Trimethylamine, N-oxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study duration: 24 MAY 2022 - 30 SEP 2022
Experimental duration: 08 JUN 2022 - 27 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 APR 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SANTE/2020/12830 Rev.1: Guidance Document on Pesticide Analytical Methods for Risk Assessment and Post-approval Control and Monitoring Purposes, 24.

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 medium of the only test concentration 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 concentration during the test period, duplicate samples from the test medium of the only test concentration and the control were collected at the end of the test (after 48 hours).
All 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 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 48 hours).

Vehicle:

no

Details on test solutions:

The test medium of the only test concentration of nominal 148 mg test item/L was prepared by dissolving 72.8 mg test item into 492 mL test water by intense stirring for 15 minutes.

The test medium was prepared just before introduction of the daphnids (= start of the test).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: clone 5
- Age at study initiation (mean and range, SD): From 1.25 to 21.75 hours old
- Method of breeding:
The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test.
Water Temperature: 18 – 22 °C Light: 16 h light : 8 h dark, < 1500 lux Medium: Elendt Medium Feeding: Daily (working days), 0.2 mg TOC/daphnid/day The daphnids are held individually. Three water-renewals per week are performed.
The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
- Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture. The origin of the brood was Noack Laboratorien GmbH.
- Age of parental stock: The age of the parental animals was 35 days.
- Feeding during test: none

ACCLIMATION: Was not necessary, since the test was performed in the same medium as the culturing.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

2.5 mmol/L (=250 mg/L) as CaCO3

Test temperature:

19.6-20.4 degrees Celsius

pH:

8.0-8.1

Dissolved oxygen:

8.8-8.9 mg/L

Nominal and measured concentrations:

Nominal 148 mg test item/L equivalent to 100 mg Trimethylamine N-Oxide Anhydrate/L.

At the start of the test 102 % of the nominal test concentration was found. After 48 hours test duration, 93 % of the nominal value was determined. During the test the daphnids were exposed to a mean of 97 % of nominal.

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: 100mL beakers
- Material, size, headspace, fill volume: Glass, 100mL containing approximately 60 mL of test medium, covered with a transparent plastic lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
- Volume of solution: 60 mL of test medium
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4 vessels with 5 organisms per vessel

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Alkalinity: 0.9 mmol/L
- Culture medium different from test medium: no (Elendt M4 medium used for culturing and testing).
- Intervals of water quality measurement: The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark
- Light intensity: light intensity: 843 to 870 lux (target: ≤ 1500 lux)

EFFECT PARAMETERS MEASURED : mobility after 24 and 48 hours.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions. The range finding test was performed in a 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):

yes

Remarks:

Potassium dichromate; purity: 99.67%

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Trimethylamine N-Oxide Anhydrate

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No sublethal effects were observed in any treatment

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: Control, 0.40, 0.60, 0.89, 1.33, and 2.0 mg reference item/L
- Limit test: no
- Dose-response test: yes
- EC50: 1.96 mg/L (1.90 - > 2.0 mg/L 95% CI)
-EC10: 1.82 mg/L (1.77 - 1.88 mg/L 95% CI)
-LOEC: 2.0 mg/L
-NOEC: 1.33 mg/L

Reported statistics and error estimates:

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

Validity criteria fulfilled:

yes

Remarks:

Control Immobilisation Rate: 0% and no daphnia showed signs of disease or stress (should not exceed 10%). DOC: ≥ 8.8 mg O2/L in all treatment groups at the end of the test (≥ 3 mg O2/L).

Conclusions:

The study was valid since all required validity criteria were met.
The toxic effect of the test item Trimethylamine N-Oxide Dihydrate to Daphnia magna was assessed in a static limit test. The 48-hour NOEC was determined to be ≥ 148 mg test item/L (≥ 100 mg Trimethylamine N-Oxide Anhydrate/L). The 48-hour LOEC was determined to be > 148 mg test item/L (> 100 mg Trimethylamine N-Oxide Anhydrate/L) and the 48-hour EC50 value was determined to be > 148 mg test item/L (> 100 mg Trimethylamine N-Oxide Anhydrate/L).

Executive summary:

The aim of the study was to determine the effect of the test item Trimethylamine N-Oxide Dihydrate on the mobility of Daphnia magna. For this purpose, young daphnids (< 24 hours old) were exposed in a static test for 48 hours to the only concentration of nominal 148 mg/L (based on the results gained during a non-GLP range-finding test) under defined conditions. The recorded effects were the mobility of the daphnids after 24 and 48 hours.

After 48 hours of exposure, no immobilisation of the test animals was observed in the control. At the only test concentration of nominal 148 mg test item/L equivalent to 100 mg Trimethylamine N-Oxide Anhydrate/L, 1 daphnid was immobile which does not represent a statistically significant difference compared to the control.  No sublethal effects were observed in any treatment.

The 48-hour NOEC was determined to be ≥ 148 mg test item/L (≥ 100 mg Trimethylamine N-Oxide Anhydrate/L). The 48-hour LOEC was determined to be > 148 mg test item/L (> 100 mg Trimethylamine N-Oxide Anhydrate/L) and the 48-hour EC50 value was determined to be > 148 mg test item/L (> 100 mg Trimethylamine N-Oxide Anhydrate/L).

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 202 (April 13, 2004) and SANTE/2020/12830 Rev.1 (February 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 202 study with Daphnia magna, trimethylamine N-oxide did not demonstrate any toxic effects and a 48 hour NOEC of 100 mg/L for the trimethylamine N-oxide anhydrate is reported.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 100 mg/L