Trimethylamine, N-oxide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Duration: 19 MAY 2022 - 27 SEP 2022
Experimental Duration: 30 MAY 2022 - 09 JUN 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:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SANTE/2020/12830 Rev.1

Version / remarks:

Feb 2021

Deviations:

no

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.

Analytical monitoring:

yes

Remarks:

Direct dilution of samples followed by analysis via LC-MS/MS. LC: Agilent Series 1200 pump and autosampler Column: Hypercarb, 100 * 4.6 mm, 5 μm Detector: Mass spectrometer API 4000

Details on sampling:

- Sampling method:

The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director.
Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control were collected at the end of the test (after 96 hours) from the approximate centre of the aquaria.
The samples were diluted by a factor of two with acetonitrile.
Additional samples of the untreated control and of the sample dilution solvent acetonitrile were taken at each sampling without any sample treatment.


- Sample storage conditions before analysis:

All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen (≤ -20 °C) and will be kept stored up to the date of the final report.

All samples were analysed within 24 hours after sample preparation without further storage.

- Analyses:

The concentrations of Trimethylamine N-Oxide Anhydrate of the test item Trimethylamine N-Oxide Dihydrate were analysed in the duplicate test media samples from all test concentrations, and in the duplicate control samples and the additional untreated control and dilution solvent samples, from both sampling times (0 and 96 hours).

Vehicle:

no

Details on test solutions:

A stock solution of 1480 mg test item/L was prepared by dissolving 2960.6 mg test item into 2000.4 mL test water by intense stirring for 25 minutes. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations.

The test media were prepared just before introduction of the test fish (= start of the test).

- Test concentration separation factor: 2.2

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Source: The test fish were obtained from Forellenzuchtbetrieb Störk, 88348 Bad Saulgau, Germany
- Length at study initiation (length definition, mean, range and SD): The mean body length of the fish* in the test was 4.15 cm ± 0.1 cm (mean ± SD)
- Weight at study initiation (mean and range, SD): the mean body wet weight* 0.53 g ± 0.1 g (mean ± SD).
* 10 fish from the test fish batch were measured before the start of the test

FEEDING DURING TEST: none

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

178.4 mg/L as CaCO3 (calculated value); target: 40 – 250 mg/L CaCO3, preferably < 180 mg/L CaCO3

Test temperature:

11.9 to 12.6 °C (target: 10 – 14 °C)

pH:

7.1 to 7.7 (Target: 6.0 - 8.5)

Dissolved oxygen:

96 to 100 % of the air saturation value (Target: at least 60 % of the air saturation value)

Nominal and measured concentrations:

Nominal: 148, 67.3, 30.6, 13.9, and 6.32 mg test item/L equivalent to 100, 45.5, 20.7, 9.4, and 4.3 mg Trimethylamine N-Oxide Anhydrate (spacing factor 2.2).

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values since the concentrations of the test item were within ± 20 % of the nominal concentrations during the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 12L glass aquaria (size approximately 28 x 20 x 22 cm) with 10 L test medium, loosely covered with a glass lid
- Material, size, headspace, fill volume: Glass, 12L vessels with 10 L test medium
- Aeration: The test media were slightly aerated during the test
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Alkalinity: 0.55 mmol/L
- Ca/mg ratio: 4 : 1 (based on molarity)
- Culture medium different from test medium: no (ISO Medium, based on ISO 6341)
- Intervals of water quality measurement: Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control were collected at the end of the test (after 96 hours) from the approximate centre of the aquaria.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 h light - 12 h dark
- Light intensity: 620 to 780 lux (target: 540 – 1000 lux)

EFFECT PARAMETERS MEASURED: The test fish were observed at test start and after approximately 2, 4, 24, 28, 48, 52, 72, 76 and 96 hours test duration for sublethal effects and mortality.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
Range finding study
- Test concentrations: 96-h static design using 148, 14.8, 1.48 and 0.148 mg test item/L.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Trimethylamine N-Oxide Anhydrate

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Other abnormalities: All fish survived until the end of the experiment and only slight signs of stress were observed:

In the control no fish died during the test.

The dissolved oxygen concentration did not fall below 96% of air saturation.

Reported statistics and error estimates:

No statistical analysis was performed due to the absence of mortality.

Sublethal observations / clinical signs:

After 24 hours:
148 mg/L: 2 fish with abnormal bottom distribution

After 48 hours:
30.6 mg/L: 1 fish with abnormal bottom distribution
67.3 mg/L: 1 fish with darkening
148 mg/L: 4 fish with darkening, 3 of which had abnormal bottom distribution

After 72 hours:
30.6 mg/L: 1 fish with darkening and abnormal bottom distribution
67.3 mg/L: 1 fish with darkening
148 mg/L: 4 fish with darkening and abnormal bottom distribution

After 96 hours:
30.6 mg/L: 1 fish with darkening and abnormal bottom distribution
148 mg/L: 3 fish with darkening and abnormal bottom distribution

Validity criteria fulfilled:

yes

Remarks:

Control mortality: 0 (req. : ≤ 1). DOC: >= 96 % of air sat'n value during the test (required: ≥ 60%). Analytical Measurement: Analytical measurements were conducted.

Conclusions:

The acute effect of the test item Trimethylamine N-Oxide Dihydrate to rainbow trout (Oncorhynchus mykiss) was assessed in a static concentration-response test. Based on the test results the 96-hour LC50 was determined to be > 148 mg test item/L (equivalent to 100 mg Trimethylamine N-Oxide Anhydrate), based on nominal concentrations. The NOEC was determined to be ≥ 148 mg test item/L (equivalent to 100 mg Trimethylamine N-Oxide Anhydrate) also based on nominal concentrations.

Executive summary:

The study evaluated the acute toxicity of the test item Trimethylamine N-Oxide Dihydrate to fish.  Juvenile rainbow trout were exposed in a static test to various concentrations under defined conditions for 96 hours. The recorded effects were mortality and sublethal effects (according to OECD 203) on the fish.

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) each containing 7 individuals.  The test fish were observed at test start and after approximately 2, 4, 24, 28, 48, 52, 72, 76 and 96 hours test duration for sublethal effects and mortality. Dead fish were removed at least once daily and discarded.  The test concentrations were: 148, 67.3, 30.6, 13.9 and 6.32 mg test item/L equivalent to 100, 45.5, 20.7, 9.4 and 4.3 mg Trimethylamine N-Oxide Anhydrate (spacing factor 2.2) and a control.

In the control and up to the highest test concentration of 148 mg test item/L (equivalent to 100 mg Trimethylamine N-Oxide Anhydrate), all fish survived until the end of the experiment and only slight signs of stress were observed.

The quantification of Trimethylamine N-Oxide Anhydrate of the test item Trimethylamine N-Oxide Dihydrate in the test samples was performed using liquid chromatography with MS/MS detection.

The study was conducted according to OECD 203 (18 JUN 2019) and SANTE/2020/12830 Rev.1 (FEB 2021).  Validity criteria were achieved.  This study was conducted in compliance with GLP and included a GLP Certificate.  This study was assessed in compliance with the study plan and the ibacon Standard Operating Procedures. This study and procedures were periodically inspected by the Quality Assurance Unit (QAU) and the dates and the phases of the inspections are included in the final report. The data presented in this final report were audited in comparison to the raw data. A quality assurance statement, signed by the Quality Assurance Unit, is included in this final report.

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 203 study with Oncorhynchus mykiss, trimethylamine N-oxide only demonstrated slight signs of stress with no mortality and a 96 hour LC50 of greater than 100 mg/L for the trimethylamine N-oxide anhydrate is reported.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 100 mg/L