4,8-Dimethyl-2,5,7,10-tetraoxaundecane

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 27 May 2020 to 03 July 2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lambiotte & Cie - 2003271010R
- Purity: 99.90%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L
- Sampling method: Samples were taken from the control and the 100 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis. A set of duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: immediate quantitative analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A nominal amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give a 100 mg/L stock. An aliquot (2 liters) of the stock solution and control were separately inoculated with algal suspension (7.8 mL) to give an initial nominal cell density of 5.00 x 103 cells/mL.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 103 cells/mL.
- Method of cultivation: The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 105 to 106 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ±1 ºC throughout the test

pH:

8.2-8.3

Nominal and measured concentrations:

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 86 to 80 mg/L (86% to 79% of nominal) respectively. As there was no significant change in the measured concentrations at 72 hours, the results are based on the 0-hour measured test concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels (250 mL glass conical flasks) in order to minimize possible losses due to volatilization.
- Type (delete if not applicable): closed
- Aeration: no
- Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate (250 mg/L) was added to the culture medium to provide a source of carbon dioxide for algal growth. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.
- Initial cells density: initial nominal cell density of 5.00 x 103 cells/mL
- No. of vessels per concentration (replicates):6
- No. of vessels per control (replicates):6

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used:
NaNO3 25.5 mg/L, MgCl2.6H2O 12.16 mg/L, CaCl2.2H2O 4.41 mg/L, MgSO4.7H2O 14.6 mg/L, K2HPO4 1.044 mg/L, NaHCO3 15.0 mg/L, H3BO3 0.186 mg/L, MnCl2.4H2O 0.415 mg/L, ZnCl2 0.00327 mg/L, FeCl3.6H2O 0.160 mg/L, CoCl2.6H2O 0.00143 mg/L, Na2MoO4.2H2O 0.00726 mg/L, CuCl2.2H2O 0.000012 mg/L, Na2EDTA.2H2O 0.30 mg/L.
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

EFFECT PARAMETERS MEASURED:
Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Range finding study: series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.
- Test concentrations: main study: 100 mg/L (nominal), 86 mg/L (measured)
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Growth data: the growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were not affected by the presence of the test item at a test concentration of 86 mg/L (based on the 0 hour measured test concentration) over the 72 hour exposure period. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Inhibition of growth rate:
ErC10 (0 to 72 hour) : >86 mg/L
ErC20 (0 to 72 hour) : >86 mg/L
ErC50 (0 to 72 hour) : >86 mg/L
There were no statistically significant differences (P≥0.05), between the control and 86 mg/L test group and therefore the NOEC based on growth rate was 86 mg/L.

Inhibition of yield:
EyC10 (0 to 72 hour) : >86 mg/L
EyC20 (0 to 72 hour) : >86 mg/L
EyC50 (0 to 72 hour) : >86 mg/L
There were no statistically significant differences (P≥0.05), between the control and test group and therefore the NOEC based on yield was 86 mg/L.

Results with reference substance (positive control):

Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.2 mg/L; 95% confidence limits 1.1 to 1.3 mg/L
EyC50 (0 to 72 hour) : 0.51 mg/L; 95% confidence limits 0.45 to 0.58 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L

Reported statistics and error estimates:

A Student’s t-test incorporating Shapiro-Wilk’s Test on Normal Distribution and by Levene’s Test on Variance Homogeneity was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/L test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using ToxRatPro software package (TOXRAT).
No statistical difference for all data collected

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Raphidocelis subcapitata to the test item gave EC50 values (for growth rate and yield) based on the 0 hour measured test concentration of greater than 86 mg/L. The No Observed Effect Concentration (NOEC) was 86 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Executive summary:

Introduction
A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) No 761/2009.
Methods
Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.
Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Results
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 86 to 80 mg/L (86% to 79% of nominal) respectively. As there was no significant change in the measured concentrations at 72 hours, the results are based on the 0 hour measured test concentrations.
Exposure of Raphidocelis subcapitata to the test item gave EC50 values (for growth rate and yield) based on the 0 hour measured test concentration of greater than 86 mg/L. The No Observed Effect Concentration (NOEC) was 86 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Description of key information

Exposure of Raphidocelis subcapitata to the test item gave EC50 values (for growth rate and yield) based on the 0 hour measured test concentration of greater than 86 mg/L. The No Observed Effect Concentration (NOEC) was 86 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

86 mg/L