N,N-dimethyl-3-(octadecyloxy)propylamine

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:

From July 23, 2007 to October 4, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethyl-3-(octadecyloxy)propylamine

EC Number:

241-516-8

EC Name:

N,N-dimethyl-3-(octadecyloxy)propylamine

Cas Number:

17517-01-0

Molecular formula:

C23H49NO

IUPAC Name:

dimethyl[3-(octadecyloxy)propyl]amine

impurity 1

Reference substance name:

Unknown impurities

IUPAC Name:

Unknown impurities

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 – R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and sored at approximately -20ºC for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

For the purpose of the definitive test, the test material was dispersed directly in culture medium.

An amount of test material (100 mg) was dispersed in culture medium with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 500 ml to give a 200 mg/l stock dispersion. A series of dilutions was made from this stock dispersion to give further stock dispersions of 20, 2.0, 0.20, 0.10, 0.050, 0.025 and 0.0125 mg/l. An aliquot (250 ml) of each of the 0.0125, 0.025, 0.050, 0.10 and 0.20 mg/l stock dispersions was separately mixed with algal suspension (250 ml) to give the required test concentration of 0.00625, 0.0125, 0.025, 0.050, 0.10 mg/l.

The stock dispersions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Desmodesmus subspicatus strain CCAP 276/20. Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory under constant aeration and constant illumination at 21±1 ºC.

Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24±1ºC until the algal cell density was approximately 10^4 – 10^5 cells/ml.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24±1ºC

pH:

The pH values of the control cultures were observed to increase from pH 7.5 at 0 hours to pH 7.7 - 7.8 at 72 hours.

Nominal and measured concentrations:

Nominal test concentrations (mg/L): 0.00625, 0.0125, 0.025, 0.050, 0.10.
Geometric Mean Measured Test Concentrations (mg/L): 0.00081, 0.0011, 0.0016, 0.022, 0.035.

Details on test conditions:

As in the range-finding test 250 ml glass conical flask were used. Six flasks each containing 100 ml of test preparation were used for the control and three flasks each containing 100 ml were used for each treatment group.

The control group was maintained under identical conditions but not exposed to the test material.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.82 x 10^5 cells/ml. This suspension was diluted to a cell density of 8.18 x 10^3 cells/ml prior to use. At initiation of the test the culture contained a nominal cell density of 4 x 10^3 cells/ml.

The flasks were plugged with polyurethane foam bungs and incubated at 24±1ºC under continuous illumination provided by warm white lighting and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 26, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

It was possible to monitor algal cells using a Coulter® Multisizer Particle Counter as at the test concentrations employed, any dispersed test material present did not interfere with the algal cell counts.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.017 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

All test and control cultures were inspected microscopically at 72 hours. These were no abnormalities detected in any of the control or test cultures at 72 hours.

At the start of the test the control cultures were observed to be clear colourless solutions. The test cultures were all observed to be clear colourless media columns with very fine particles dispersed throughout. After the 72-Hour test period all control, 0.00625, 0.0125 and 0.025 mg/l test cultures were observed to be green dispersions. The 0.050 mg/l test cultures were observed to be pale green dispersions whilst the 0.10 mg/l test cultures were observed to be clear colourless solutions.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to be in the range of 87% to 94% of nominal. Analysis of the test preparations at 72 hours showed a decline in measured test concentrations with values in the range of less than the limit of quantitation (LOQ) of the analytical method employed which was assessed down to 0.0023 mg/l to 20% of nominal.

This decline was considered to be due to a combination of both the slightly light unstable nature of the test material as was seen in the preliminary stability analyses conducted and adsorption to the algal cells present. Whilst the recovery analyses conducted in the presence of algal cells indicated that no immediate adsorption occurred this does not preclude long-term adsorption over the test period.

It was therefore considered justifiable to base the results on the geometric mean measured test concentrations. In cases where the measured concentration was less than the LOQ of the analytical method, a value of half the LOQ was used to enable calculation of the geometric mean measured concentration.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference material. The test concentrations were 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

ErC50 (0-72h) was 0.49 mg/l and NOEC based on growth rate was 0.125 mg/l. The results from the positive control with potassium dichromate were within the normal range for this reference material.

Any other information on results incl. tables

See attached Table 3-5 and Figure 1 and 5 for details on results

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave an ErC50 (0-72h) of 0.026 mg/l; 95% confidence limits 0.024-0.027 mg/l and an ErC10 (0-72h) of 0.017 mg/l based on the geometric mean measured test concentrations. The LOEC was 0.022 mg/l, and the NOEC was 0.0016 mg/l based on the geometric mean measured test concentrations.

Executive summary:

The study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed was the OECD 201 referenced as Marthod C.3.

 

Following preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous dispersion of the test material at concentrations of 0.00625, 0.0125, 0.025, 0.050 and 0.10 mg/L (three replicates flasks per concentration) for 72 hours, under constant illumination and shaking a temperature of 24 ± 1ºC.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

 

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentration of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24±1ºC.

 

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an ErC50 (0-72h) of 0.026 mg/l; 95% confidence limits 0.024-0.027 mg/l and an ErC10 (0-72h) of 0.017 mg/l based on the geometric mean measured test concentrations. The LOEC was 0.022 mg/l, and the NOEC was 0.0016 mg/l based on the geometric mean measured test concentrations.