1-Propanaminium, N,N,N-trimethyl-3-(octadecyloxy)-, chloride (1:1)

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 July 2007 to 19 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21 August 2007 Date of Signature: 15 October 2007

Test material

Test material information

Constituent 1

Reference substance name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

EC Number:

700-414-8

Cas Number:

23328-71-4

Molecular formula:

C24H52ClNO

IUPAC Name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

Constituent 2

Reference substance name:

3-Octadecyloxypropyl-N, N, N-trimethylammonium chloride

IUPAC Name:

3-Octadecyloxypropyl-N, N, N-trimethylammonium chloride

Details on test material:

- Name of test material (as cited in study report): 3-Octadecyloxypropyl-N,N,N-trimethylammonium chloride
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: white solid
- Physical state: solid
- Analytical purity: Not reported
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: Not reported
- Isomers composition: Not reported
- Purity test date: Not reported
- Lot/batch No.: Exp. I-070518
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Instability of the test material in water. Preliminary stability analysis indicated that the test material may be unstable over 48 hours.
- Storage condition of test material: room temperature in the dark.
- Other: Not reported.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not reported.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.0018, 0.0032, 0.0056, 0.010, 0.018, 0.032, 0.056, 0.10 and 0.18 mg/l.
- Sampling method: Water samples were taken from the control (replicates R1 – R2 pooled) and each test group (replicates R1 – R2 pooled) at 0 hours (fresh media), 24 hours (fresh and old media) and 48 hours (old media) for quantitative analysis.
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20 deg C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
An amount of test material (100 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 45 minutes and the volume adjusted to 1 litre to give a 100 mg/l stock solution from which serial dilutions were prepared in reconstituted water to give further stock solutions of 10, 1.0 and 0.10 mg/l. Aliquots (18, 32 and 56 ml) of the 0.10 mg/l stock solution were each separately diluted in reconstituted water and the volume adjusted to 1 litre to give the test concentrations of 0.018, 0.032 and 0.056 mg/l. Similarly, aliquots (10, 18, 32, 56, 100 and 180 ml) of the 1.0 mg/l stock solution were each separately diluted in reconstituted water and the volume adjusted to 1 litre to give the test concentrations of 0.010, 0.018, 0.032, 0.056, 0.10 and 0.18 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Preliminary stability analysis indicated that the test material may be unstable over 48 hours. Therefore, in an attempt to minimise losses of test material the test media were freshly prepared at 0 and 24 hours prior to dosing using a semi-static test regime.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 hours (fresh media), 24 hours (fresh and old media) and 48 hours (old media)
- Differential loading: Not applicable.
- Controls: The control group was maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The control and all test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea.
- Strain: Not reported.
- Source: In-house laboratory cultures.
- Age at study initiation (mean and range, SD): 1st instar.
- Weight at study initiation (mean and range, SD): Not applicable.
- Length at study initiation (length definition, mean, range and SD): Not applicable.
- Valve height at study initiation, for shell deposition study (mean and range, SD): Not applicable.
- Peripheral shell growth removed prior to test initiation: Not reported.
- Method of breeding:
Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.
- Feeding during test: Not fed during test: Each culture was fed daily with a suspension of algae (Chlorella sp.). The diet is considered not to contain any contaminant that would affect the integrity or outcome of the study.

ACCLIMATION
Not applicable

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Tempertaure was recorded throughout the test, maintained at 21 deg C .
Some of the temperatures during the range-finding tests were observed to be slightly in excess of the range given in the protocol of 20 ± 1°C. This deviation was considered not to have affected the outcome or the validity of the test as the control daphnia during testing showed no sub-lethal effects or immobilisation over the test period.

pH:

7.8 - 8.0

Dissolved oxygen:

8.5 - 9.0 mg O2/l.
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

Salinity:

Not reported.

