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

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:

20 June 1995 to 16 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD 202

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1300 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

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 and 48 hours.
- Sampling method: Water samples were taken from the solvent control and the 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 microlitres/litres)

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test material were each separately dissolved in dimethylformamide with the aid of ultrasonic disruption 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: The control and solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 50 µL/L of dimethylformamide.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution): NDA
- Evidence of undissolved material (e.g. precipitate, surface film, etc): NDA

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: 1st instar Daphnia magna
- Source: Laboratory culture from a strain supplied by the Institut National de Recherche Chimique Appliquee (I.R.CH.A), France
- Age at study initiation (mean and range, SD): Adult
- Weight at study initiation (mean and range, SD): N/A
- Length at study initiation (length definition, mean, range and SD): N/A
- Valve height at study initiation, for shell deposition study (mean and range, SD): N/A
- Peripheral shell growth removed prior to test initiation: N/A
- Method of breeding: Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated 24 hours prior to the initiation of the test, the young daphnids produced overnight were then removed for testing.
- Feeding during test
- Food type: The daphnids received no food during the exposure period.
- Amount: N/A
- Frequency: N/A


ACCLIMATION
- Acclimation period: NDA. The dosing apparatus was set up approximately 18.5 h prior to the start of the study to allow equilibration of test concentrations.
- Acclimation conditions (same as test or not): Same as test conditions
- Type and amount of food: Mixed algae (predominantly Chlorella spp.)
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): NDA


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:

48 h

Post exposure observation period:

N/A

Test conditions

Hardness:

104 mg CaCO3/L

Test temperature:

Water temperature: 21 °C

pH:

0 h: 7.6 (mean)
48 h: 7.5 (mean)

Dissolved oxygen:

0 h: 8.2 mg O2/L
48 h: 8.3 mg O2/L

Salinity:

NDA

Nominal and measured concentrations:

Nominal concentrations: 1.0, 1.8, 3.2, 5.6 and 10 mg/L
Mean measured test concentration: 0.533, 0.849, 0.792, 2.875 and 4.301 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: Glass exposure vessels containing a nominal volume of 250 mL of test solution were used. Daphnids were held in the test vessel by a fine stainless stell mesh in the base of the exposure vessel.
- Aeration: None
- Type of flow-through: Peristaltic
- Renewal rate of test solution (frequency/flow rate): The diluent supply was pumped at a rate of 30 mL/minute with solvent stock solutions dosed at a rate of 0.09 mL/h to give the test series.
- No. of organisms per vessel: 10
- 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 tap water
- Total organic carbon: 1.0 mg C/L
- Particulate matter: 1.8 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 animals.
- Intervals of water quality measurement: Water temperature was recorded daily and dissolved oxygen concentrations and pH were recorded at the start and termination of the study.


OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: NDA


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: NDA
- Justification for using less concentrations than requested by guideline: The use of only 5 test concentrations instead of the 9 that were agreed in the protocol was considered to be appropriate for this study given that during both the static and dynamic, continuous flow range-finding studies, the immobilisation data showed no immobilisation at a concentration of 1.0 mg/L after 48 h exposure and 100 % immobilisation at a concentration of 10 mg/L after 48 h. Thus it was considered justifiable to use only 5 test concentrations as the test range assigned to the study ensured that 24 and 48 h EC50 values and a 48 h NOEC were achieved and that this deviation was not considered to affect the integrity or validity of the study.
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed no immobilisation at the test concentrations of 0.10 and 1.0 mg/L. However, immobilisation was observed at 10 and 100 mg/L. Based on this information 5 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:

EC50

Effect conc.:

2.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL: 1.9-3.1 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL: 1.9-2.6 mg/L

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

10 mg/L

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.85 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: zero immobilisation

Details on results:

- Behavioural abnormalities: There were no adverse reactions to exposure.
- Observations on body length and weight: N/A
- Other biological observations: None
- Mortality of control: 0 % immobilisation
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: NDA
- Effect concentrations exceeding solubility of substance in test medium: NDA

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The EC50 values and associated confidence limits were calculated by the moving average method of Thompson (1947).

Any other information on results incl. tables

Temperature was maintained at 20 ± 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 32-53 % of nominal at 0 hours and 17-63 % of nominal at 48 h. 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 immobilisation data in the definitive study

Nominal concentration (mg/L)	Cumulative immobilised Daphnia (initial population = 10 per replicate)
	24 hours				48 hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
Solvent control	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0
1.8	0	0	0	0	0	0	0	0
3.2	0	0	0	0	3	1	4	20
5.6	2	4	6	30	5	6	11	55
10	9	8	17	85	10	10	20	100

R1 and R2 = replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48 h EC50 value for OS 114451A was 2.2 mg/L (95 % confidence limits 1.9-2.6 mg/L) based on mean measured test concentrations. The NOEC was 0.85 mg/L.

Executive summary:

The 48–hr-acute toxicity of OS 114451A toDaphnia Magnawas studied under flow through conditions.  Daphnids 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 for 48 hr.  Mortality/immobilization and sublethal effects were observed daily.  The 48– hour EC₅₀was 4.9 mg a.i./L (nominal) and 2.2 mg a.i./L (measured concentration).  The 48– hr NOEC based on mortality/immobilization was 1.8 mg a.i./L (nominal) and 0.85 mg a.i./L (measured concentration).  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.