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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 August 2007 to 5 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:

2007

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection 30 August 2005, Date of signature 21 November 2005

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

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: The test samples were tested for stability without prior mixing (sonication) of the test sample bottles to assess for losses due to adsorption and/or insolubility.
The bottom level light and dark and middle level dark samples were just above the normal acceptance limit of 120%. However as most of the other values were just below 120% the difference is not considered significant and the test samples have been considered to be stable in the test medium.
The 0.10 mg/l unsonicated stability vessel showed evidence of insolubility or adherence to glass.
- 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:

Water samples were taken from the control and all surviving test groups at 0 (fresh media), 24 and 96 (old media) hours for quantitative analysis.
Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (fresh and old media) were taken and stored at approximately -20°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:
For the purpose of the definitive test the test material was dissolved directly in dechlorinated tap water.
An amount of test material (100 mg) was dissolved in dechlorinated tap water with the aid of ultrasonication, when appropriate, for approximately 60 minutes and the volume adjusted to 1 litre to give a 100 mg/l stock solution. Aliquots (2.0, 3.6, 6.4, 11.2 and 20 ml) of the 100 mg/l stock solution were each separately dispersed in a final volume of 20 litres of dechlorinated tap water and stirred with a flat bladed mixer for approximately 1 minute to give the 0.010, 0.018, 0.032, 0.056 and 0.10 mg/l test concentrations respectively.
The stock solution at 0, 24 and 72 hours was inverted several times to ensure adequate mixing and homogeneity.
- Controls:
The control group was maintained under identical conditions but not exposed to the test material.
- Loading rate:
Based on the mean weight value this gave a loading rate of 0.46 g bodyweight/litre (static volume).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc):
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout.
- Strain: Not reported.
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK.
- Age at study initiation (mean and range, SD): Not reported.
- Length at study initiation (length definition, mean, range and SD): Not reported (a mean standard length of 4.6 cm (sd = 0.2) at the end of the definitive test).
- Weight at study initiation (mean and range, SD): Not reported (a mean weight of 1.30 g (sd = 0.27) at the end of the definitive test).
- Method of breeding: Not applicable.
- Feeding during test: No feeding during test.

ACCLIMATION
- Acclimation period: From 19 September 2007 to 1 October 2007.
- Acclimation conditions: Same as test.
- Type and amount of food: The stock fish were fed commercial trout pellets.
- Feeding frequency: Not reported (feeding was discontinued approximately 24 hours prior to the start of the definitive test).
- Health during acclimation (any mortality observed): There was less than 1% mortality in the 7 days prior to the start of the test.

ACCLIMATION
Not applicable.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

A total hardness of approximately 140 mg/l as CaCO3.

Test temperature:

Approximately 14°C.


Temperature was maintained at approximately 14deg C throughout the test

pH:

7.7 - 8.2.

Dissolved oxygen:

9.1 - 10.3 mg O2/l.
The oxygen concentration in the 0.10 mg/l test concentration at 24 hours was observed to be greater than the air saturation value (ASV) thereby resulting in a value of 101%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent.

Salinity:

Not reported.

Nominal and measured concentrations:

Initial range-finding test: Nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l.
Second range-finding test: Nominal test concentrations of 0.0010 and 0.010 mg/l.
Definitive test: 0.010, 0.018, 0.032, 0.056 and 0.10 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed.
- Material, size, headspace, fill volume: In the definitive test 20 litre glass exposure vessels were used for each test concentration.
- Aeration: The test vessels were aerated via narrow bore glass tubes.

- Type of flow-through: Not applicable
- Renewal rate of test solution (frequency/flow rate): A daily renewal.
- No. of organisms per vessel: 7 fish at the start of the definitive test.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: Not recorded.

WATER PARAMETERS
- Source/preparation of dilution water:
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
- Total organic carbon: (Average) 1.429 mg/l.
- Particulate matter: Not reported.
- Metals: (Average)
Al <10.596 µg/l
Pb <1.113 µg/l
Fe <28.038 µg/l
Cd <0.213 µg/l
Cr <0.551 µg/l
Cu <0.021 mg/l
Ni <2.325 µg/l
Hg <0.013 µg/l
Mn <1.723 µg/l

- Pesticides: (Average) 0.019 µg/l
- Chlorine: (Average) 0.274 mg/l
- Alkalinity: pH 7.655
- Ca/mg ratio: Not reported.
- Conductivity: (Average) 403.577 µS/cm at 20°C
- Water different from test medium: No
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Spacing of 1.7 - 1.8 with concentrations of 0.010, 0.018, 0.032, 0.056 and 0.10 mg/l.
- Justification for using less concentrations than requested by guideline: Not applicable.

