Trimethylamine, N-oxide

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:

Study Duration: 07 APR 2022 - 21 SEP 2022
Experimental Duration: 15 MAY 2022 - 15 JUL 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Dihydrate form of the registered substance Trimethylamine N-oxide (CAS 1184-78-7)

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

May 30, 2008 (EEC Publication No. L 142/496, May 2008)

Deviations:

yes

Remarks:

Aliquot of prepared flasks frozen at -20 +/- 5C until determination of nitrite and nitrate. Temperatures reached -6.4C. No presumed effect on the study - samples remained frozen the whole time.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

yes

Remarks:

Aliquot of prepared flasks frozen at -20 +/- 5C until determination of nitrite and nitrate. Temperatures reached -6.4C. No presumed effect on the study - samples remained frozen the whole time.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Dihydrate form of the registered substance Trimethylamine N-oxide (CAS 1184-78-7)

SOURCE OF TEST MATERIAL
-Source: Sigma-Aldrich
-Batch number of test material: BCCF8795
-Titration with HClO4: 97.9%
- Purity (TLC): 100.0%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At 20 degrees Celsius +/- 5 degrees Celsius, in the dark.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

Source: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf (Groß-Zimmerner Str. 30, 64380 Rossdorf), Germany.

Preparation: The aerobic activated sludge used for this study was deposited for 15 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. 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 3.5 g dry material per litre were mixed with test water (see 6.5) and aerated overnight. This suspension was used for the experiment. The suspension was stored under light/dark regime of 12/8 hours and room temperature (20 ± 5°C) prior to use.

Duration of test (contact time):

28 d

Initial conc.:

101.6 mg/L

Based on:

act. ingr.

Remarks:

Trimethylamine N-Oxide Dihydrate

Initial conc.:

68.6 mg/L

Based on:

other: Potential degradable Trimethylamine-Oxide Anhydrate

Initial conc.:

117 mg/L

Based on:

ThOD

Remarks:

ThOD(NH4)

Initial conc.:

175.5 mg/L

Based on:

ThOD

Remarks:

ThOD(NO3)

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Oxygen uptake of the microorganisms determined by change of pressure in the test flasks which was measured by means of a manometric method (BSB-Sensomat-System©).

Details on study design:

TEST CONDITIONS
- Composition of medium: The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used, but the solutions were dispersed by stirring to achieve a homogeneous solution of the test item.
- Test temperature: 22C +/- 1C
- pH: 7.4 (start); 6.8-7.6 (end)
- pH adjusted: yes
- Suspended solids concentration: 3.5 g dry material per litre of washed sludge preparation was added to test water.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL.
- Number of culture flasks/concentration: 2 test item flasks
- Method used to create aerobic conditions: prepared sludge was aerated overnight prior to use.
- Measuring equipment: BSB-Sensomat-System©
- Measurement of oxygen: The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB-Sensomat-System©. The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

SAMPLING
- Sampling frequency and method: The change of pressure in the test flasks was measured daily by means of a manometric method (BSB-Sensomat-System©).

CONTROL AND BLANK SYSTEM
- Inoculum blank: activated sludge and test water.
- Abiotic sterile control: test item, CuSO4, and test water.
- Toxicity control: test item, reference item (sodium benzoate), activated sludge, and test water.
- Procedure control: reference item, activated sludge, and test water.

DETERMINATION OF NITRIFICATION
On exposure day 0 a sufficient aliquot was withdrawn from the bottles containing the test item and inoculum, from the inoculum control (after measurement of pH) for analysis of nitrate and nitrite using Continuous Flow Analysis and stored deep frozen (-20 ± 5 °C) until nitrate determination was done. At day 28 an aliquot from both inoculum controls, and both test item flasks was taken for analysis. Due to an increase in the concentration of nitrate and nitrite in the test bottles, the oxygen uptake by nitrification was calculated as described in Annex V of the test guidelines.

Amounts of 14.8 mg and 72.2 sodium nitrite and potassium nitrate, respectively, were diluted in 200 mL (sodium nitrite) and 100 mL (potassium nitrate) 0.1 M KCl to prepare the standard stock solutions for nitrite-N and nitrate-N determination. Appropriate aliquots of the stock solutions were automatically diluted by the dilution unity with 0.1 M KCl to prepare 6 standard solutions at a range of 0.5 mg/L to 3.0 mg/L for nitrite-N and 7 standard solutions at a range of 1.0 mg/L to 12.0 mg/L for nitrate-N determination. Before photometric determination, frozen soil extracts were thawed.

The samples of day 28 were diluted 1:2 in 0.1 M KCl before determination.

