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Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
See principles of methods
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. (ECETOC, 1996 and OECD 2019). A series of solvent stock solutions was prepared in dimethylformamide in a series of nominal concentrations . An aliquot of each solvent stock solution was separately dispersed in test water with the aid of magnetic stirring fto give the desired nominal test concentrations.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
Samples were taken from the sovent control and each test group from the bulk test at the start, from additional media run alongside the test at 24 and 48 hours, and from the pooled replicates at 72 hours for inalysis. The samples were placed in vials containing 5 mL of Tetrahydrofuran and stored until analysis. Additional samples of each test concentration were incubated alongside the test to provide samples for analysis at 24 and 48 hours. Samples were either analysed on the day or frozen until analysis
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Range finder tests were carried out with and without solvent. The method preferred for the definitive test used Dimethyl formamide as the vehicle as the substance alone could not be suitably solubilised using a water accomodated fraction approach. Aliquots of stock solution were diluted in test medium to give required final test concentrations.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethyl formamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 10g/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

- Acclimation period: 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 103 cells/mL. The plugged flasks were agitated under illumination until the algal cell density was approximately 105 to 106 cells/mL.
- Culturing media and conditions (same as test or not): yes, except for the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with conducting the test in an enclosed system.
- Any deformed or abnormal cells observed: not reported
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
not reported
Test temperature:
24 ±1 ºC
7.6 at 0 hours increasing to pH 10.0 at 72 hours.
Dissolved oxygen:
not reported
not applicable
not reported
Nominal and measured concentrations:
Nominal: 0.01, 0.026, 0.064, 0.16, 0.40mg/L. Concentrations declined during the study. Geometric means of the measured concentrations at 24, 48 and 72 hours were 0.0061, 0.0074, 0.035, 0.12, 0.25mg/L
Details on test conditions:
- Test vessel: Conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass, 250mL completely filled
- Aeration: no
- Initial cells density: 5000 cells/mL
- Control end cells density: ~600000cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3

- Standard medium used: similar:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L

- No data

- Sterile test conditions: sterile conditions not specifically mentioned
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 7000lux warm white light 380-730nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Counter multisizer particle counter, three determinations per sample.

- Range finding study: Two initial tests run using a water accomodated fraction approach with nominal loadings of 1, 10, 100mg/L. A second study was run at nominal loadings of 0.001, 0.01, 0.1 and 1.0mg/L. Due to inconsistenc of results using an initial WAFapproach and test concentrations of 0.04, 0.09, 0.2, 0.5 and 1.2mg/L, the approach was taken to use a DMFvehicle 'solvent spike' approach and a further range finder was run using nominal concentrations of 0.001, 0.1 and 1.0mg/L via a DMF solution. This proved acceptable so this approach was used for the definitive study.
Reference substance (positive control):
A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test. The last one performed (July 2019) was satisfactory.
72 h
Dose descriptor:
Effect conc.:
0.052 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
0.035 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
0.007 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- No unusual observations reported that might invalidate the results
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 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
Reported statistics and error estimates:
Growth rate 95% confidence limit 0.043 - 0.062mg/L
Yield 95% confidence limit 0.013 - 0.018mg/L
Executive summary:

In a guideline OECD201 Freshwater algae growth inhibition test, the substance THI TM exhibited a 72 hr NOEC of 0.0074mg/L and an EC50 of 0.054mg/L based on growth rate. The substance was solubilised using DMF as a vehicle.

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
0.052 mg/L

Additional information