- Range finding study
In the initial range-finding test fish were exposed to a series of nominal test concentrations of 0.10, 1.0, 10, and 100 mg/l. The test material was dissolved directly in water. Due to the observation of 100% mortality in all the test concentrations employed the test was terminated after 48 hours exposure. Therefore a second range-finding test was conducted.
In the second range-finding test fish were exposed to a series of nominal test concentrations of 0.0010 and 0.010 mg/l. The test material was dissolved directly in water. In the range-finding tests 3 fish were added to each 20 litre test and control vessel and maintained at approximately 14°C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions.
The results (see tables 3 and 4) showed no mortalities at the test concentrations of 0.0010 and 0.010 mg/l in the second range-finding test. However, mortalities were observed at 0.10, 1.0, 10 and 100 mg/l In the initial range-finding test.
After approximately 24 hours exposure a single fish was observed to be moribund at 0.10 mg/l. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) this fish was killed and classed as a mortality for the following observational time point. Based on this information test concentrations of 0.010, 0.018, 0.032, 0.056 and 0.10 mg/l were selected for the definitive test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.051 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

other:

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits of 0.043 – 0.061 mg/l

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.075 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits of 0.056 – 0.10 mg/l.

Effect concentrations 4

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Mortality: Cumulative mortality data from the exposure of rainbow trout to the test material during the definitive test are given in Table 5.
- Behavioural abnormalities: Not examined.
- Observations on body length and weight: Not reported.
- Other biological observations: Not reported.
- Mortality of control: 0%
- Other adverse effects control: Not reported.
- Abnormal responses: Not examined.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Sub-lethal effects of exposure were observed at the test concentration of 0.056 mg/l (Table 6). This response was loss of equilibrium.
- Effect concentrations exceeding solubility of substance in test medium:
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.
- Verification of test concentrations: Initial samples were analysed at 0 (fresh media) 24 and 96 hours (old media). The results of the samples at 0 hours showed the majority of measured test concentrations to range from 98% to 113% of nominal with the exception of the 0.032 mg/l concentration at 0-hours which showed a measured concentration of 1820% of nominal. This very high measured concentration for the 0.032 mg/l sample at 0-hours was considered to be erroneous therefore a frozen duplicate sample was analysed. This showed a measured concentration of 61% of nominal confirming the original analysis to be erroneous. The results of the 24 and 96 hour samples showed measured test concentrations to range from 46% to 67% of nominal.
Due to the low results obtained for the 24 and 96-hour old media, frozen samples of the test concentrations at 24 (fresh media), 48 (fresh and old media) and 72 hours (old and fresh media) were analysed. The results of these analyses were also shown to be variable. Careful examination of the data did not reveal a cause for the variability. However this was considered to be possibly due to adsorption/desorption to and from the glassware used in the test given that the test material was a quaternary ammonium compound. Also the results of the stability analysis performed for the Acute Toxicity to Daphnia magna test (Safepharm laboratories Project Number 0140/1413) showed evidence of instability of the test material.
Overall inspection of the data showed a general trend for a decline in measured concentrations. Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations of test media to give a "worst case" analysis of the data.
The time weighted mean measured concentrations were calculated to be 0.0087, 0.014, 0.020, 0.043 and 0.061 mg/l.
Analysis of the mortality data by the geometric mean method at 48, 72 and 96 hours based on the time weighted mean test concentrations gave the results shown in Table 7.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Not reported.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 3 Cumulative Mortality Data in the Range-finding Tests

Nominal Concentration (mg/l)	Cumulative Mortality (Initial Population = 3)
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control*	0	0	0	0	0	0
0.0010	0	0	0	0	0	0
0.010	0	0	0	0	0	0
0.10	0	0	0**	3	-	-
1.0	3	3	3	3	-	-
10	3	3	3	3	-	-
100	3	3	3	3	-	-

*The results observed for the controls during both range-finding tests were observed to be identical and therefore were not separately tabulated.

** After approximately 24 hours exposure a single fish was observed to be moribund. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) this fish was killed and classed as a mortality for the 48-Hour time point.

- initial range-finding test terminated after 48 hours due to 100% mortality in all test concentrations employed.

