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Ecotoxicological information

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:
2017-12-12 to 2018-08-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
Referenced as Method C.3 of Commission Regulation (EC) 761/2009
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
-
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Japan/ Batch No.: 7J13AA
- Expiration date of the lot/batch: 15.10.2018
- Purity test date: 2018


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in dark
- Stability under test conditions: Stable under normal conditions
Hazardous reactions : Reactions with strong acids.
Hazardous decomposition products: None with proper storage and handling.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Prior to start of the test sufficent master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10exp3 cells/mL.
The flasks were plugged with polyurethane foam stoppers and kept under constant illumination at 24+/-1 °C until the algal cell density was approximately 10 Exp4 to 10DExp cells/mL.

- Final dilution of a dissolved solid, stock liquid or gel:
-A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 30 minutes 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, 0.10, 0.032, 0.010, 0.0032 and 0.0010 mg/L. An aliquot (900 mL) of each of the 0.0010, 0.0032, 0.010, 0.032 and 0.10 mg/L stock solutions was separately inoculated with algal suspension (4.1 mL) to give the required test concentrations of 0.0010, 0.0032, 0.010, 0.032 and 0.10 mg/L.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: [nominal Test concentrationsin mg/L]/ 0,0010, 0,0032, 0,010, 0,032,0,10.
- Sampling method: flask metod
- Sample storage conditions before analysis: RT in the dark.

Test solutions

Vehicle:
not specified

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
- Strain: CCAP278/4
- Source (laboratory, culture collection): Obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

ACCLIMATION
The master cultures were maintained under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ± 1°C

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Growth rate and yield were affected after 72 h of exposure.
Observations on Cultures:
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0,00082, 0,0019 and 0,0081 mg/L, however no algae cells were observed to be present in the test cultures at 0,022 and 0,082 mg/L.

Test conditions

Hardness:
Not stated
Test temperature:
24 ± 1
pH:
7,5-8,2
Dissolved oxygen:
Not stated
Salinity:
Not stated
Nominal and measured concentrations:
Nominal test concentrations:[mg/L]
0,00010, 0,0010, 0,010 and 1.0 mg/L for a period of 72 hours.
Geometric Mean Measured Test concentrations:[mg/L]
0,00082, 0,0019, 0,0081, 0,0022, 0,082.
Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open [conical flasks]
- Material, size, headspace, fill volume: glass, 100 mL
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 10 Exp 3 Cells/mL
- Control end cells density: Nominal cell density of control at 0 hours: 5.00X10 Exp 3 cells per mL
-Mean cell density of control at 72 hours: 1, 19 x 10 Exp 3 cells per mL
- No. of organisms per vessel: Nominally inoculated cell concentration: 5, 00 X 10 Exp 3 cells per mL)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5
- No. of vessels per vehicle control (replicates):

GROWTH MEDIUM
- Standard medium used: yes
- Light intensity and quality: continuous illumination ( intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm)
TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentration than requested by guideline: N/A
- Range finding study The results of the range finding tests showed no effect on growth and the test concentrations of 0,0000010 and 0,010 mg/L and 0,0010 mg/L.

- Test concentrations:
Based on the results of the fourth range-finding test the following test concentrations were assigned to the definitive test: 0.0010, 0.0032, 0.010, 0.032 and 0.10 mg/L

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.009 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Statistical analysis of the growth rate data was carried out for the control and all test concentrations . There were no statistically significant differences (P≥0.05), between the control, 0.00082 and 0.0019 mg/L test concentrations.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.004 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: Statistical analysis of the growth rate data was carried out for the control and all test concentrations . There were no statistically significant differences (P≥0.05), between the control, 0.00082 and 0.0019 mg/L test concentrations.
Key result
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
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.008 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
the following results were determined from the data based on the geometric mean measured test concentrations:

Inhibition of Growth Rate


ErC10 (0 to 72 hour): 0.0053 mg/L

ErC20 (0 to 72 hour) : 0.0065 mg/L

ErC50 (0 to 72 hour) : 0.0090 mg/L; 95% confidence limits 0.0081 to 0.010 mg/L
where ErCx is the test concentration that reduced growth rate by x%.

