N,N,N,2,2,6,6-heptamethylpiperidin-4-aminium chloride

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Study period:

2018-06-04 to 2018-07-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[2,2,6,6-tetramethyl-4-(trimethylazaniumyl)piperidin-1-yl]oxidanyl chloride

EC Number:

814-375-0

Cas Number:

67036-09-3

Molecular formula:

C12H26N2OCl

IUPAC Name:

[2,2,6,6-tetramethyl-4-(trimethylazaniumyl)piperidin-1-yl]oxidanyl chloride

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal test substance concentrations of 100, 31.3, 9.77, 3.05, 0.954 mg/L and control
- Sampling method: 1 mL analytical samples were taken at 0 h and 72 h.
- Sample storage conditions before analysis: stored deep frozen

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 1000 mg/L was prepared by weighing the test substance on a weighing scoop and transfering to a flask. Test medium was added and the solution homogenised by shaking. Lower test concentrations were prepared by dillution of the stock solution.
- Test medium: Test concentrations were prepared with AAP-medium.
- Controls: Test medium without test substance, 6 replicates
- Evidence of undissolved material: The stock solution was clear and yellowish.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: P. subcapitata Hindak, SAG-61.81
- Source: commercial supplier
- Age of inoculum (at test initiation):
- Method of cultivation: Cultivation in sterile cultures in the laboratory with periodical replacement of old medium. Stock cultures are ordered regularly from commercial supplier.

ACCLIMATION
- Acclimation period: 3 to 4 days before start of the test, test medium was inoculated with the test organism for a pre-culture.
- Culturing media and conditions: same

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.5 – 23.3 °C

pH:

The pH-value of the control ranged from 7.54 to 8.38 during the test period

Nominal and measured concentrations:

nominal test substance concentrations of 100, 31.3, 9.77, 3.05, 0.954 mg/L (spacing factor: 3.2) and control. The measured initial concentration was in the range of 94-95%, the concentration in aged medium was in the range of 99% to 101% of the nominal concentration. Therefore, the effect values are based on the nominal concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks
- Type: closed with aluminium caps
- Material, size, headspace, fill volume: 100 mL flask volume, 50 mL fil/test volume
- Aeration: continuous agitation
- Renewal rate of test solution: static test
- Initial cells density: 0.5*10^4 cells/mL
- Control end cells density: 4.354 *10^5 cells/mL
- No. of organisms per vessel: 2.5*10^5 cells/test vessel
- No. of vessels per concentration (replicates): 3 replicates per concentration
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP -medium, according to guideline, Annex 3

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no. The pH of the test medium was adjusted to 7.5 before the test.
- Photoperiod: Continuously
- Light intensity and quality: 88.1 µEm-2s-1 (mean)


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Fluorescence measurements in a microplate reader at 670 nm, in duplicate, at 0 h, 24, 48, and 72 h
- Other: Morphological appearance was examined microscopically at the test end.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study
- Test concentrations: 100, 10, 1.0, and 0.1 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Substance specific analytic show good recovery. Therefore, the results were based on nominal concentrations.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Substance specific analytic show good recovery. Therefore, the results were based on nominal concentrations.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Substance specific analytic show good recovery. Therefore, the results were based on nominal concentrations.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Substance specific analytic show good recovery. Therefore, the results were based on nominal concentrations.

Basis for effect:

cell number

Remarks:

yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Substance specific analytic show good recovery. Therefore, the results were based on nominal concentrations.

Basis for effect:

cell number

Remarks:

yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Substance specific analytic show good recovery. Therefore, the results were based on nominal concentrations.

Basis for effect:

cell number

Remarks:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The cells were considered normal for the control and for the test substance concentrations.

The growth conditions (pH and temperature) during the test were within the range specified in the guideline.

After 72 h at termination of the test no inhibition of growth rate or yield was observed for test item concentrations up to and including 31.3 mg/L. At the highest test item concentration of 100 mg/L the inhibition of growth rate peaked at 10.8 % and the inhibition of yield peaked at 36.3 %

The measured initial concentrations of the test substance ranged from 94 % to 95 % of nominal. In the aged samples the measured concentrations were between 99 % and 101 % of nominal. Therefore toxicological endpoints were evaluated using the nominal concentrations of the test item.

Results with reference substance (positive control):

ErC50= 1.6 mg/L (nominal)

Reported statistics and error estimates:

Due to an inhibition of all parameters (growth rate, yield) below 50 % the database was weak for probit analysis, hence the EC10- value for growth rate and yield as well as the EC20-value for yield was not determined. The EC20-value for growth rate and EC50-value for growth rate and yield was considered to be > 100 mg/L (nominal).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

After 72 h no inhibition of growth rate or yield was observed for test item concentrations up to and including 31.3 mg/L. Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 100 mg/L (nominal). The overall LOEC was therefore determined to be 100 mg/L (nominal), the corresponding NOEC was set at 31.3 mg/L (nominal).
Due to an inhibition of all parameters (growth rate, yield) below 50 % the database was weak for probit analysis, hence the EC10- value for growth rate and yield as well as the EC20-value for yield was not determined. The EC20-value for growth rate and EC50-value for growth rate and yield was considered to be > 100 mg/L (nominal).