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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 February 2018 - 15 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch: 53715080
Purity: not supplied
Physical state/Appearance: colorless slightly viscous liquid
Analytical monitoring:
yes
Details on sampling:
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 item under test conditions. All samples were stored frozen prior to analysis.
Vehicle:
no
Details on test solutions:
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control group and 3 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 item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 9.49 x 10e5 cells per mL. Inoculation of 900 mL of test medium with 4.7 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10e3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were kept under constant agitation by orbital shaker (100 to 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 10e4 to 10e5 cells/mL.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not specified
Test temperature:
24 ±1 °C.
pH:
7.7 - 8.1
Dissolved oxygen:
not specified
Salinity:
not specified
Conductivity:
not specified
Nominal and measured concentrations:
nominal: 0.1, 1, 10, 100 % v/v
measured: 0.28, 1, 3.2, 11, 33 % v/v
Details on test conditions:
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 1.0, 3.2, 10 and 32% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with 4.7 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution. The stock solution and each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours

250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control group and 3 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 item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 9.49 x 10e5 cells per mL. Inoculation of 900 mL of test medium with 4.7 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10e3 cells per mL and had no significant dilution effect on the final test concentration. The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 20, 45 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 10e3 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.
The pH of the control and each 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.
Samples were taken from the control and each test group from the bulk test preparation at 0 hours, at 24 and 48 hours from the media which was run alongside the test 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.


Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 4.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.28 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.:
ca. 1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.28 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences:
- Flocculation: not specified
- Adherence to test vessels: not specified
- Aggregation of algal cells: not specified
- Other: not specified
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not specified
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 0.28, 1.0 and 3.2 mg/L, however sparse but intact cells were present in the test cultures at 11 and 33 mg/L.
Results with reference substance (positive control):
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:
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control and the 0.28 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the No Observed Effect Concentration (NOEC) based on growth rate was 0.28 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 1.0 mg/L.
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 = 4.5 mg/L
- NOEC = 0.28 mg/L
- LOEC = 1.0 mg/L

Yield:
- EC50 = 2.0 mg/L
- NOEC = 0.28 mg/L
- LOEC = 1.0 mg/L

Description of key information

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 = 4.5 mg/L

- NOEC = 0.28 mg/L

- LOEC = 1.0 mg/L

Yield:

- EC50 = 2.0 mg/L

- NOEC = 0.28 mg/L

- LOEC = 1.0 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
4.5 mg/L

Additional information