Inhibition of Yield

EyC10 (0 to 72 hour) : 0.0015 mg/L

EyC20 (0 to 72 hour) : 0.0021 mg/L

EyC50 (0 to 72 hour) : 0.0036 mg/L; 95% confidence limits 0.0030 to 0.0043 mg/L

Where EyCx is the test concentration that reduced yield by x%.

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.00082 and 0.0019 mg/L test cultures were observed to be green dispersions. The 0.0081 mg/L test cultures were extremely pale green dispersions whilst the 0.022 and 0.082 mg/L test cultures were observed to be clear colorless solutions.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50:

ErC10 (0 to 72 hour) : 0.0053 mg/L

ErC20 (0 to 72 hour): 0.0065 mg/L

ErC50 (0 to 72 hour) : 0.0090 mg/L; 95% confidence limits 0.0081 to 0.010 mg/L

0.0053 mg/L.

where ErCx is the test concentration that reduced growth rate by x%.

Any other information on results incl. tables

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 84% to 122% of nominal. An overall decline in measured test concentrations was observed after each 24-Hour period, in the range of 65% to 96% of nominal at 24 hours, 48% to 79% of nominal at 48 hours, and from 48% to 71% of nominal at 72 hours, suggesting that the test item was unstable under the conditions of the test.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be 0.00082, 0.0019, 0.0081, 0.022 and 0.082 mg/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

 Response variable  EC50 (mg/L)  95% Confidence Limits (mg/L)  No Observed Effect Concentration (NOEC) (mg/L)  Lowest Observed Effect Concentration (LOEC) (mg/L)
 Growth Rate  0.0090  0.0081 - 0.010  0.0019  0.0081
 Yield  0.0036  0.0030 - 0.0043  0.0019  0.0081

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:
Growth Rate: EC50 (mg/L)= 0.0090 95% Confidence Limits (mg/L): 0.0081 - 0.010 No Observed Effect Concentration (NOEC) (mg/L): 0.0019  Lowest Observed Effect Concentration (LOEC) (mg/L):  0.0081
Yield: EC50 mg/L= 0.0036 95% Confidence Limits (mg/L): 0.0030 - 0.0043 No Observed Effect Concentration (NOEC) (mg/L: 0.0019 Lowest Observed Effect Concentration (LOEC) (mg/L):  0.0081
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga 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" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Range-Finding Test

The analytical methodology was not validated prior to the analysis of range-finding samples; full validation was later conducted at a suitable concentration.

Definitive Test

Validation of the Analytical Method Specificity

The biological control samples and an analyzed analytical blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.

Linearity

The calibration data for the calibration standards of the test item is given in Table 2 and Figure 1. The data was found to have a quadratic correlation within the calibration range of

0 to 0.0016 mg/L. The R² fit of the calibration curve to the data was 0.9976, and was considered to be acceptable.

Accuracy (Recovery) and Precision

A set of recovery samples accurately fortified at a relevant concentration of test item was prepared five-fold and analyzed.

The method was considered to be sufficiently accurate and precise for the purposes of this test. The test sample results were not corrected for recovery.

Limit of Quantification

The LOQ was determined by calculating the sample concentration that gave a peak equivalent to ten times the baseline noise. Using this method the LOQ was determined to be 0.000049 mg/L.

Results:

Response variable  EC50 (mg/L)  95% Confidence Limits (mg/L)  No Observed Effect Concentration (NOEC) (mg/L)  Lowest Observed Effect Concentration (LOEC) (mg/L)
 Growth Rate  0.0090  0.0081 - 0.010  0.0019  0.0081
 Yield  0.0036  0.0030 - 0.0043  0.0019  0.0081