Nominal and measured concentrations:

Initial range-finding test: 0.10, 1.0, 10 and 100 mg/l (nominal).
Second range-finding test: 0.00010, 0.0010, 0.010 and 0.10 mg/l (nominal).
Initial experiment: 0.010, 0.018, 0.032, 0.056, 0.10, 0.18, 0.32, 0.56 and 1.0 mg/l (nominal).
Definitive test: 0.0018, 0.0032, 0.0056, 0.010, 0.018, 0.032, 0.056, 0.10 and 0.18 mg/l (nominal).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: Closed. The test vessels were then covered to reduce evaporation
- Material, size, headspace, fill volume: In the definitive test 250 ml glass jars containing approximately 250 ml of test preparation were used
- Aeration: Not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate):For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.
- No. of organisms per vessel: 10 daphnids in each test and control vessel at random.
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: Not reported.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water.

Stock Solutions
a) CaCl2.2H2O : 11.76 g/l
b) MgSO4.7H2O: 4.93 g/l
c) NaHCO3: 2.59 g/l
d) KCl: 0.23 g/l

Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

- Total organic carbon: Not reported.
- Particulate matter: Not reported.
- Metals: Not reported.
- Pesticides: Not reported.
- Chlorine: Not reported.
- Alkalinity: pH of 7.8 ± 0.2.
- Ca/mg ratio: Not reported.
- Conductivity: <5 µS cm-1.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Adjustment of pH: The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
- Photoperiod: Photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Sapcing facor of 1.7 - 1.8 (definitive study).
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations: Initial range-finding test: 0.10, 1.0, 10 and 100 mg/l (nominal), Second range-finding test: 0.00010, 0.0010, 0.010 and 0.10 mg/l (nominal).
- Results used to determine the conditions for the definitive study:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding tests are given in Table 2.
In the initial range-finding test, immobilisation was observed at all test concentrations. Therefore a second range-finding study was conducted in order to obtain a No Observed Effect Concentration (NOEC).
In the second range-finding test, no immobilisation was observed at the test concentrations of 0.00010, 0.0010 and 0.010 mg/l. However, immobilisation was observed at 0.10 mg/l.
Based on this information test concentrations of 0.010, 0.018, 0.032, 0.056, 0.10, 0.18, 0.32, 0.56 and 1.0 mg/l were selected for the initial experiment.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.01 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits of 0.0080 – 0.011 mg/l

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.003 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.017 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits of 0.015 – 0.020 mg/l

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.006 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Not reported.
- Observations on body length and weight: Not reported.
- Other biological observations: Not reported.
- Mortality of control: Not examined.
- Other adverse effects control: Not reported.
- Abnormal responses: Not reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
Verification of test concentrations
Analysis of the test preparations (see Appendix 2) at 0 hours showed measured test concentrations to range from 60% to 100%. These low measured test concentrations were considered to be due to possible adsorption to the glassware used in the preparation of the test series, given that the test material was a quaternary ammonium compound. Further declines in the old test media were considered to be due to instability of the test material in water and were in line with the stability analysis performed (see Appendix 2). Similar results were obtained for the 24-hour fresh and 48-hour old test media. Given that all test concentrations were analysed throughout the study it was considered that the lower than nominal concentrations at 0 and 24 hour fresh media did not affect the results of the test as measured concentrations were determined for all concentrations and it was considered appropriate to base the results on the time weighted mean measured test concentrations.
The time weighted mean measured concentrations were calculated to be 0.0015, 0.0014, 0.0026, 0.0053, 0.010, 0.017, 0.033, 0.055 and 0.098 mg/l.
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the time weighted mean measured test concentrations gave the following results:
Time (h) EC50 (mg/l) 95% Confidence limits
(mg/l)
24 h EC50 0.026 mg/l 95% Confidence limits of 0.022 – 0.029
48 h EC50 0.0096 mg/l 95% Confidence limits of 0.0080 – 0.011
The No Observed Effect Concentrations after 24 and 48 hours exposure were 0.010 and 0.0026 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slopes and their standard errors of the response curves at 24 and 48 hours were 9.6 (SE = 2.0) and 6.3 (SE = 1.22) respectively.
The use of time weighted mean measured test concentrations was considered not to significantly affect the results.
- Effect concentrations exceeding solubility of substance in test medium: The control and all test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Mortality: Not reported.
- EC50/LC50:
Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the geometric mean method at 48 hours based on the nominal test concentrations gave the following results:
Time (h) EC50 (mg/l)
(mg/l)
3 h EC50 > 3.2 mg/l 95% Confidence limits -
24 h EC50 1.2 mg/l 95% Confidence limit of 1.1 - 1.4
48h EC50 0.75 mg/l 95% Confidence limit of 0.56 - 1.0 (Concentrations resulting in 0% and 100% immobilisation respectively)
- Other:
The No Observed Effect Concentration after 24 and 48 hours was 0.56 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slope and standard error of the response curve at 24 hours was 6.9 (SE = 1.4). Due to the unsuitable nature of the data it was not possible to calculate the slope and standard error of the response curve at 48 hours.
The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.81 mg/l (sd = 0.21).