The nitrogen determination was done using a AA3 Continuous Flow Analyzer. The limit of quantification was determined to be 0.021 mg/L and 0.172 mg/L for nitrite-N and nitrate-N; in case of complete nitrification, the oxygen taken up by 14 g of nitrogen is 64 g and thus the oxygen consumed in nitrate formation is 4.57 x increase of nitrate-N concentration.

Reference substance:

benzoic acid, sodium salt

Remarks:

(99.9%)

Test performance:

Deviation:
According to the Study Plan:
An aliquot of the separate prepared flasks for pH measurement (control and test item treated flask) will be sampled at test start and stored deep frozen (-20 °C ± 5 °C) until the determination of nitrite and nitrate.

Deviation to the Study Plan:
Storage temperature deviated from the required range for more than 2 hours. Temperatures reached -6.4°C, and temperatures were higher than -15°C for 2.5 hours.

Presumed Effect on the Study:
None, samples remained frozen the whole time.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNO3

Value:

91

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNO3

Value:

10

Sampling time:

4 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNO3

Value:

86

Sampling time:

14 d

Details on results:

The concentrations of nitrite-N and nitrate-N over the complete incubation time was evaluated to define if the results should be based on ThODNH4 or ThODNO3. The nitrite-N+nitrate-N formation rate after 28 days of incubation in the controls was 2.115 mg/L (mean). The nitrite-N+nitrate-N formation rate in the test item treated vessels was 15.482 mg/L (mean). In both test item replicates a formation of oxidized nitrogen above the inoculum control level was observed. The measured values indicated that the complete nitrogen of the test item was oxidized over time. Based on these results, the ThODNO3 was used for evaluation.

The mean biodegradation of at least 10% of Trimethylamine N-Oxide Anhydrate was reached at day 4 (ThODNO3). At the end of the 10-day window at day 14, the mean degradation of Trimethylamine N-Oxide Anhydrate was 86% (ThODNO3) and therefore the 10 day window criterion was passed. The mean biodegradation at test end after 28 days was 91% (ThODNO3).

If no nitrification is considered, the mean biodegradation of at least 10% of Trimethylamine N-Oxide Anhydrate was reached at day 4 (ThODNH4). At the end of the 10-day window at day 14, the mean degradation of Trimethylamine N-Oxide Anhydrate was 128% (ThODNH4) and therefore the 10 day window criterion was passed. The mean biodegradation at test end after 28 days was 137% (ThODNH4).

TOXICITY CONTROL
In the toxicity control containing both, the test item and the reference item sodium benzoate, 92% (ThODNH4) biodegradation was noted within 14 days and 88% (ThODNH4) biodegradation after 28 days of incubation (76% and 73% based on ThODNO3).

The test item can be assumed to be not inhibitory.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 81% after 14 days and to 84% after 28 days of incubation.

Validity criteria fulfilled:

yes

Remarks:

Inoculum Con: 30 mg O2/L (<60 mg O2/L) pH: 6.8 & 7.3 (6.0-8.5) Ref Item: >60% deg after 3 days (<= 14 days) Test Item: difference of deg. at end of test and day 14 was 12% and 11% (<20%) Toxicity Con: deg: 92% ThODNH4 and 76% ThODNO3 at day 14 (>=25%)

Interpretation of results:

readily biodegradable

Remarks:

The mean biodegradation of at least 10% of Trimethylamine N-Oxide Anhydrate was reached at day 4 (ThODNO3). At the end of the 10-day window at day 14, the mean degradation of Trimethylamine N-Oxide Anhydrate was 86% (ThODNO3) and therefore the 10 day window criterion was passed. The mean biodegradation at test end after 28 days was 91% (ThODNO3). The test item can therefore be considered as readily biodegradable.

Conclusions:

The test item is readily biodegradable meeting the 10-day window.

Executive summary:

The test method was chosen based on the test item properties (good water solubility, not volatile, and no expected toxicity to activated sludge bacteria). Appropriate reference control was used (sodium benzoate). Test item loading rate (initial concentration) was determined as ThODNH4 and ThOD NO3. As the potential biodegradable part of the test item contains nitrogen, the concentrations of nitrite-N and nitrate-N over the complete incubation time was evaluated to define if the results should be based on ThODNH4 or ThODNO3. The measured values indicated that the complete nitrogen of the test item was oxidized over time. Based on these results, the ThODNO3 was used for evaluation. Test item was readily biodegradable. Biodegradation of sodium benzoate confirmed the suitability of the aerobic activated sludge inoculum used. The test item was assumed not to be inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

The study was conducted in compliance with EC C.4-D (2008) and OECD 301F and is GLP compliant with a Quality Assurance statement. Based on the test methods used and the level of detail in the study report, there are no concerns regarding deficiencies.

Description of key information

Based on the findings of a GLP-compliant OECD 301F study, trimethylamine N-oxide is considered to be readily biodegradable meeting the 10-day window criteria.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater