectively a ThODNH4 of about 5.01 mg O2/L).

STATISTICAL METHODS:

Reference substance:

other: sodium benzoate

Key result

Parameter:

% degradation (O2 consumption)

Value:

5.1

Sampling time:

28 d

Details on results:

Under the test conditions the percentage biodegradation of 3-Tert Butyladipic Acid reached a mean of 5.1 % after 28 days based on its ThODNH4. The highest biodegradation value of 5.4 % was noticed on the 14th day of the experiment. The obtained biodegradation values were minimal throughout the test, within the biological variability range of the applied test system.
The pass level for ready biodegradability is the removal of 60 % ThODNH4 in a 10-day window. Minimal biodegradation of the test item occurred in this study; however, the value remained far below the pass level; therefore, the test item was considered to be not ready biodegradable.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to a mean of 72.8 % after 14 days, and to a mean of 75.0% after 28 days of incubation, based on its ThODNH4. The biodegradability reached its plateau on about the 7th day and from this day the slight changes were considered as being within the biological variability range of the applied test system.

Validity of the Study

Inoculum control: The oxygen depletion in the inoculum control did not exceed 1.5 mg O2/L after 28 days. It was 1.27 mg O2/L in average.

Oxygen concentration: The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any time. (The lowest value was 2.55 mg O2/L, it was measured on the 28th in the toxicity control.)

Parallels: The difference of duplicate values for the degradation at the plateau, at the end of the test or at the end of the 10-d window was not greater than 20 %.

The highest difference (17.1 %) between the duplicate values from the 7th to the 28th day of the test (this period was taken into consideration as biodegradation plateau) or at the end of the test for degradation was calculated in the test item group, observed on the 12th day of the test.

Reference item: 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 72.8 % on the 14th day.)

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this test, the test item is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % ThODNH4 in a 10-day window. 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 group was higher than 25 % within 14 days.

Executive summary:

The ready biodegradability of the test item was determined in a study according to OECD Guideline 301D (Closed Bottle Test). 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 was tested simultaneously under the same conditions as the test item and functioned as a procedure control.

The test item 3-Tert Butyladipic Acid was investigated at the concentration of 3.0 mg/L. The test item concentration was chosen based on the theoretical oxygen demand of the test item (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 1.98 mg O2/mg. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met.

Under the test conditions the percentage biodegradation of the test substance reached a mean of 5.1 % after 28 days based on its theoretical oxygen demand. The highest biodegradation value of 5.4 % was noticed on the 14th day of the experiment. The obtained biodegradation values were minimal throughout the test, within the biological variability range of the applied test system. The reference item sodium benzoate was sufficiently degraded to a mean of 72.8 % after 14 days, and to a mean of 75.0 % after 28 days of incubation, based on ThODNH4. (The biodegradability reached its plateau on about the 7th day and from this day the slight changes were considered as being within the biological variability range of the applied test system.) In the toxicity control containing both, the test item and the reference item, a mean of 34.1 % biodegradation was noted within 14 days and 42.8 % biodegradation after 28 days of incubation.

Under the conditions of this test, the test item was considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is 60% of its ThODNH4. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. 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 group was higher than 25 % within 14 days.

Description of key information

The test substance is considered to be not ready biodegradable (TOXI-COOP, 2017).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The ready biodegradability of the test item was determined in a study according to OECD Guideline 301D (Closed Bottle Test). 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 was tested simultaneously under the same conditions as the test item and functioned as a procedure control.

The test item 3-Tert Butyladipic Acid was investigated at the concentration of 3.0 mg/L. The test item concentration was chosen based on the theoretical oxygen demand of the test item (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 1.98 mg O2/mg. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met.

Under the test conditions the percentage biodegradation of the test substance reached a mean of 5.1 % after 28 days based on its theoretical oxygen demand. The highest biodegradation value of 5.4 % was noticed on the 14th day of the experiment. The obtained biodegradation values were minimal throughout the test, within the biological variability range of the applied test system. The reference item sodium benzoate was sufficiently degraded to a mean of 72.8 % after 14 days, and to a mean of 75.0 % after 28 days of incubation, based on ThODNH4. (The biodegradability reached its plateau on about the 7th day and from this day the slight changes were considered as being within the biological variability range of the applied test system.) In the toxicity control containing both, the test item and the reference item, a mean of 34.1 % biodegradation was noted within 14 days and 42.8 % biodegradation after 28 days of incubation.

Under the conditions of this test, the test item was considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is 60% of its ThODNH4. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. 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 group was higher than 25 % within 14 days.