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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:
10 - 13 October 2017 (range-finding test), 07 - 10 November 2017 (definitive test)
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:
Adopted March 2006, Annex 5 corrected July 2011.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source: The Sponsor (Osaka Soda Co. Ltd.). Lot/batch no. 161120-01
- Expiration date of the lot/batch: 20 November 2018
- Purity test date: 97% (by HPLC (CofA))

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (15 ± 10°C)
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Negligible solubility in water; 0.001 g/L (1 mg/L)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: n/a
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 50 mg/l
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) Dissolved in liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) n/a

OTHER SPECIFICS: n/a
Analytical monitoring:
no
Details on sampling:
n/a
Vehicle:
no
Remarks:
Both the preliminary range-finding test and the definitive test were prepared by the direct addition of measured amounts of test substance to dilution water and stirred.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The definitive test concentrations were prepared by measuring 10, 20, 40, 80 and 160 ml of stock solution. The stock solution was prepared by weighing 0.0500 g of homogenised test substance and added to 1000 ml nutrient growth medium. The test concentrations (expressed as loading rates) were 0.5, 1, 2, 4, 8 mg/l and the stock solution was 50mg/l (±5% tolerance by weight). These were stirred for 22 hours and 50 minutes and settled for 5 hours and 20 minutes. After settling the first 100 ml (approximately) of aqueous phase was removed (avoiding all settled and floating material) and discarded after filtering through a Whatman 54 filter. The remaining aqueous phase was filtered to provide sufficient volumes for water quality measurements and testing.
- Eluate: n/a
- Differential loading: n/a
- Controls: Yes. 0 mg/L test item (6 replicates)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The dissolved concentrations were likely to be lower than stated in this report as Chinomethionate has negligible solubility in water.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa from a pre-culture growing in exponential phase
- Age of inoculum (at test initiation): Not stated
- Method of cultivation: Not specified

ACCLIMATION
- Acclimation period: Not specified.
- Culturing media and conditions (same as test or not): Yes. The stocks of algal culture were maintained, and the tests performed, in nutrient growth medium.
- Any deformed or abnormal cells observed: Not specified
Test type:
not specified
Water media type:
other: Sterilised deionised water with added nutrients according to the OECD 201 standard
Total exposure duration:
72 h
Post exposure observation period:
n/a
Hardness:
n/a
Test temperature:
20.3 – 20.9°C
pH:
7.66 – 8.24
Dissolved oxygen:
n/a
Salinity:
n/a
Conductivity:
n/a
Nominal and measured concentrations:
Nominal concentrations - 0.5, 1, 2, 4 and 8 mg/L
The Sponsor specified that analytical confirmation of exposure concentrations was not required. All effect concentrations have, therefore, been calculated from nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250ml conical flask
- Type (delete if not applicable): Not specified
- Material, size, headspace, fill volume: 250 mL
- Aeration: Not specified
- Type of flow-through (e.g. peristaltic or proportional diluter): Not specified
- Renewal rate of test solution (frequency/flow rate): Not specified
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 62.5 x 10^4 cells/mL
- No. of organisms per vessel: n/a
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n/a

GROWTH MEDIUM
- Standard medium used: yes (OECD 201 standard)
- Detailed composition if non-standard medium was used: n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The stocks of algal culture were maintained, and the tests performed, in nutrient growth medium
(OECD 201) which was prepared by adding appropriate amounts of nutirent stocks to deionised water at a pH of 8.13.
- Total organic carbon: Not specified
- Particulate matter: Not specified
- Metals: Not specified
- Pesticides: Not specified
- Chlorine: Not specified
- Alkalinity: Not specified
- Ca/mg ratio: Not specified
- Conductivity: Not specified
- Culture medium different from test medium: No
- Intervals of water quality measurement: Not specified

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Not specified
- Light intensity and quality: 5840 - 7910 lux continuous white light.
- Salinity (for marine algae): n/a

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microscopically by direct cell counts using a haemocytometer and microscope
- Chlorophyll measurement: n/a
- Other: n/a

