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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
Study period:
2015-09-25 to 2015-10-24
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 201 (Alga, Growth Inhibition Test)
according to
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Source and lot/batch No.of test material:
* batch: M14IB5308
* WIL research test item number: 206568/B
- Expiration date of the lot/batch: 2016-02-03 (retest date)
- Purity test date: 2015-04-23

- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water 0.26 g/L
Analytical monitoring:
Details on sampling:
- Sampling method: samples were taken before the start of the test and after 72 hours from each test medium and from the control. 2.0 mL of volume was taken. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: stored in a freezer until analysis. Addionally, reserve samples of 2.0 mL were taken for possible analysis
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of test solutions started by dissolving test item in medium with a concentration of 100 mg/L applying 2 days of magnetic stirring in the dark to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixture was filtered through a preconditioned 0.45 µm membrane filter (Whatman) The obtained filtrate was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the saturated solution (SS) in test medium
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): final test solutions were clear and colourless
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

- Acclimation period: 3 days pre-culture before start of the test
- Culturing media and conditions (same as test or not): same as the test

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
24 mg CaCO3/L
Test temperature:
t=0h: 8.0
t=72h: 7.9
Dissolved oxygen:
not reported
not relevant
Nominal and measured concentrations:
Final test:
nominal test concentrations : 0.10, 0.32, 1.0, 3.2, 10, and 100% of the SS prepared at 100 mg/L
measured test concentration t= 0 h (mg/L): 0.111, 0.344, 1.07, 3.33, 3.44, 10.7, 33.9, 108
measured test concentration t= 72h (mg/L): 0.105, 0.354, 1.12, 3.46, 3.60, 10.8, 35.4, 112
Details on test conditions:
- Test vessel: 100 mL all-glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Initial cells density: 10,000 cells/mL
- Control end cells density: 151.0 x 10,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

- Source/preparation of dilution water: M2, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

- Sterile test conditions: no information
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: using TLD-lamps (60 to 120 µE/m²/s)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] At the beginning, cells were counted using a microscope and a counting chamber. thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength = 20mm).
- effect calculated parameters: specific growth rate and yield

- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 1.0, 10 and 100% of the SS
- Results used to determine the conditions for the definitive study: yes.
Reference substance (positive control):
potassium dichromate
Key result
72 h
Dose descriptor:
Effect conc.:
12 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: not determined
Key result
72 h
Dose descriptor:
Effect conc.:
1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: clear test solution
- EC50 (yield) = 1.5 mg T001159 /L (95% CL 1.4 - 1.7 mg/L)
- EC10 (growth rate) = 8.6 mg T001159 / L (95% CLnot determined)
- EC10 (yield) = 0.04 mg T001159 / L (95% CL 0.03 - 0.05 mg/L)
- NOEC (yield) = <0.10 mg T001159 / L.
Results with reference substance (positive control):
- Results with reference substance valid? yes (should be between 0.3 and 1.0 mg/L)
- EC50: 72-h EC50 (growth rate) = 1.2 mg/L (95% C.L.: 1.1 to 1.2 mg/L)
Reported statistics and error estimates:
NOEC and EC values were determined using a Williams Multiple Sequential t-test Procedure (alpha=0.05, one-sided, smaller).
Calculation of ECx values was based on probit analysis using linear max. likelihood regression.
Validity criteria fulfilled:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance T001159 according to the OECD guideline 201 (GLP conditions).
Under the test conditions with Pseudokirchneriella subcapitata, T001159 inhibited growth rate of this fresh water algae species significantly. The 72-h ECr50 was determined to be 12 mg/ and the NOEC was 1 mg/L.
The results of the test can be considered reliable without restrictions.

Description of key information

The study of Bouwman (2016), investigating the acute toxicity of T001159 to Pseudokirchnierella subcapititata according to OECD guideline 201, was considered as the key study for endpoint coverage. The measured 72-hour ErC50 was determined to be 12 mg/L.

Key value for chemical safety assessment

Additional information