Reported statistics and error estimates:

Not reported.

Any other information on results incl. tables

Table 3  Cumulative Immobilisation Data in the Initial Experiment

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.010	0	0	0	0	5	7	12	60
0.018	0	1	1	5	9	7	16	80
0.032	4	5	9	45	10	9	19	95
0.056	6	7	13	65	10	10	20	100
0.10	7	8	15	75	10	10	20	100
0.18	10	10	20	100	10	10	20	100
0.32	10	10	20	100	10	10	20	100
0.56	10	10	20	100	10	10	20	100
1.0	10	10	20	100	10	10	20	100

R₁– R₂ = Replicates 1 and 2

Table 4 Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.0018	0	0	0	0	0	0	0	0
0.0032	0	0	0	0	0	0	0	0
0.0056	0	0	0	0	0	0	0	0
0.010	0	0	0	0	0	2	2	10
0.018	0	0	0	0	4	5	9	45
0.032	0	1	1	5	10	10	20	100
0.056	9	8	17	85	10	10	20	100
0.10	10	10	20	100	10	10	20	100
0.18	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 0.017 mg/l with 95% confidence limits of 0.015 – 0.020 mg/l. The No Observed Effect Concentration at 48 hours was 0.0056 mg/l.
Based on the time weighted mean measured test concentrations of the test media the 48-Hour EC50 was 0.0096 mg/l with 95% confidence limits of 0.0080 – 0.011 mg/l and the No Observed Effect Concentration was 0.0026 mg/l.

Executive summary:

Introduction. A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Directive 92/69/(which constitutes Annex V of Council Directive 67/548/).

Methods.

Following preliminary range-finding tests, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 0.0018, 0.0032, 0.0056, 0.010, 0.018, 0.032, 0.056, 0.10 and 0.18 mg/l for 48 hours at a temperature of 21°C under semi-static test conditions. In an effort to maintain near nominal test concentrations semi-static test conditions were used during the definitive test as preliminary stability analysis suggested that the test material was unstable over 24 hours. The number of immobilised Daphnia were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of 21°C to 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results.

The 48-Hour EC₅₀for the test material to Daphnia magna based on nominal test concentrations was 0.017 mg/l with 95% confidence limits of 0.015 – 0.020 mg/l. The No Observed Effect Concentration was 0.0056 mg/l.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 60% to 100%. These low measured test concentrations were considered to be due to possible absorption to the glassware used in the preparation of the test series, given that the test material was a quaternary ammonium compound. Further declines in the old test media were considered to be due to instability of the test material in water and were in line with the stability analysis performed. Similar results were obtained for the 24-hour fresh and 48-hour old test media. Given that all test concentrations were analysed throughout the study it was considered that the lower than nominal concentrations at 0 and 24 hour fresh media did not affect the results of the test as measured concentrations were determined for all concentrations and it was considered appropriate to base the results on the time weighted mean measured test concentrations.

The time weighted mean measured concentrations were calculated to be 0.0015, 0.0014, 0.0026, 0.0053, 0.010, 0.017, 0.033, 0.055 and 0.098 mg/l.

The 48-Hour EC₅₀for the test material to Daphnia magna based on time weighted mean measured test concentrations was 0.0096 mg/l with 95% confidence limits of 0.0080 – 0.011 mg/l. The No Observed Effect Concentration was 0.0026 mg/l.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.75 mg/l with 95% confidence limits of 0.56 – 1.0 mg/l. The No Observed Effect Concentration was 0.56 mg/l.