Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

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:

14 June 1995 to 22 September 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed to a guideline and used GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

92/69/EEC, C1

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1400 (Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

EC Number:

421-660-1

EC Name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

Molecular formula:

Hill formula: C50 H96 N2 O10 CAS formula: C42 H74 O8. 2(C4 H11 N O)

IUPAC Name:

bis((2-hydroxyethyl)dimethylazanium) (4E)-3-{[2-({3-carboxylato-3-[(2E)-hexadec-2-en-2-yl]propanoyl}oxy)ethoxy]carbonyl}-4-methyloctadec-4-enoate

Constituent 2

Reference substance name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

IUPAC Name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0, 24 and 96 hours.
- Sampling method: Water samples were taken from the solvent control and all surviving test groups (replicates pooled) for quantitative analysis.
- Sample storage conditions before analysis: NDA

Test solutions

Vehicle:

yes

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: Dimethylformamide (50 microlites/litre)

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test material were each separately dissolved in dimethylformamide and the volume adjusted to 25 mL to give 0.50, 0.90, 1.60, 2.80 and 5 g of test material/ 25 mL solvent stock solutions. They were freshly prepared each day prior to dosing.
- Eluate: N/A
- Differential loading: NDA
- Controls: A normal control (i.e. without the test material) and a solvent control were used and were maintained under identical test conditions to the test samples.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): NDA
- Evidence of undissolved material (e.g. precipitate, surface film, etc): NDA

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Parkwood Trout Farm, Wigmore, Kent, UK
- Age at study initiation (mean and range, SD): Juvenile
- Length: Mean standard length of 4.2 cm (s.d. = 0.2) at the end of the definitive study
- Weight: Mean weight of 0.59 g (s.d. = 0.14) at the end of the definitive study
- Method of breeding: NDA
- Feeding during test
- Food type: Commercial trout pellets
- Amount: NDA
- Frequency: Feeding was discontinued 48 hours prior to the start of the definitive study.


ACCLIMATION
- Acclimation period: 11 September 1995 to 18 Spetember 1995
- Acclimation conditions (same as test or not): Test conditions
- Type and amount of food: Commercial trout pellets. Amount not specified.
- Feeding frequency: NDA
- Health during acclimation (any mortality observed): Less than 1 % mortality


QUARANTINE (wild caught)
- Duration: N/A
- Health/mortality: N/A

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

N/A

Test conditions

Hardness:

109 mg CaCO3/L

Test temperature:

Water temperature: 14 °C

pH:

7.4

Dissolved oxygen:

Dissolved oxygen: greater than or equal to 9.8 mg O2/L

Salinity:

NDA

Nominal and measured concentrations:

Nominal test concentrations: 1.0, 1.8, 3.2, 5.6 and 10 mg/L
Mean measured concentrations: 1.047, 1.430, 3.014, 3.733, 5.868 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: 20 L glass exposure vessels were used
- Aeration: No auxiliary aeration was used; only the diluent supply was aerated.
- Type of flow-through: Peristaltic
- Renewal rate of test solution (frequency/flow rate): The diluent supply was pumped at a rate of 120 mL/minute with solvent stock solutions dosed at a rate of 0.36 mL/h to give the test series.
- No. of organisms per vessel: 10 fish
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: NDA


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated laboratory tap water
- Total organic carbon: 2.0 mg C/L
- Particulate matter: 0.80 mg/L
- Metals: Various
- Pesticides: <0.08 µg/L
- Chlorine: 0.22 mg/L total chlorine, 0.17 mg/L free chlorine
- Alkalinity: pH 7.4
- Ca/mg ratio: 43 mg Ca/L, 7 mg Mg/L
- Conductivity: 382 M1CSM
- Culture medium different from test medium: The test water used for the studies was the same as that used to maintain the stock fish.
- Intervals of water quality measurement: Water temperature, pH and dissolved oxygen concentrations were measured daily.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: NDA


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


TEST CONCENTRATIONS
- Spacing factor for test concentrations: NDA
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: Nominal concentrations 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed no mortalities at the test concentrations of 1.0 mg/L. However, mortalities were observed at 10 and 100 mg/L. Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were selected for the definitive study.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 1.9-2.3 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 1.5-1.9 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 1.0-1.4 mg/L (resulting in 0 and 100 % mortalities, respectively)

Effect concentrations 4

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 1.0-1.4 mg/L (resulting in 0 and 100 % mortalities, respectively)

Effect concentrations 5

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 6

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: Zero mortalities and the absence of any behavioural responses to exposure

Details on results:

- Behavioural abnormalities: Behavioural resposnes to exposure (other than death) were observed at test concentrations of > 1.0 mg/L. These responses were swimming at the surface, swimming at the bottom of test vessels, loss of equilibrium and the presence of moribund fish.
- Observations on body length and weight: NDA
- Other biological observations: None
- Mortality of control: 0 %
- Other adverse effects control: None
- Abnormal responses: NDA
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The LC50 values and associated confidence limits were calculated by the moving average method of Thompson (1947).

Any other information on results incl. tables

Sublethal observations / clinical signs:

Temperature was maintained at 14 ± 1 °C throughout the study, while there were no treatment related differences for oxygen concentration or pH.

Analysis of the test preparations showed the measured concentrations to range between 52 -116 % of nominal. The low and variable results obtained are considered to be due to deficiencies in the analytical method and not due to losses during dosing.

Table 1 Cumulative mortality data in the definitive study

Nominal concentration (mg/L)	Cumulative mortality (initial population = 10)						% mortality
	3 h	6 h	24 h	48 h	72 h	96 h	96 h
Control R1 R2	0 0	0 0	0 0	0 0	0 0	0 0	0 0
Solvent control R1 R2	0 0	0 0	0 0	0 0	0 0	0 0	0 0
1.0 R1 R2	0 0	0 0	0 0	0 0	0 0	0 0	0 0
1.8 R1 R2	0 0	0 0	0 0	5 7	10 10	10 10	100 100
3.2 R1 R2	0 0	0 0	9 6	10 10	10 10	10 10	100 100
5.6 R1 R2	0 0	0 0	10 10	10 10	10 10	10 10	100 100
10 R1 R2	2 5	9 10	10 10	10 10	10 10	10 10	100 100

R1 and R2 = replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 96 h LD50 value for OS 114451A was 1.2 mg/L (95 % confidence limits 1.0-1.4 mg/L) based on mean measured test concentrations. The NOEC was 1.0 mg/L.

Executive summary:

In a 96-h acute toxicity study, Rainbow Trout (Oncorhyncus mykiss)were exposed to OS 114451A at nominal concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg a.i./L in dimethylformamide under flow through conditions.  The 96-h LC₅₀was 1.3 mg a.i./L (nominal) and 1.2 mg a.i./L (measured concentration).  The NOEC value, based on sublethal effects, was 1.0 mg a.i./L.  Sublethal effects of swimming at the surface, swimming at the bottom of the test vessels, loss of equilibrium and the presence of moribund fish were observed in the groups exposed to concentrations of greater than 1 mg/L of OS 114451A. Based on the results of this study, OS 114451A would be classified as toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment, in accordance with Annex III of Directive 67/548/EEC.