2-ethoxyethyl ethyl carbonic acid ester

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 June 2018 to 10 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Range Finding Test
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test material under test conditions. All 0-Hour samples were stored frozen prior to analysis. Only the concentration to be employed in the definitive test was provided for analysis.

- Definitive Test
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 quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

The test material was dissolved directly in culture medium.

- Range Finding Test
A nominal amount of test material (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (2.2 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

- Definitive Test
A nominal amount of test material (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution. This stock solution was inoculated with algal suspension (4.5 mL) to give the required test concentration of 100 mg/L.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland

CULTURE CONDITIONS
Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C.

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 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1°C

pH:

7.9 - 8.4

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): closed. Test vessels were plugged with polyurethane foam bungs to reduce evaporation.
- Fill volume: 100 mL
- Aeration: test vessels were constantly shaken at approximately 150 rpm for 72 hours
- Initial cells density: 5.00 x 10³ cells per mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

CULTURE MEDIUM
- Standard medium used: yes
- Detailed composition of culture medium: 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 deionised water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Culture medium different from test medium: no. The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Intervals of water quality measurement: The pH of the control and 100 mg/L test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination provided by warm white lighting (380 to 730 nm)
- Light intensity and quality: approximately 7000 lux

EFFECT PARAMETERS MEASURED
Samples were taken at 27, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10³ cells/mL) was taken as the starting cell density.
To determine the potential effect of the test material 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
- Range finding study:
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

DATA EVALUATION
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the following equation:

µ = (ln Nn - ln N1) / (tn - t1)
where
μ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test material vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
In addition the section by section specific growth rate (Days 0 to 1, 1 to 2 and 2 to 3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.
Percentage inhibition of growth rate for each replicate test material vessel was calculated using the following equation:

Ir = [(µc - µt) / µc] x 100
where
Ir = percentage inhibition of average specific growth rate
μc = mean average specific growth rate for the control cultures
μt = average specific growth rate for the test culture

Yield is calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn - N0
where
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = [(Yc - Yt) / Yc] x 100
where
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

> Range Finding Test

The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.
Based on this information a single test concentration of six replicates, of 100 mg/L was selected for the definitive test.
Chemical analysis of the 100 mg/L test preparations at 0 and 72 hours showed measured test concentrations of 96 % and 93 % of nominal respectively were obtained indicating that the test material was stable under test conditions.


> Definitive Test

- Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of 96 % and 88 % of nominal respectively were obtained and so the results are based on nominal test concentrations only.

- Growth Data
The 72-hour ErC50 was determined to be > 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

- Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 353 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 1.76 x 10^6 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 5% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35 %.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 1 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

- Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

- Observations on Test Material Solubility
At the start of the test, all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

A positive control (study number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.6 mg/L; 95 % confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour) : 0.77 mg/L; 95 % confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) 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 the SAS computer software package (SAS, 1999 - 2001).

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) based on growth rate was 100 mg/L. The No Observed Effect Concentration (NOEC) based on yield was less than 100 mg/L.

Executive summary:

The toxicity of the test material to the freshwater algae, Pseudokirchneriella subcapitata, was investigated in a study which was conducted in accordance with the standardised guidelines OECD 201 and EU Method C.3, under GLP conditions.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test material 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.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of 96 % and 88 % of nominal respectively were obtained and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test material gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) based on growth rate was 100 mg/L. The No Observed Effect Concentration (NOEC) based on yield was less than 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Description of key information

The effect of the test material on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) based on growth rate was 100 mg/L. The No Observed Effect Concentration (NOEC) based on yield was less than 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The toxicity of the test material to the freshwater algae, Pseudokirchneriella subcapitata, was investigated in a study which was conducted in accordance with the standardised guidelines OECD 201 and EU Method C.3, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test material 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.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of 96 % and 88 % of nominal respectively were obtained and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test material gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) based on growth rate was 100 mg/L. The No Observed Effect Concentration (NOEC) based on yield was less than 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.