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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:
January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test. Adopted 23 March 2006.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Nominal Nominal
Fyrolflex RDP n=1 Component
Negative Control Negative Control
Solvent Control Solvent Control
1.9 μg/L 1.3 μg/L
3.8 μg/L 2.7 μg/L
7.5 μg/L 5.3 μg/L
15 μg/L 11 μg/L
30 μg/L 21 μg/L



- Sampling method:Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the test substance. At each sampling interval samples were collected in duplicate with one set of samples processed immediately for analysis and the other set of samples were stored under refrigerated conditions for potential future analysis. Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to distribution into the test chambers. At exposure termination, samples were collected from the pooled replicates from each treatment and control group. At test termination the abiotic replicate included in the highest treatment group was also sampled. At each interval, 10 mL samples were collected into 20 mL glass scintillation vials containing 10 mL of acetonitrile.

- Sample storage conditions before analysis: The other set of samples were stored under refrigerated conditions for potential future analysis.

Vehicle:
yes
Remarks:
N,N-Dimethylformamide (DMF)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions in freshwater AAP medium were prepared by diluting 50 μL of each respective stock to a final volume of 500 mL with freshwater AAP medium. The test solutions were inverted to mix and appeared clear and colorless with no visible particulates or surface-slicks.
- Controls: A solvent control was prepared by diluting 50 μL of DMF to a final volume of 500 mL with freshwater AAP medium. The negative control solution consisted of freshwater AAP medium without test substance or solvent added.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): The solvent concentration in the solvent control and all Fyrolflex RDP treatment groups was 0.1 mL DMF/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
- Other relevant information: A primary stock solution was prepared by dissolving 0.0300 g of Fyrolflex RDP in 100 mL of N,N-dimethylformamide (DMF) to achieve a nominal concentration of 300 μg/mL. The primary stock was mixed by inversion and appeared clear and colorless at the time of preparation. No particulates or surface slicks were observed. Four additional stock solutions were prepared in DMF at target nominal concentrations of 18.8, 37.5, 75.0, and 150 μg/mL by serial dilution of the 300 μg/mL primary stock.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Freshwater Alga (Raphidocelis subcapitata)
- Strain:
- Source (laboratory, culture collection): culture identification No. 17-01. Eurofins Cultures Easton, Maryland 21601. Original algal cultures were obtained from the University of Texas at Austin and have been maintained in culture medium at Eurofins, Easton, Maryland since June 2017. Algal cells used in this test were obtained from Eurofins-Easton cultures that had been actively growing in culture medium under the same environmental conditions as used in this test for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh medium three days prior to test initiation.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23
pH:
day 0: 7.3-7.5
Day 3: 9.3-9.7
Nominal and measured concentrations:
TEST CONCENTRATIONS:
Geometric Mean, Geometric Mean,
Nominal Nominal Measured n=1 Measured
Fyrolflex RDP n=1 Component Component Fyrolflex RDP2
Negative Control Negative Control Solvent Control Solvent Control 1.9 μg/L 1.3 μg/L 0.83 μg /L 1.2 μg/L
3.8 μg/L 2.7 μg/L 1.8 μg/L 2.6 μg/L
7.5 μg/L 5.3 μg/L 3.5 μg/L 5.0 μg/L
15 μg/L 11 μg/L 6.7 μg/L 9.5 μg/L
30 μg/L 21 μg/L 13 μg/L 18 μg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:250 mL glass Erlenmeyer flasks plugged with sterile foam stoppers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL of test or control medium
- Aeration: no
- Initial cells density: At test initiation, an inoculum of the algal cells was added to each test chamber to achieve a nominal concentration of approximately 10,000 cells/mL
- Control end cells density: 1,905,196 cells/mL
- No. of vessels per concentration (replicates):Three replicate test chambers were maintained in each treatment group.
- No. of vessels per control (replicates): Six replicates were maintained in the negative control group.
- No. of vessels per vehicle control (replicates):Three replicate test chambers were maintained in the solvent control.

