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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:
2017-02-10 to 2017-02-17
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
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Analytical purity: assay 101.1%
- Source and lot/batch No.of test material: M16FB2273
- Expiration date of the lot/batch: 2017-06-14 (retest date)
- Purity test date: 2016-09-19 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: <0.1 g/L
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 36 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.
- 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, saturated solutions (SS) were prepared. The preparation of test solutions started with a loading rate of 100 mg/L applying 3 days of magnetic stirring at room temperature to ensure maximum dissolution of the test item in test medium. This resulted in a clear and colourless dispersion that contained undissolved material. The obtained mixture was filtered through a 0.45 µm membrane filter (Whatman; RC55) to remove the undissolved fraction. 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
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algal 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. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before start of the test)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
23°C
pH:
7.5-8.0
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: n.d., n.d., n.d., n.d. mg/L
measured test concentration final test t= 24h: n.d., n.d., n.d., n.d. mg/L
measured test concentration final test t= 72h: n.d., n.d., n.d., n.d. 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: 211.8 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: 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
Remarks on result:
other: C.L. 95%: 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:
- EC50 (yield) = >100 mg T003421 / L
- EC10 (growth rate) = >100 mg T003421 / L
- EC10 (yield) = >100 mg T003421 / L
- NOEC (yield) = 100 mg T003421 / L.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 0.48 mg/L
Reported statistics and error estimates:
Statistical analyses of the data was not needed as the effects recorded at the limit concentration showed stimulation rather than inhibition of growth.
No EC50 values could be calculated because the test item proved to be non-toxic (EC50 > maximum concentration tested).
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).
Validity criteria fulfilled:
yes
Conclusions:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance T003421 according to the OECD guideline 201 (GLP conditions).
Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of JNJ-42808389-AAA (T003421) tested. The EC50 for both growth rate (72-h ErC50) and yield inhibition (72-h EyC50) was beyond the range tested, i.e. exceeded the maximum solubility of test item in medium, obtained in a saturated solution prepared at a loading rate of 100 mg/L. The results of the test can be considered reliable without restrictions.

Description of key information

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance T003421 according to the OECD guideline 201 (Tobor-Kaplon, 2017).

Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of JNJ-42808389-AAA (T003421) tested. The EC50 for both growth rate (72-h ErC50) and yield inhibition (72-h EyC50) was beyond the range tested, i.e. exceeded the maximum solubility of test item in medium, obtained in a saturated solution prepared at a loading rate of 100 mg/L. The results of the test can be considered reliable without restrictions.

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Additional information