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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
effects on growth of green algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Jan to 18 Feb 2022
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Mitsuboshi Chemical Co., Ltd. Bx 10378Q
- Purity, including information on contaminants, isomers, etc.: 99.8% as per CoA

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: stable
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: stable
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not reactive

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): None
- Preliminary purification step (if any): None
- Final concentration of a dissolved solid, stock liquid or gel: 100 mg/L
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution:

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
- Description of the formulation, e.g. formulated product for foliar application; formulated product soil application; solution in organic solvent for soil application; formulated product seed treatment; solution in organic solvent seed treatment:

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added:
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 0.1, 1.0, 10.0, 100.0 mg/L
- Sampling method: Two samples per test concentrations in which the algal cells are exposed will be taken for analytical verification at the start and end of the definitive test. One of this sample will be injected at the HPLC (approximately 20μL are injected, depending of the method development), and the other one will be retained for repeat analysis. At the end of the 72-hour exposure period algal cells will be removed from the samples by centrifugation for 20 minutes at 3,000rpm prior to analysis. Test substance specific analysis of each test concentration will be carried out as soon as possible after sampling, with samples being stored frozen until analysis. Where samples are stored before analysis the stability of the samples will be confirmed by a suitable test of storage stability. Where it is not possible to measure actual concentrations in the test system, results will be based on nominal concentrations.
- Sample storage conditions before analysis:
Vehicle:
yes
Remarks:
water
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test concentrations (loading rates) will be stirred on magnetic stirrers for 24±2 hours, followed by a period of settling (at least 4 hours). After settling the first 100-200ml of the aqueous phase will be removed and discarded prior to testing (and be used to precondition filters Whatman 54 (or equivalent) should the aqueous phase and test sample be difficult to separate (including the control)). Concentrations being tested under 10mg/L will be made by dilution of the lowest prepared WAF concentration. Due to the limited confidence in accuracy of weights under this value.
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Test concentration separation factor: 3
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):
- Other relevant information:
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: n/a
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, SAMS Research Services Ltd, Scottish Marine institute, OBAN, Argyll, PA37 1QA Scotland, United Kingdom.
- Age of inoculum (at test initiation):
- Method of cultivation:

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Standard exposure length
Test temperature:
21 to 24°C
pH:
8.1±0.1
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 or 300 ml conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: fill volume 100 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 1 x 10E4 cell/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates):

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- 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
- Adjustment of pH: yes
- Photoperiod: continuous
- Light intensity and quality: 6000 - 8000 lux
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Approximately every 24 hours (± 2h) during the test, samples will be removed from each flask to
determine algal growth microscopically by haemocytometer. At least six fields on the haemacytometer slide are counted after 24-hours for the control and three for test concentrations. Thereafter three fields are counted at 48 and 72 hours (all ±2 hours), in all concentrations.
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: At least one preliminary (range-finding) test at 0 (Control), 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:

CULTURING APPARATUS
-Details on culturing apparatus used:
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
9.047 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Determined by Bonferroni t Test
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 9.047 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
yield
Remarks on result:
other: Determined by Bonferroni t Test
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.096 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
23.224 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1 099.58 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.113 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC20
Effect conc.:
5.876 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
11 271.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.012 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.116 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.559 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
48 h
Dose descriptor:
EC20
Effect conc.:
0.005 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
22.344 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
yield
Details on results:
- Exponential growth in the control (for algal test): yes/no
- Observation of abnormalities (for algal test):
- Unusual cell shape:
- Colour differences:
- Flocculation:
- Adherence to test vessels:
- Aggregation of algal cells:
- Other:
- Any stimulation of growth found in any treatment:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium:
Validity criteria fulfilled:
yes
Conclusions:
The results of this study give NOEC 72h 9.047 mg/L and ErC50 72h 1099.58 mg/L for a readily degradable substance. Therefore this substance is not classified.

Description of key information

The results of this study give NOEC 72h 9.047 mg/L and ErC50 72h 1099.58 mg/L for a readily degradable substance. Therefore this substance is not classified.

Key value for chemical safety assessment

EC50 for freshwater algae:
1 099.58 mg/L
EC10 or NOEC for freshwater algae:
9.047 mg/L

Additional information

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