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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 June 1995 to 29 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:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)

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.
- Sampling method: Water samples were taken from the control and 100 mg/L test group (replicates pooled) at 0 and 96 hours for quantitative analysis.
- Sample storage conditions before analysis: NDA

Test solutions

Vehicle:

no

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: No auxillary solvent was used.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was prepared by a direct dispersion in culture medium. An amount of test material (100 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 200 mg/L stock solution. This stock solution was mixed with 500 mL of algal suspension to give the test concentration of 100 mg/L.
- Eluate: NDA
- Differential loading: NDA
- Controls: The control was maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc): NDA

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures were obtained from the Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Cumbria.
- Age of inoculum (at test initiation): The culture conditions gave an algal suspension in log growth phase growth charactersied by an absorbance of 0.534 (at 665 nm). This suspension was diluted to an absorbance of 0.025 prior to use.
- Method of cultivation: NDA


ACCLIMATION
- Acclimation period: NDA
- Culturing media and conditions (same as test or not): The culture medium used for the studies was the same as that used to maintain the stock culture.
- Any deformed or abnormal cells observed: NDA

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

N/A

Test conditions

Hardness:

NDA

Test temperature:

24 ± 1 °C

In the first 35 minutes of the study the temperature in the incubator dropped to 21.9-22.9 °C but this was caused by the incubator being opened to allow the test vessels to be placed in the incubator.

pH:

The pH of the culture medium after equilibration with air was approximately 7.5.

Mean pH of control at 0h: 8.0
Mean pH of control at 96 h: 9.9

Mean pH of test sample flasks at 0 h: 8.1
Mean pH of test sample flasks at 96 h: 10.1

Dissolved oxygen:

NDA

Salinity:

NDA

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Mean measured concentration at 0 h: 80 mg/L
Mean measured concentration at 96 h: 63 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Closed - the flasks were covered with aluminium foil
- Material, size, headspace, fill volume: Six flasks each containing 100 mL of solution were prepared for the treatment group
- Aeration: The flasks were constantly shaken at 100 rpm for 96 hours
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 10^4 cells per mL
- Control end cells density: Mean cell density of control at 96 h: 2.24x10^6 cells/mL
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 6 vessels
- No. of vessels per control (replicates): 3 vessels
- No. of vessels per vehicle control (replicates): N/A


GROWTH MEDIUM
- Standard medium used: Yes
- Detailed composition if non-standard medium was used: The composition of the culture medium was identical to that given in the OECD guideline 201 with the following exceptions:
Na2EDTA·2(H2O) was not present
FeCl3·6(H2O) was present at 0.08 mg/L instead of 0.064 mg/L



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: NDA
- Total organic carbon: NDA
- Particulate matter: NDA
- Metals: NDA
- Pesticides: NDA
- Chlorine: NDA
- Alkalinity: NDA
- Ca/mg ratio: NDA
- Conductivity: NDA
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of each control and test flask was determined at initiation of the study and after 96 h exposure.


OTHER TEST CONDITIONS
- Sterile test conditions: NDA
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: Intensity approximately 7000 lux
- Salinity (for marine algae): N/A


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined by direct counting with the aid of a haemocytometer.
- Chlorophyll measurement: None
- Other: None


TEST CONCENTRATIONS
- Spacing factor for test concentrations: NDA
- Justification for using less concentrations than requested by guideline: See below
- Range finding study
- Test concentrations: Nominal 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 effect on growth at all concentrations tested. Based on the result of the range finding study a limit test was conducted for the definitive study at a test concentration of 100 mg/L to confirm that at the maximum test concentration given in the test guidelines, no effect on algal growth was observed.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 63 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 63 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 63 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 63 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): The cell concentration of the control cultures increased by a facotr of 36 after 72 hours and by a factor of 182 after 96 h.
- Observation of abnormalities (for algal test): There were no abnormalities detected in any of the control or test cultures.
- Any stimulation of growth found in any treatment: Neither the growth (r) or the biomass (b) of Selenastrum capricornutum was affected by the presence of 100 mg/L of the test material over the 96 h exposure period.
- 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):

N/A

Reported statistics and error estimates:

NDA

Any other information on results incl. tables

Table 1 Inhibition of growth

Nominal concentration (mg/L)	Area under curve at 72 h	% inhibition	Area under curve at 96 h	% inhibition	Growth rate (0-24 h)	% inhibition
Control	1.17x10^7	-	4.36x10^7	-	0.078	-
100	1.39x10^7	[19]	4.62x10^7	[6]	0.085	[9]

[increase in growth as compared to control]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EC values based on the 96 h mean measured test concentration were greater than 63 mg/L. Correspondingly the NOEC was greater than or equal to 63 mg/L.

Executive summary:

In a 96 hour acute toxicity study, the cultures of the green alga Selenastratum capricornutum were exposed to OS 114451A at a nominal concentration of 100 mg a.i./L under static conditions in accordance with Method C.3 of Commission Directive 92/69/EEC.  The NOEC and EC₅₀values based on cell density were 100 and >100 mg a.i./L (nominal) and 63 and >63 mg a.i./L (measured concentration), respectively.  There were no compound related phytotoxic effects.