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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:
2015-12-04 to 2015-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: I15FB2768
- Expiration date of the lot/batch: 01 July 2017 (retest date)
- Purity: 100% (based on base titration assay)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: no data
Analytical monitoring:
yes
Details on sampling:
- Sampling method: samples were taken before the start of the test, after 24 hours 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. Additionally, reserve samples of 2.0 mL were taken for possible analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to an expected low water solubility of the test substance saturated solutions (SS) were prepared. The preparation of test solutions started with a loading rate of 100 mg/L applying a two-day period of magnetic stirring at room temperature, mostly in the dark. The obtained mixture was filtered through a 0.45 μm membrane (RC55, Whatman) to remove the undissolved fraction. The filter was pre-conditioned with a small volume of test solution that was discarded. The obtained saturated solution (SS) was used as the highest test concentration and was of a very light yellow colour. Lower test concentrations were prepared by subsequent dilutions of the 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:
TEST ORGANISM
- 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
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as the test
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
21-22°C
pH:
t=0h: 7.9
t=72h: 7.7-7.9
Dissolved oxygen:
not reported
Salinity:
not relevant
Nominal and measured concentrations:
nominal test concentrations : 1.0, 10, and 100 mg/L of the SS prepared at 100 mg/L
measured test concentration t= 0 h: 0.0355 mg/L, 0.0332 mg/L (without algae)
measured test concentration t= 24 h: 0.030 mg/L, 0.0348 mg/L (without algae)
measured test concentration t= 72h: 0.0110 mg/L, 0.0169 mg/L (without algae)
Details on test conditions:
TEST SYSTEM
- 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: 101.1 x 10,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- 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

TEST MEDIUM / WATER PARAMETERS
- 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.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: using TLD-lamps (with a light intensity within the range of 78 to 90 µE.m-2.s-1)

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 680 nm using a spectrophotometer with immersion probe (pathlength = 20mm).
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- 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):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: not determined
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 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
- 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) = >23 µg T001325 /L
- EC10 (growth rate) = >23 µg T001325 / L (95% not determined)
- EC10 (yield) = >23 µg T002325 / L
- NOEC (yield) = 23 µg T001325 / 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) = 1.5 mg/L (95% C.L. 1.4 to 1.6 mg/L)
- EC50: 72-h EC50 (yield) = 0.51 mg/L (95% C.L. 0.49 to 0.53 mg/L)
Reported statistics and error estimates:
No EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration).
Validity criteria fulfilled:
yes
Conclusions:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance T001325 according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had no inhibitory effect on the growth of Pseudokirchneriella subcapitata after the test period of 72 hours up to and including the highest test concentration of 23 µg/L (calculated as the TWA concentration in the undiluted SS) and corresponding with a loading rate of 100 mg/L. This concentration was the highest measured concentration of the test item that could be dispersed in the test water. The results of the test can be considered reliable without restrictions.

Description of key information

The study of Migchielsen (2016) investigating the acute toxicity of T001325 to algae according to OECD guideline 201, was considered as the key study for endpoint coverage. The test item T001325 had no inhibitory effect on the growth of Pseudokirchneriella subcapitata after the test period of 72 hours up to and including the highest test concentration of 23 µg/L (calculated as the TWA concentration in the undiluted SS) and corresponding with a loading rate of 100 mg/L. This concentration was the highest measured concentration of the test item that could be dispersed in the test water.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information