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: for detailed information on dilusion water used in the study please see appendix 4 in the attached report.
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes, the medium was sterilized by filtration (0.22 µm) prior to use.
- Adjustment of pH:The pH of the medium was adjusted to 7.5 ± 0.1 with 10% hydrochloric acid
- Photoperiod: The algae were held under continuous cool-white fluorescent lighting throughout the test.
- Light intensity and quality:5,974 ± 248.9 lux
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Cell counts were performed using an electronic particle counter (Coulter Electronics, Inc.).
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x2
- Justification for using less concentrations than requested by guideline:
- Range finding study:A preliminary range-finding test was conducted.
- Test concentrations: 1.9, 3.8, 7.5, 15 and 30 μg/L
- Results used to determine the conditions for the definitive study: A preliminary range-finding test was conducted with nominal concentrations of 0.30, 3.0, and 30 μg/L, and yielded -2, 2, 5% inhibition of mean cell density after 72 hours of exposure
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Remarks:
For Fyrolflex RDP
Effect conc.:
> 30 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EC50
Remarks:
For n = 1 component
Effect conc.:
> 13 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
NOEC
Remarks:
For Fyrolflex RDP
Effect conc.:
> 30 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Remarks:
For n = 1 component
Effect conc.:
> 13 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): After 72 hours of exposure, flocculations or aggregations of cells were not observed in the negative control, solvent control, or any of the treatment groups. Adherence of cells to the test chambers was not observed in the negative control, solvent control, or any of the treatment groups. Cells in the Fyrolflex RDP treatment groups and solvent control appeared normal when compared to cells present in the negative control.
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other:
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The measured concentrations of the n=1 component of Fyrolflex RDP in test solution samples did not remain constant during the study, this is thought to be due to the hydrophobic nature of the test substance
- Effect concentrations exceeding solubility of substance in test medium: Alhough, the study was performed at concentrations above solubility of the test material, no effects were detected.
Reported statistics and error estimates:
Statistical analyses was conducted using “The SAS System for Windows, Version 8.2”
The mean treatment group responses were compared to the pooled control response using Dunnett’s one-tailed t-test (α = 0.05).
Validity criteria fulfilled:
yes
Conclusions:
The freshwater alga, Raphidocelis subcapitata, was exposed to a geometric series of five treatment levels of Fyrolflex RDP ranging from 0.83 to 13 μg/L, based on geometric mean, measured concentrations of the n = 1 component of Fyrolflex RDP. All control validity criteria were achieved. Toxicity of Fyrolflex RDP to R. subcapitata was assessed based on effects on growth rate and yield relative to the pooled control groups. The 72-hour EC50 values for growth rate and yield were determined to be >13 μg/L (>30 μg/L based on nominal Fyrolflex RDP concentrations), for both endpoints. The 72 hour NOEC value was determined to be 13 μg/L (30 μg/L based on nominal Fyrolflex RDP concentrations), the highest concentration tested. Even though a solvent used for increasing substance solubility no toxicity was observed under the experimental conditions.

Executive summary:

The freshwater alga, Raphidocelis subcapitata, was exposed to a five treatment levels of Fyrolflex RDP ranging from 0.83 to 13 μg/L, measured concentrations of the n = 1 component of Fyrolflex RDP. The 72-hour EC50 values for growth rate and yield were determined to be >13 μg/L (>30 μg/L based on nominal Fyrolflex RDP concentrations), for both endpoints.  The 72 hour NOEC value was determined to be 13 μg/L (30 μg/L based on nominal Fyrolflex RDP concentrations), the highest concentration tested. Even though a solvent used for increasing substance solubility no toxicity was observed under the experimental conditions.

Description of key information

In a key study according to OECD guideline 201 under GLP, both the 72h-NOEC and 72h-EC50 of Fyrolflex RDP towards the alga Pseudokirchneriella subcapitata were found to be >30 µg/L. Even though a solvent used for increasing substance solubility no toxicity was observed under the experimental conditions.

Key value for chemical safety assessment

EC50 for freshwater algae:
30 µg/L
EC10 or NOEC for freshwater algae:
30 µg/L

Additional information