Trimethylamine, N-oxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Duration: 24 MAY 2022 - 30 SEP 2022
Experimental Duration: 20 JUN 2022 - 28 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006, corrected July 28, 2011.

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:

February 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 (without algae) of all test concentrations and from 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 (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
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 were 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 72 hours).

Vehicle:

no

Details on test solutions:

The test medium of the highest test concentration of nominal 148 mg test item/L was prepared by dissolving 74.6 mg test item into 504 mL test water by intense stirring for 20 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.

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

Spacing factor = 3.16

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Raphidocelis subcapitata (KORSHIKOV), Strain No. 61.81 SAG, recently renamed from Pseudokirchneriella subcapitata and formerly known as Selenastrum capricornutum.
- Source: The algae were originally supplied by the "Culture Collection of Algae at Goettingen University (SAG)", 37073 Göttingen, Germany. The algae are at ibacon since 2017.
- Method of cultivation:
The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines:
Water Temperature: 21 – 24 °C Light: Continuous illumination, 4440-8880 lux Medium: OECD Medium (see 2.5) pH of medium: 8.1 ± 0.1
No acclimation needed to be considered before test start.
The flasks in the sterile culture were continuously stirred by magnetic stirrers in a water bath. Algae were transferred twice per week into fresh medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.

Test temperature:

Time (hrs) Measured Temp (Celsius)
0 21.9
24 23.0
48 23.3
72 23.4

pH:

The test water was adjusted with 1 M NaOH-solution to pH 8.1

Nominal and measured concentrations:

Nominal test material:
1.5, 4.7, 14.8, 46.8, 148 mg/L
Nominal active ingredient (Trimethylamine N-Oxide Anhydrate)
1.014, 3.177, 10.005, 31.637, 100.048 mg/L
Average Measured active ingredient (Trimethylamine N-Oxide Anhydrate)
1.120, 3.213, 9.450, 30.184, 91.548 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50mL Erlenmeyer flasks
- Type: covered with watch glasses
- Material, size, headspace, fill volume: Glass, 50mL flasks with 50 mL nominal test medium
- Aeration: Stirring
- Initial cells density: nominal 5000 algal cells/mL test medium
- Control end cells density: 518,540 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: analytical grade salts added as nominal concentrations to deionised
water
- Conductivity: ≤ 10 μS cm-1 (deionized water prior to salt addition)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: 1 M NaOH-solution to pH 8.1
- Photoperiod: Continuous illumination
- Light intensity and quality:
The light intensity was measured once during the test at 6 positions distributed over the experimental area
at the surface of the test media (target: 4440 – 8880 lux, ± 15 % of mean value).
Mean light intensity: 5777 lux (range: 5340 to 6100 lux).

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: spectrophotometric observation at 24, 48, and 72 hours of exposure

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: 72 hrs; static design; 148, 14.8, 1.48, and 0.148 mg test item/L
- Test concentrations: 148, 46.8, 14.8, 4.7, and 1.5 mg test item/L equivalent to 100, 32, 10, 3.2 and 1.0 mg Trimethylamine N-Oxide Anhydrate/L.
- Results used to determine the conditions for the definitive study: yes

CULTURING APPARATUS
-Details on culturing apparatus used: The flasks in the sterile culture were continuously stirred by magnetic stirrers in a water bath. Algae were transferred twice per week into fresh medium.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, purity: 99.67%

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Trimethylamine N-Oxide Anhydrate

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Trimethylamine N-Oxide Anhydrate

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

148 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities (for algal test):
The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 148 mg test item/L (100 mg Trimethylamine N-Oxide Anhydrate/L) and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- 72h EC50:
-Yield: 0.652 mg/L (0.613 - 0.695 95% CI)
-Growth Rate: 1.17 mg/L (1.12 - 1.23 95% CI)

Reported statistics and error estimates:

Dunnett's t-test, α = 0.05, one-sided smaller
No statistically significant toxicity was observed for growth inhibition or yield.

The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Validity criteria fulfilled:

yes

Remarks:

Cell Density (Control): 103.7x incr. within 72 hrs (required: ≥ 16-fold) Coefficient of Var. of Sectional (Daily) Growth Rates in Control: 11.8 % (required: ≤ 35 %) Coefficient of Var. of Avg. Growth between Control Replicates: 1.3 % (required: ≤ 7%)

Conclusions:

the 72-hour ErC10 of the active ingredient & registered substance is > 100 mg/L

Executive summary:

Exponentially growing cultures of Raphidocelis subcapitata were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.

The study included 6 treatment groups (5 concentrations of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometric measurement.

The effect of the test item on yield and growth rate were determined, using test concentrations of 148, 46.8, 14.8, 4.7 and 1.5 mg test item/L equivalent to 100, 32, 10, 3.2 and 1.0 mg Trimethylamine N-Oxide Anhydrate/L (spacing factor 3.16) and a control.  The 72-hour EC50 values for both yield and growth rate were determined to be >148 mg test item/L (>100 mg Trimethylamine N-Oxide Anhydrate/L).  The 72-hour NOEC values for both yield and growth rate were determined to be ≥148 mg test item/L (≥100 mg Trimethylamine N-Oxide Anhydrate/L).

The initial concentrations and the maintenance of the exposure concentrations during the test were verified via LC-MS. As the concentrations of the test item were within ± 20% of the nominal concentrations during the test, all reported results referred to nominal values.

The study was conducted according to OECD 201 (23 Mar 2006, corrected 28 Jul 2011) 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 201 study with Raphidocelis subcapitata, trimethylamine N-oxide did not demonstrate any toxic effects and a 72 hour ErC10 > 100 mg/L for the trimethylamine N-oxide anhydrate is reported.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L