Table 4  Sub-lethal Effects of Exposure in the Range-finding Tests

Nominal Concentration (mg/l)	Sub-lethal Effects	Time (Hours)
		3	6	24	48	72	96
Control*	No abnormalities detected
0.0010	No abnormalities detected
0.010	No abnormalities detected
0.10	Moribund			1/3*[1]	A/D
1.0	No abnormalities detected	A/D
10	No abnormalities detected	A/D
100	No abnormalities detected	A/D

*The results observed for the controls during both range-finding tests were observed to be identical and therefore were not separately tabulated.

[1]* After approximately 24 hours exposure a single fish was observed to be moribund. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) this fish was killed and classed as a mortality for the 48-Hour time point.

A/D= All fish dead

 

Table 5 Cumulative Mortality Data in the Definitive Test

Nominal Concentration (mg/l)	Cumulative Mortality (Initial Population =7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
0.010	0	0	0	0	0	0	0
0.018	0	0	0	0	0	0	0
0.032	0	0	0	0	0	0	0
0.056	0	0	0	0	0	0	0
0.10	0	0	0	7	7	7	100

Table 6 Sub-lethal Effects of Exposure in the Definitive Test

Nominal Concentration (mg/l)	Sub-lethal Effects	Time (Hours)
		3	6	24	48	72	96
Control	No abnormalities detected
0.010	No abnormalities detected
0.018	No abnormalities detected
0.032	No abnormalities detected
0.056	Loss of equilibrium						1/7
0.10	No abnormalities detected				A/D

A/D= All fish dead

Table 7 Time weighted mean measured concentrations

Time (h)	LC50(mg/l)	95% Confidence limits (mg/l)
3	>0.061	-
6	>0.061	-
24	>0.061	-
48	0.051	0.043 - 0.061*
72	0.051	0.043 - 0.061
96	0.051	0.043 - 0.061

*Concentrations resulting in 0% and 100% mortalities respectively

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 value of 0.075 mg/l with 95% confidence limits of 0.056 – 0.10 mg/l. The No Observed Effect Concentration was 0.032 mg/l.
Based on the time-weighted mean measured test concentrations of the test media the acute toxicity of the test material to rainbow trout gave a 96-Hour LC50 value of 0.051 mg/l with 95% confidence limits of 0.043 – 0.061 mg/l. The No Observed Effect Concentration was 0.020 mg/l.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.

Following preliminary range-finding tests, fish were exposed, in groups of seven, to an aqueous solution of the test material over a range of concentrations of 0.010, 0.018, 0.032, 0.056, and 0.10 mg/l for a period of 96 hours at a temperature of approximately 14°C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results.

The 96-Hour LC₅₀ based on nominal test concentrations was 0.075 mg/l with 95% confidence limits of 0.056 – 0.10 mg/l. The No Observed Effect Concentration was 0.032 mg/l.

Initial samples were analysed at 0 (fresh media) 24 and 96 hours (old media). The results of the samples at 0 hours showed the majority of measured test concentrations to range from 98% to 113% of nominal with the exception of the 0.032 mg/l concentration at 0-hours which showed a measured concentration of 1820% of nominal. This very high measured concentration for the 0.032 mg/l sample at 0-hours was considered to be erroneous therefore a frozen duplicate sample was analysed. This showed a measured concentration of 61% of nominal confirming the original analysis to be erroneous. The results of the 24 and 96 hour samples showed measured test concentrations to range from 46% to 67% of nominal.

Due to the low results obtained for the 24 and 96-hour old media, frozen samples of the test concentrations at 24 (fresh media), 48 (fresh and old media) and 72 hours (old and fresh media) were analysed. The results of these analyses were also shown to be variable. Careful examination of the data did not reveal a cause for the variability. However this was considered to be possibly due to adsorption/desorption to and from the glassware used in the test given that the test material was a quaternary ammonium compound. Also the results of the stability analysis performed for the Acute Toxicity to Daphnia magna test (Safepharm laboratories Project Number 0140/1413 - see 6.1.3 Short term toxicity in aquatic invertebrates) showed evidence of instability of the test material.

Overall inspection of the data showed a general trend for a decline in measured concentrations. Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations of test media to give a "worst case" analysis of the data.

The time weighted mean measured concentrations were calculated to be 0.0087, 0.014, 0.020, 0.043 and 0.061 mg/l.

The 96-Hour LC₅₀ based on the time-weighted mean measured test concentrations of the test media was 0.051 mg/l with 95% confidence limits of 0.043 – 0.061 mg/l. The No Observed Effect Concentration was 0.020 mg/l.