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Double
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: 0 (control), 0.1, 1.0, 10, 100 mg/l.
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (deterined in a separate reference study ENV 11396) from 16 - 19 January 2017. The ErC50 obtained was 1.06 mg/l and all validity criteria were met
Key result
Duration:
48 h
Dose descriptor:
other: EC(r)50
Effect conc.:
ca. 5.26 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
48 h
Dose descriptor:
other: EC(y)50
Effect conc.:
ca. 2.54 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
other: EC(r)50
Effect conc.:
ca. 6.18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
other: EC(y)50
Effect conc.:
ca. 2.01 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
other: NOEC(r)
Effect conc.:
ca. 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The algal cells were examined microscopically during the determination of cell concentration. All cells within the control and test concentrations appeared normal, no abnormalities were observed
- Unusual cell shape: n/a
- Colour differences: When observed visually, the control conical flasks appeared clear and colourless at 0 and 24 hours and slightly green after 48 and 72 hours. All test concentrations appeared clear and colourless at 0 and 24 hours. 0.5, 1 and 2 mg/l appeared slightly green and 4 and 8 mg/l clear and colourless at 48 hours. 0.5, 1, and 2 mg/l appeared normal but green, 4 mg/l appeared normal but slightly green and 8mg/l clear and colourless after 72 hours.
- Flocculation: n/a
- Adherence to test vessels: n/a
- Aggregation of algal cells: n/a
- Other: n/a
- Any stimulation of growth found in any treatment: n/a
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: n/a
- Effect concentrations exceeding solubility of substance in test medium: n/a
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 1.06 mg/L
- Other: A separate reference study (ENV 11396) was run from 16 to 19 January 2017 using potassium dichromate (K1r2O7) as a means of checking the test procedure and sensitivity of the test species
Reported statistics and error estimates:
Statistical methods used in ToxCalc v5.0: Maximum Likelihood-Logit with 95% confidence limits calculated
Validity criteria fulfilled:
yes
Conclusions:
The 48 hour EC(r)50 and EC(y)50 of Chinomethionate to Pseudokirchneriella subcapitata were 5.26 mg/l and 2.54 mg/l respectively.
The 72 hour EC(r)50 and EC(y)50 of Chinomethionate to Pseudokirchneriella subcapitata were 6.18 mg/l and 2.01 mg/l respectively.
The 0 to 72-hour NOEC(r) was 1 mg/l and the 0-72 hour LOEC was 2 mg/l
Executive summary:

The effect of the test item Chinomethionate to Pseudokirchneriella subcapitata was investigated in an alga growth inhibition study to determine the 72-hour EC50.

Results of the preliminary (range-finding) test dictated the conditions of the definitive test, where test item concentrations of 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L were studied in triplicate, along with 6 control samples. The stocks of algal culture were maintained, and the tests performed, in nutrient growth medium (OECD 201) which was prepared by adding appropriate amounts of nutirent stocks to deionised water at pH 8.13. Cell densities were measured microscopically by direct cell counts on each test and control replicate, in sextuplicate at 24 hours for the control and triplicate thereafter, and in triplicate at 24, 48 and 72 hours (±2h) for the test concentrations.

The 48 hour EC(r)50 and EC(y)50 of Chinomethionate to Pseudokirchneriella subcapitata were 5.26 mg/l and 2.54 mg/l respectively. The 72 hour EC(r)50 and EC(y)50 of Chinomethionate to Pseudokirchneriella subcapitata were 6.18 mg/l and 2.01 mg/l respectively. The 0 to 72-hour NOEC(r) was 1 mg/l and the 0-72 hour LOEC was 2 mg/l.

Description of key information

The 48 hour EC(r)50 and EC(y)50 of Chinomethionate to Pseudokirchneriella subcapitata were 5.26 mg/l and 2.54 mg/l respectively.

The 72 hour EC(r)50 and EC(y)50 of Chinomethionate to Pseudokirchneriella subcapitata were 6.18 mg/l and 2.01 mg/l respectively.

The 0 to 72-hour NOEC(r) was 1 mg/l and the 0-72 hour LOEC was 2 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:
6.18 mg/L

Additional information