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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:
2017-01-27 to 2017-02-07
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
Principles of method if other than guideline:
Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: samples were taken before the start of the test and after 72 hours from all test concentrations and from the control. A volume of 2.0 mL was taken. The filter used for preparation of the SS was kept for possible analysis of the residue. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Reserve samples of 2.0 mL were taken from all test solutions for possible analysis.
- Sample storage conditions before analysis: stored in a freezer (< -15°C) until 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 item, a saturated solution (SS) was prepared. The preparation of the SS started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring at room temperature to ensure maximum dissolution of the test item in test medium. This resulted in a hazy dispersion containing undissolved material. The obtained dispersion was filtered through a 0.45 µm membrane filter (Whatman; RC55). The filter was pre-conditioned with a small volume of test solution that was discarded. The resulting SS was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
- Controls: yes
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- 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. 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
21-23°C
pH:
7.8-8.2
Dissolved oxygen:
not reported
Salinity:
not relevant
Nominal and measured concentrations:
nominal test concentrations final test: 1.0, 10 and 100% of a SS prepared at 100 mg/L
measured test concentration final test t= 0 h: <0.0020, <0.0020, <0.0020 mg/L
measured test concentration final test t= 24h: n.d., <0.0020, <0.0020 mg/L
measured test concentration final test t= 72h: n.d., <0.0020, <0.0020 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 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
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system applied
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 10,000 cells/mL
- Control end cells density: 176.8 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

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: no
- Intervals of water quality measurement: temperature measured continuously, pH at the beginning and the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm, using TLD-lamps.

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.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x 10

- Range finding study
- Test concentrations: 1.00, 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
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:
- EC50 (yield) = >100 mg T003066 /L
- EC10 (growth rate) = >100 mg T003066 / L
- EC10 (yield) = >100 mg T003066 / L
- NOEC (yield) = 100 mg T003066 / L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.2 mg/L
Reported statistics and error estimates:
Statistical analysis of the data was not needed as no inhibition of growth was recorded. No EC10, EC20 and EC50 values could be calculated because the observed effects were below 10%.
Validity criteria fulfilled:
yes
Conclusions:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance T003066 according to the OECD guideline 201 (GLP conditions).
Due to the very low solubility of JNJ-39453349-AAA (T003066) in test medium, concentration levels that might be toxic for algae could not be reached. The 72h-NOEC for inhibition of both growth rate and yield was at the concentration obtained in 100% of a saturated solution prepared at a loading rate of 100 mg/L. This concentration was considered to be the maximum solubility of the test item in test medium and was below the limit of detection.
The 72h-EC50 for both growth rate inhibition (ERC50) and yield inhibition (EYC50) was beyond the range tested, i.e. exceeded the maximum solubility of test item in test medium.
The results of the test can be considered reliable without restrictions.

Description of key information

The toxicity of T003066 to green algae Pseudokirchneriella subcapitata was determined in a 72-h growth inhibition test according to the OECD guideline 201 (GLP conditions). Due to the very low solubility of JNJ-39453349-AAA (T003066) in test medium, concentration levels that might be toxic for algae could not be reached. The 72h-NOEC for inhibition of both growth rate and yield was at the concentration obtained in 100% of a saturated solution prepared at a loading rate of 100 mg/L. This concentration was considered to be the maximum solubility of the test item in test medium and was below the limit of detection. The 72h-EC50 for both growth rate inhibition (ERC50) and yield inhibition (EYC50) was beyond the range tested, i.e. exceeded the maximum solubility of test item in test medium.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information