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Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-10 to 2017-03-21
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
according to guideline
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): JNJ-7982559-AAA (T001329)
- Physical state: solid (powder)
- Appearance: Grey-brown powder

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method: samples were taken before the start of the test, after 24h and after 72h from all test concentrations and from the control. Additionally, reserve samples of 2.0 mL were taken for possible analysis. 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

Test solutions

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 with a loading rate of 100 mg/L applying 20 minutes of ultrasonication followed by 3 days of magnetic stirring at room temperature. The obtain mixture was filtred through a 0.45 µm membrane filter (Whatman; RC55). The resulting SS was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. 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 provinding a cell density of 10^4 cells/mL.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All final test solutions were clear and colourless

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: fresh water algae
- Strain: NIVA CHL 1
- 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.
- 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 1 x 10¨4 cells/mL. The cell density was measured immediately before use.

- Acclimation period: not relevant

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

24 mg CACO3/L
Test temperature:
7.9 - 8.1
Dissolved oxygen:
not applicable
not relevant
not relevant
Nominal and measured concentrations:
nominal test concentrations: 100 % SS, 100% SS without algae
measured test concentrations t=0 h: 1.49, 1.32 mg/L
measured test concentrations t=24 h: 0.794, 0.607 mg/L
measured test concentrations t=72 h: 0.730, 0.355 mg/L

A combined limit/range-finding test was performed, during which six replicates of exponentially growing algal cultures, per group, were exposed to a control and to the undiluted SS. In addition, three replicates per group were exposed to 1.0 and 10% of the SS. Samples taken from the undiluted SS were analysed.
Details on test conditions:
- Test vessel: 100 mL all glass
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL of test solution
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 101.6 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- 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
- Light intensity and quality: 92 µE/m²/s 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 of the test, 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 (path length = 20 mm).
- effect calculated parameters: specific growth rate and yield

- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: combined limit/range-finding test
- Test concentrations: 1.0, 10 and 100 % of the SS
Reference substance (positive control):
Potassium Dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
> 100 other: % of a Saturated Solution prepared at 100 mg test substance/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
>= 100 other: % of a Saturated Solution prepared at 100 mg test substance/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
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- 72-h EC50 (yield and growth rate) >0.87 mg/L
- 72-h EC10 (yield and growth rate) >0.87 mg/L
- 72-h NOEC (yield and growth rate) = 0.87 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72-h EC50 (growth rate) = 1.2 mg/L (95% C.L. 1.1 to 1.2 mg/L)
- 72-h EC50 (yield) = 0.43 mg/L (95% C.L. 0.43 to 1.1 mg/L)
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used.
Two-sample t-test Procedure, alpha=0.05, one-sided, smaller).
The calculations were performed with ToxRat Professional v. 3.2.1.

Applicant's summary and conclusion

Validity criteria fulfilled:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance T001329 according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had no statistically significant inhibitory effect on the growth rate of Pseudokirchneriella subcapitata at any of the concentrations of T001329 tested during the 72-hour test period. The 72-h EC50 (growth rate and yield) were beyond the range tested, i.e. exceeded an average exposure concentration of 0.87 mg/L, corresponding to a 100% SS prepared at a loading rate of 100 mg/L. The 72-h NOEC (growth rate and yield) was determined to be >= 0.87 mg/L. The results of the test can be considered reliable without restrictions.